wenz iD proefscrift - Tesse Leunissen by wenz iD

Towards tailored antiplatelet therapy in vascular disease Tesse Charlotte Leunissen

Towards tailored antiplatelet therapy in vascular disease Tesse Leunissen

Towards tailored antiplatelet therapy in vascular disease Copyright ÂŠ T.C. Leunissen 2016

ISBN: 978-90-9030058-0 Layout and Design: Wendy Schoneveld || wenz iD || www.wenzid.nl Printed by: Gildeprint Drukkerijen, Enschede

Part of this thesis was supported by a grant from CSL Behring Publication of this thesis was financially supported by: Bayer, Olympus, the Surgical Company and Stago NL Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged

Towards tailored antiplatelet therapy in vascular disease Richting gepersonaliseerde behandeling met bloedplaatjesremmers in vaatziekten (met een samenvatting in het nederlands)

Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 20 december 2016 des middags te 12.45 uur. door

Tesse Charlotte Leunissen geboren op 19 januari 1990 te Breda

Promotoren:

Prof. dr. F. L. Moll Prof. dr. GJ. de Borst

Copromotoren: Dr. R. T. Urbanus Dr. M. Roest

Contents PART I Introduction and background Chapter 1

Introduction, objectives and outline

Chapter 2

The effect of P2Y12 inhibition on platelet activation assessed by aggregation- and flow cytometry based assays Accepted for publication in Platelets

Part II Platelet reactivity in peripheral arterial disease Chapter 3

Personalized antiplatelet therapy following endovascular revascularization in peripheral artery occlusive disease: A novel concept Eur J Vas Endovas Surg Short Reports, 2015 Oct; Issue 29, p11-17

Chapter 4

High on-treatment platelet reactivity in peripheral arterial disease: A pilot study to find the optimal test and cut off values Eur J Vas Endovas Surg, 2015 May; Volume 52, Issue 2, p198-204

Part III Platelet reactivity in carotid artery disease Chapter 5

The role of perioperative antiplatelet therapy and platelet reactivity testing in carotid revascularization: Overview of the evidence J Cardiovasc Surg, 2015 April; Volume 56, Issue 2, p 156-172

Chapter 6

Validation of the automated electronic micro emboli detection system in patients undergoing carotid endarterectomy Conditionally accepted for publication in Eur. Journal of Ultrasound

Chapter 7

Clopidogrel is the strongest suppressor of platelet reactivity and perioperative solid micro emboli during carotid endarterectomy

107

Chapter 8

Clinical risk factors and plaque characteristics associated with new development of contralateral stenosis in patients undergoing carotid endarterectomy Cerebrovascular Diseases, 2016 April; Volume 42, p 122-130

127

Part IV Platelet reactivity in cardiac disease Chapter 9

The use of platelet reactivity testing in patients on antiplatelet therapy for prediction of bleeding events after cardiac surgery Vasc Pharmacology, 2016 Feb; Volume 77, p 19-27

145

Chapter 10 Lower platelet reactivity is associated with presentation of unstable coronary artery disease Accepted for publication in the Int J of Angiology

167

Chapter 11 Reticulated platelets as predictor of postoperative troponin elevation and 30-day mortality Submitted to British Journal of Sugery

187

Part V Summary and discussion Chapter 12 General discussion and summary

207

Chapter 13 Nederlandse samenvatting

217

Part VI Appendix Review committee Dankwoord Authorâ&#x20AC;&#x2122;s list of publications Curriculum Vitae

228 229 234 236

PART I Introduction and background

Chapter 1 Introduction, objectives and outline

Chapter 1

Vascular disease Atherothrombotic diseases are an extensive healthcare problem 1 and responsible for >25% of total mortality worldwide, most commonly in low- and middle income countries.2 It can affect the entire vascular bed, from cerebral to peripheral arteries resulting in a range of conditions, such as stroke, myocardial infarction and peripheral arterial disease (PAD). In atherothrombotic disease, the vascular system is threatened by atherosclerosis; the lesions of atherosclerosis occur principally in large and medium-sized elastic and muscular arteries and may present throughout a person’s lifetime. The earliest type of lesion, the ‘’fatty streak’’ occurs already during foetal development 3 and contains primarily cholesterol, recruiting inflammatory cells as macrophages and T- lymphocytes.4 Consequent activation of these inflammatory cells leads to the release of hydrolytic enzymes, cytokines, chemokines, and growth factors, 5 which can induce further damage and eventually lead to core necrosis. The advanced, complicated atherosclerotic lesion develops when formation of fibrous tissue leads to further enlargement and restructuring of the lesion, so that it becomes covered by a fibrous cap. At some point, the atherosclerotic lesion intrudes the lumen of the artery and decreases the blood flow. Moreover the lesion can destabilize and rupture, leading to thrombosis on the ruptured surface of the lesions or form emboli that end more distally in the arterial bed, leading to clinical symptoms. High-risk lesions have a large lipid-rich necrotic core with an overlying thin fibrous cap infiltrated by inflammatory cells and diffuse calcification. Formation of new fragile and leaky vessels that invade the expanding intima contributes to enlarging the necrotic core and thereby increasing the vulnerability of the plaque. 6 Multiple cardiovascular risk factors as male sex, smoking, diabetes, hypertension, inactivity, high-fat diet, hypertension and positive family history for cardiovascular disease are known and form the basis for primary prevention. Patients at low risk for clinical events are unlikely to gain substantial benefits from pharmaceutical interventions and therefore should thus be managed with lifestyle modifications.7 Conversely patients at high risk for events are more likely to benefit from pharmacologic interventions and therefore are appropriate candidates for intensive risk factor modification efforts. Platelets in arterial thrombosis The involvement of platelets in atherothrombotic disease and the subsequent formation of occlusive thrombi depend on platelets adhesive properties and the ability to respond to stimuli with rapid activation. Platelets circulate in blood in a resting state and are cleared by macrophages in spleen and liver. Platelet are the monitors of the integrity of the vessel wall and are continuously searching for vessel wall injury. When vessel wall injury occurs and the extracellular matrix is exposed to the blood, platelets bind to collagen, followed by platelet–platelet interaction to form a plug that effectively seals the injured vessel wall to prevent excessive blood loss. The aggregated platelets form a surface for adherence of coagulation factors and form fibrin to stabilize the platelet plug. Activation of platelets is carefully regulated since spontaneous or continuous activation of platelets may lead to clinically thrombotic events. In normal physiology, the

Introduction

endothelium prevents platelet activation by secretion of nitric oxide, prostacyclin and exonucleotidases. 8 This physiological mechanism is hindered when an atherosclerotic plaque ruptures and a platelet plug is formed inside the intact artery. 9 Antiplatelet therapy Although arterial thrombosis is a multifactorial disease with diverse genetic and acquired predisposing risk factors, the importance of platelets in this process is widely recognized. As a result, many people are treated with antiplatelet therapies for secondary prevention of thrombotic cardiovascular events (CVE), such as myocardial infarction, cerebrovascular accident and transient ischemic attack. Since the 1970s, the effect of aspirin (reducing thromboxane formation by COX-1) as a platelet inhibitor has been studied extensively. 10 Aspirin has been very efficient in the prevention of secondary CVE; a meta- analysis of randomized studies of various antiplatelet drugs showed a 25% decrease in thrombotic vascular outcomes in patients with pre-existing conditions on aspirin.11 With the addition of alternative and more potent P2Y12 inhibitors such as clopidogrel, prasugrel and ticagrelor, the prevention of secondary CVE has become even more effective, however associated with increased bleeding risk. 12,13 Antiplatelet therapy (APT) using monotherapy aspirin or in combination with P2Y12 inhibitors has become standard treatment as primary, secondary and periprocedural prevention of CVE. 14 However, some patients still develop CVE despite treatment with antiplatelet therapy. 15 Pooled analysis showed that high platelet reactivity despite aspirin treatment was present in 22.2% of the patients with associated increased risk of developing CVE (RR: 2.09, 95%CI: 1.77-2.47). In 40.4% of the patients, high platelet reactivity despite clopidogrel treatment was present with even a higher associated increased risk for CVE (RR: 2.80: 95%CI: 2.40-3.27). In some patients, this phenomenon can be caused by a mutation in the genes coding for cytochrome P450 2C19 (CYP2C19) activity, a liver enzyme that converts the clopidogrel prodrug into its active metabolite. 16 Other independently related factors to high platelet reactivity despite clopidogrel treatment are non-compliance, diabetes mellitus, renal failure, and non-smoking. 17,18 Tailoring antiplatelet therapy The association of high platelet reactivity with increased risk of thrombotic events sparked the concept of tailoring APT based on platelet reactivity testing. The basis of the concept is that switching APT (to a higher dosage or different type APT) with the aim to achieve lower platelet reactivity would result in less thrombotic events, especially in the high-risk patients, as patients with diabetes mellitus or renal failure. Multiple studies suggest that not only high platelet reactivity should be an indicator to adjust APT; but that low platelet reactivity should also be considered as a reason to adapt APT, due to the increased risk of bleeding complications, such as gastro-intestinal and intra-cerebral bleeding complications.19,20 These studies suggest the presence of a therapeutic window for platelet reactivity with HPR at one end and LPR at the other end of the spectrum. Laboratory testing of ones platelet reactivity would enable guiding optimal antiplatelet

Chapter 1

therapy and thus reduce the risk of secondary cardiovascular events, but also the risk of bleeding events. Currently multiple tests are available for measuring in vitro platelet reactivity; these tests measure diverse pathways of thrombus formation and the reproducibility and applicability vary strongly. 21,22 So far none of the available tests has proven its superiority in predicting thrombotic events. However the Verify Now, a commercially available point-of-care test, shows congruent results with the Light Transmittance Aggregometry and is widely studied in clinical trials. 15 Up to now the clinical benefit of tailoring APT based on platelet function tests in patients undergoing PCI, showed disappointing results. Two major trials (GRAVITAS, n=2,21423 and ARCTIC, n=2,24024) showed no difference in their primary composite endpoint (death of cardiovascular causes, nonfatal acute myocardial infarction and ST-elevation) or bleeding complications after 6 months in GRAVITAS and 1 year in ARCTIC, when comparing standard APT with tailored APT. However a few smaller studies did show a beneficial effect of tailored vs. standard APT on a composite endpoint (e.g. cardiac death, stent thrombosis, recurrent ACS, re PCI< 1 year), without difference in bleeding complications.25-28 Currently two studies (TAILOR-PCI and POPULAR GENETICS) investigate the potential benefit of tailoring antiplatelet therapy based on CYP2C19 genotyping in patients with respectively acute coronary symptoms or stable angina pectoris and patients with STEMI. These studies are enrolling patients. Outline of this thesis The main objective of this thesis was to evaluate the role of platelet reactivity testing in vascular disease as stepping stone towards tailored antiplatelet therapy. We developed a platelet reactivity test for testing multiple pathways and markers of platelet activation, analysed the prognostic value of this test for different patient populations and compared the test results with the clinical golden standard, the VerifyNow. A future aim is to further optimize platelet reactivity testing (regarding timing of testing and suitable platelet reactivity test), defining cut-off values per patient population and define the appropriate therapy change for patient with high on treatment platelet reactivity. Part I gives a general introduction about vascular disease, antiplatelet therapy, platelet reactivity testing and tailoring antiplatelet therapy. A detailed background of the flowcytometry platelet activation test (PACT) can be found in chapter 2. Part II evaluates platelet reactivity testing in patients with peripheral arterial disease. Chapter 3 gives an example of the application of tailored antiplatelet therapy with an overview of the literature. In chapter 4 we investigate the proportion of patients with high on clopidogrel platelet reactivity in patient with PAD, different platelet reactivity tests, their correlation and the optimal timing for these tests as a stepping stone for a future trial investigating the potential benefit of tailored antiplatelet therapy in PAD patients. Part III starts with an overview of the evidence for antiplatelet therapy in carotid revascularization (chapter 5). We have studied the prognostic value of the PACT

Introduction

compared to the VerifyNow in association with the number of micro embolic signals measured with transcranial Doppler, in patients undergoing carotid endarterectomy. To quantify the amount of micro embolic signals, we validate the automatic emboli detection software against human experts (chapter 6). The results of the validation study are shown in chapter 7. Next we evaluate carotid plaque characteristics for the development of new contralateral stenosis (chapter 8). Part IV evaluates platelet reactivity measurement in patients undergoing cardiac surgery or angiography. Chapter 9 focusses on low platelet reactivity in contrary to the other chapters and gives an overview of platelet reactivity testing for prediction of bleeding events after cardiac surgery. Next we show in chapter 10 that in patients undergoing angiography, not taking P2Y12 inhibitors, low platelet reactivity is related to presentation of unstable coronary artery disease. Finally we end the thesis with chapter 11 where we investigate a potential new marker of cardiovascular risk: the young, reticulated platelets.

Chapter 1

References 15. 1. 2.

5. 6.

10.

11.

12.

13.

14.

Ross R. Atherosclerosis--an inflammatory disease. The New England journal of medicine. Jan 1999 Shanthi Mendis TA, Douglas Bettcher, Francesco Branca, Jeremy Lauer, Cecile Mace, Shanthi Mendis, Vladimir Poznyak, Leanne Riley, Vera Da Costa E Silva, Gretchen Stevens. Global status report on noncommunicable diseases 2014. Switzerland 2014. Napoli C, D’Armiento FP, Mancini FP, et al. Fatty streak formation occurs in human fetal aortas and is g re a t l y enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions. The Journal of clinical investigation. Dec 1997 Stary HC, Chandler AB, Glagov S, et al. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. May 1994 Ross R. Cellular and molecular studies of atherogenesis. Atherosclerosis. Jun 1997 Badimon L, Vilahur G. Thrombosis formation on atherosclerotic lesions and plaque rupture. Journal of internal medicine. Dec 2014 Andrus B, Lacaille D. 2013 ACC/AHA guideline on the assessment of cardiovascular risk. Journal of the American College of Cardiology. Jul 2014 van Hinsbergh VW. Endothelium--role in regulation of coagulation and inflammation. Seminars in immunopathology. Jan 2012 de Groot PG, Urbanus RT, Roest M. Platelet interaction with the vessel wall. Handbook of experimental pharmacology. 2012 Evans G, Packham MA, Nishizawa EE, Mustard JF, Murphy EA. The effect of acetylsalicyclic acid on platelet function. The Journal of experimental medicine. Nov 1968 Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. Jan 2002 Tang XF, Fan JY, Meng J, Jin C, Yuan JQ, Yang YJ. Impact of new oral or intravenous P2Y12 inhibitors and clopidogrel on major ischemic and bleeding events in patients with coronary artery disease: a meta-analysis of randomized trials. Atherosclerosis. Apr 2014 Gouya G, Arrich J, Wolzt M, et al. Antiplatelet treatment for prevention of cerebrovascular events in patients with vascular diseases: a systematic review and meta-analysis. Stroke; Feb 2014 Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-Society Consensus

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for the Management of Peripheral Arterial Disease (TASC II). Journal of vascular surgery. Jan 2007 Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 Scott SA, Sangkuhl K, Gardner EE, et al. Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450-2C19 (CYP2C19) genotype and clopidogrel therapy. Clinical pharmacology and therapeutics. Aug 2011 Serebruany V, Cherala G, Williams C, et al. Association of platelet responsiveness with clopidogrel metabolism: role of compliance in the assessment of “resistance”. American heart journal. Dec 2009 Gurbel PA, Bliden KP, Logan DK, et al. The influence of smoking status on the pharmacokinetics and pharmacodynamics of clopidogrel and prasugrel: the PARADOX study. Journal of the American College of Cardiology. Aug 2013 Sibbing D, Schulz S, Braun S, et al. Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. Journal of thrombosis and haemostasis : JTH. Feb 2010 Cuisset T, Cayla G, Frere C, et al. Predictive value of post-treatment platelet reactivity for occurrence of post-discharge bleeding after non-ST elevation acute coronary syndrome. Shifting from antiplatelet resistance to bleeding risk assessment? EuroIntervention : journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology. Aug 2009 Breet NJ, van Werkum JW, Bouman HJ, et al. Comparison of platelet function tests in predicting clinical outcome in patients undergoing coronary stent implantation. JAMA Feb 2010 Janssen PW, ten Berg JM. Platelet function testing and tailored antiplatelet therapy. Journal of cardiovascular translational research. Jun 2013 Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA : Mar 2011 Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 Siller-Matula JM, Francesconi M, Dechant C, et al. Personalized antiplatelet treatment after percutaneous coronary intervention: the MADONNA study. International journal of cardiology. Sep 2013 Wang XD, Zhang DF, Zhuang SW, Lai Y. Modifying clopidogrel maintenance doses according to

Introduction

vasodilator-stimulated phosphoprotein phosphorylation index improves clinical outcome in patients with clopidogrel resistance. Clinical cardiology. May 2011 27. Bonello L, Camoin-Jau L, Arques S, et al. Adjusted clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index decrease rate of major adverse cardiovascular events in patients with clopidogrel resistance: a multicenter randomized prospective study. Journal of the American College of Cardiology. Apr 2008 28. Valgimigli M, Campo G, de Cesare N, et al. Intensifying platelet inhibition with tirofiban in poor responders to aspirin, clopidogrel, or both agents undergoing elective coronary intervention: results from the double-blind, prospective, randomized Tailoring Treatment with Tirofiban in Patients Showing Resistance to Aspirin and/or Resistance to Clopidogrel study. Circulation. Jun 2009

Chapter 2 The effect of P2Y12 inhibition on platelet activation assessed by aggregation- and flow cytometry based assays Accepted for publication in Platelets

T. Leunissen, PP. Wisman, T. van Holten, P. de Groot, S. Korporaal, A. Koekman, F. Moll, M. Teraa, M. Verhaar, GJ. de Borst , R. Urbanus and M. Roest

Chapter 2

Abstract Patients on P2Y12 inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, only response to ADP does not necessarily reflect the effect of P2Y12 inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity towards other physiologically relevant agonists could provide more insight in the efficacy of P2Y12 inhibitors. The effect of in vitro and in vivo P2Y12 inhibition on ÎąIIbÎ˛3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12 inhibitors was compared to VerifyNow results. P2Y12 inhibitors showed strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12 inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed by flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response, identifies different patients as non-responders to P2Y12 inhibitors, compared to only ADP- induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.

Differential platelet activation

Introduction Platelets are the first to respond when the integrity of the vasculature is breached. They quickly adhere to sites of vascular damage, where they aggregate and form a platelet plug that prevents further blood loss. During platelet activation, intracellular alpha and dense granules fuse with the platelet membrane, leading to P-selectin expression and the release of multiple pro-inflammatory and vasoactive substances into the local environment. This causes secondary platelet activation and promotes interaction between the activated platelets and the endothelium or white blood cells. 1-3 Although the formation of a platelet plug is essential for the prevention of bleeding, an excessive platelet response can lead to vascular occlusion and downstream tissue ischemia. For this reason, patients with severe atherosclerosis are treated with different classes of platelet inhibitors, ranging from relatively mild anti-platelet drugs (COX-1 inhibitors) to a combination of COX-1 inhibitors and P2Y12 inhibitors, which induces stronger platelet inhibition. Clopidogrel is the mildest and most commonly prescribed P2Y12 inhibitor, whereas prasugrel, ticagrelor and cangrelor are prescribed when stronger platelet inhibition is required. Currently there is no evidence based approach for choosing the best P2Y12 inhibitor in individual patients. Although large-scale clinical studies have shown that P2Y12 inhibitors reduce thrombosis incidence in severe atherosclerotic patients, some patients are still insufficiently protected and develop thromboembolic events, while other patients on P2Y12 inhibitors may develop bleeding complications. 4,5 This suggests that there is a therapeutic window for platelet reactivity with insufficient platelet inhibition at one end of the spectrum and excessive platelet inhibition at the other end. Monitoring of platelet reactivity in patients on platelet inhibitors may prevent future thromboembolic and bleeding complications, although level I evidence from large randomized controlled trials is lacking. The most widely used platelet reactivity test in clinical trials and daily practice is the VerifyNow P2Y12 assay. 6,7 This assay specifically addresses the effect of treatment with P2Y12 inhibitors on the platelets adenosine diphosphate (ADP) pathway through two separate aggregation measurements: 1) ADP/prostaglandin E1 (PGE1)- induced aggregation to determine the effect of P2Y12 inhibition on platelet function and 2) thrombin receptor-activating peptide (TRAP)- induced platelet aggregation to determine baseline P2Y12 inhibitor-free platelet activation. 8 These two measurement are then used to calculate the percentage of platelet inhibition by clopidogrel. The premise of this system is that TRAP-induced platelet activation is unaffected by P2Y12 inhibitors. However, multiple studies have shown inhibitory effects of these drugs on PAR-1 and PAR-4 mediated platelet aggregation, both in platelet rich plasma (PRP) and in whole blood. 8-11 So far, there is no convincing evidence that tailoring antiplatelet therapy based on platelet reactivity tests is effective.12-14 This is not surprising, as the effect of P2Y12 blockade on ADP-induced platelet aggregation does not necessarily reflect the platelet response towards either collagen or thrombin, which are physiologically essential during platelet plug formation. Loss of thrombin activity greatly inhibits the formation of the thrombus core region and thus attenuates full platelet activation. 15

Chapter 2

Here, we investigated whether assessment of the platelet response to thrombin or collagen-mimetics allows identification of poor responders to P2Y12 inhibitors. To this end, we compared the effects of P2Y12 inhibition on several activation pathways and validated our findings in a cohort of patients receiving antiplatelet therapy.

Methods Reagents Adenosine 5’-diphosphate (ADP), apyrase and phosphoenolpyruvate (PEP) were from Sigma-Aldrich, (A2754-1G, A6132-1KU and 10108294001, Zwijndrecht, the Netherlands). Pyruvate kinase (PK) was from Roche (10128155001, Almere, the Netherlands). PAR-1 agonist SFLLRN was from Bachem (H-2936, Weil am Rhein, Germany) and PAR-4 agonist AYPGKF was synthesized at the Dutch Cancer Institute (NKI-AVL, Amsterdam, the Netherlands). Cross-linked Collagen-Related Peptide (CRP-xl) was a generous gift from Professor Richard Farndale (Cambridge, UK). HORM collagen was obtained from Hormon Chemie (10500, Boom BV, Meppel, the Netherlands). Phycoerythrin (PE) labeled mouse anti-human P-selectin (AK-4) and Alexa Fluor 647 (Alexa647) labeled mouse anti-human CD63 (H5C6), were from BD Pharmingen™ (55524 and 561983 Franklin Lakes, US). Fluorescein Isothiocyanate (FITC) labeled anti-human fibrinogen antibodies were obtained from Dako (F0111, Glostrup, Denmark). P2Y12 antagonist AR-C69931MX (cangrelor) was kindly provided by AstraZeneca, (Loughborough, UK). Optilyse-B was from Beckman Coulter (IM1400, Woerden, the Netherlands). Antibodies against human platelet factor 4 (PF4), β-thromboglobulin (β-TG), chemokine ligand 5 (RANTES/CCL5) and platelet derived growth factor (PDGF) A/B were from R&D systems (AF795, BAF393, AB-278-NA and DY222, Minneapolis, United States). Prostacyclin was from Cayman Chemicals (18220, Michigan, US). Patients Blood of 72 consecutive patients on P2Y12 inhibitors visiting the vascular surgery department of the University Medical Center Utrecht, was collected in 0.105 M tri-sodium citrate tubes (454321,Greiner Bio-One Vacuette) via venipuncture. Both VerifyNow responses and platelet reactivity as determined with flow cytometry were measured in the same sample. The type of P2Y12 inhibitor and indication for monitoring was recorded. Data was processed anonymously. Similar measurements for flow cytometry were performed for 43 consecutive healthy controls. Flow cytometry measurements of the 72 patients were normalized to the results of the 43 healthy controls. In the absence of an evidence-based cutoff value for the ideal platelet inhibition measured by VerifyNow, we arbitrarily adopted PRU<208 for clopidogrel as indicators of good response to clopidogrel, based on previous studies. 16-18. Blood from the 43 healthy controls, who claimed not to have taken antiplatelet therapy, was obtained through venipuncture and collected in 0.105 M tri-sodium citrate (367714,

Differential platelet activation

Becton Dickinson, Temse, Belgium) vacuum tubes. Approval for this study was obtained from the medical ethics review board of the UMC Utrecht. Informed consent requirement for patients on P2Y12 inhibitors was waived by the Institutional Review Board. All healthy donors gave written informed consent in accordance with the declaration of Helsinki. Assessment of platelet activation with flow cytometry Blood was diluted 1:10 in a reaction mixture containing a platelet agonist and FITCconjugated antibodies against fibrinogen (1:100), PE-conjugated antibodies against P-selectin (1:25) and Alexa647 conjugated antibodies against CD63 (1:25). Platelets were activated with ADP, CRP-xl, PAR-1 agonist SFLLRN or PAR-4 agonist AYPGKF in the presence or absence of 1 µM AR-C69931MX or 0.2 U/mL apyrase. After 20 minutes, samples were fixed in buffer containing 0.148% formaldehyde, 137 mM NaCl, 2.7 mM KCl, 1.12 mM NaH2PO4, 1.15 mM KH2PO4, 10.2 mM NaHPO4, and 4 mM EDTA (pH 6.8) and analyzed on a BD FACS Canto II flow cytometer (BD-biosciences, San Jose, United States) on the same day of processing. Single platelets were gated based on forward and side scatter properties. Fibrinogen binding was used as a measure of αIIbβ3 activation and P-selectin- and CD63 expression as markers of granule release. Data are expressed as median fluorescence intensity (MFI) for dose-response curves, or normalized to the median MFI values obtained in 43 healthy controls. The normalized data were used to calculate the percentage inhibition of platelet reactivity in P2Y12 inhibitor users as compared to healthy controls without antiplatelet therapy. We used an in-house cut-off for poor platelet reactivity, set at the 2.5th percentile of the response in 122 healthy donors. Light transmission aggregometry Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 160g for 15 minutes at room temperature. The supernatant was collected in a new tube. The remaining blood was centrifuged for 10 minutes at 2000g and this supernatant (plasma) was used to adjust the PRP to a platelet count of approximately 200-250 x109/L. Platelet aggregation was assessed after addition of ADP (2 or 10 µM), PAR-1 agonist (2 or 10 µM), PAR-4 agonist (100 or 250 µM) and CRP-xl (12.5 or 200 ng/ml) in the presence or absence of 1 μM AR-C69931MX on a Chronolog Optical Lumi-Aggregometer (Havertown, PA, USA). Aggregation was monitored for fifteen minutes at 37 degrees Celsius at 900 rpm Release of alpha granule content Washed platelets were stimulated with 156 µM PAR-1 agonist with or without 1 µM ARC69931MX for 20 minutes. Platelets were centrifuged at 4000g for 2 minutes and the supernatant was collected. ELISA’s were performed on the supernatant for PF-4, β-TG, RANTES/CCL5 and PDGF-A/B as described before. 19 In short, plates were coated with capture antibodies overnight at 4°C and subsequently blocked with 1% BSA in PBS. Samples were diluted and incubated on the ELISA plates. Bound factors were detected

Chapter 2

with biotin coupled antibodies raised against PF-4, β-TD, RANTES and PDGF-A/B in PBS with 1% bovine serum albumin (BSA). Plates were washed five times with 0.05% Tween20 in PBS pH 7.4 between each subsequent incubation step. Biotin coupled antibodies were detected with Super Signal West Pico Chemiluminescent substrate (Westburg, Leusden, the Netherlands) and read with a Spectramax L Luminometer (Molecular Devices, Sunnyvale, CA, USA). Sample concentrations were deduced from a calibration curve of normal pooled serum with known concentrations. Platelet ADP/ATP release Washed platelets from 4 healthy donors and 3 patients on P2Y12 inhibitors were stimulated with vehicle, 10 µM PAR-1 agonist or 100 ng/mL CRP-xl, with or without 1µM ARC69931MX. After 20 minutes, reactions were stopped with EDTA (0.5 mM final concentration) and prostacyclin (10 ng/ml), after which platelet suspensions were centrifuged at 500g for 10 minutes. The supernatant was used for ATP/ADP quantification. Supernatants were divided into two fractions. In the first fraction, ADP was converted into ATP by incubation with 95 µM PEP and 25 µg/mL PK in 0.2 M Tris-Maleate, 10 mM KCl, 15 mM MgSO4, pH 7.4 for 15 minutes at 37°C. Reactions were stopped by heating the samples at 80°C for 10 minutes. ATP levels in both fractions were determined with the ATPLite 1 step kit (6016731, Perkin Elmer, Waltham, MA, USA) according to the instructions of the manufacturer. ATP levels were deduced from an ATP calibration curve. ADP levels were calculated by subtracting the ATP levels in the second fraction from those in the first. Statistical analysis Differences in final aggregation with and without P2Y12 inhibitor were calculated per agonist concentration with the non-parametric Wilcoxon signed-Rank test. Differences in αIIbβ3 activation, P-selectin and CD 63 expression with and without apyrase or ARC69931MX, assessed with flow cytometry and differences in alpha- and dense granule release by presence and absence of P2Y12 inhibition, were also calculated with the nonparametric Wilcoxon signed-Rank test. Differences in αIIbβ3 activation after stimulation with ADP, PAR-1 agonist or CRP-xl between responders and non-responders were calculated with the Mann-Whitney U test for independent samples. All statistic tests were performed using IBM SPSS Statistics Version 21 (Armonk, New York, USA).

Results P2Y12 inhibitors strongly inhibit αIIbβ3 activation after PAR-1, PAR-4 and CRP-xl stimulation, despite mild inhibition of platelet aggregation Full platelet activation requires both activation of the fibrinogen receptor integrin αIIbβ3 and the secretion of alpha and dense granule content into the extracellular environment. To compare the effects of P2Y12 inhibition on ADP mediated platelet activation with those

Differential platelet activation

on thrombin and collagen mediated platelet activation, we first studied the effects of P2Y12 inhibition on platelet aggregation. P2Y12 inhibition completely prevented ADP-induced platelet aggregation, but only partially attenuated aggregation induced with low doses of PAR-1 agonist, PAR-4 agonist or CRP-xl (figure 1a and 1b). P2Y12 inhibition had no effect on platelet aggregation induced with higher concentrations of the same agonists. Next, we studied the effect of P2Y12 inhibition on αIIbβ3 activation as measured with flow cytometry. As expected, addition of cangrelor or apyrase to platelets stimulated with ADP almost completely abrogated αIIbβ3 activation (figure 1c). Addition of cangrelor or apyrase to platelets stimulated with PAR-1 agonist, PAR-4 agonist or CRP-xl resulted in strong inhibition of αIIbβ3 activation (figure 1c and Supplemental figure 1).

Figure 1. Fibrinogen binding after stimulation in the presence of P2Y12 antagonism. A Light transmission aggregation was recorded after stimulation with high and low concentrations of ADP, PAR1 and PAR-4 agonist and CRP-xl in the presence and absence of cangrelor (AR-C) in four healthy donors. Mean and standard error of mean are shown for the final amplitude of aggregation after 14 minutes. B Representative aggregation traces. C. Platelets were stimulated with increasing concentrations of ADP, PAR-1, PAR-4 agonist or CRP-xl in presence of cangrelor (dark grey), apyrase (light grey) or buffer only (black) and fibrinogen binding was analyzed with flow cytometry as a measure of αIIbβ3 activation. Area under the curve of αIIbβ3 activation in presence of cangrelor or apyrase are expressed as a percentage of mock treated platelets. Mean and standard error of the mean are shown. Measurements were performed in 5 healthy donors. (*: p<0.05)

Chapter 2

Dense granule secretion is more strongly reduced than the release of α-granule content by P2Y12 inhibitors Next, we analyzed the effect of P2Y12 inhibition on platelet granule secretion. Whereas ADP-induced P-selectin expression was completely prevented by addition of cangrelor or apyrase, P-selectin expression was only mildly reduced in platelets stimulated with PAR-1 agonist, PAR-4 agonist or CRP-xl (figure 2a) as measured with flow cytometry (Supplemental figure 1. Dose response curves for all agonists). Since P-selectin is present in both alpha and dense granules, we next investigated the effect of P2Y12 inhibition on PAR-1-mediated release of alpha granule content. PAR-1 induced secretion of RANTES (figure 2b), PF4 (figure 2c), β-TG (figure 2d), or PDGF A/B (figure 2e) was similar between platelets treated with cangrelor and untreated platelets, which suggests that P2Y12 inhibition scarcely influences PAR-1-mediated alpha granule secretion. We therefore investigated whether P2Y12 inhibition influences dense granule release. As expected, ADP induced CD63 expression was completely abolished by addition of cangrelor or apyrase (figure 3a). However, CD63 expression in platelets stimulated with PAR-1 agonist, PAR-4 agonist or CRP-xl was mildly reduced, as to a similar extent as P-selectin expression, in platelets treated with P2Y12 inhibitors (Supplemental figure 1). Interestingly, both ATP (figure 3b, c) and ADP release (figure 3d, e) was significantly reduced in platelets treated with P2Y12 inhibitor after stimulation with PAR-1 agonist (figure 3b, d) or CRP-xl (figure 3c, e). Multiple good responders to P2Y12 inhibitors, identified with VerifyNow, show normal αIIbβ3 activation after stimulation with PAR-1 and CRP-xl Because the response to ADP differed substantially from the response to either PAR-1 agonist, PAR-4 agonist, or CRP-xl in platelets treated with P2Y12 inhibitors in vitro, we determined the platelet response (agonist induced fibrinogen binding and P-selectin expression) during antiplatelet therapy in a cross-sectional cohort of 72 patients that visited the outpatient clinic of the vascular surgery department (figure 4a-c). Antiplatelet therapy is shown in table 1. Platelet responses were normalized on the median response of a group of 43 healthy individuals. The threshold of whether or not adequate platelet inhibition was achieved was set at the 2.5th percentile of the normal response. The VerifyNow P2Y12 assay identified 18 patients (25.0%) as non-responders with the cut-off set at PRU < 208. The majority of patients on P2Y12 inhibitors had a strongly reduced response to ADP (figure 4a). After ADP stimulation only four patients (5.6%) showed normal remaining αIIbβ3 activation and seven patients (9.7%) showed normal remaining P-selectin expression. In concordance with our in-vitro data, the inhibition of αIIbβ3 activation was stronger than inhibition of granule release. 10% of patients had normal fibrinogen binding after stimulation with PAR-1 agonist, but P-selectin expression remained normal in 45.8% of the patients (Figure 4b). Activation of αIIbβ3 after stimulation with CRP-xl was normal in 31.9% of patients and P-selectin expression remained normal in 66.7% of the patients (figure 4c).

Differential platelet activation

Figure 2. Alpha- granule release after stimulation in the presence of P2Y12 antagonism. Platelets were stimulated with increasing concentrations of ADP, PAR-1, PAR-4 agonist or CRP-xl in presence of cangrelor (AR-C) (dark grey), apyrase (light grey) or buffer only (black) and P-selectin expression was analyzed with flow cytometry as a measure of granule secretion. A. Area under the curve of P-selectin expression in the presence of cangrelor or apyrase are expressed as a percentage of mock treated platelets. Mean and standard error of the mean are shown. Measurements were performed in 5 healthy donors. B. Platelets were activated with PAR-1 agonist with or without cangrelor and concentrations of RANTES, C platelet factor 4 (PF-4), D β-thromboglobulin (β-TG) and E PDGF-A/B were determined with ELISA. Data are expressed as mean ± SEM and standard error of the mean are shown. (*: P<0.05).

Chapter 2

There was little agreement in the identification of responders and non-responders to P2Y12 inhibitors between VerifyNow and platelet reactivity assessment with flow cytometry. However, fibrinogen binding assessed with flow cytometry was lower in VerifyNow-identified responders than in non-responders after stimulation with ADP (p=0.001) (figure 4d). No difference in fibrinogen binding was found after stimulation with PAR-1 agonist and CRP-xl. P-selectin expression was similar between VerifyNowresponders and non-responders after stimulation with all agonists (data not shown).

Table 1. Characteristics and test results of anonymous patients on P2Y12 inhibitors. N =72

VN PRU post

VN % inhibition

ADP (fibrinogen binding, %)

PAR-1 (fibrinogen binding, %)

CRP-xl (fibrinogen binding, %)

Clopidogrel monotherapy

180 (161-219)

29 (19-41)

8.3 (5.3-16.5)

18.5 (13.3-35.6)

37.5 (23.8-63.2)

Aspirin & clopidogrel

160 (107-286)

29 (0-60)

18.9 (10.3-33.3)

24.7 (17.8-39.5)

40.1 (20.4-87.4)

Prasugrel

128 (64 -165)

65 (44-78)

10.4 (5.8-17.9)

20.9 (12.- 32.5)

26.4 (21.9-48.6)

Ticagrelor

227 (191-262)

39 (38-40)

12.6 (9.7-15.5)

25.7 (7.4-44.0)

39.2 (11.8-66.7)

Results are displayed as median with interquartile range. ADP adenosine diphosphate, CRP â&#x20AC;&#x201C; xl crosslinked collagen related peptide, PAR protease activated receptor,PRU P2Y12 Reactive Units, VN VerifyNow

Discussion The efficacy of antiplatelet therapy with P2Y12 inhibitors for thromboprophylaxis is mostly monitored with tests that measure the platelet response to P2Y12-ligand ADP, ignoring the response to other physiologically relevant platelet agonists. This study shows that P2Y12 inhibitors not only attenuate the response to ADP, but also have a strong inhibitory effect on the response of platelets to collagen and thrombin. Measurement of ÎąIIbÎ˛3 activation after stimulation with collagen or thrombin in addition to the response to ADP provides insight in the inhibitory status of patients receiving antiplatelet therapy and might allow identification of patients with suboptimal platelet inhibition that benefit from more stringent antiplatelet therapy. There are marked differences in the effects of P2Y12 inhibition on the ADP pathway and those on the collagen or thrombin pathways of platelet activation. P2Y12 inhibitors completely abrogate the capacity to aggregate after ADP stimulation. In contrast, P2Y12 inhibition only mildly attenuates the platelet capacity to aggregate after stimulation with low dose thrombin-receptor agonist or CRP-XL. Interestingly, platelet aggregation is normal at high concentrations of these agonists, despite a greatly reduced capacity of the platelets to bind fibrinogen. P2Y12 inhibition has been shown to reduce thrombus

Differential platelet activation

Figure 3. Dense granule release after stimulation in the presence of P2Y12 antagonism. Platelets were stimulated with increasing concentrations of ADP, PAR-1, PAR-4 agonist or CRP-xl in presence of cangrelor (dark grey), apyrase (light grey) or buffer only (black) and CD63 expression was analyzed with flow cytometry as a measure of dense granule secretion. A. Area under the curve of CD63 expression in the presence of cangrelor or apyrase are expressed as a percentage of mock treated platelets. Mean and standard error of the mean are shown. Measurements were performed in 5 healthy donors. ATP release (B, D) and ADP release (C, E) was measured in healthy donors (n=4) in the presence and absence of cangrelor and in blood of 3 patients on P2Y12 inhibitors after stimulation with PAR-1 agonist (B, C) and CRP-xl (3D ,E). Data are expressed as mean Âą SEM and standard error of the mean are shown. (*= p<0.05)

Chapter 2

growth after vascular injury, leaving platelet granule release unaffected. 15 This is in line with the attenuated response towards collagen- and thrombin mimetics after P2Y12 inhibition that we and others observed with flow cytometry, 20 but is at odds with the lack of effect of P2Y12 inhibition we observed in aggregation experiments with the same agonists. This disparity between flow cytometric analysis and aggregation based analysis of platelet reactivity might be explained by a threshold in αIIbβ3 activation above which platelet aggregation can take place. When platelet stimulation results in αIIbβ3 activation exceeding this threshold, aggregation-based assays lose their sensitivity for the effects of platelet inhibiting drugs, whereas flow cytometric quantification of platelet activation can still differentiate between good and poor responses to stimuli. However, currently LTA is still accepted as gold standard for laboratory platelet reactivity testing: high platelet reactivity after 5 or 20 µmol ADP increased the risk of MACE with resp. 3.25 [95%CI: 1.86–5.67] or 2.98 [95%: 1.88–4.72]. 6 Sensitivity and specificity of the LTA ADP 5 and 20 µmol for prediction of atherothrombotic events was 54.6- 60.2% and 53.1-60.9%. 14

Figure 4. Platelet reactivity testing in patients on P2Y12 inhibitors. Inhibition of αIIbβ3 activation and P- selectin expression measured in 72 patients on P2Y12 inhibitors, normalized against the response in 43 healthy donors. Black symbols represent responders to P2Y12 inhibitors according to the VerifyNow P2Y12 assay (based on cut-off PRU < 208 ) and grey symbols represent non-responders A Platelets were stimulated with 31.25 µM ADP , B 100 µM PAR-1 agonist or C 1000 ng/mL CRP-xl (5c). The dotted lines represent the lowest 2.5th percentile of fibrinogen binding and P-selectin expression of the healthy donors. D Percentage comparison of fibrinogen binding after stimulation with ADP, PAR-1 agonist or CRP-xl between healthy donors (controls), responders to P2Y12 inhibition and non-responders. (*= p<0.05).

Differential platelet activation

In this study , the influence of differential set-up conditions between flow cytometry and LTA might also play part; whole blood diluted 1:10 was used for flow cytometry, diminishing GPIIbIIIa outside-in signalling and potentially the sustained fibrinogen binding. LTA is performed with vigorous stirring of the sample, which may activate platelets, which does not occur in flow cytometry assays. Lastly, the temperature of the LTA (37 ° Celsius) might have induced full platelet activation, in contrast to room temperature of flow cytometry. While ADP-induced granule release is fully prevented by P2Y12 inhibitors, granule release after stimulation of the thrombin or collagen pathways is affected to a lesser extent. This is in line with previous studies by other groups, which reported only moderate reduction in P-selectin expression after stimulation of the thrombin pathway. 21-23 Our data might indicate that the reduction in P-selectin expression is mainly due to the inhibition of dense granule release, while alpha granule release remains largely unaffected. Alpha granules are the major transporters of VEGF, PDGF, TNF-α, TGF-β, PF-4, β-TG, RANTES and thrombospondin through the blood and can release their content at high concentrations at the site of injury. Physiologically, this may be important for the regulation of wound healing and neo vascularization. Our findings that P2Y12 inhibitors have minor impact on the expression of P-selectin indicate that the platelets keep their capacity to regulate interactions between platelets and endothelial cells, white blood cells and progenitor cells via P-selectin-PSGL-1 interaction or CD40L-CD40 interaction.24,25 Large clinical trials have shown that treatment with P2Y12 inhibitors may significantly reduce the number of secondary cardiovascular events on population level. 26-29 However, on individual patient level there is still recurrence of thrombotic events, while other patients develop bleeding complications.4,5 A meta-analysis by our group summarized that high on treatment platelet reactivity is an important predictor for secondary thrombotic events. 6 Both the thromboembolic and bleeding complication rate might be further reduced by tailored adjustment of treatment intensity, based on adequate platelet reactivity monitoring of individual patients. However, clinical trials with patients undergoing percutaneous coronary interventions found that tailored treatment adjustment based on VerifyNow platelet reactivity monitoring did not reduce the recurrence rate of arterial thrombosis, when compared to patients who were not monitored. 13,30 Although these first attempt were disappointing, this does not completely rule out the potential benefit of tailored antiplatelet therapy. Platelet reactivity testing is still in its infancy and the VerifyNow has severe limitations, including a varying sensitivity in studies for the prediction of thrombotic events.31-33 More importantly, the read-out of the VerifyNow is restricted to the P2Y12 activation pathway and does not quantify the other major physiological platelet activation pathways. Our data show that platelet reactivity measurements based on multiple activation pathways provide more insight in platelet inhibition status than measurements limited to the ADP pathway. In fact, a large percentage of responders according to the VerifyNow retained normal fibrinogen binding after stimulation with PAR-1 agonist and CRP-xl (inducing the

Chapter 2

strongest primary platelet activation pathways) despite strongly reduced responses to ADP, suggesting poor platelet inhibition. Overall, this study showed little agreement between the results of the VerifyNow P2Y12 assay and the flow cytometric analysis of ÎąIIbÎ˛3 binding in patients receiving P2Y12 inhibitors; although there were differences in fibrinogen binding between VerifyNow responders and non-responders, these differences were small and there was considerable overlap between groups. It is therefore doubtful that these differences can be used to guide treatment in clinical practice. A number of groups all over the world are currently developing comprehensive assays for the detection of platelet dysfunction in bleeding disorders. Amongst these are assays based on flow cytometric analysis of microaggregate formation after stimulation with various agonists.34 This assay requires much lower numbers of platelets than conventional LTA, which makes the assay suitable for thrombocytopenic samples. Another approach is based on the analysis of various stages of thrombus formation after perfusion of whole blood over arrays of 52 microspotted adhesive surface at high shear stress.35 A third example is the Optimul assay, a 96-well plate-based assay that allows an appraisal of 7 platelet activation pathways.36 The assay is based on light absorbance through stirred PRP and resembles the traditional method of the LTA. Yet, the Optimul is faster and requires less blood than LTA. All of these assays could be used for monitoring of antiplatelet therapy, although none of these assays have been used for this purpose to this date. A point of care platelet reactivity test that is further along in its development is the Multiplate, which detects the increase of electrical impedance resulting from the adhesion and aggregation of platelets on two metal sensor wires. To stimulate the aggregation, multiple agonists can be used to test different platelet activation pathways. An attempt has been made to determine a therapeutic window for APT dosage based on the Multiplate results in patients with coronary artery disease. 37 However, measuring platelet function by global aggregation measure approach is usually less specific to evaluate the effect of specific antiplatelet drug. One of the limitations of our study is that we were not able to investigate the association between platelet fibrinogen binding after stimulation with ADP, thrombin receptor agonists or collagen mimetics and clinical outcome during follow-up. Future clinical studies should indicate whether assessment of additional platelet activation pathways will improve the predictive value of antiplatelet therapy monitoring on the individual patient level. In summary, we show that P2Y12 inhibition affects not only the ADP platelet activation pathway, but also the thrombin and collagen pathways. Flow cytometry based assays are more sensitive for decreases in the platelets response to collagen and thrombin than aggregation based assays. Additional insight in the inhibitory status of patients receiving antiplatelet therapy can be obtained by measurements of platelet fibrinogen binding after stimulation of thrombin- or collagen receptors. These additional insights can improve identification of patients with suboptimal platelet inhibition, who might benefit from more stringent antiplatelet therapy.

Differential platelet activation

References 1.

Stellos K, Gawaz M. Platelet interaction with progenitor cells: potential implications for regenerative medicine. Thrombosis and haemostasis. Nov 2007 2. Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. The Journal of clinical investigation. Dec 2005 3. Ghasemzadeh M, Hosseini E. Platelet-leukocyte crosstalk: Linking proinflammatory responses to procoagulant state. Thrombosis research. Mar 2013 4. Sibbing D, Schulz S, Braun S, et al. Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. Journal of thrombosis and haemostasis : JTH. Feb 2010 5. Cuisset T, Cayla G, Frere C, et al. Predictive value of post-treatment platelet reactivity for occurrence of post-discharge bleeding after non-ST elevation acute coronary syndrome. Shifting from antiplatelet resistance to bleeding risk assessment? EuroIntervention : journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology. Aug 2009 6. Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 7. Bouman HJ, Parlak E, van Werkum JW, et al. Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel. Journal of thrombosis and haemostasis : JTH. Mar 2010 8. Lordkipanidze M, Pharand C, Nguyen TA, Schampaert E, Diodati JG. Assessment of VerifyNow P2Y12 assay accuracy in evaluating clopidogrel-induced platelet inhibition. Therapeutic drug monitoring. Jun 2008 9. Behan MW, Fox SC, Heptinstall S, Storey RF. Inhibitory effects of P2Y12 receptor antagonists on TRAP-induced platelet aggregation, procoagulant activity, microparticle formation and intracellular calcium responses in patients with acute coronary syndromes. Platelets. March 2005 10. Badr Eslam R, Lang IM, Koppensteiner R, Calatzis A, Panzer S, Gremmel T. Residual platelet activation through protease-activated receptors (PAR)-1 and -4 in patients on P2Y12 inhibitors. International journal of cardiology. Sep 2013 11. Jakubowski JA, Zhou C, Egan B, et al. Modification of the VerifyNow(R) P2Y12 test BASE channel to accommodate high levels of P2Y(12) antagonism. Platelets. 2011 12. Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary

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intervention: the GRAVITAS randomized trial. JAMA March 2011 Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 Breet NJ, van Werkum JW, Bouman HJ, et al. Comparison of platelet function tests in predicting clinical outcome in patients undergoing coronary stent implantation. JAMA Feb 2010 Stalker TJ, Traxler EA, Wu J, et al. Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood. March 2013 Trenk D, Stone GW, Gawaz M, et al. A randomized trial of prasugrel versus clopidogrel in patients with high platelet reactivity on clopidogrel after elective percutaneous coronary intervention with implantation of drug-eluting stents: results of the TRIGGER-PCI (Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy With Prasugrel) study. Journal of the American College of Cardiology. Jun 2012 Storey RF, Angiolillo DJ, Bonaca MP, et al. Platelet Inhibition With Ticagrelor 60 mg Versus 90 mg Twice Daily in the PEGASUS-TIMI 54 Trial. Journal of the American College of Cardiology. Mar 2016 Alexopoulos D, Stavrou K, Koniari I, et al. Ticagrelor vs prasugrel one-month maintenance therapy: impact on platelet reactivity and bleeding events. Thrombosis and haemostasis. Sep 2014 van Holten TC, Bleijerveld OB, Wijten P, et al. Quantitative proteomics analysis reveals similar release profiles following specific PAR-1 or PAR-4 stimulation of platelets. Cardiovascular research. Jul 2014 Nylander S, Mattsson C, Ramstrom S, Lindahl TL. The relative importance of the ADP receptors, P2Y12 and P2Y1, in thrombin-induced platelet activation. Thrombosis research. 2003 Storey RF, Sanderson HM, White AE, May JA, Cameron KE, Heptinstall S. The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity. British journal of haematology. Sep 2000 Storey RF, Newby LJ, Heptinstall S. Effects of P2Y(1) and P2Y(12) receptor antagonists on platelet aggregation induced by different agonists in human whole blood. Platelets. Nov 2001 Klinkhardt U, Bauersachs R, Adams J, Graff J, Lindhoff-Last E, Harder S. Clopidogrel but not aspirin reduces P-selectin expression and formation of platelet-leukocyte aggregates in patients with atherosclerotic vascular disease. Clinical pharmacology and therapeutics. Mar 2003

Chapter 2

24. Lievens D, Zernecke A, Seijkens T, et al. Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis. Blood. Nov 2010 25. Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature. Feb1998 26. Yusuf S, Zhao F, Mehta SR, Chrolavicius S, Tognoni G, Fox KK. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. The New England journal of medicine. Aug 2001 27. Lee M, Wu YL, Saver JL, et al. Is clopidogrel better than aspirin following breakthrough strokes while on aspirin? A retrospective cohort study. BMJ open. 2014 28. Oâ&#x20AC;&#x2122;Gara PT, Kushner FG, Ascheim DD, et al. 2013 ACCF/AHA guideline for the management of ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines. Circulation. Jan 2013 29. Clark MG, Beavers C, Osborne J. Managing the acute coronary syndrome patient: Evidence based recommendations for anti-platelet therapy. Heart & lung : the journal of critical care. Jan 2015 30. Price MJ, Angiolillo DJ, Teirstein PS, et al. Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: a timedependent analysis of the Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety (GRAVITAS) trial. Circulation. Sep 2011 31. Gremmel T, Steiner S, Seidinger D, Koppensteiner R, Panzer S, Kopp CW. Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation. Thrombosis and haemostasis. Feb 2009 32. Flechtenmacher N, Kammerer F, Dittmer R, et al. Clopidogrel Resistance in Neurovascular Stenting: Correlations between Light Transmission Aggregometry, VerifyNow, and the Multiplate. AJNR. American journal of neuroradiology. Aug 2015 33. Zhang HZ, Kim MH, Jeong YH. Predictive values of post-clopidogrel platelet reactivity assessed by different platelet function tests on ischemic events in East Asian patients treated with PCI. Platelets. 2014 34. De Cuyper IM, Meinders M, van de Vijver E, et al. A novel flow cytometry-based platelet aggregation assay. Blood. Mar 2013 35. de Witt SM, Swieringa F, Cavill R, et al. Identification of platelet function defects by multi-parameter assessment of thrombus formation. Nature communications. 2014 36. Lordkipanidze M, Lowe GC, Kirkby NS, et al. Characterization of multiple platelet activation pathways in patients with bleeding as a highthroughput screening option: use of 96-well Optimul assay. Blood. Feb 2014

37. Petricevic M, Milicic D, White A, et al. Development of a concept for a personalized approach in the perioperative antiplatelet therapy administration/ discontinuation management based on multiple electrode aggregometry in patients undergoing coronary artery surgery. Journal of thrombosis and thrombolysis. Jul 2015

Supplemental figure 1. Dose response curve for fibrinogen binding, P-selectin and CD63 expression per agonist. The dose response curve of ÎąIIbÎ˛3 activation (A, B, C and D), P-selectin (E, F, G and H) and CD63 (I, J, K and L) expression are shown after platelet stimulation with increasing concentrations of ADP, PAR-1, PAR-4 agonist or CRP-xl in presence of cangrelor (AR-C) (dark grey), apyrase (light grey) or buffer only (black) and measured with flow cytometry. Response is expressed in arbitrary units (AU).

Differential platelet activation

Supplemental

PART II Platelet reactivity in peripheral arterial diseas

Chapter 3 Personalized antiplatelet therapy following endovascular revascularization in peripheral artery occlusive disease: A novel concept Eur J Vas Endovas Surg Short Reports, 2015 Oct; Issue 29, p11-17

T. Leunissen*, S. Peeters Weem*, M. Teraa, E.J. Vonken, GJ. de Borst, F. Moll * Authors contributed equally to the manuscript

Chapter 3

Abstract Case A 73 year old patient with a longstanding history of peripheral artery occlusive disease (PAOD) presented with an acute on chronic progression of symptoms, based on a long occlusion of the superficial femoral artery (SFA), which was treated by thrombosuction, percutaneous transluminal angioplasty, and SFA stenting. Post-procedural dual antiplatelet therapy was initiated and subsequently adjusted based on platelet reactivity testing. Discussion Increasingly complex arterial lesions are treated by an endovascular approach; however, long-term patency rates are often disappointing. In order to optimize the patency rates (dual) antiplatelet therapy is initiated. It is known that a substantial proportion of patients have high platelet reactivity despite the use of antiplatelet drugs. Several methods have been published to test the individual response to different antiplatelet drugs. There is evidence that adjusting antiplatelet therapy based on platelet reactivity testing results in a reduction of cardiovascular events and bleeding complications; however, the optimal test and the exact role of personalized antiplatelet therapy in PAOD is currently unknown. Conclusion Although some important hurdles should be overcome before routine implementation, the concept of post-procedural antiplatelet therapy in patients with PAOD is advocated in order to optimize the results of endovascular interventions, as apparent from the presented case.

Personalised antiplatelet therapy

Case A 73-year old male with acute progression of chronic peripheral arterial disease (PAD) Rutherford stage 5, visited the outpatient clinic of a tertiary vascular referral center. The patient had a medical history comprising kidney transplantation, stage 3 chronic kidney disease (CKD), deep venous thrombosis complicated by a pulmonary embolism and intermittent claudication for which he underwent stenting of both common iliac arteries 10 years earlier and was prescribed aspirin. A digital subtraction angiography (DSA) was performed, which revealed a 23cm long occlusion of the superficial femoral artery (SFA) and the proximal anterior tibial artery (Figure 1A). Thrombosuction (AngioJet, Boston Scientific) and subsequently percutaneous transluminal angioplasty (PTA) of the SFA were performed (Figure 1B). Because of multilevel residual stenosis, three self-expandable stents and a balloon expandable stent were placed in the SFA over a total length of 27cm, with satisfactory results (no residual stenosis >30%). After the procedure dual antiplatelet therapy (DAPT) was initiated; clopidogrel (loading dose 300mg) was added to the aspirin. The next day a VerifyNow P2Y12 assay (Accumetrics, San Diego, USA) and a CYP2C19 polymorphism DNA test (Spartan RX CYP2C19, Spartan Bioscience Inc., Ottawa, Canada) were performed, which showed 0% platelet inhibition and two loss-of-function CYP2C19 alleles, respectively, which suggested that clopidogrel was not effective. Therefore clopidogrel was switched to the stronger P2Y12 inhibitor, prasugrel. A VerifyNow showed an effective platelet inhibition on prasugrel (41% inhibition, PRU 171). At six months follow-up the patient did not report any pain, the duplex ultrasound confirmed patency of the stents without restenosis. Endovascular revascularization Nowadays, minimally invasive endovascular techniques are often the first line of therapy in PAD. The choice of the specific endovascular technique depends on various factors, like the cause, length and degree of stenosis or occlusion, and the duration of the occlusion. Mechanical thrombectomy or thrombosuction is most effective in removing fresh thrombi, causes minimal peripheral embolization and is less time-consuming than medicinal thrombolysis.1 PTA is usually the preferred choice in limited disease such as stenosis or occlusions up to 10 cm in length and provisional stenting is advised when there is a residual stenosis of â&#x2030;Ľ30-50% or a flow-limiting dissection.2,3 One-year primary patency rates of balloon angioplasty in long femoral occlusions vary from 27 to 43.5% 4, which is improved by the use of drug-eluting balloons (primary patency to 76.1%).5 Primary patency at one year for stenting of long femoral occlusions varies from 64.8 to 66% and secondary patency rates vary between 70 and 83%, which depends on the characteristics of the atherosclerotic lesion and type of stent.5-8. Because of the acute on chronic presentation in the current case, due to a total and likely recent occlusion of the SFA, there was opted for mechanical thrombectomy followed by PTA and provisional stenting due to residual multilevel stenosis.

Chapter 3

Figure 1. A. Digital subtraction angiography (DSA), which shows an occlusion of 23 cm in the superficial femoral artery. B. DSA after two times thrombosuction with the Angiojet catheter. C. DSA after PTA and stenting.

Post-procedural antiplatelet therapy The fast and continuous evolution of endovascular technologies allows for successful revascularization of increasingly complex atherosclerotic lesions. Life-long antiplatelet therapy (APT) is generally recommended to promote patency after peripheral endovascular interventions of femoropopliteal arteries, although no evidence-based guideline exists.1 Nowadays, many different types of antiplatelet regimens are used (table 1). Studies on APT in patients with PAD have major limitations due to small study populations and potential risk of bias.9 This lack of high quality evidence leads to a great (inter)national variety in the administered APT, duration of treatment, and platelet reactivity test used to assess the effect of APT.10 In cardiology trials, a relative risk reduction of secondary cardiovascular events (cardiovascular death, myocardial infarction or any revascularization) of around 30% is seen when adding clopidogrel to aspirin after PCI.11-13 It is not exactly known whether this is the case in PAOD, however a similar risk reduction is assumed. Prior to PTA and stenting of femoropopliteal arteries, a loading dose of clopidogrel is often added to aspirin and DAPT is continued for one to three months post-PTA. The rationale for this practice is largely based on extrapolation of data derived from percutaneous coronary interventions (PCI), and is not supported by international guidelines.1,14 The patient in this case received a loading dose of 300mg clopidogrel after the procedure followed by a maintenance dose of 75mg once daily for six months. Several recent cardiology trials suggest that a loading dose of 600mg might be more effective in preventing major adverse cardiac events, without increasing the risk of bleeding. 15

Personalised antiplatelet therapy

However, for PAOD patients there is no evidence that suggests superiority of a higher loading dose. In analogy with observations in patients undergoing PCI, a natural variation in response on APT can be expected among patients undergoing peripheral revascularization. A recently published review showed that high-on-aspirin platelet reactivity (HAPR) occurred in 22.2% of the patients in a pooled analysis of 102 studies containing a total of 44,098 patients with coronary artery, cerebrovascular or peripheral arterial disease who were treated with aspirin with or without clopidogrel.16 Patients with HAPR had an increased risk of future cardiovascular events (combined endpoint of cardiovascular death, myocardial infarction, stent thrombosis, stroke, acute limb ischemia, peripheral revascularization and acute peripheral occlusion; RR 2.09, 95%CI: 1.77-2.47). The amount of non-responders for clopidogrel was even higher: 40.4% of patients was diagnosed with high-on-clopidogrel platelet reactivity (HCPR), which was also associated with an increased risk of cardiovascular events (RR 2.80, 95%CI: 2.40-3.27). Furthermore, the incidence of HAPR and HCPR is significantly higher in patients with chronic kidney disease and diabetes mellitus, both common risk factors in PAD.17,18 Platelet reactivity testing Multiple tests are available for measuring platelet reactivity (see table 2); the different tests measure diverse pathways of thrombus formation and the reproducibility and applicability vary strongly between these tests.19 So far, none of the available tests has proven its superiority in predicting thrombotic or bleeding events. However the Verify Now, a commercially available point-of-care test, shows congruent results with the Light Transmittance Aggregometer and is widely studied in clinical trials.16 Personalized antiplatelet therapy The association of HAPR and HCPR with increased risk of thrombotic events sparked the concept of tailoring APT based on platelet reactivity measurements. The basis of the concept is that switching APT (to a higher dose or other APT) with the aim to achieve lower platelet reactivity would result in less thrombotic events, especially in high-risk patients, such as patients with diabetes mellitus or renal failure. Multiple studies suggest that not only high platelet reactivity (HPR) should be an indicator to adjust APT; but that low platelet reactivity (LPR) should also be considered as a reason to adapt APT, due to the increased risk of bleeding complications, such as gastrointestinal and intra-cerebral bleeding complications.20,21 These data suggest the presence of a therapeutic window for platelet reactivity with HPR at one end and LPR at the other end of the spectrum. The data on HPR and LPR might elicit the idea that simply testing platelet reactivity in response to antiplatelet agents and adjusting the regimen based on the results, will lead to improved clinical outcomes. However, the clinical benefit of tailoring APT based on platelet function tests in patients undergoing PCI, showed disappointing results thus far.

44 Same mechanism of action as clopidogrel/ ticlopidine. Needs transformation to an active metabolite via the CYP system in a similar manner to clopidogrel

Same mechanism of action as clopidogrel/ prasugrel

Prasugrel

Ticlopidine

Blocks GPIIb/IIIa, the platelets receptor for fibrinogen and von Willebrand factor

Same mechanism of action as cilostazol

Dipyridamole

Abciximab Eptifibatide Tirofiban

Inhibits phosphodiesterase enzymes that break down cAMP, (resulting in increasing cellular cAMP levels and thereby blocking of platelet aggregation response to ADP) and/or cGMP

Same mechanism of action as ticagrelor

Cangrelor

Cilostazol

Reversible P2Y12 receptor antagonists. Unlike the thienopyridines, ticagrelor is not a prodrug and does not require metabolic activation

Ticagrelor

Rapid onset of action

Effects of dipyridamole and aspirin on platelet behaviour are additive

Effective in increasing pain free walking distance in patient with CI

Fast acting drug, suitable for bridging surgery patients who require P2Y12 inhibition

More effective than thienopyridines in prevention of vascular death, MI or stoke

Suitable when aspirin has failed or is not tolerated.

Faster onset of action than clopidogrel No effect of CYP2C19 genotype on clinical cardiovascular event rates

Economical

Economical Also analgesic, antiinflammatory and antioxidant properties

Pros

IV administration

High dose causes vasodilatation Twice daily administration

Dangerous with heart failure

IV administration

Twice daily administration Increased bleeding risk

Rare, but serious side effects of neutropenia and TTP

Increased bleeding risk in patients >75 years, <60 kg and/or with CVA/TIA in medical history More expensive than clopidogrel

Requires CYP2C19 for its metabolization Slow onset of action interindividual variability

Risk of gastrointestinal bleeding Resistance

Cons

ADP: adenosinediphosphate, cAMP: Cyclic adenosine monophosphate, cGMP: Cyclic guanosine monophosphate, CI: claudication intermittence, COX: Cyclooxygenase inhibitors, CVA: cerebrovascular accident, CYP: Cytochromes P450, GPIIb/IIIa: Glycoprotein IIb/IIIa, IV intravenous, TIA Transient ischemic accident, TTP Thrombotic thrombocytopenic purpura.

Glycoprotein IIb/IIIa inhibitors

Phosphodiesterase inhibitors

P2Y12 inhibitors Nucleotide analogues

Binds irreversibly to the ADP receptors (P2Y12 receptors), ensuring a inhibition of the ADPpathway Prodrug that needs to be metabolized via the CYP by the liver

Clopidogrel

P2Y12 inhibitors Thienopyridines

Irreversibly inhibits the enzyme COX, resulting in reduced platelets production of thromboxane A2 (powerful vasoconstrictor that lowers cyclic AMP and acts as secondary platelet activator)

Aspirin Carbasalate calcium Triflusidal

Mechanism of action

Drugs

Cyclooxygenase inhibitors

Table 1. Different types of antiplatelet regimens.

Class

Chapter 3

Platelets are stimulated with agonists, causing platelet activation and precipitating to the fibrinogen coated beats in the cartridges. Degree of platelet is based on remaining optical light transmittance through the cartridges.

Citrated whole blood is centrifuged, resulting PRP and PPP. After stimulation of the platelets with agonists, formed aggregates modify optical density of the samples, expressed as % platelet aggregation.

Blood is aspirated at high shear rates through cartridges with either collagen and epinephrine or collagen and ADP, which induce platelet adhesion, activation and aggregation. This leads to rapid occlusion of the aperture and cessation of blood flow, defined as closure time.

Activated platelets, after stimulation with agonists, adhere to metal sensor wires and electrical resistance increases.

ADP inhibits VASP (an intracellular platelet protein) phosphorylation through the P2Y12 receptor. Persistent VASP phosphorylation, as measured with flow cytometry, correlates with P2Y12 receptor inhibition, reflecting the effect of the P2Y12 inhibitor.

VerifyNow

Light transmittance aggregometry (LTA)

Platelet function analyser (PFA)

Multiplate

Vasodilator-stimulated phosphoprotein (VASP)phosphorylation assay

Standardized kit

Aspirin and P2Y12 monitoring

Rapid and automatic

Golden standard due to long lasting experience

asy and rapid point-of-care test E Aspirin, P2Y12 and GPIIb/IIIa inhibitor monitoring

Pros

ADP adenosinediphosphate; APT antiplatelet therapy; PRP platelet rich plasma; PPP platelet poor plasma

Technique

Platelet reactivity testing

Table 2. Commonly used platelet reactivity test.

Only applicable for monitoring of P2Y12 inhibitors Time consuming

Semi-automatic; preparation of the agonists and pipetting is necessary to perform the tests

Originally designed as screening instrument for thrombopathy and thus better standardized compared to monitoring of APT

Manually conducted test therefore reproducibility between laboratories is low Time consuming

ut off values differ per patient C population Low correlation with golden standard (LTA)

Cons

Personalised antiplatelet therapy

Chapter 3

Two major trials (GRAVITAS, n=2,21422 and ARCTIC, n=2,24023) showed no difference in their primary composite endpoint (death of cardiovascular causes, nonfatal acute myocardial infarction and ST-elevation) or bleeding complications after 6 months in GRAVITAS and 1 year in ARCTIC, when comparing standard APT with tailored APT. However, a few smaller studies showed a beneficial effect of tailored vs. standard APT on a composite endpoint (e.g. cardiac death, stent thrombosis, recurrent ACS, re PCI< 1 year), without difference in bleeding complications.24-27 The two largest trials included a low risk population, counteracted HPR primarily with a higher dose of the same APT instead of switching APT, and used a single platelet reactivity test, which could have resulted in the lack of effect of tailored APT. PAPT in patients with PAOD Most research regarding personalized antiplatelet therapy (PAPT) has been performed in patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). Randomized control trials to investigate the potential benefit of PAPT in patients with PAOD are mandatory. However, several questions need to be answered before this treatment strategy can be implemented in clinical trials.28 1. What is the incidence of HAPR and HCPR in patients with PAOD? 2. Is high and low platelet reactivity in patients with PAOD related to the occurrence of respectively thrombotic- or bleeding events during follow-up? 3. What are the optimal cut-off values of different platelet reactivity tests to predict thrombotic or bleeding events? 3. Does HPR change over time and what is the optimal moment for platelet reactivity testing? 4. What is the optimal alternative APT for patients with PAOD displaying HAPR or HCPR? Importantly, the low patency rates after PTA leave room for randomized control trials to prove significant benefit of PAPT compared to standard care after the abovementioned questions are answered. Since the risk of hemorrhagic complications might increase by the use of novel and dual APT, the use of a combined clinical benefit endpoint, comprising both thrombotic and bleeding events, would be advised in potential future trials

Conclusion Technological improvements in the endovascular armentarium have made increasingly complex atherosclerotic lesions eligible for endovascular interventions, which has led to a primarily endovascular first strategy in PAD. However, the patency rates of endovascular procedures still leave room for improvement and optimization of post-procedural medical therapy, including personalized APT, is therefore essential. Current evidence regarding the optimal APT after peripheral endovascular revascularization is inconclusive. It is reasonable to expect that personalized APT, based on platelet reactivity measurements, may improve outcomes. However, some important conditions should be fulfilled prior to routine

Personalised antiplatelet therapy

implementation of personalized APT in clinical practice, such as determination of the optimal platelet reactivity test, and the optimal type and duration of (D)APT. In conclusion, despite the fact that some important hurdles should be overcome prior to routine implementation, we want to advocate the concept of individualized medicine regarding the type of endovascular intervention, but especially the post-procedural APT in patients with PAD in order to optimize the results of endovascular interventions, as apparent from the presented case.

Chapter 3

References 1.

Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). Journal of vascular surgery. Jan 2007 2. Tetteroo E, van der Graaf Y, Bosch JL, et al. Randomised comparison of primary stent placement versus primary angioplasty followed by selective stent placement in patients with iliacartery occlusive disease. Dutch Iliac Stent Trial Study Group. Lancet. Apr 1998 3. Hirsch AT, Haskal ZJ, Hertzer NR, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease Circulation. Mar 2006 4. Kougias P, Chen A, Cagiannos C, Bechara CF, Huynh TT, Lin PH. Subintimal placement of covered stent versus subintimal balloon angioplasty in the treatment of long-segment superficial femoral artery occlusion. American journal of surgery. Nov 2009 5. Zeller T, Rastan A, Macharzina R, et al. Drugcoated balloons vs. drug-eluting stents for treatment of long femoropopliteal lesions. Journal of endovascular therapy : an official journal of the International Society of Endovascular Specialists. Jun 2014 6. Bosiers M, Deloose K, Callaert J, et al. Results of the Protege EverFlex 200-mm-long nitinol stent (ev3) in TASC C and D femoropopliteal lesions. Journal of vascular surgery. Oct 2011 7. Ansel GM, Lumsden AB. Evolving modalities for femoropopliteal interventions. Journal of endovascular therapy : an official journal of the International Society of Endovascular Specialists. Apr 2009 8. Davaine JM, Azema L, Guyomarch B, et al. Oneyear clinical outcome after primary stenting for Trans-Atlantic Inter-Society Consensus (TASC) C and D femoropopliteal lesions (the STELLA “STEnting Long de L’Artere femorale superficielle” cohort). European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Oct 2012 9. Robertson L, Ghouri MA, Kovacs F. Antiplatelet and anticoagulant drugs for prevention of restenosis/ reocclusion following peripheral endovascular treatment. The Cochrane database of systematic reviews. 2012 10. Allemang MT, Rajani RR, Nelson PR, Hingorani A, Kashyap VS. Prescribing patterns of antiplatelet agents are highly variable after lower extremity endovascular procedures. Annals of vascular surgery. Jan 2013 11. Mehta SR, Yusuf S, Peters RJ, et al. Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet. Aug 2001

12. Brener SJ, Steinhubl SR, Berger PB, Brennan DM, Topol EJ. Prolonged dual antiplatelet therapy after percutaneous coronary intervention reduces ischemic events without affecting the need for repeat revascularization: insights from the CREDO trial. The Journal of invasive cardiology. Jul 2007 13. Steinhubl SR, Berger PB, Mann JT, 3rd, et al. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial. JAMA Nov 2002 14. Alonso-Coello P, Bellmunt S, McGorrian C, et al. Antithrombotic therapy in peripheral artery disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. Feb 2012 15. Vyas A, El Accaoui R, Blevins A, Karrowni W. Outcome comparison of 600 mg versus 300 mg loading dose of clopidogrel for patients with ST-elevation myocardial infarction: a metaanalysis. Postgraduate medicine. Sep 2014 16. Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 17. Spiliopoulos S, Pastromas G, Diamantopoulos A, Katsanos K. Efficacy of clopidogrel treatment and platelet responsiveness in peripheral arterial procedures. Expert opinion on pharmacotherapy. Oct 2014 18. Droppa M, Tschernow D, Muller KA, et al. Evaluation of Clinical Risk Factors to Predict High On-Treatment Platelet Reactivity and Outcome in Patients with Stable Coronary Artery Disease (PREDICT-STABLE). PloS one. 2015 19. Leunissen TC, De Borst GJ, Janssen PW, Ten Berg JM. The role of perioperative antiplatelet therapy and platelet reactivity testing in carotid revascularization: overview of the evidence. The Journal of cardiovascular surgery. Apr 2015 20. Sibbing D, Schulz S, Braun S, et al. Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. Journal of thrombosis and haemostasis : JTH. Feb 2010 21. Cuisset T, Cayla G, Frere C, et al. Predictive value of post-treatment platelet reactivity for occurrence of post-discharge bleeding after non-ST elevation acute coronary syndrome. Shifting from antiplatelet resistance to bleeding risk assessment? EuroIntervention : journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology. Aug 2009 22. Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA Mar 2011

Personalised antiplatelet therapy

23. Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 24. Siller-Matula JM, Francesconi M, Dechant C, et al. Personalized antiplatelet treatment after percutaneous coronary intervention: the MADONNA study. International journal of cardiology. Sep 2013 25. Wang XD, Zhang DF, Zhuang SW, Lai Y. Modifying clopidogrel maintenance doses according to vasodilator-stimulated phosphoprotein phosphorylation index improves clinical outcome in patients with clopidogrel resistance. Clinical cardiology. May 2011 26. Bonello L, Camoin-Jau L, Arques S, et al. Adjusted clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index decrease rate of major adverse cardiovascular events in patients with clopidogrel resistance: a multicenter randomized prospective study. Journal of the American College of Cardiology. Apr 2008 27. Valgimigli M, Campo G, de Cesare N, et al. Intensifying platelet inhibition with tirofiban in poor responders to aspirin, clopidogrel, or both agents undergoing elective coronary intervention: results from the double-blind, prospective, randomized Tailoring Treatment with Tirofiban in Patients Showing Resistance to Aspirin and/or Resistance to Clopidogrel study. Circulation. Jun 2009 28. Siller-Matula JM, Jilma B. Why have studies of tailored anti-platelet therapy failed so far? Thrombosis and haemostasis. Oct 2013

Chapter 4 High on-treatment platelet reactivity in peripheral arterial disease: A pilot study to find the optimal test and cut off values Eur J Vas Endovas Surg, 2015 May; Volume 52, Issue 2, p198-204

T. Leunissen*, S. Peeters Weem*, R. Urbanus, H. den Ruijter, F. Moll, F. Asselbergs, GJ. de Borst * Authors contributed equally to the manuscript

Chapter 4

Abstract Objective Restenosis and stent thrombosis after endovascular intervention in patients with peripheral arterial disease (PAD) are potentially tackled by more intensive antiplatelet therapy, such as dual antiplatelet therapy (DAPT) consisting of aspirin and P2Y12 inhibitor. Despite clopidogrel treatment, some patients still display high platelet reactivity (HCPR). Tailored antiplatelet therapy, based on platelet reactivity testing, might overcome HCPR. However, more data regarding the proportion of patients with HCPR in the PAD population, different platelet reactivity tests, their correlation and optimal timing for these tests is warranted as stepping stone for a future trial investigating the potential benefit of tailored antiplatelet therapy in PAD patients. Methods

Thirty patients on DAPT after percutaneous transluminal angioplasty underwent platelet reactivity testing by VerifyNow, vasodilator-stimulated phosphoprotein (VASP)- and platelet activation assay, and CYP2C19-polymorphism testing. Results Proportion of patients with HCPR measured by VerifyNow varied between 43.3 and 83.3%, depending on the used cut-off values. Testing â&#x2030;¤24 hours after initiation of DAPT lead to a higher proportion of HCPR than testing >24 hours. According to DNA testing, 14.8% was CYP2C19*2 homozygote, 22.2% heterozygote and 63% was CYP2C19*2 negative. VASP-assay revealed 24% HCPR. The highest HCPR-rate was found with VerifyNow cut-off â&#x2030;¤40% inhibition, while the lowest HCPR-rate was found with the VASP-assay. There was a low correlation between the tests. Conclusion HCPR is present in PAD patients and research on HCPR is needed in this population; timing of the tests is relevant and standardization of tests is needed. The optimal conditions for platelet function testing should be determined.

High on-treatment platelet reactivity

Introduction Restenosis and stent thrombosis remain the main challenges after endovascular treatment of peripheral arterial disease (PAD). The one and five year patency of PTA alone is 71% and 49% and with additional stent placement the patency rates increase to 74% and 65% after one and three years.1-6 Because of the leading part of platelets in restenosis and stent thrombosis, antiplatelet therapy (APT) is given to prevent these complications. Numerous publications from the Antithrombotic Trialistsâ&#x20AC;&#x2122; Collaboration have concluded the use of aspirin in patients with cardiovascular disease will result in a 25% odds reduction in subsequent cardiovascular events (CVE).7 The prescription of dual antiplatelet therapy (DAPT), consisting of aspirin and P2Y12 inhibitor, for patients undergoing endovascular treatment has increased, although the evidence for DAPT after peripheral endovascular procedures is lacking.1,8 Clopidogrel, a P2Y12 inhibitor, is often the first choice APT added to aspirin, although previous trials have shown that 40% of patients show high platelet reactivity, despite additional clopidogrel treatment.9 The few trials reporting on high on-clopidogrel platelet reactivity (HCPR) in PAD patients indicate an even higher incidence. 10-12 This phenomenon can in some patients be caused by a mutation in the genes coding for cytochrome P450 2C19 (CYP2C19) activity, a liver enzyme that converts the clopidogrel pro-drug into its active metabolite.13 Other causes include non-compliance, diabetes mellitus, renal failure and non-smoking.14-17 The existence of HCPR has led to the concept of tailored APT, the idea that simply testing platelet reactivity in response to APT and adjusting the regimen based on the results, will lead to improved clinical outcomes.18 Most research on this concept is performed in cardiac patients undergoing percutaneous coronary intervention. Up to now, clinical trials on tailored APT showed diverging results. Two large clinical trials (GRAVITAS19, n=2214 and ARCTIC20, n=2240) showed no difference in primary outcome (composite of cardiovascular death, nonfatal acute myocardial infarction and ST-elevation) or bleeding complications after tailored APT compared to standard therapy. However, few smaller studies did show a beneficial effect of tailored APT compared to standard therapy.21-23 No studies regarding tailored APT for PAD patients have been performed. Currently, there is a large need for randomized trials investigating the benefit of (tailored) APT in PAD patients after endovascular treatment. To perform such trials, more knowledge is warranted concerning the proportion of patients with HCPR in this population, the optimal timing, test and cut-off values to identify HCPR . Therefore, the aim of this pilot study was to display the proportion of patients with HCPR in the PAD population and to evaluate different platelet reactivity tests, their correlation and optimal timing for these tests.

Chapter 4

Materials and methods Study design The present study was a prospective, observational pilot study with 30 patients. Since no previous trial results were available regarding PAD patients with HCPR, an adequate power calculation was not possible. The study was conducted with approval of the local ethics committee and in accordance with the declaration of Helsinki. All patients gave written informed consent prior to the procedure. Patient selection Patients were included if they were adults planned to undergo an endovascular revascularization (PTA, percutaneous transluminal angioplasty) of the superficial femoral artery or popliteal artery and were on aspirin treatment prior to the intervention. Exclusion criteria were treatment with heparin, oral anticoagulants or P2Y12 inhibitors at the time of the PTA, since these patients would not be suitable to receive DAPT post intervention. Procedures Participating patients underwent regular PTA with or without additional stenting. Procedures were performed by either the interventional radiologist or vascular surgeon and aspirin was continued during endovascular treatment. Blood was drawn from the arterial access sheath directly prior to the angioplasty for platelet reactivity testing. Prescription of DAPT after intervention was left to the treating physicianâ&#x20AC;&#x2122;s discretion. Patients with an indication for DAPT received a loading dose (LD) of 300mg clopidogrel at the same day of the procedure and platelet reactivity tests were performed between 1 and 5 days after the LD. In addition to the platelet reactivity tests, a CYP2C19 polymorphism DNA test was performed using the Spartan RX CYP2C19 DNA testing system (Spartan Bioscience Inc, Ottawa, Canada), to determine the presence of CYP2C19*2 loss of function alleles. Platelet reactivity measurements VerifyNow Platelet reactivity was assessed using the VerifyNow P2Y12 assay (AccumetricsÂŽ, San Diego, CA, USA), which is a cartridge-based optical detection system utilizing whole blood. Blood was collected in a Greiner Bio-One 3.2% citrate Vacuette tube. Although the VerifyNow is a widely used point-of-care test, different cut-off values are used in clinical and research setting. Some researchers advocate the use of P2Y12 reaction units (PRU), while others use the percentage of platelet inhibition as measurement for sufficient response to P2Y12 inhibitors. In current literature the most frequent used cut-off value is post PRU <235,24 although the manual of the VerifyNow advises a cut-off value of <208 PRU.25 A third commonly used cut-off value is >40% inhibition.23,26 We therefore compared these three cut-offs to evaluate the differences in (non)-responders to clopidogrel. At

High on-treatment platelet reactivity

the other end of the spectrum, low platelet reactivity due to clopidogrel treatment, increases the risk of bleeding. Low platelet reactivity is defined as PRU <95 by the VerifyNow manual. VASP assay Vasodilator-stimulated phosphoprotein (VASP) is an intracellular platelet protein that is not phosphorylated when the P2Y12 receptors are active. Persistent VASP phosphorylation, as measured with flow cytometry, correlates with P2Y12 receptor inhibition, reflecting the effect of antiplatelet therapy. Blood was collected in a 0.105 M tri-sodium citrate tube. Flow-cytometry analysis of VASP phosphorylation was performed using a commercial kit (PLT VASP/P2Y12 Test Kit, Biocytex, Marseille, France) and FACS Canto flow cytometer (BD Biosciences, San Jose, USA). Platelet reactivity index (PRI) was calculated and expressed as continuous percentage value (%). PRI >50% is regarded to predict major adverse cardiac events of clinical interest with sensitivity of 100% and is therefore used as cut-off of (non)-responder in this study.27,28 PACT The platelet activation (PACT) assay is a flow cytometry based test, stimulating platelets in whole blood with increasing concentrations of the agonists adenosine diphosphate (ADP) (Roche, Almere, the Netherlands) and SFLLRN (TRAP-6) (Bachem, Weil am Rhein, Germany) in a hydroxyethyl-piperazineethane-sulfonic (HEPES) buffered saline mixture which contains a fixed concentration (1:25) R-Phycoerythrin (RPE)-conjugated anti-Pselectin (BD Pharmingen™, Franklin Lakes, NJ, USA) and (1:50) fluorescein isothiocyanate (FITC)-conjugated antifibrinogen (Dako, Glostrup, Denmark). Per agonist, wells were filled with a 50 µl assay mixture wherein 5 µl whole blood was pipetted. The mix was homogenised and incubated at room temperature. The reaction was stopped after 20 minutes by pipetting 500 µl fixative solution (0.148% formaldehyde, 137 mM NaCl, 2.7 mM KCl, 1.12 mM NaH2PO4, 1.15 mM KH2PO4, 10.2 mM NaHPO4, 4 mM EDTA, pH 6.8) and analysed on a BD FACS Canto II flow cytometer (BD-biosciences, San Jose, United States) on the same day of processing. Single platelets were gated based on forward and side scatter properties. Fibrinogen binding was used as a measure of IIbβ3 activation and P-selectin expression as marker of granule release. The median fluorescent intensity (MFI) of the highest concentration agonist was used to calculate the percentage inhibition between platelet reactivity assessed prior and post intervention (and consequently the LD clopidogrel). End points Primary end point was the proportion of patients with high platelet reactivity, based on different platelet reactivity tests and different cut-off values as explained in the previous paragraphs. Secondary aim was to determine the correlation between the different tests.

Chapter 4

Statistical analysis Data were analyzed using Statistical Package for Social Science (IBM SPSS, version 22, IBM Corp., Armonk, NY, USA). Statistical significance was considered at a double-sided p < .05. Non-normally distributed data were displayed as median (interquartile range, IQR) and normally distributed data were displayed as mean (standard deviation, SD). Correlation between different platelet reactivity tests was tested using Spearman’s rankorder correlation coefficient (rho). Inhibition rates after ADP and TRAP-6 stimulation in the PACT test were compared using the Wilcoxon signed rank test. Results of the CYP2C19 test were considered as ordinal data, with 0 being CYP2C19*2 loss of function allele negative and 2 being two CYP2C19*2 loss-of-function alleles. Bonferroni’s correction was used to adjust for multiple testing, when necessary.

Results Patients and procedures Thirty-five out of 50 consecutive patients undergoing endovascular treatment for superficial femoral or popliteal atherosclerosis received postoperative DAPT. Thirty of them were tested for platelet reactivity and could be included in the analyses. Median time between LD and platelet reactivity testing was 1 day (IQR 1 - 3). A summary of baseline characteristics is displayed in table 1. VerifyNow Proportion of patients with high on clopidogrel platelet reactivity The cut-off value PRU ≥235 identified 13 out of 30 included patients (43.3%) as nonresponders. The cut-off value PRU ≥208 identified 18 patients (60.0%) as non-responders while the cut-off value ≤40% identified 25 patients (83.3%) as non-responders (table 2). Proportion of patients with low platelet reactivity (PRU <95) Three patients displayed low platelet reactivity (PRU 30, 30 and 35 and inhibition 89%, 90% and 85%, respectively). Timing of platelet reactivity measurements VerifyNow was mostly performed within 24 hours after LD clopidogrel (60%). Of the 18 patients tested at day one, 8 patients (44.4%) were non-responder with cut-off value PRU ≥235, 13 patients (72.2%) with cut-off value PRU ≥208 and all 18 patients (100%) were non-responder when applying the cut-off level of 40% inhibition. Of the 12 patients measured beyond day one, five patients (41.7%) were non-responders with both cut-off values PRU ≥235 and PRU ≥208 and seven patients (58.3%) were non-responder with cut-off value ≤40% inhibition. Exact numbers of non-responders at different time points are displayed in table 3. The three patients with low platelet reactivity were measured at day 3, 4 and 5 post intervention.

High on-treatment platelet reactivity

Table 1. Baseline characteristics of included patients (n=30). Male sex, n (%)

Age, years (SD)

70.5

8.61

Cardiovascular risk factors, n (%) Diabetes mellitus Dyslipidemia

Hypertension

Smoking, current or previous

History of coronary artery disease

Fontaine IIa

Fontaine IIb

Fontaine III

Fontaine IV

100

Statins

ACE-inhibitors

Î˛-blockers

Diuretics

Proton pump inhibitors

History of CVA / TIA

Fontaine classification, n (%)

Medication at baseline, n (%) Aspirin Dipyridamole

Table 2. Numbers of non-responders with different cut-off values of VerifyNow. Cut-off method

Responders (%)

Non-responders (%)

Cut-off PRU < 235

17 (56.7)

13 (43.3)

Cut-off PRU < 208

12 (40.0)

18 (60.0)

5 (16.7)

25 (83.3)

Cut-off inhibition > 40% PRU indicates P2Y12 reactive units

Four patients were tested at two different time intervals. The first measurement was on the first day postoperative and the second VerifyNow between day 5 and 21. They were tested twice because there was a suspicion that the interval between the LD and VerifyNow was insufficient and the treating physician didnâ&#x20AC;&#x2122;t want to switch regime based on these test results. In three patients, the second test showed substantially more inhibition than the first test, the fourth patient persistently displayed 0% inhibition after 21 days. Results of patients tested at different intervals are summarized in table 4. All four patients were CYP2C19*2 negative.

Chapter 4

Table 3. Days between start dual antiplatelet therapy and VerifyNow with corresponding numbers of non-responders and responders. Days between start DAPT and VerifyNow Cut-off method PRU <235

PRU<208

non-responder

responder

non-responder

responder >40% inhibition

non-responder responder

PRU indicates P2Y12 reactive units

Table 4. VerifyNow results of patients tested at different intervals. Day

VN baseline

VN post

VN inhibition

Day

VN baseline

VN post

VN inhibition

226

222

289

81%

272

249

255

187

27%

223

277

252

253

255

273

228

187

18%

VN indicates VerifyNow

Vasodilator-stimulated phosphoprotein (VASP) assay Proportion of patients with high on clopidogrel platelet reactivity In the 25 tested patients, the Platelet Reactivity Index varied widely (median 71.3%, IQR 53.2 – 84.5). Only six patients (24%) were identified as good responders to clopidogrel. From these responders, three patients were acknowledged by VerifyNow cut-off value inhibition ≤40%, five by cut-off PRU ≥235 and four PRU ≥208 as good responders. One patient showed very low platelet reactivity (PRI=0%), which was confirmed by VerifyNow PRU 35 and inhibition of 85%. PACT results The median inhibition of fibrinogen binding was 53.8% (IQR 14.5 - 75.1%) after stimulation with ADP and 40.3% (IQR 21.7- 61.6%) after stimulation with TRAP-6, which is not significantly different (p=0.459). Granule release, measured as P-selectin expression, after stimulation with ADP was inhibited significantly stronger (median inhibition 40.7%, IQR 12.5 - 69.6%) than granule release after stimulation with TRAP-6 (median 1.5%, IQR 0.034.0%, P<0.001). Despite the P2Y12 treatment, no inhibition of granule release after TRAP6 stimulation was observed in 11 patients (42.3%). The inhibitory effect of clopidogrel was more evident in the P2Y12 signaling pathway than in the thrombin signaling pathway.

High on-treatment platelet reactivity

CYP2C19*2 polymorphism CYP2C19*2 polymorphism DNA testing was performed in 29 patients. Two patients displayed inconclusive results and were counted as missing values. Of 27 remaining patients, 17 had no CYP2C19*2 variant (63.0 %), six patients were heterozygous for the CYP2C19 *2 allele (22.2 %), and 4 patients were homozygous for CYP2C19*2 (14.8 %). Results of the platelet reactivity and DNA tests for all included patients are displayed in supplementary table 1. Strategies in patients with HCPR Ten out of 30 included patients switched to prasugrel during follow-up, based on the VerifyNow results with cut-off level <40% inhibition. Two of them had two CYP2C19*2 alleles, four had one CYP2C19*2 allele, three were negative and one was missing. Correlation between different platelet reactivity test results There was a significant correlation between the percentage inhibition of the VerifyNow and inhibition of fibrinogen binding after TRAP stimulation, measured with PACT (rho = 0.541, p = 0.004, table 5), but no significance was found after correction for multiple testing. No other significant correlations between VerifyNow inhibition and PACT test results could be demonstrated. A significant correlation was seen between the results of the VASP assay and fibrinogen binding after ADP stimulation (rho = -0.606, p = 0.002, table 5 and figure 1). Within the PACT, there was a significant correlation between inhibition in fibrinogen binding after ADP stimulation and all three other PACT tests separately (ADP stimulated granule relase: rho= 0.931, P <0.001; fibrinogen binding after TRAP stimulation: rho= 0.458, p=0.019 and TRAP stimulated granule release: rho= 0.464, p=0.017). After correction for multiple testing only the correlation between inhibition on fibrinogen and granule release after ADP stimulation remained statistically significant. Results of the correlation tests are summarized in table 5.

Discussion Our prospective explorative study on thirty patients with PAD undergoing endovascular treatment with platelet reactivity testing revealed that the proportion of HCPR was highly dependent on the utilized platelet reactivity tests and cut-off values. Prevalence of HCPR varied between 43.3-83.3% using different cut-offs for the VerifyNow. The instructions state that the optimal cut-off value is PRU ≥208,25 but in current literature ≥234 or ≥235 are more common.10,11,24,29 The VASP-assay displayed HCPR in 76% of the patients. Based on this pilot study, HCPR is common in PAD patients undergoing endovascular treatment. An extensive meta-analysis evaluating platelet reactivity in various patients on antiplatelet therapy, assessed with numerous platelet reactivity tests showed HCPR in 40.4% of the pooled patients, based on 59 studies with 34.776 patients. 9 The few studies investigating

Chapter 4

Table 5. Correlation between different platelet reactivity tests according to Spearmanâ&#x20AC;&#x2122;s correlation coefficient (rho) VN (%)

VN (%)

ADP ADP fibrinogen granule release

TRAP TRAP fibrinogen granule release

CYP2C19 DNA test

VASP

Rho

0.369

0.385

0.541

-0.019

-0.476

-0.224

0.063

0.052

0.004

0.926

0.014

0.282

N ADP fibrinogen

ADP granule release

TRAP fibrinogen

TRAP granule release

CYP2C19 DNA test

VASP

Rho

0.369

0.931

0.458

0.464

-0.394

-0.606

0.063

<0.001

0.019

0.017

0.057

0.002

Rho

0.385

0.931

0.335

0.537

-0.456

-0.493

0.052

<0.001

0.094

0.005

0.025

0.017

Rho

0.541

0.458

0.335

0.010

-0.231

-0.351

0.004

0.019

0.094

0.960

0.277

0.101

Rho

-0.019

0.464

0.537

0.010

-0.124

-0.491

0.926

0.017

0.005

0.960

0.564

0.017

Rho

-0.476

-0.394

-0.456

-0.231

-0.124

0.272

0.014

0.057

0.025

0.277

0.564

0.209

Rho

-0.224

-0.606

-0.493

-0.351

-0.491

0.272

0.282

0.002

0.017

0.101

0.017

0.209

VN indicates VerifyNow; VASP, vasodilator-stimulated phosphoprotein phosphorylation

platelet reactivity in PAD, all showed a higher incidence of HCPR compared to patients with coronary artery disease, confirming the relevance of HCPR in PAD patients.10-12 In this study, the test results seemed to be highly dependent on timing after LD clopidogrel. Most patients were tested <24 hours after the LD of 300 mg clopidogrel, according to the advised minimum of 8 hours after LD.25 This advice is based on a single study examining platelet inhibition after different loading dosages of clopidogrel, tested hourly up to 7 hours post LD and daily for 5 days after discontinuation of daily treatment. However, when re-evaluating these study results, we agree that maximum platelet inhibition was reached after 6 hours post loading with 600 or 900 mg clopidogrel, but we disagree that maximum platelet inhibition is reached in patients after loading with 300 mg. 30 However, it is unclear whether maximum inhibition is necessary for clinically effective inhibition.

High on-treatment platelet reactivity

Figure 1. Correlation between the results of the VASP assay and fibrinogen binding after ADP stimulation.

This study shows that most non-responders were diagnosed in patients tested <24 hours after LD and even reached 100% non-responders with cut-off value <40% inhibition. However, four patients displaying 0% inhibition at day 1, were tested a few days later and three showed notably higher inhibition levels (PRU<208). We suspect that testing <24 hours after LD clopidogrel does not reflect maximal achieved platelet inhibition. Different time periods of platelet reactivity testing need to be investigated. Since restenosis mostly occurs within the first six months after PTA, adequate platelet inhibition during the first days after intervention is of particular importance.31,32 We therefore suggest trials investigating platelet reactivity testing within 3 to 5 days after DAPT initiation. The different tests performed in this study showed negligible correlation. Partially this might be explained by suboptimal timing and lack of power, but even more by the different mechanisms of action. Previous studies have evaluated the agreement between VerifyNow and VASP-assay with divergent conclusions. 33,34 Although both tests reflect P2Y12-receptor blockage, the tests results might not be interchangeable, since different aspects of platelet activation are measured. A trial investigating the superiority of either of these tests is desirable. Furthermore, a combination of tests might be necessary for accurate monitoring of the platelet function. Platelets do not only play part in thrombus formation but also in wound healing, angiogenesis and inflammation. In the acute phase after intervention, the main goal of antiplatelet therapy is preventing thrombus formation. However, APT is also prescribed to prevent secondary CVEs.35,36 With regard to the latter, other expressions of

Chapter 4

platelet activation than aggregate formation (VerifyNow) might be more relevant and should be tested in platelet reactivity testing. More studies on differential platelet activation and the clinical consequences should be assessed. The study holds some limitations: since the study design was observational, there was no consistent policy for the patients with HCPR. Some patients switched to prasugrel and some continued the clopidogrel treatment without adjustment, according to physicianâ&#x20AC;&#x2122;s discretion. This study is underpowered for conclusions about the optimal treatment strategy in HCPR. Previous studies showed that increasing the dose of clopidogrel is less effective than switching to prasugrel in patients with HCPR, so switching to another drug is recommended.21,37

Conclusion This pilot study in patients with PAD undergoing PTA shows a high percentage of HCPR. Large variance in proportion HCPR exists between the different platelet reactivity tests (VerifyNow, VASP-assay and PACT). Future studies are needed to determine the timing of testing and the optimal combination of platelet reactivity testing before studies regarding tailored antiplatelet therapy in PAD patients can be performed.

High on-treatment platelet reactivity

References 1.

Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). Journal of vascular surgery. Jan 2007 2. Schillinger M, Sabeti S, Loewe C, et al. Balloon angioplasty versus implantation of nitinol stents in the superficial femoral artery. The New England journal of medicine. May 2006 3. Krankenberg H, Schluter M, Steinkamp HJ, et al. Nitinol stent implantation versus percutaneous transluminal angioplasty in superficial femoral artery lesions up to 10 cm in length: the femoral artery stenting trial (FAST). Circulation. Jul 2007 4. Dick F, Ricco JB, Davies AH, et al. Chapter VI: Follow-up after revascularisation. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Dec 2011 5. Laird JR, Katzen BT, Scheinert D, et al. Nitinol stent implantation versus balloon angioplasty for lesions in the superficial femoral artery and proximal popliteal artery: twelve-month results from the RESILIENT randomized trial. Circulation. Cardiovascular interventions. Jun 2010 6. Rastan A, Krankenberg H, Baumgartner I, et al. Stent placement versus balloon angioplasty for the treatment of obstructive lesions of the popliteal artery: a prospective, multicenter, randomized trial. Circulation. Jun 2013 7. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. Jan 2002 8. Peeters Weem SM, van Haelst ST, den Ruijter HM, Moll FL, de Borst GJ. Lack of Evidence for Dual Antiplatelet Therapy after Endovascular Arterial Procedures: A Meta-analysis. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Aug 2016 9. Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 10. Spiliopoulos S, Pastromas G, Katsanos K, Kitrou P, Karnabatidis D, Siablis D. Platelet responsiveness to clopidogrel treatment after peripheral endovascular procedures: the PRECLOP study: clinical impact and optimal cutoff value of ontreatment high platelet reactivity. Journal of the American College of Cardiology. Jun 2013 11. Pastromas G, Spiliopoulos S, Katsanos K, et al. Clopidogrel responsiveness in patients undergoing peripheral angioplasty. Cardiovascular and interventional radiology. Dec 2013

12. Kliger C BA, Shah B, Feith F, Slater J, Attubato M. Dual antiplatelet therapy responsiveness in patients undergoing percutaneous revascularization for peripheral arterial occlusive disease. Journal of American college of cardiology. 2012 13. Scott SA, Sangkuhl K, Gardner EE, et al. Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450-2C19 (CYP2C19) genotype and clopidogrel therapy. Clinical pharmacology and therapeutics. Aug 2011 14. Serebruany V, Cherala G, Williams C, et al. Association of platelet responsiveness with clopidogrel metabolism: role of compliance in the assessment of â&#x20AC;&#x153;resistanceâ&#x20AC;?. American heart journal. Dec 2009 15. Mangiacapra F, Patti G, Peace A, et al. Comparison of platelet reactivity and periprocedural outcomes in patients with versus without diabetes mellitus and treated with clopidogrel and percutaneous coronary intervention. The American journal of cardiology. Sep 2010 16. Morel O, Muller C, Jesel L, Moulin B, Hannedouche T. Impaired platelet P2Y12 inhibition by thienopyridines in chronic kidney disease: mechanisms, clinical relevance and pharmacological options. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association European Renal Association. Aug 2013. 17. Gurbel PA, Bliden KP, Logan DK, et al. The influence of smoking status on the pharmacokinetics and pharmacodynamics of clopidogrel and prasugrel: the PARADOX study. Journal of the American College of Cardiology. Aug 2013 18. Peeters Weem SM, Leunissen T.C, Teraa M, Vonken EJ, de Borst GJ, Moll FL. Personalized Antiplatelet Therapy Following Endovascular Revascularization in Peripheral Artery Occlusive Disease: A Novel Concept. European Journal of Vascular and Endovascular Surgery: Short reports. 2015 19. Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA Mar 2011 20. Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 21. Siller-Matula JM, Francesconi M, Dechant C, et al. Personalized antiplatelet treatment after percutaneous coronary intervention: the MADONNA study. International journal of cardiology. Sep 2013 22. Bonello L, Camoin-Jau L, Arques S, et al. Adjusted clopidogrel loading doses according to vasodilator-stimulated phosphoprotein

Chapter 4

23.

24.

25. 26.

27.

28.

29.

30.

31.

phosphorylation index decrease rate of major adverse cardiovascular events in patients with clopidogrel resistance: a multicenter randomized prospective study. Journal of the American College of Cardiology. Apr 2008 Valgimigli M, Campo G, de Cesare N, et al. Intensifying platelet inhibition with tirofiban in poor responders to aspirin, clopidogrel, or both agents undergoing elective coronary intervention: results from the double-blind, prospective, randomized Tailoring Treatment with Tirofiban in Patients Showing Resistance to Aspirin and/or Resistance to Clopidogrel study. Circulation. Jun 2009 Price MJ, Endemann S, Gollapudi RR, et al. Prognostic significance of post- clopidogrel platelet reactivity assessed by a point-of-care assay on thrombotic events after drug-eluting stent implantation. European heart journal. Apr 2008 Accumetrics. Pocket Guide VerifyNow. San Diego, California, USA 2013. Lee DH, Arat A, Morsi H, Shaltoni H, Harris JR, Mawad ME. Dual antiplatelet therapy monitoring for neurointerventional procedures using a pointof-care platelet function test: a single-center experience. AJNR. American journal of neuroradiology. Aug 2008 Barragan P, Bouvier JL, Roquebert PO, et al. Resistance to thienopyridines: clinical detection of coronary stent thrombosis by monitoring of vasodilator-stimulated phosphoprotein phosphorylation. Catheterization and cardiovascular interventions : official journal of the Society for Cardiac Angiography & Interventions. Jul 2003 Campo G, Fileti L, Valgimigli M, et al. Poor response to clopidogrel: current and future options for its management. Journal of thrombosis and thrombolysis. Oct 2010 Karnabatidis D, Spiliopoulos S, Pastromas G, et al. Prevalence of nonresponsiveness to aspirin in patients with symptomatic peripheral arterial disease using true point of care testing. Cardiovascular and interventional radiology. Jun 2014 Price MJ, Coleman JL, Steinhubl SR, Wong GB, Cannon CP, Teirstein PS. Onset and offset of platelet inhibition after high-dose clopidogrel loading and standard daily therapy measured by a point-of-care assay in healthy volunteers. The American journal of cardiology. Sep 2006 Connors G, Todoran TM, Engelson BA, Sobieszczyk PS, Eisenhauer AC, Kinlay S. Percutaneous revascularization of long femoral artery lesions for claudication: patency over 2.5 years and impact of systematic surveillance. Catheterization and cardiovascular interventions : official journal of the Society for Cardiac Angiography & Interventions. Jun 2011

32. Thukkani AK, Kinlay S. Endovascular intervention for peripheral artery disease. Circulation research. Apr 2015 33. Varenhorst C, James S, Erlinge D, et al. Assessment of P2Y(12) inhibition with the point-of-care device VerifyNow P2Y12 in patients treated with prasugrel or clopidogrel coadministered with aspirin. American heart journal. Mar 2009 34. Bal Dit Sollier C, Berge N, Boval B, Dubar M, Drouet L. Differential sensitivity and kinetics of response of different ex vivo tests monitoring functional variability of platelet response to clopidogrel. Thrombosis and haemostasis. Sep 2010 35. Joseph P, Teo K. Optimal medical therapy, lifestyle intervention, and secondary prevention strategies for cardiovascular event reduction in ischemic heart disease. Current cardiology reports. Aug 2011 36. Simmons BB, Yeo A, Fung K. Current guidelines on antiplatelet agents for secondary prevention of noncardiogenic stroke: an evidence-based review. Postgraduate medicine. Mar 2010 37. Dridi NP, Johansson PI, Clemmensen P, et al. Prasugrel or double-dose clopidogrel to overcome clopidogrel low-response--the TAILOR (Thrombocytes And IndividuaLization of ORal antiplatelet therapy in percutaneous coronary intervention) randomized trial. Platelets. 2014

High on-treatment platelet reactivity

Supplementary Supplementary table 1. Results of different platelet reactivity tests in all included PAD patients No

CYP2C19 *2 alleles*

VASP (PRI)

VN PRU post <208†

VN PRU post <235†

VN % inhibition†

Postoperative days to VN

+/+

N/A

+/+

N/A

+/+

+/-

-/-

Inconclusive

+/+

+/-

-/-

N/A

-/-

N/A

Inconclusive

VN indicates VerifyNow; VASP, vasodilator-stimulated phosphoprotein phosphorylation; PRU, Platelet reactive units. *+/+ homozygous for CYP2C19*2 , +/- heterozygous for CYP2C19*2, -/- CYP2C19*2 not present, † - Bad responder to clopidogrel, + Good responder to clopidogrel

PART III Platelet reactivity in carotid artery disease

Chapter 5 The role of perioperative antiplatelet therapy and platelet reactivity testing in carotid revascularization: Overview of the evidence J Cardiovasc Surg, 2015 April; Volume 56, Issue 2, p 156-172

T. Leunissen, GJ. de Borst, P. Janssen and J. ten Berg

Chapter 5

Abstract Antiplatelet therapy has reduced the incidence of thromboembolic events for patients undergoing carotid revascularization.Â However, the platelet inhibitory effect of aspirin and clopidogrel, the most common used P2Y12 receptor inhibitors, is variable between patients. Patients displaying high platelet reactivity despite aspirin or clopidogrel treatment are at higher risk for thromboembolic events during and after carotid revascularization. In order to reduce the incidence of high platelet reactivity, more potent P2Y12 receptor inhibitors as prasugrel are used. However, this strategy increases the risk of bleeding. As there is evidence of a therapeutic window for platelet inhibition, platelet function tests could be helpful for tailoring antiplatelet therapy based on the patientâ&#x20AC;&#x2122;s thrombotic and bleeding risk. In this evidence overview, we describe the most commonly used platelet inhibitors, platelet function tests and the current evidence for tailoring of antiplatelet therapy in patients undergoing carotid revascularization.

Antiplatelet therapy in carotid revascularisation

Introduction Carotid artery stenosis is an important cause of stroke and represents a key target in stroke prevention. Roughly 20% of all cerebral infarctions are caused by an extracranial carotid stenosis. Multiple randomized trials have shown the efficacy of carotid endarterectomy (CEA) as secondary prevention for symptomatic patients with a stenosis of 70-99% in the carotid artery and in primary prevention for asymptomatic men aged <75 years with a degree of carotid artery stenosis â&#x2030;Ľ 50%. CEA has been challenged by carotid artery stenting (CAS) and although procedural outcome in terms of combined stroke and death rates is significantly better with CEA as compared with CAS within the first 30 days after treatment, the outcomes from 30 days and further have been shown to be comparable between the two treatments. As technical improvements with stenting are to be expected, the safety of CAS will undoubtedly improve in the near future. Independent of the type of intervention, the benefit of carotid revascularization in general is hampered by a risk of stroke related to the procedure itself. One of the feared pathophysiological mechanisms of perioperative stroke is a heightened thrombotic state leading to thromboembolization of the operated arterial tract. Thrombus formation at the injured endothelium of the endarterectomy site and the site of the clamp after restoration of the blood flow, is probably due to the exposure of the subendothelial extracellular matrix to the circulation, causing activation of the platelet aggregation. To some extent, this physiological mechanism happens in all patients that undergo carotid revascularization, fortunately it leads to micro-emboli and new postoperative cerebral deficits in only a minority of patients. Micro- embolic signals (MES) can be detected by Transcranial Doppler in the ipsilateral middle cerebral artery and adequate measures can be taken to prevent further damage. However, ideally, patients at increased risk of thrombotic complications are detected prior to the intervention to be able to undertake timely individualized preventive measures. Antiplatelet therapy in carotid revascularization All patients undergoing cardiovascular revascularization and needing secondary prevention are prescribed antiplatelet therapy (APT), which is not only fundamental in the prophylaxis of cardiovascular events in patients with carotid atherosclerosis, but also in the perioperative management of patients undergoing carotid revascularization. Historically, the choice of APT in carotid revascularization has been based on evidence in coronary interventions, as opposed to carotid interventions and standard perioperative antithrombotic therapy was originally carried out using an anticoagulant regimen of heparin or warfarin and aspirin monotherapy. However, sub-acute stent thrombosis still occurred as a serious complication in approximately 5-20% of patients after percutaneous coronary interventions (PCI). Studies comparing dual antiplatelet therapies (DAPT) and anticoagulant therapies were performed in the late 1990s, and showed the superiority of DAPT with thrombosis rate of respectively 0.0% and 5.0% (p<.001) and DAPT is now recommended as the gold standard treatment with regard to periprocedural management in PCI1.

Chapter 5

In the beginning CEA was performed without any form of APT, but the benefit of postoperative aspirin treatment as prevention of secondary strokes and prolongation of postoperative survival was soon picked up from multiple cardiology trials2. The next step was to start aspirin pre-operatively and stop several days prior to intervention, however this was not supported by any evidence. Thereafter, because of thrombosis related complications, aspirin was continued during and after carotid revascularization. Nowadays, DAPT is also used as perioperative medical treatment but this treatment strategy is not evidence based and still little consensus exists regarding the optimal combination of APT. In 2009, a survey of European vascular surgeons showed that 5% of the surgeons would stop aspirin before the procedure and 43% would stop clopidogrel before the procedure in symptomatic patients undergoing CEA. Of the surgeons who would stop clopidogrel, 49% would do this â&#x2030;Ľ7 days before surgery. In contrast, 3% would prescribe a single dose of 75 mg clopidogrel 12 hours before surgery3. In 2007, a clinical expert consensus document on CAS advised the combined administration of aspirin and clopidogrel 4 days prior to the procedure. After the procedure, clopidogrel should be continued for at least 30 days and aspirin should be continued lifelong4. This advice is mainly based on studies evaluating APT as secondary prevention after stroke and TIA and studies regarding APT during coronary interventions, because only few prospective studies have evaluated different regimens of APT in patients undergoing carotid revascularization. In CEA and CAS, the dose and regimen of the newer and stronger antiplatelet agents, such as P2Y 12 inhibitors (prasugrel, ticagrelor and cangrelor), cilostazol and GP IIb/IIIa-antagonists, have not yet been established. Testing platelet reactivity In analogy with observations in patients undergoing PCI, a natural variation in response on APT can be expected between individual patients undergoing carotid revascularization. A recently published review showed that High on Aspirin Platelet Reactivity (HAPR) occurred in 22.2% of the patients in a pooled analysis of 102 studies containing a total of 44,098 patients with coronary artery-, cerebrovascular- and peripheral artery disease who were treated with aspirin and/or clopidogrel. This analysis revealed that patients with HAPR had an increased risk of cardiovascular events (combined endpoint of cardiovascular death, myocardial infarction, stent thrombosis, stroke, acute limb ischemia, peripheral revascularization and acute peripheral occlusion) (RR 2.09, 95% CI: 1.77-2.47). The amount of non-responders for clopidogrel was even higher: 40.4% of patients was diagnosed with High on Clopidogrel Platelet Reactivity (HCPR), which was also associated with an increased risk of cardiovascular events (RR 2.80, 95%CI: 2.40-3.27)5. Studies evaluating HAPR and HCPR report a wide variation in the incidence of nonresponders. This variation can be explained by differences in the use of platelet reactivity tests (PRT), threshold values and antiplatelet medicaments in the various studies and the differences between patient populations in which the response to APT was assessed.

Antiplatelet therapy in carotid revascularisation

The different tests measure diverse pathways of thrombus formation and the reproducibility and applicability vary strongly between these tests. We will briefly summarize the most commonly used tests: VerifyNow® System The VerifyNow System provides an easy and rapid point-of-care test to measure the platelet inhibition in response to aspirin, P2Y12 receptor inhibitors and GP IIb/IIIainhibitors. It uses cartridges that contain different agonists to stimulate platelets in whole blood. The platelets activate, bind to fibrinogen coated beats, and precipitate in the cartridge and the degree of inhibition is subsequently determined, based on optical light transmittance through the cartridge. Results from the P2Y12 -assay are reported in P2Y12 Reaction Units (PRU), a BASE value and a percentage of inhibition. Results from the Aspirin assay are reported in Aspirin Reactive Units (ARU). Higher PRU and ARU levels indicate higher levels of residual platelet reactivity. The results are congruent with the LTA and cut-off values of ≥208 PRU have been defined for the prediction of thrombotic events following PCI. Light Transmittance Aggregometry Light Transmittance Aggregometry (LTA) is often referred to as the ‘’golden standard’’ because of the long lasting experience with the use of this technique. It uses optical detection of the formation of aggregates in platelet-rich-plasma stimulated with manually added agonists. Citrated whole blood is centrifuged in two steps, resulting in PlateletRich plasma (PRP) and Platelet- Poor plasma (PPP). The amount of light that shines through the PPP is defined as 100% aggregation, while the amount of light shining though the PRP is defined as 0% aggregation. The formed aggregates modify the optical density of the samples and this can be expressed as a percentage aggregation. Because the LTA is a manually conducted test, the reproducibility between laboratories is low and the test is time consuming. The Multiplate® The Multiplate detects the increase of electrical impedance resulting from the adhesion and aggregation of platelets on two metal sensor wires in the Multiplate test cell. When activated, platelets adhere to the sensor wires and the electrical resistance between the wires increases. This increase in resistance is recorded and reported as aggregation units (AU) and plotted against time (AU*min). To stimulate the aggregation, multiple agonists are used. The Multiplate can be used for both aspirin and P2Y12 monitoring. The test is semi-automatic since preparation of the agonists and pipetting is necessary to perform the tests. Vasodilator-stimulated Phosphoprotein Phosphorylation Assay The Vasodilator-stimulated phosphoprotein (VASP) is an intracellular platelet protein that is not phosphorylated under normal conditions and which phosphorylation is regulated

Chapter 5

by cyclic adenosine monophosphate (cAMP) cascade. While prostaglandin E1 (PGE1) activates cyclic adenosine monophosphate (cAMP), Adenosine diphosphate (ADP) inhibits VASP phosphorylation through the P2Y12 receptor. As a result, persistent VASP phosphorylation (VASP-P), as measured with flow cytometry, correlates with P2Y12 receptor inhibition, reflecting the effect of the medical treatment. Therefore, the VASP-P assay is only applicable for monitoring of P2Y12 inhibitors. The test has a standardized diagnostic assay kit, is time consuming, and requires experienced laboratory personnel. Platelet Function Analyser-100. The Platelet Function Analyser (PFA)-100 aspirates whole blood at high shear rates, mimicking injured endothelium, through cartridges containing an aperture with a membrane coated with either collagen and epinephrine or collagen and ADP. The agonists induce aggregation leading to rapid occlusion of the aperture and cessation of blood flow whereby the speed represents the outcome; closure time. The test is quick, relatively cheap and easy to use. However the PFA-100 is mainly intended as screening instrument for issues related to primary haemostasis with a high negative predictive value and not as monitoring instrument for antiplatelet therapy. Tailoring antiplatelet therapy in coronary artery disease The association of HAPR and HCPR with increased risk of thrombotic events has resulted in the idea of tailoring APT based on the platelet reactivity response. In extension, it has been hypothesized that switching APT to achieve a lower level of platelet reactivity in high risk patients would result in less adverse events. But not only high platelet reactivity (HPR) can be an indicator to change APT; multiple studies suggest a relation of low platelet reactivity (LPR) to the risk of bleeding in patients undergoing coronary stenting procedures6,7. This may suggests that there is a therapeutic window for platelet reactivity with HPR at one end of the spectrum and LPR at the other end. The potential benefit of tailoring APT on clinical outcome has been investigated in patients undergoing PCI (table 1). Disappointing results from two major trials (GRAVITAS, n=2,2148 and ARCTIC, n=2,240 9) show no difference in primary composite endpoint (death of cardiovascular causes, nonfatal acute myocardial infarction (AMI) and STelevation) or bleeding events after 6 months in GRAVITAS and 1 year in ARCTIC, when comparing standard APT or tailored APT. However, a few smaller studies (MADONNA n=805 10, Wang et al. n=306.11, Bonello et al. n=162 12and Valgimigli et al. n= 13613.) do show a decrease in composite thrombotic endpoint and no difference in bleeding events. Although the major trials were adequately performed and of course have the most power, they also had some weaknesses related to their design, such as the lack of an adequate treatment to permanently combat HPR and low risk populations. Therefore, there is still some belief in the potential of tailoring APT. Tailoring antiplatelet therapy in carotid revascularization Patients undergoing carotid revascularization might also benefit from tailoring APT based on platelet reactivity testing, as the rates of per- and postoperative MES, TIA, cerebral infarction and death still leave room for improvement. To discuss the available literature

Antiplatelet therapy in carotid revascularisation

on tailoring APT in carotid revascularization we performed a PubMed search for available prospective studies regarding ‘’platelet reactivity testing’’ and ‘’carotid revascularization’’ in humans. We wish to emphasize that this overview is intended as a topical summary of the available data and our assessment should not be considered as a systematic review or meta-analysis of all the literature available on the utility of platelet function testing. The articles we found have been divided by the performed intervention; carotid endarterectomy (table 2) and carotid artery stenting (table 3). A total of 16 relevant studies were found, of which three studies adjusted the antiplatelet therapy based on platelet reactivity test results; the largest study performed the VerifyNow-P2Y12 assay in 2014 patients with DAPT 1 hour before CAS14. Based on the results of this test, 99 patients with HCPR (PRU≥230) were randomly assigned to receive low dose clopidogrel (75 mg/d, n= 50) or to high dose clopidogrel (150 mg/d, n= 49). After 30 days, the second VerifyNow-P2Y12 assay was performed. The results of this study could not confirm a benefit for high dose clopidogrel in reducing platelet reactivity since the differences between median 30-day PRU (p=.483) and percentage of change of PRU (p=.442) were not significant. In a historical cohort analysis patients were measured with VerifyNow P2Y12 assay prior to neurovascular stent placement (51% CAS)15; initially 49 patients received 75 mg clopidogrel in the observational phase (2006-2008) and in the interventional phase (2008-2011) 47 patients were given tailored clopidogrel (maintenance dose 150-600 mg) with a goal inhibition of >20% . Overall 36.5% had HCPR (≤20% inhibition) and periprocedural thromboembolic complications were seen in 7 patients (7.3%). No significant decrease in thromboembolic complications were seen in the interventional group as compared to the observation group. In another historical cohort analysis with patients undergoing CAS, a benefit for tailoring APT was seen when adding cilostazol prior to intervention in patients with HCPR16; in period I (2010-2011) 28 patients, under which 12 with HCPR (PRU≥240 measured by VerifyNow P2Y12 assay) were all treated with DAPT and in period II (2011-2013) 36 patients all received DAPT including 13 patients with HCPR, who received extra cilostazol 200 mg daily prior to CAS. PRU was significantly lower (300 ± 36 and 240 ± 62; p=.006) and % inhibition was significantly higher (8.8 ± 8.7 and 28 ± 18; p=.005) in period II. Unfortunately no specified information about platelet reactivity in the 13 HCPR patients receiving extra cilostazol is reported. On diffusion-weighted imaging a significant decrease in new ischemic lesions was seen in period II compared to period I (p=.034). None of the patients had haemorrhagic or thromboembolic adverse events. Six other studies investigated the correlation of platelet reactivity levels and clinical outcome after CEA or CAS without adapting antiplatelet therapy based on the measured platelet reactivity; results showed higher platelet reactivity being linked to either more postoperative micro-embolic signals in CEA17, more new cerebral ischemic lesions after CAS18 or higher percentage of the composite endpoint ‘’ischemic events’’ (stroke, in-stent stenosis and neurologic deficit)15,19,20. This finding seems more evident in HCPR than in HAPR19.

Aim

Assessing the utility of individualized APT based on MEA

Assessing the effect of tailoring antiplatelet therapy in patients with HAPR and/ or HCPR

Evaluate the utility of prasugrel compared with clopidogrel in patient with HCPR after PCI

Assessing the effect of high dose clopidogrel as compared to low dose clopidogrel

Authors (study, ref.)

Siller- Matula et al. 2013 (MADONNA) (10)

Collet et al. 2012 (ARCTIC) (14)

Trenk et al. 2012 (TRIGGER-PCI) (15)

Price et al. 2011 (GRAVITAS) (8)

Double blind multicentre RCT

Single centre RCT

Multi- centre RCT

Prospective cohort study

Study design

1105 patient without LD + MD 75 mg clopidogrel vs. 1109 patients with LD 600 mg + MD 150 mg clopidogrel

211 patients with LD 600 mg + MD 75 mg/d clopidogrel vs. 212 patients with LD 60 mg + MD 10 mg prasugrel

1227 patients with LD 600 mg clopidogrel + MD left to operator’s discretion vs. 1213 patients with GPIIb/ IIIa- inhibitor + extra LD 600 mg and MD 150 mg clopidogrel or LD 60 mg and MD 10 mg prasugrel

395 patients with LD 600 mg clopidogrel vs. 410 patients with repeated LD 600 mg prasugrel or LD 600 mg clopidogrel up to 4 times, guided by MEA results

Medication

Table 1. Platelet reactivity testing and percutaneous coronary intervention.

VerifyNow P2Y12 assay

VerifyNow Aspirin and P2Y12 assay

Multiple electrode aggregometry (ADP test)

PRT

P2Y12: PRU ≥230

P2Y12: PRU >208

Aspirin: ≥550 ARU P2Y12: ≥235 PRU

≥50 U

Used cut off values for HAPR/ HCPR

• No difference in death from cardiovascular causes (p=.14), nonfatal AMI (p=.42) and stent thrombosis (p=.72) • HCPR was only reduced in 60% of patients at 30-day follow up

• Prematurely halted: primary endpoint event rate was lower than expected (2.3% vs 7%) • Early analyses: PRU decreased more in prasugrel group compared to control (p=.001)

• At 1 year no difference in composite endpoint (34.6 vs 31.1% HR: 1.13, 95%CI: 0.98-1.29) • After 2-4 weeks 15.6% of study participants still had HAPR/ HCPR

• Stent thrombosis and ACS occurred less often in guided group vs nonguided group (p=.027 and p=.001) • No differences in cardiac death and major bleeding (p=.422 and p=.186)

Main findings

Chapter 5

Assessing the effect of the addition of tirofiban on clinical outcome in patients with HAPR and/ or HCPR

Adjusted loading dose clopidogrel according to VASP-PRI

Valgimigli et al. 2009 (3T/2R) (13)

Bonello et al. 2008 (12)

Multi- centre RCT

Double blind multicentre RCT

Monocentric RCT

84 patients with LD 600 mg clopidogrel vs. 78 patients with added max 3 boluses 600 mg clopidogrel to reach VASP-PRI<50%

132 patients with standard aspirin and clopidogrel vs. 131 patients with standard aspirin and clopidogrel + additional tirofiban (All patients with HAPR and/or HCPR)

156 patients with 75 mg clopidogrel vs. 150 patients with additional 75 mg clopidogrel up to 3 times

VASP

VerifyNow Aspirin and P2Y12 assay

VASP

• The rate of combined endpoint (cardiovascular death, stent thrombosis, recurrent ACS, re PCI <1 year) was higher in control group • No difference in bleeding • Periprocedural AMI in 20.4% of tirofiban vs. 35.1% in the controls (RR: 0.58, 95%CI, 0.39 to 0.88; p=.0089) • No difference in bleeding (p=.99) • 86% of the VASP guided group the VASP- index was significantly decreased (p<0.001) • Adverse events occurred less in the VASP guided group than in controls (p=.007)

VASP-PRI ≥50%

Aspirin: ≥550 ARU P2Y12: <40% inhibition

VASP-PRI>50% after loading dose of 600 mg clopidogrel

ACS acute coronary syndrome, ADP adenosine diphosphate, AMI acute myocardial infarction, APT antiplatelet therapy, CAS coronary artery disease, HAPR high on aspirin platelet reactivity, HCPR high on clopidogrel platelet reactivity, LD loading dose, LTA light transmittance aggregometry, MD maintenance dose, MEA multiple electrode aggregometry, PCI percutaneous coronary intervention, VASP (-PRI) vasodilatator- stimulated phosphoprotein (-platelet reactivity index)

Assessing the effect of modifying clopidogrel dosage based on VASP on clinical outcome

Wang et al. 2011 (11)

Antiplatelet therapy in carotid revascularisation

Cohort study

Assessing aspirin- induced COX- inhibition

Assessing the effect of clopidogrel on PR and amount of MES

Assessing the effect of aspirin on platelet aggregation during CEA

Assadian et al. 2007 (17)

Payne et al. 2004 (18)

Payne et al. 2004 (19)

Single- centre prospective cohort analysis

Double-blind RCT

Assessing the effect of clopidogrel on PR pre- and post-CEA

Payne et al. 2007 (16)

Study design

Aim

Authors (ref.)

Table 2. Platelet reactivity testing and carotid endarterectomy.

50 CEA patients + 18 controls, all on aspirin

46 patients with aspirin + clopidogrel vs. 54 patients with aspirin + placebo

86 patients with history of CEA on aspirin + 29 patients with carotid artery stenosis not treated with aspirin

27 patients with aspirin + clopidogrel vs. 29 patients with aspirin + placebo

Population

LTA (AA 2.5, and 5 mmol/L. ADP 0.05, 0.1, 0.2 and 0.4 μmol/L, collagen 0.5 and 1.0 μg/ml and TRAP 6 μmol/L)

1. LTA (AA 2.5 mmol/L) 2. Flow cytometry (ADP 1 μmol/L)

1. FACS flow cytometry (AA 80 μmol/L, ADP 2.5 μmol/L) 2. PFA-100 (CEPI - and CADP cartridge)

1. Flow cytometry (ADP 0.1, 1, 10 μmol/L ) 2. 4 channel aggregometry (ADP 0.5, 1, 2, and 4 μmol/L, AA 2.5 mmol/L).

PRT and agonist

N.a.

HAPR: >20% of maximum platelet aggregation in response to AA

1. FACS flow cytometry: <15% CP62p- positive platelets. 2. PFA-100: CEPI-CT of <165 seconds

HAPR: >20% of maximum platelet aggregation in response to AA

Used cut off values for HAPR/ HCPR

• Increased aggregation on AA (570%) despite aspirin treatment during CEA • ADP, TRAP and collagen increased to a smaller extent (<40%)

• Clopidogrel showed a small reduction of platelet response to ADP compared to placebo (p=.03), but a major reduction in number of patients with >20 MES. (OR 10.23 95%CI: 1.3 to 83.3; p=.01)

• PFA-100 showed ‘’aspirin resistance’’ in 14/86 (16%) patients, while flow cytometry showed significant COX- inhibition for all patients (0%)

• Percentage MPA’s during surgery increased in the placebo- but not in the clopidogrel-treated group • Percentage fibrinogen binding in response to ADP was significantly lower in clopidogrelgroup (p=.002) • Highest baseline ADP response showed greatest reduction in PR on clopidogrel

Main findings

Chapter 5

Evaluate PR during CEA and determine the effect of Dextran- 40

Robless et al. 1999 (21)

Single- centre prospective cohort analysis

34 patients on aspirin + 6 patients on aspirin and postoperative Dextran infusion

128 patients on single aspirin therapy

1. Flow cytometry (no agonists used) 2. Whole blood aggregometry with Chronolog (ADP 5 μmol/L)

LTA (ADP: 0.5, 1, 2, and 4 μmol/L), collagen (10, 20, and 50 mg/mL) and arachidonic acid (3 or 6 μmol/L). 2. Flow cytometry (ADP 0.1, 1, 10 μmol/L and thrombin 0.02, 0.04, 0.08, 0.16 μ/mL)

• Higher platelet response to ADP in high risk group (>25 emboli) vs. low risk group (≤25 emboli) measured by aggregometry (p=.0012) and flow cytometry (p=.0018). • Same effect in CRP, but not in thrombin of AA • Increase of ADPinduced platelet activation up to 1 hour postoperative despite aspirin therapy (p<0.001) • Dextran reduces Pselectine expression and number MES (p=.01 and p=.01)

N.a.

HAPR: >50 MES /30 min as indication for additional infusion of Dextran

AA arachidonic acid, ADP adenosine diphosphate, CADP-CT collagen/adenosine diphosphate closure time, CEPI-CT collagen/epinephrine closure time, CPA Cone and platelet analyzer, COX cyclo- oxogynase, CRP collagen-related peptide, DAPT dual antiplatelet therapy, FACS Fluorescence Activated Cell Sorting, HAPR high on aspirin platelet reactivity, HCPR high on clopidogrel platelet reactivity, MES micro embolic signals, MPA monocyte platelet aggregate, N.a. not applicable, PFA-100 platelet function analyser, PR platelet reactivity, PRT platelet reactivity test, RCT randomized controlled trial, TRAP thrombin receptor agonist peptide

Assessing the relationship between PR and the amount of MES perioperative

Hayes et al. 2003 (20)

Antiplatelet therapy in carotid revascularisation

Prospective double-blind RCT

Single- centre prospective cohort analysis

Assessing the effect of the addition of cilostazol to DAPT on PR

Assessing the relationship between preoperative PR and clinical outcome after CAS

Assessing the relationship between HAPR and HCPR and clinical outcome after CAS and assessing the effect of adapting doses of clopidogrel in HCPR

Assessing the effect of 300 mg or 600 mg LD of clopidogrel on number of MES

Nakagawa et al. 2014 (23)

Sorkin et al. 2014 (24)

Fifi et al. 2013 (25)

Heyden van der et al. 2013 (26)

Maruyama Assessing the effect of the et al. 2011 (27) addition of cilostazol to DAPT on PR

Single-centre historical cohort analysis

Single-centre RCT

Assessing the effect of dose adjustment of clopidogrel (75 or 150 mg) on PR

Gonzalez et al. 2014 (22)

Design

Aim

Authors (ref.)

62 patients with clopidogrel vs. 15 patients with clopidogrel + cilostazol

19 patients with 300 mg LD clopidogrel vs. 16 patients with 600 mg LD clopidogrel

49 patients with standard clopidogrel vs. 47 patients with tailored clopidogrel (maintenance dose 150 up to 600 mg)

449 patients on aspirin and clopidogrel

12 HCPR patients with DAPT vs. 13 HCPR patients with DAPT and cilostazol

50 HCPR patients with 75 mg clopidogrel vs. 49 HCPR patients with 150 mg clopidogrel

Population

Table 3. Platelet reactivity testing and carotid artery stenting (CAS).

VerifyNow P2Y12 assay

1. VerifyNow P2Y12 assay 2. LTA (ADP 5 and 20 mmol/L)

VerifyNow Aspirin and P2Y12 assay

VerifyNow P2Y12 assay

PRT

P2Y12: ≤20% inhibition

N.a.

Aspirin: ≥550 ARU P2Y12: ≤20% inhibition

Aspirin: ≥550 ARU P2Y12: ≥237 PRU

PRU ≥240

PRU ≥230

Used cut off values for HCPR

• Percentage inhibition higher in cilostazol + clopidogrel vs clopidogrel group (p=.005)

• No significant differences in platelet aggregation in patients with vs. without MES (VerifyNow p=0.7704 and aggregometry p=.8449)

• 16.7 % of nonresponders had thrombotic adverse event (vs. 1.6% in responders group) • Events occurred in 10.3% of standardvs. 4.5% of tailored clopidogrel therapy (p=.38)

• Event free survival regarding ischemic events was longer with cut-off PRU ≤198 • ARU values were not related to primary outcomes after CAS

• Significant lower PRU in DAPT + cilostazol than in DAPT after one week (p=.044)

• No difference after 30 days in median PRUvalues (p=.483) or percentage of change (p=.442)

Main findings

Chapter 5

Assessing the effect of clopidogrel and aspirin on PR and evaluating the relationship between HAPR and HCPR and clinical outcome

Assessing the relationship between HAPR and HCPR and the occurrence of new cerebral ischemic lesions

To assess the correlation between LTA and FACS analysis in patients with Abciximab (GPIIb/IIIainhibitor)

Prabhakaran et al. 2008 (29)

Song et al. 2002 (30)

Shenkman et. Al. 2001 (31)

Single- centre prospective cohort analysis

Retrospective cohort analysis

Single- centre prospective cohort analysis

16 patients with aspirin and Abciximab

76 patients on clopidogrel and aspirin therapy

76 patients: 71 with aspirin + 55 with clopidogrel

50 patients with clopidogrel + 50 healthy donors as controls

1. LTA (ADP 5 mM) 2. Flow cytometry (TRAP 25 mM) 3. CPA

VerifyNow Aspirin and P2Y12 assay

Multiplate- analyzer (ADP test)

• 28% of clopidogrel treated patients had HCPR • 41% of patients with HCPR (vs. 0% in responders) had adverse thrombotic events • No relationship between platelet inhibition and clinical outcome. • Age >55 years and diabetes were independent predictors for HCPR. • After CAS, 59.2% of patients developed new ischemic lesions, of whom 15.8% had HAPR and 65.8% had HCPR. • Patients with new ischemic lesions had more HCPR then patients without new lesions (82.2 vs 41.9%, p=.001) • 30 min after bolus administration CPA and ADP- induced aggregation showed decrease in PR of 90-98% • ADP- induced aggregation and CPA had high correlation (P<.01 and r2=0.64).

>52 U

Aspirin: ≥550 ARU P2Y12: ≤40% inhibition

Aspirin: ≥550 ARU P2Y12: ≥240 PRU

N.a.

AA arachidonic acid, ADP adenosine phosphate, CEP-CT collagen- epinephrine closure time, CPA Cone and platelet analyzer, COX cyclooxygenase, CRP collagen-related peptide, DAPT dual antiplatelet therapy, HAPR high on aspirin platelet reactivity, HCPR high on clopidogrel platelet reactivity, MES micro embolic signals, MPA monocyte platelet aggregate, N.a. not applicable, PFA-100 platelet function analyzer, PR platelet reactivity, PRT platelet reactivity test, RCT randomized controlled trial, TRAP thrombin receptor agonist peptide

Assessing the effect of clopidogrel on PR and the relationship between HCPR and clinical outcome

MüllerSchunk et al. 2008 (28)

Antiplatelet therapy in carotid revascularisation

Chapter 5

Contrasting one prospective cohort analysis in CAS, including 76 patients measured with VerifyNow Aspirin and P2Y12 assay, could not show any relation between HAPR (ARU≥550) or HCPR (PRU≤40% inhibition)21. In this study age≥ 55 (b=-16.3, p=.020) and diabetes (b=26.8, p=.015) were found as independent predictors for HCPR. None of the studies evaluated the risk of low platelet reactivity on haemorrhagic events. Multiple studies aimed to evaluate the effect of aspirin22,23, clopidogrel24-26, cilostazol16,27 or dextran-4028 on platelet reactivity and clinical outcome. These studies focussed mainly on the efficacy and safety of the APT and thereby no adjustments in APT were made. One study observed the absolute inhibitory capacity of aspirin in 50 CEA patients on monotherapy aspirin; the inhibitory effect on platelet reactivity seems to be severely diminished during CEA but becomes partly restored at the end of surgery without adding aspirin therapy22. Platelet reactivity was measured prior to surgery (7.6 ± 5.5%), during surgery (73.8 ± 7.2%) and postoperative (45.9 ± 7.4%) with arachidonic acidinduced aggregometry. Another study measured the course of platelet reactivity during CEA in 34 patients with monotherapy aspirin, receiving a bolus of 5000 units unfractionated heparin before carotid artery clamping28; p-selectin expression and ADP-induced platelet aggregation increased after carotid clamping (p< .01 and p< .01) and clamp release (p < .05 and p< .01) compared to baseline despite antiplatelet therapy. Two randomized controlled trials by the same author evaluated the effect of single dose 75 mg clopidogrel + aspirin compared to placebo + aspirin prior to CEA: the clopidogrel group showed major reduction in number of patients with >20 MES postoperative (OR:10.23; 95% CI; 1.3 to 83.3; p=.01)25 and decrease in platelet reactivity stimulated by ADP compared to baseline (resp. p=.002)26. An increase in time from flow restoration to skin closure was noticed (p=.04), but no significant difference in bleeding complications due to the low patient numbers. Two studies showed that adding cilostazol to either DAPT16 or clopidogrel27 in patients undergoing CAS resulted in stronger inhibition of platelet reactivity measured with VerifyNow P2Y12-assay; the first study reported lower PRU (p=.044) in DAPT + cilostazol and the second higher percentage of inhibition (p=.005). Patients with DAPT + cilostazol also showed less new ischemic lesions (p=.034) without increasing the risk of bleeding complications. Dextran-infusion in 6 patients on aspirin therapy after CEA with >50 MES/30 min in the postoperative period, resulted in a reduction of P-selectine expression after stimulation with ADP (p=.01) and amount of MES after 24 hours (p=.01). Two studies evaluated platelet reactivity within different test methods; a newer method, Cone and Platelet Analyser (CPA) and ADP- induced platelet aggregometry was used for measuring platelet reactivity in 16 patients undergoing CAS with Abciximab29. A high correlation between the CPA and aggregometry was seen (p<.01 and r2=.64), thereby suggesting that CPA could be a quick, easy and accurate method in the clinic to monitor platelet reactivity in patients with GPIIb/IIIa- antagonists. In a different study, HAPR was

Antiplatelet therapy in carotid revascularisation

found in resp. 16% and 0% of 86 patients with a history of CEA when measured with PFA-100 or flow cytometry, thereby showing a bad correlation between both platelet reactivity tests23.

Discussion The currently available evidence on the potential benefit of tailoring APT in patients undergoing carotid revascularization is very scarce. Therefore, at this stage, we cannot make any statements towards the utility of PRT in carotid revascularization. More research has been done in patients with coronary artery disease (CAD) undergoing PCI and the presence of HAPR and HCPR is clearly associated with an increased risk of thrombotic events, however no benefit has been shown with respect to the adjustment of APT based on these measurements. At this time, it is not recommended to routinely measure platelet reactivity in patients with stable CAD in daily clinical practice. Platelet reactivity only plays a role in patients at high risk for stent thrombosis30; There is a need for randomized control trials to investigate the potential benefit of tailoring APT based on platelet reactivity in patients undergoing carotid revascularization. However, multiple questions need to be answered before this concept can be assessed in clinical trials: 1. What is the incidence of HAPR and HCPR in patients undergoing carotid revascularization? 2. Are HAPR and HCPR in patients undergoing carotid revascularization related to the occurrence of thrombotic and/or bleeding events during follow-up and what are the optimal cut-off values to predict these events? 3. Does HAPR or HCPR change over time and can we accurately predict the occurrence of events with just a single platelet reactivity test measurement? 4. What is the optimal alternative antiplatelet therapy for patients undergoing carotid revascularization with HAPR or HCPR? Apart from these remaining questions, one encounters many difficulties during the development of a proper study design31. We would like to mention the biggest problems and challenges: Most importantly; we do not know what APT is the most suitable for non-responders. In GRAVITAS, patients who underwent PCI with HCPR were randomized to a higher loading- and maintenance dose clopidogrel. However, over 40% of the patients remained low- or non-responder despite high dose of clopidogrel. In contrast, when patients with HCPR were switched to prasugrel in the MADONNA- trial, the amount of non-responders, stent thrombosis and acute coronary syndrome decreased. The non-respons to clopidogrel could potentially be explained by genetic varieties in the CYP2C19-allele that results in a decreased metabolization of clopidogrel by the CYP2C19 enzyme: clopidogrel is a pro-drug which needs to be metabolized into an active metabolite before

Chapter 5

it has an effect on platelet reactivity. Therefore, higher doses of clopidogrel in nonresponders are of no use and future studies are recommended to change the antiplatelet drug in patients with HCPR; other P2Y12-inhibitors, such as prasugrel and ticagrelor or additional cilostazol could be a proper alternative. However, prasugrel and ticagrelor are associated with an increased bleeding rate and costs, which makes them less suitable as standard medical therapy. Currently, ST-elevation myocardial infarction (STEMI)patients are recruited for ‘’the Patient Outcome after primary PCI (POPular) Genetics study (ClinicalTrials.gov Identifier, NCT01761786)’’ in which APT is tailored to the CYP2C19 metabolizer status 32. Patients are randomized to either routine treatment with ticagrelor or prasugrel or APT based on CYP2C19 genotyping; *1/*1 (wild-type) patients receive clopidogrel, and patients who carry 1 or 2 loss-of-function allele(s) (CYP2C19*2 or *3) receive ticagrelor or prasugrel33. The next problem to deal with is that many different platelet reactivity tests are used in trials and in daily clinical practice. There is no evidence showing superiority of one test, although the VerifyNow, PFA-100 and VASP are widely available. Because of this heterogeneity in platelet reactivity tests, there is no clear definition of HAPR and HCPR and comparing trials becomes nearly impossible. For each individual platelet reactivity tests, there is no consensus about the usage of arterial or venous blood. Recently, a study compared platelet reactivity measured with LTA, VerifyNow and PFA-100 in blood from the coronary artery-, femoral artery and femoral vein, showing wide differences between tests used as well as between coronary and venous blood34. Another problem for the interpretation of the values of platelet reactivity tests in patients with carotid atherosclerosis is that the commonly used thresholds for HAPR and HCPR were determined in patients with CAD. Other threshold values could improve selection of high risk patients undergoing carotid revascularization. In patients undergoing CAS, a new threshold for HCPR as assessed with the VerifyNow was already proposed by Sorkin et al, based on retrospective analysis of 449 patients 19. In the future, threshold values for HAPR and HCPR in patients undergoing CEA and CAS should be determined. However, we should not only focus on High platelet reactivity, but also on low platelet reactivity. This is especially important in patients undergoing CEA, as this is a more invasive procedure with a higher bleeding risk: the therapeutic window could be much smaller in this population as compared to patients undergoing stenting procedures. The timing of platelet reactivity testing is also important. In our opinion, timing of testing platelet reactivity and switching APT should ideally be before or very early after intervention, since the thrombotic and bleeding risks are highest in the early phase after intervention and HAPR and/or HCPR might play an important role in this early phase. However, it is probably not possible to prevent all mechanical complications related to the procedure with adequate platelet inhibition, as is suggested by the results from the ARCTIC- trial35 in PCI patients and the IMPACT- trial in CAS patients24. Finally, the combined event rate of stroke and death after carotid revascularization is relatively low (about 10% in CAS and 4% in CEA) resulting in the need of a large number of patients to prove significant benefit of new treatment strategy compared to standard

Antiplatelet therapy in carotid revascularisation

care. With the introduction of new antiplatelet drugs, we expect that the thrombotic event rates will become lower. However, the risk of hemorrhagic complications might increase, therefore we would advise the use of a combined clinical benefit endpoint, comprising both thrombotic and bleeding events, in future trials. This holds true for the cardiology population as well, since no definitive proof for tailoring APT has been found in this population either. To conclude; based on evidence from studies in patients undergoing carotid revascularization, we cannot make any statement towards the best medical APT and the utility of platelet reactivity testing in this group of patients. Dual antiplatelet therapy with aspirin and clopidogrel seems to be promising in CEA and CAS, but the role of the relatively newer P2Y12- inhibitors (prasugrel, ticagrelor and cangrelor), cilostazol and GPIIb/IIIa-antagonists has not yet been studies. Various platelet reactivity tests are available to investigate a patientâ&#x20AC;&#x2122;s platelet response on APT. Even though the correlation between high platelet reactivity despite APT and an increased amount of MES and thrombotic events in patients undergoing carotid vascularization is proven, there is no evidence that tailoring antiplatelet therapy based on platelet reactivity tests can improve clinical outcomes. Many more studies have to be conducted to gain more insight on this matter.

Chapter 5

References 1.

Schomig A, Neumann FJ, Kastrati A, et al. A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary-artery stents. The New England journal of medicine. Apr 1996 2. Goodnight SH, Coull BM, McAnulty JH, Taylor LM. Antiplatelet therapy--Part II. The Western journal of medicine. May 1993 3. Hamish M, Gohel MS, Shepherd A, Howes NJ, Davies AH. Variations in the pharmacological management of patients treated with carotid endarterectomy: a survey of European vascular surgeons. European journal of vascular and endovascular surger. Oct 2009 4. Bates ER, Babb JD, Casey DE, Jr., et al. ACCF/ SCAI/SVMB/SIR/ASITN 2007 clinical expert consensus document on carotid stenting: a report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents (ACCF/SCAI/SVMB/SIR/ASITN Clinical Expert Consensus Document Committee on Carotid Stenting). Journal of the American College of Cardiology. Jan 2007 5. Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 6. Sibbing D, Schulz S, Braun S, et al. Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. Journal of thrombosis and haemostasis : JTH. Feb 2010 7. Cuisset T, Cayla G, Frere C, et al. Predictive value of post-treatment platelet reactivity for occurrence of post-discharge bleeding after non-ST elevation acute coronary syndrome. Shifting from antiplatelet resistance to bleeding risk assessment? EuroIntervention : journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology. Aug 2009 8. Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA Mar 2011 9. Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 10. Siller-Matula JM, Francesconi M, Dechant C, et al. Personalized antiplatelet treatment after percutaneous coronary intervention: the MADONNA study. International journal of cardiology. Sep 2013 11. Wang XD, Zhang DF, Zhuang SW, Lai Y. Modifying clopidogrel maintenance doses according to

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vasodilator-stimulated phosphoprotein phosphorylation index improves clinical outcome in patients with clopidogrel resistance. Clinical cardiology. May 2011 Bonello L, Camoin-Jau L, Arques S, et al. Adjusted clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index decrease rate of major adverse cardiovascular events in patients with clopidogrel resistance: a multicenter randomized prospective study. Journal of the American College of Cardiology. Apr 2008 Valgimigli M, Campo G, de Cesare N, et al. Intensifying platelet inhibition with tirofiban in poor responders to aspirin, clopidogrel, or both agents undergoing elective coronary intervention: results from the double-blind, prospective, randomized Tailoring Treatment with Tirofiban in Patients Showing Resistance to Aspirin and/or Resistance to Clopidogrel study. Circulation. Jun 2009 Gonzalez A, Moniche F, Cayuela A, GonzalezMarcos JR, Mayol A, Montaner J. Antiplatelet effects of clopidogrel dose adjustment (75 mg/d vs 150 mg/d) after carotid stenting. Journal of vascular surgery. Aug 2014 Fifi JT, Brockington C, Narang J, et al. Clopidogrel resistance is associated with thromboembolic complications in patients undergoing neurovascular stenting. AJNR. American journal of neuroradiology. Apr 2013 Nakagawa I, Wada T, Park HS, et al. Platelet inhibition by adjunctive cilostazol suppresses the frequency of cerebral ischemic lesions after carotid artery stenting in patients with carotid artery stenosis. Journal of vascular surgery. Mar 2014 Hayes PD, Box H, Tull S, Bell PR, Goodall A, Naylor AR. Patientsâ&#x20AC;&#x2122; thromboembolic potential after carotid endarterectomy is related to the plateletsâ&#x20AC;&#x2122; sensitivity to adenosine diphosphate. Journal of vascular surgery. Dec 2003 Song TJ, Suh SH, Min PK, et al. The influence of anti-platelet resistance on the development of cerebral ischemic lesion after carotid artery stenting. Yonsei medical journal. Mar 2013 Sorkin GC, Dumont TM, Wach MM, et al. Carotid artery stenting outcomes: do they correlate with antiplatelet response assays? Journal of neurointerventional surgery. Jun 2014 Muller-Schunk S, Linn J, Peters N, et al. Monitoring of clopidogrel-related platelet inhibition: correlation of nonresponse with clinical outcome in supra-aortic stenting. AJNR. American journal of neuroradiology. Apr 2008 Prabhakaran S, Wells KR, Lee VH, Flaherty CA, Lopes DK. Prevalence and risk factors for aspirin and clopidogrel resistance in cerebrovascular stenting. AJNR. American journal of neuroradiology. Feb 2008

Antiplatelet therapy in carotid revascularisation

22. Payne DA, Jones CI, Hayes PD, Webster SE, Ross Naylor A, Goodall AH. Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance? Thrombosis and haemostasis. Jul 2004 23. Assadian A, Lax J, Meixner-Loicht U, Hagmuller GW, Bayer PM, Hubl W. Aspirin resistance among long-term aspirin users after carotid endarterectomy and controls: flow cytometric measurement of aspirin-induced platelet inhibition. Journal of vascular surgery. Jun 2007 24. Van Der Heyden J, Van Werkum J, Hackeng CM, et al. High versus standard clopidogrel loading in patients undergoing carotid artery stenting prior to cardiac surgery to assess the number of microemboli detected with transcranial Doppler: results of the randomized IMPACT trial. The Journal of cardiovascular surgery. Jun 2013 25. Payne DA, Jones CI, Hayes PD, et al. Beneficial effects of clopidogrel combined with aspirin in reducing cerebral emboli in patients undergoing carotid endarterectomy. Circulation. Mar 2004 26. Payne DA, Jones CI, Hayes PD, Naylor AR, Goodall AH. Therapeutic benefit of low-dose clopidogrel in patients undergoing carotid surgery is linked to variability in the platelet adenosine diphosphate response and patientsâ&#x20AC;&#x2122; weight. Stroke; Sep 2007 27. Maruyama H, Takeda H, Dembo T, et al. Clopidogrel resistance and the effect of combination cilostazol in patients with ischemic stroke or carotid artery stenting using the VerifyNow P2Y12 Assay. Intern Med. 2011 28. Robless PA, Tegos TJ, Okonko D, et al. Platelet activation during carotid endarterectomy and the antiplatelet effect of Dextran 40. Platelets. Jun 2002 29. Shenkman B, Schneiderman J, Tamarin I, KotevEmeth S, Savion N, Varon D. Testing the effect of GPIIb-IIIa antagonist in patients undergoing carotid stenting: correlation between standard aggregometry, flow cytometry and the cone and plate(let) analyzer (CPA) methods. Thrombosis research. May 2001 30. Janssen PW, ten Berg JM. Platelet function testing and tailored antiplatelet therapy. Journal of cardiovascular translational research. Jun 2013 31. Siller-Matula JM, Jilma B. Why have studies of tailored anti-platelet therapy failed so far? Thrombosis and haemostasis. Oct 2013 32. Bergmeijer TO, Janssen PW, Schipper JC, et al. CYP2C19 genotype-guided antiplatelet therapy in ST-segment elevation myocardial infarction patients-Rationale and design of the Patient Outcome after primary PCI (POPular) Genetics study. American heart journal. Jul 2014 33. Steg PG, James SK, Atar D, et al. ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation. European heart journal. Oct 2012 34. Hu YF, Lu TM, Wu CH, et al. Differences in high

on-treatment platelet reactivity between intracoronary and peripheral blood after dual antiplatelet agents in patients with coronary artery disease. Thrombosis and haemostasis. Jul 2013 35. Collet JP, Cuisset T, Range G, et al. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. The New England journal of medicine. Nov 2012

Chapter 6 Validation of the automated electronic micro emboli detection system in patients undergoing carotid endarterectomy Conditionally accepted for publication in the European Journal of Utrasound, 2016

T. Leunissen, D. van Vriesland, H. de Ruijter, F. Moll, W. Mess and GJ. de Borst

Chapter 6

Abstract Objectives To assess the diagnostic values of automatic embolus detection software (AEDS) in transcranial doppler (TCD) monitoring for detection of solid microemboli in patients at risk of perioperative stroke and/or mortality during carotid endarterectomy (CEA). Methods in 50 patients undergoing CEA, perioperative TCD registration was recorded. All recorded events, identified and saved by the AEDS, were off-line analysed in twofold by two human experts (HE), within a timeframe of > 4 months. Inter- and intraobserver variability were assessed. The overall agreement with HE, sensitivity, specificity, negative- and positive predicting value (NPV and PPV) of the AEDS was computed for different cut-offs (patient displaying perioperative 5, 10, 20, 25, or 50 microemboli). Results 77,233 events were analyzed. The inter- and intraobserver variability was good (min κ= 0.72, max κ=0.79). AEDS and HE identified respectively 760 and 470 solid emboli. Agreement of AEDS and HE for solid emboli detection was poor (κ=0.24, SE= 0.016). Specificity and NPV were high (99.2% and 99.6%) but sensitivity and PPV were low (30.6% and 19.8%). Applying a threshold of > 20 microemboli resulted in the best sensitivity (100.0%), specificity (84.4%), PPV (42.7%), NPV (100.0%) and area under the curve (0.898). However, 58.3% of the patients was false positive classified by AEDS. Conclusion In this validation cohort, AEDS holds an insufficient agreement with HE in the identification of solid emboli. AEDS and HE disagree in pointing out specific patients at risk, therefore AEDS cannot be used as stand-alone system to identify patients at risk of perioperative stroke and/or mortality during CEA.

Validation of micro emboli detection software

Introduction Multiple randomized trials have shown the efficacy of carotid endarterectomy (CEA) as secondary prevention for symptomatic patients with a stenosis of 70-99% in the carotid artery and as primary prevention for asymptomatic men aged <75 years with a degree of carotid artery stenosis â&#x2030;Ľ 50%.1,2 Paradoxically, the benefit of CEA is hampered by a risk of stroke related to the procedure itself, occurring in approximately 2-3% of the patients.3-5 Several underlying pathophysiological mechanisms for the occurrence of stroke due the revascularization procedure have been identified, of which thrombotic occlusion and hemodynamic disturbance have been shown the most frequent. 3,6 It is of practical relevance that these strokes are potentially preventable. A thorough understanding of the mechanisms is a prerequisite for applying preventive measures, which is essential in our ongoing aim to further minimize CEA related complications. During dissection, atherothrombotic debris may be spontaneous released or by manipulation of the plaque. 6 Platelets circulate in the blood in a resting state, but react immediately upon vessel wall injury by adhering to the exposed collagen, followed thrombus formation to seal the injured vessel wall. 7 After CEA, thrombus formation can occur at the injured endothelium of the endarterectomy- and clamp site after restoration of the blood flow. These physiological mechanisms occur to some extent in all patients that undergo CEA, but it leads to new postoperative cerebral deficits in a minority of patients. Perioperative monitoring with the Transcranial Doppler (TCD) may further reduce CEA related complications. By using TCD microemboli in the middle cerebral artery (MCA) can be detected. The microemboli can be either of gaseous or particulate nature and give rise to a typical signal within the Doppler spectogram, the so-called microembolic signal (MES). TCD monitoring is clinically relevant during CEA since spontaneously occurring MES are associated with unstable plaque characteristics and an increased risk of infarction.8,9 In case an abundant number of MES in combination with a decrease of the peak systolic velocity compared to previous measurements is seen in the MCA, acute thrombo-embolization of the ipsilateral internal carotid artery can be diagnosed.10 Intraoperative TCD monitoring can provide immediate feedback to the treating physician allowing prompt corrections in tissue handling.11 Using TCD, the amount of ultrasound being reflected depends on the size and the acoustic impedance of the embolus. The greater the difference between the acoustic impedance of the embolus and surrounding blood, the more intense the MES appears in the Doppler spectogram. Solid emboli emerge as short unidirectional Doppler shifts, gaseous emboli as uni- or bidirectional Doppler shifts with a high intensity and artefacts as bidirectional Doppler shifts with lower intensity.12 Most Doppler systems offer automatic software for detecting MES, but a major problem during operative interventions is that these systems do not differentiate between solid and gaseous emboli. The neurological consequences of larger solid emboli are more serious than the consequences of gaseous emboli.

Chapter 6

In a clinical setting solid emboli during dissection can be detected by TCD monitoring performed by a trained technician. For research purposes, the Doppler signal is recorded and evaluated offline by an expert panel. Both the monitoring as the evaluation is time consuming and a mentally strenuous procedure. In addition, the interobserver agreement for differentiation of a given signal as gaseous or solid microembolus or artefact is not always sufficient despite a comprehensive consensus concerning the interpretation of MES. 13 Therefore, multiple attempts have been made to develop automated embolus detection software in order to reduce the burden for the examiners and to deliver unambiguous results. The performance of these systems varies: sensitivities ranging from 44-97% when compared to an expert panel have been reported. 14, 15 One of the latest developments is the â&#x20AC;&#x2DC;Versatile Embolic Detection System (EDS; Keunen Medical Software BV, the Netherlands). 16 Unlike other automatic emboli detection programs, the algorithm of EDS uses the information given by the zero crossing frequency of the audio Doppler signal and only uses the fast Fourier transform (FFT) as visual support for later analysis by human experts. Classification of audio signals is performed within a multi-layer neural network, examining the duration and intensity of the audio signal and the zero- crossing characteristics. 16 All events are recorded and can be viewed and analysed retrospectively. This software has been validated in postoperative CEA patients who were conscious and able to follow instructions. 16 Up to now, as we are aware, the EDS software has not yet been validated for perioperative TCD monitoring in which gaseous emboli and procedure related artefacts can be expected. The primary aim of this study was to validate the EDS for perioperative solid microemboli detection in 50 patients undergoing CEA against the human expert opinion. Secondly, we assessed the optimal settings of the EDS for detecting solid microemboli.

Methods Patients The present sub analysis was performed using the initial 50 TCD recordings from the ongoing â&#x20AC;&#x2DC;High thrombocyte reactivity testing in association with increase of perioperative MicroEmbolic Signals measured by Transcranial doppler during Carotid EndArterectomy (MESCEA)â&#x20AC;&#x2122; study (NL33061.041.10) that validates a new platelet reactivity test against the VerifyNow (gold standard). The institutional review board of the University Medical Center Utrecht approved the study protocol, the study was conducted according to the Declaration of Helsinki, and all patients provided written informed consent. The TCD recordings were performed between 2012-2013 in the University Medical Centre Utrecht, the Netherlands. Inclusion criteria were 1) indication for CEA according to recommendations of the Asymptomatic Carotid Atherosclerosis Study and Asymptomatic Carotid Surgery Trial studies for asymptomatic patients and the North American Symptomatic Carotid Endarterectomy Trial and European Carotid

Validation of micro emboli detection software

Surgery Trial studies for symptomatic patients,17-19 2) accessible temporal window for TCD registration. Patients on preprocedural Vitamin K antagonists and requiring blood transfusion were excluded. Carotid endarterectomy Patients underwent standard CEA with an longitudinal arteriotomy under general anaesthesia with standard neuromonitoring with electro-encephalography (EEG) and TCD measurements. Surgery was executed by an experienced vascular surgeon or a vascular trainee under supervision. A shunt or patch was selectively used. In all patients, heparin (5.000 IU) was administered before cross-clamping. Other clinical cofounders were recorded. Transcranial Doppler recordings We used a Delica UMS-9UA system equipped with a 1.6-MHz probe in a special head frame (SMT Medical, Wurzburg, Germany) to allow hands-off monitoring of the MCA ipsilateral to the carotid artery that was operated on. TCD monitoring was performed to detect solid microemboli and intraoperative cerebral hypoperfusion. The perioperative TCD monitoring started when the patients arrived in the operating room (prior induction) and ended when patients were fully awake from anaesthesia. Depth settings were between 48 and 56 mm, depending on pre-operative TCD measurement. Sample volume was maintained at 10 mm. The gain of the input signal was manually adjusted to achieve an optimal stable envelop curve of the Doppler spectrum (reflecting maximal velocity) at the lowest gain to prevent overload of the preamplifier. Emboli detection system From the audio output of the Delica UMS-9UA, the signal was sampled into the Embolic Detection System (EDS, SMT Medical, WĂźrzburg, Germany) and stored. The EDS allows off-line analysis of the intensity, duration and zero crossing index of all recorded MES.16 The amplitude intensity threshold for recording events was set for a minimum of 2.5 decibel (dB). Technical details of intraoperative20 and postoperative21 monitoring have been described previously. All recordings were performed by experienced technicians. All TCD recordings were coded and analysed by human experts (HE), blinded to a patients identity (Figure 1). International Consensus criteria were used by HE to identify solid emboli according to their characteristic spectral and acoustic appearance.13 All recorded perioperative events, classified by the EDS as solid- or gaseous emboli or artefacts were analysed. To examine the reliability of the HE as gold standard, both HE scored the TCD recordings independently, in twofold, with a timeframe of > 4 months. The cohenâ&#x20AC;&#x2122;s kappa was calculated for inter- and intra-observer variability. The scoring results of the HE with the best intra-observer agreement was used for further analysis. All events regarded as gaseous emboli by the EDS, were scored as artefacts by the human exerts, since the primary aim of this study was to validate the capacity of the EDS to identify perioperative solid emboli.

Chapter 6

Figure 1. Validation protocol.

The HE did not only listen to the actual sound of the recorded events but was supplied with all available data of the audio signal (as ZCI) and FFT (as duration, intensity, and velocity display). Within the display of the ZCI a zoom function was available to see all details of the event at different time scales. Statistical analysis SPSS version 20.0 (SPSS Inc, Chicago, Illinois) was used for all statistical analyses. The kappa value for the agreement between the first and second scoring round, regarding all recorded events of the 50 patients, was determined for both observers separately (intra-observer variability). We also calculated the kappa value for the agreement between both observers both for the first and second scoring rounds, again regarding all recorded events of the 50 patients (inter-observer variability). The sensitivity, specificity, positive and negative predicting value (PPV and NPV) of the EDS was calculated, based on different thresholds for the definition of â&#x20AC;&#x2DC;patient at riskâ&#x20AC;&#x2122; of

Validation of micro emboli detection software

postoperative CVA. Patients were regarded to be ‘at risk’ when displaying 5, 10, 20, 25 or 50 perioperative solid MES. Per threshold the area under the curve (AUC) was calculated, based on the receiver operating characteristic (ROC)-curve. To circumvent that EDS does display the right number of patients at risk, but is not capable to identify the exact patients being at risk, we investigated the percentage true and false positive patients plus the percentage of missed ‘patients at risk’ by the EDS. To investigate the optimal intensity threshold to achieve the highest sensitivity and specificity, we compared the mean and maximum intensity (dB) of the artefacts and solid emboli according to the EDS by independent sample t-test.

Results Patient characteristics The cohort consisted of 50 patients, 37 men (74.0%), mean age 71.5 years (SD 9.1), who underwent CEA preceded by stroke (28.0%), transient ischemic attack (38.0%), ocular symptoms (28.0%) or no symptoms (6.0%). Most patients were treated preoperatively with aspirin (86.0%) or dipyridamol (78.0%), a minority received clopidogrel (6.0%) or oral anticoagulants (12.0%). Preoperatively 87.5% was treated with a statin. A summary of baseline characteristics is displayed in table 1. Agreement in scoring of events by human experts A two- by two table was constructed with classification of solid emboli or artefact by HE1 and HE 2 (Table 2). Both the inter-observer as intra-observer agreement was substantial (min κ=0.720, max κ=0.791). The highest intra-observer agreement was seen within the scoring of human expert 1 within both rounds (HE 1, κ=0.791, SE=0.029). Because of the highest intra-observer agreement in HE1 and the good overall interobserver agreement (round 1 κ =0.724 and round 2 κ= 0.720), we used the data of HE1 for further analysis, as gold standard. Validation of EDS A total amount of 77.233 recorded events by EDS were analysed. Overall, the patients showed 726 solid emboli according to the EDS and 470 solid emboli according to HE (table 3A). As described previously, we considered the events classified by EDS as gaseous emboli and artefacts both as artefacts, since our primary outcome was the capacity of the EDS to detect solid emboli (Table 3A). Here, the specificity and NPV are high (99.2% [95%CI: 99.2-99.3] and 99.6% [99.5-99.6]) due to the large number of true artefacts but the sensitivity and PPV are low (30.6% [95% CI: 26.5-35.0] and 19.8% [95% CI: 17.0-22.9]). Considering the events classified by the EDS as gaseous emboli and artefact both as artefacts, might influence the diagnostic values of the EDS. Therefore, we also calculated the NPV, PPV, sensitivity and specificity of the events (solid emboli and artefact according to EDS), excluding the events classified as gaseous emboli by EDS, what resulted in almost

Chapter 6

equal results (Table 3B). Overall, the specificity and NPV remain high (99.2% [95% CI: 99.1-99.2] and 99.7% [95% CI: 99.7-99.8]) and the sensitivity and PPV remain low (43.2% [95% CI: 37.9-48.8] and 19.8%). The agreement of EDS and HE on classification of all events was considered poor (κ = 0.235, SE: 0.016). 22

Table 1. Patient characteristics. N=50 Age, mean (±SD)

71.5 (9.2)

Men, n (%)

37 (74.0)

Antiplatelet therapy, n (%) Acetylsalicylic acid

43 (86.0)

Dipyridamol

39 (78.0)

Clopidogrel

3 (6.0)

OAC

6 (12.0)

Index event, n (%) Asymptomatic

3 (6.0)

Stroke

14 (28.0)

Transient ischemic attack

19 (38.0)

Ocular

14 (28.0)

Medical History, n (%) History of CAD

16 (32.0)

History of CVA

19 (38.0)

History of PAD

11 (22.0)

Ipsilateral stenosis grade, n (%) 50-690% 70-99%

6 (12.0) 43 (86.0)

Contralateral stenosis grade, n (%) 0-49%

23 (46.0)

50-100%

17 (34.0)

Risk factors, n (%) Hypercholesterolemia (statin use)

41 (81.0)

Hypertension

37 (74.0)

Diabetes

18 (36.0)

Current smoker

16 (32.0)

Body Mass Index, kg/m2, mean (±SD)

25.9 (4.5)

eGFR, ml/min/1.73m2, mean (±SD)

82.8 (91.2)

CAD coronary artery disease, CVA cerebrovascular accident, eGFR estimated glomerular filtration rate, OAC oral anticoagulation, PAD peripheral artery disease, SD standard deviation

Validation of micro emboli detection software

Table 2 Inter- and intra- observer agreement

Intra-observer agreement HE 1

Kappa

Standard Error

0.791

0.029

Intra-observer agreement HE 2

0.778

0.031

Inter-observer agreement Round 1

0.724

0.033

Inter-observer agreement Round 2

0,720

0.033

Table 3A. Classification of all events, gaseous emboli included as artefact. Human expert EDS*

Emboli

Artefact

Total

Emboli

144

582

726

Artefact

326

76181

76507

Total

470

76763

77233

* EDS embolic detection software

Table 3B. Classification of all events, gaseous emboli excluded as artefact. Human expert

EDS*

Emboli

Artefact

Total

Emboli

144

582

726

Artefact

189

69549

69738

Total

333

70131

* EDS embolic detection software

Determination of optimal threshold to identify patients at risk Based on the total amount of detected emboli according to HE, patients could be regarded ‘at risk’. To determine the sensitivity, specificity, PPV and NPV of different thresholds of risk classification, we constructed 2x2 matrices. Patients were identified as being ‘at risk’ after displaying 5, 10, 20, 25 or 50 perioperative emboli. Per threshold a ROC-curve was calculated (Table 4). The AUC, sensitivity and NPV is the highest at a threshold of 20 microemboli (resp. 0.90 (0.81-0.98), 100.00% [95% CI: 47.8-100.0] and 84.40% [95% CI: 70.5-93.5]), although at expense of the PPV. With this threshold only 41.67% of the emboli detected by EDS is true positive. Capacity of embolic detection system to identify patients at risk To verify whether the EDS does not only display the correct total amount of patients at risk, but also points out the exact patients who are at risk, a table was constructed with the classified patients according to the EDS compared to HE (Table 5). The EDS classifies a large percentage of patients false as being ‘at risk’ (43.5-100.0%) when using different thresholds. When applying lower thresholds, the EDS holds a small percentage of falsenegatives (0.0-7.1%). However, at the highest threshold the EDS misclassifies all patients at risk (false positive 100.0%) and not at risk (false negative 100.0%).

Chapter 6

Table 4. Diagnostic value of different emboli thresholds. Threshold (emboli)

PPV*

NPV**

Sensitivity (%)

Specificity (%)

AUCâ&#x20AC;

46.0

92.3

94.4

37.5

0.760

(29.5-63.1)

(64.0-99.8)

(72.7- 99.9)

(21.1-56.3)

(0.623-0.896)

56.5

96.3

92.9

72.2

0.818

(34.5-76.8)

(81.0-99.9)

(66.1-99.8)

(54.8-85.8)

(0.700-0.937)

41.7

100.0

84.4

0.898

(15.2-72.3)

(90.8-100.0)

(47.8-100.0)

(70.5-93.5)

(0.812-0.984)

22.2

95.1

50.0

84.8

0.875

(2.8-60.0)

(83.5-99.4)

(6.8-93.2)

(71.1-93.6)

(0.779-0.971)

0.0

93.6

0.0

93.6

0.88

(0.0-70.7)

(82.5-98.7)

(0.0-70.7)

(82.5-98.7)

(0.781-0.978)

* PPV positive predicting value, ** NPV negative predicting value â&#x20AC; Area under the Curve

In this series of 50 patients one patient developed a procedural stroke, presenting by left sided facial drop, hemianopia and paresis of the upper extremity. The computed tomography brain showed new ischemia of area supplied by the middle cerebral artery, most likely due to per-or direct postoperative thromboemboli. The patient was adequately treated with acetylsalicylic acid and dipyridamole (confirmed with platelet reactivity testing). For this patient, the HE detected perioperative 52 solid MES and the EDS 33 solid MES (Figure 2). Most emboli were detected prior to clamping. The EDS missed 28 solid emboli that were detected by HE and misclassified 9 MES as solid emboli. No other patients experienced neurological deterioration due to peri-procedural cerebral ischemia.. The perioperative stroke rate (2.0%) of this cohort was not decreased with the use of TCD and the AEDS.

Figure 2. Perioperative occurrence of solid emboli in single patient displaying postoperative hemiparesis.

Validation of micro emboli detection software

Table 5. Capacity of EDS to detect patients at risk, at different emboli thresholds. Patients at risk, according to human expert (N)

True positive patients, according to EDS (n, %)

False positive patients, according to EDS (n, %)

Missed patients at risk, by EDS (n, %)

17 (45.9%)

20 (54.1%)

1 (5.6%)

13 (56.5%)

10 (43.5%)

1 (7.1%)

5 (41.7%)

7 (58.3%)

0 (0.0%)

2 (22.2%)

7 (77.8%)

2 (50.0%)

0 (0%)

3 (100%)

EDS emboli detection software

Figure 3. Distribution of maximum intensity of the emboli detected by human expert and EDS.

Emboli intensity The maximum intensity of artefacts according to EDS was significantly higher as compared to the maximum intensity of emboli according to the EDS (3.49 vs. 3.21 dB, p<0.001). The max intensity of false negative (missed) emboli and true positive (true) emboli was not significantly different (3.29 vs. 3.21 dB, p=0.35). A histogram of the maximum intensity of the solid emboli according to EDS and HE illustrated that most solid emboli are observed in the range of 2.5-3.5 dB (Figure 3). However, more false positive events are observed this range, compared to the ranges >3.5 dB.

Discussion In the present study in a perioperative setting of 50 consecutive patients undergoing CEA, the agreement in classifying recorded events as solid emboli or artefacts between HE and EDS was poor. When applying different threshold for regarding patients at risk, the threshold of 20 solid emboli showed the best balance between sensitivity and specificity, resp. 100.0% and 84.4 %, at the expense of the PPV. When applying this

Chapter 6

threshold only 41.2% of the patients regarded as at risk, were truly at risk. Increasing the threshold resulted in missed patients at risk (false negatives) and therefore at present we consider the automatic EDS unsuitable for perioperative solid emboli detection. Previous research has shown that the presence of MES predicts both stroke and the combined endpoint of stroke/TIA in patients with symptomatic (resp. OR 9.57, 95%CI 1.54-59.3, p=0.02 and OR 6.36, 95%CI 2.90-13.96) and asymptomatic carotid stenosis (resp. OR 7.46, 95%CI 2.24-24.89, p<0.001 and OR 12.00, 95%CI 2.43-59.34). 9,23 During all stages of CEA MES have been detected. However, only a high frequency of MES during dissection (OR 14.97, 95%CI: 5.15-42.47; p<0.00001) or immediately after CEA does (OR 24.54, 95%CI: 7.88-76.43; p<0.00001) predicts an increased postoperative risk of stroke.9 Repeated measurements in individual patients with symptomatic carotid stenosis showed stability of recordings over time, in contrast to asymptomatic patients with carotid stenosis. This may be relevant for detection of ‘’high risk’’ asymptomatic patients who might benefit from CEA, suggesting that repeated TCD measurements are mandatory in this patient category. 24 Nowadays stronger platelet inhibitors are prescribed for patients after stroke or TIA. Most patients undergoing CEA are on combination aspirin/ dipyridamole or monotherapy clopidogrel. Using these strong platelet inhibitors, one can expect that less solid emboli occur during and after CEA. Multiple studies have shown the potential efficacy of clopidogrel to reduce (a)symptomatic embolization in patients with symptomatic carotid artery stenosis before and during CEA.25,26 A previous study from 1997 reported ≥ 10 perioperative solid emboli in 24% of the patients treated with monotherapy aspirin. 27 Our study shows 28% of the patients displaying ≥ 10 perioperative solid emboli, despite stronger platelet inhibitors. This lack of major reduction indicates that perioperative TCD monitoring is still essential to minimize the risk of complications. An earlier study with the Versatile EDS, evaluating all signals ≥3 dB in patients post CEA, showed an overall agreement of EDS and HE of 96%. 16 Missed solid emboli were characterised by short duration, low intensity (≤ 4dB) and intermediate ZCI value. Equally, our study also shows a large number of solid emboli ≤ 4 dB pointed out by both the EDS as HE1 (Figure 2). However, no difference in intensity between the missed and registered solid emboli was found. Misclassification by the EDS can thus not be explained by the intensity of the signals. Further data on the clinical significance of low intensity- compared to high intensity solid emboli needs to be obtained. Moreover, improvement of this AEDS could be realised by an improved rejection of these low intensity artefacts. MES occurring after arterial opening and before vessel closure may contribute to either gaseous or solid emboli. After arterial opening, small air bubbles can be introduced, resulting in high intensity MES, a receiver overload and potential degree of aliasing. 28 The reflection of ultrasound is dependent of the acoustic impedance: air has an acoustic impedance of 1/4000th of whole blood, resulting in an extremely large reflection. The acoustic impedance of solid emboli is more similar to whole blood and therefore results in much weaker reflection.

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In this study we have evaluated both all recorded events (artefact, solid and gaseous emboli) by the Versatile EDS as well as only the events classified as artefacts or solid emboli by EDS. Disregarding gaseous emboli (according to EDS) from analysis did not improve the diagnostic value of the software. Not only gaseous emboli complicate automatic detection of MES during CEA, but also artefacts due to CEA as diathermy, movements of the surgeon and artefacts of surrounding electronical devices in the operation room. So far, no solution in the algorithm of the software has been suggested as a solution to the problem. Our study has several limitations. First, only events recorded by EDS were analysed by HE. It might be possible that solid emboli were missed by the EDS and thus excluded from further analysis by HE. Secondly, we did not split the analysis of the TCD monitoring into different stages of CEA, thus we cannot make any statement towards when MES appeared. However, our primary aim was to investigate the diagnostic quality of EDS to classify MES into solid emboli or artefacts and we believe our study approach was adequate for this purpose. Secondly, we have used a 1.6 MHz transducer, in contrast to a 2.0 MHz transducer which is applied in most studies on MES detection. This might result in a higher embolus-to-blood ratio and hence a higher MES intensity. Nonetheless, we found that MES intensities did not differ between those signals that were correctly identified by the EDS and those that were missed. The MES amplitude seemed not be a critical factor in the performance of the system. Finally a higher embolus-to-blood ratio should rather favour a better than a worse performance of the EDS. Apart from the Versatile EDS, different approaches for solid emboli detection have been suggested, such as multi-frequency TCD that insonates simultaneously with 2.5 and 2.0 MHz.12,29 Solid emboli reflect more high frequency ultrasound than lower frequency, whereas the opposite is the case for gaseous microemboli. Compared to intensity threshold approach, this approach discriminates more accurately between solid and gaseous emboli, but still holds serious limitations. 30,31 The multi-gate TCD traces a moving embolus at two or more insonation depths. Theoretically an solid embolus should appear sequentially between the two channels, with a time delay, whereas an artefact should appear simultaneously in the two gates. 32 So far, this technique is not suitable for clinical use. Another possible approach is a trained neural network, that uses a pattern-recognition-procedure to discriminate soli emboli. The network is trained by presenting many characteristic Doppler signals and after sufficient training the network could apply the learned pattern recognition to signals of new/ clinical recordings. 14,33 However, trained networks can be â&#x20AC;&#x2DC;over trainedâ&#x20AC;&#x2122; (loss of generalization), perpetuate mistakes introduced by their teacher files and can misdiagnose signals that are not trained often enough. 27,34 Concluding, to our knowledge, all attempts to develop an automatic detection system failed to match the accuracy of human experts.

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Conclusion In this validation cohort, regarding all registered events, the EDS holds a poor agreement in classification of solid emboli or artefacts, compared to the human expert. The EDS and human expert do not agree in identifying specific patients at risk for peri-operative thrombo-embolic events. At present EDS cannot be used as a stand-alone neuromonitoring system for detection of perioperative embolization.

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References 1.

Endarterectomy for asymptomatic carotid artery stenosis. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study. JAMA : the journal of the American Medical Association. May 1995 2. Halliday A, Mansfield A, Marro J, et al. Prevention of disabling and fatal strokes by successful carotid endarterectomy in patients without recent neurological symptoms: randomised controlled trial. Lancet. May 2004 3. de Borst GJ, Moll FL, van de Pavoordt HD, Mauser HW, Kelder JC, Ackerstaf RG. Stroke from carotid endarterectomy: when and how to reduce perioperative stroke rate? European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Jun 2001 4. Radak D, Popovic AD, Radicevic S, Neskovic AN, Bojic M. Immediate reoperation for perioperative stroke after 2250 carotid endarterectomies: differences between intraoperative and early postoperative stroke. Journal of vascular surgery. Aug 1999 5. Bonati LH, Dobson J, Featherstone RL, et al. Longterm outcomes after stenting versus endarterectomy for treatment of symptomatic carotid stenosis: the International Carotid Stenting Study (ICSS) randomised trial. Lancet. Feb 2015 6. Huibers A, Calvet D, Kennedy F, et al. Mechanism of Procedural Stroke Following Carotid Endarterectomy or Carotid Artery Stenting Within the International Carotid Stenting Study (ICSS) Randomised Trial. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Sep 2015 7. de Groot PG, Urbanus RT, Roest M. Platelet interaction with the vessel wall. Handbook of experimental pharmacology. 2012 8. Van Lammeren GW, Van De Mortel RH, Visscher M, et al. Spontaneous preoperative microembolic signals detected with transcranial Doppler are associated with vulnerable carotid plaque characteristics. The Journal of cardiovascular surgery. Jun 2014 9. King A, Markus HS. Doppler embolic signals in cerebrovascular disease and prediction of stroke risk: a systematic review and meta-analysis. Stroke; a journal of cerebral circulation. Dec 2009 10. Horn J, Naylor AR, Laman DM, et al. Identification of patients at risk for ischaemic cerebral complications after carotid endarterectomy with TCD monitoring. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Sep 2005 11. Pennekamp CW, Moll FL, de Borst GJ. The potential benefits and the role of cerebral monitoring in carotid endarterectomy. Current opinion in anaesthesiology. Dec 2011

12. Russell D, Brucher R. Online automatic discrimination between solid and gaseous cerebral microemboli with the first multifrequency transcranial Doppler. Stroke; a journal of cerebral circulation. Aug 2002 13. Ringelstein EB, Droste DW, Babikian VL, et al. Consensus on microembolus detection by TCD. International Consensus Group on Microembolus Detection. Stroke; a journal of cerebral circulation. Mar 1998 14. Van Zuilen EV, Mess WH, Jansen C, Van der Tweel I, Van Gijn J, Ackerstaff GA. Automatic embolus detection compared with human experts. A Doppler ultrasound study. Stroke; a journal of cerebral circulation. Oct 1996 15. Devuyst G, Darbellay GA, Vesin JM, et al. Automatic classification of HITS into artifacts or solid or gaseous emboli by a wavelet representation combined with dual-gate TCD. Stroke; a journal of cerebral circulation. Dec 2001 16. Keunen RW, Hoogenboezem R, Wijnands R, Van den Hengel AC, Ackerstaff RG. Introduction of an embolus detection system based on analysis of the transcranial Doppler audio-signal. Journal of medical engineering & technology. Jul 2008 17. Naylor AR, Rothwell PM, Bell PR. Overview of the principal results and secondary analyses from the European and North American randomised trials of endarterectomy for symptomatic carotid stenosis. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Aug 2003 18. Mayberg MR, Wilson SE, Yatsu F, et al. Carotid endarterectomy and prevention of cerebral ischemia in symptomatic carotid stenosis. Veterans Affairs Cooperative Studies Program 309 Trialist Group. JAMA : the journal of the American Medical Association. Dec 1991 19. Randomised trial of endarterectomy for recently symptomatic carotid stenosis: final results of the MRC European Carotid Surgery Trial (ECST). Lancet. May 1998 20. Jansen C, Vriens EM, Eikelboom BC, Vermeulen FE, van Gijn J, Ackerstaff RG. Carotid endarterectomy with transcranial Doppler and electroencephalographic monitoring. A prospective study in 130 operations. Stroke; a journal of cerebral circulation. May 1993 21. van der Schaaf IC, Horn J, Moll FL, Ackerstaff RG. Transcranial Doppler monitoring after carotid endarterectomy. Annals of vascular surgery. Jan 2005 22. Joseph Fleiss BL, Myunghee Cho Paik. Statistical Methods For Rates And Proportions: John Wiley And Sons Ltd; 2003. 23. Markus HS, MacKinnon A. Asymptomatic embolization detected by Doppler ultrasound predicts stroke risk in symptomatic carotid artery stenosis. Stroke; a journal of cerebral circulation. May 2005

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24. Mackinnon AD, Aaslid R, Markus HS. Ambulatory transcranial Doppler cerebral embolic signal detection in symptomatic and asymptomatic carotid stenosis. Stroke; a journal of cerebral circulation. Aug 2005 25. Tsivgoulis G, Kerasnoudis A, Krogias C, et al. Clopidogrel load for emboli reduction in patients with symptomatic carotid stenosis undergoing urgent carotid endarterectomy. Stroke; a journal of cerebral circulation. Jul 2012 26. Payne DA, Jones CI, Hayes PD, et al. Beneficial effects of clopidogrel combined with aspirin in reducing cerebral emboli in patients undergoing carotid endarterectomy. Circulation. Mar 2004 27. Levi CR, Roberts AK, Fell G, et al. Transcranial Doppler microembolus detection in the identification of patients at high risk of perioperative stroke. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Sep 1997 28. Cullinane M, Reid G, Dittrich R, et al. Evaluation of new online automated embolic signal detection algorithm, including comparison with panel of international experts. Stroke; a journal of cerebral circulation. Jun 2000

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29. Markus HS, Punter M. Can transcranial Doppler discriminate between solid and gaseous microemboli? Assessment of a dual-frequency transducer system. Stroke; a journal of cerebral circulation. Aug 2005 30. Schoenburg M, Baer J, Schwarz N, et al. EmboDop: insufficient automatic microemboli identification. Stroke; a journal of cerebral circulation. Feb 2006 31. Evans DH. Embolus differentiation using multifrequency transcranial Doppler. Stroke; a journal of cerebral circulation. Jul 2006 32. Molloy J, Markus HS. Multigated Doppler ultrasound in the detection of emboli in a flow model and embolic signals in patients. Stroke; a journal of cerebral circulation. Sep 1996 33. Georgiadis D, Kaps M, Siebler M, et al. Variability of Doppler microembolic signal counts in patients with prosthetic cardiac valves. Stroke; a journal of cerebral circulation. Mar 1995 34. Kemeny V, Droste DW, Hermes S, et al. Automatic embolus detection by a neural network. Stroke; a journal of cerebral circulation. Apr 1999

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Chapter 7 Clopidogrel is the strongest suppressor of platelet reactivity and perioperative solid micro emboli during carotid endarterectomy

T. Leunissen, PP. Wisman, D. van Vriesland, L. Schropp, S. Korporaal, F. Moll, G. Pasterkamp, M. Roest, R. Urbanus and GJ. de Borst

Chapter 7

Abstract Background Monitoring platelet reactivity and adjusting dosage or class of antiplatelet drugs may be beneficial to reduce thrombotic and bleeding complications. Currently available platelet reactivity tests based on aggregometry lack sensitivity and specificity to show benefit of tailoring antiplatelet therapy. The aim of this observational study was to compare the association of an aggregation based- (VerifyNow) versus a flow cytometry (FC) based platelet activation assay in a human in vivo model for arterial thrombosis. Methods Platelet reactivity was measured with 1) FC after stimulation with adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP) -6 and 2) VerifyNow-Aspirin and P2Y12 assay in patients undergoing carotid endarterectomy. Primary outcome was solid micro emboli (reflecting arterial thrombosis) frequency measured by Transcranial Doppler as surrogate marker for perioperative stroke and thereby platelet reactivity. Secondary outcome were major adverse cardiovascular events (MACE) Results In total 197 patient were included (83 treated with P2Y12 inhibitor). High responder status according to FC-ADP showed an increased risk of displaying â&#x2030;Ľ20 solid micro emboli or occurrence of MACE ( OR 5.732 (1.314-25.003), p=0.020 and OR 2.346 (1.126- 4.890), p=0.023). This may be explained since clopidogrel treatment resulted in decreased platelet reactivity after ADP and TRAP-6 stimulation (5 083 vs. 18 939 MFI, p< 0.001 and 6 850 vs. 20 479 MFI, p<0.001 ), less solid micro emboli (2.3 vs. 6.6, p= 0.001) and less MACE (14.6% vs 26.3%, p=0.049). Conclusion Only high platelet reactivity assessed by FC- ADP showed increased risk for displaying â&#x2030;Ľ20 micro emboli during CEA. Perioperative clopidogrel treatment showed the strongest effect on reducing platelet reactivity and micro emboli frequency during CEA. If clopidogrel treatment results in a reduction of clinical stroke events remains to be investigated.

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Effect of clopidogrel on platelet reactivity and MES

Introduction Platelets are pivotal in the pathogenesis of atherothrombosis as well as in the initiation and progression of atherosclerosis. 1,2 Consequently patients with severe atherosclerosis are treated with platelet inhibitors, ranging from relatively mild anti-platelet drugs such as the cyclooxygenase (COX)-1 inhibitor acetylsalicylic acid to stronger platelet inhibition with a combination of COX-1 inhibitors and inhibitors of the platelet adenosine diphosphate (ADP) receptor P2Y12. Currently there is no evidence based approach for choosing the appropriate antiplatelet therapy (APT) in individual patients. 3 Although the benefit of APT in reduction of secondary cardiovascular events has been proven, some patients develop thrombotic events during treatment while others develop bleeding complications. 4 High platelet reactivity, despite treatment with antiplatelet drugs, is associated with an increased risk of cardiovascular events. 5 Monitoring platelet reactivity and adjusting dosage or class of antiplatelet drugs may be beneficial to reduce over- and undertreatment and finally reduce thrombotic and bleeding complications. However up to now, tailoring APT based on platelet reactivity testing has not been proven effective. 6,7 This could be due to the lack of sensitivity and specificity of currently available platelet reactivity tests. 8 Currently, a wide range of platelet reactivity tests is available. The gold standard is classical optical light transmittance aggregometry (LTA) mainly because of the long lasting experience with this technique. However, LTA is a time consuming laborious test and thus unsuitable as point-of-care test. A meta-analysis identified the VerifyNow, an aggregation- based assay, as most promising point-of-care platelet reactivity test for analyzing platelet reactivity in patients on aspirin or P2Y12 inhibitors. 5 However, the predicting value of the VerifyNow seems only moderate and further research is required to determine optimal cut-off values for (non-) responders, assess the reproducibility and to improve standardization. 8,9 Previous in vitro- and in vivo results from our group showed that flow-cytometry based assays were more sensitive for detecting a decrease in platelet reactivity than aggregationbased assays.10 The flow-cytometry based assay is based on platelet activation induced by addition of a specific agonists to whole blood. 11 The next step is to investigate the predicting value of these two different platelet reactivity tests on clinically present arterial thrombosis. In the present study we compared a flow cytometry based assay with the VerifyNow in a human in vivo model for arterial thrombosis; patients with carotid stenosis undergoing carotid endarterectomy (CEA) display microembolic signals (MES) during the intervention, registered by Transcranial Doppler (TCD). Nowadays, standard perioperative antiplatelet therapy is treatment with both aspirin and clopidogrel. This advice is among other studies, based on an audit showing that a single tablet 75 mg clopidogrel administered the night before intervention, reduced perioperative embolization. 12 The MES consist mainly of platelet aggregates 13,14 and high number of MES is correlated with an increased ischemic stroke risk.15,16 Perioperative stroke and death occurs in up to 5% of carotid interventions17,

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leading to an unfeasible large sample size, if chosen as primary outcome. MES frequency might therefore be an acceptable surrogate marker for perioperative stroke and thus for the efficacy of APT on platelet reactivity. 18 Therefore, in this study we aimed to compare the association of an aggregation based(VerifyNow) versus a flow cytometry based platelet activation assay with the number of solid micro emboli, detected by the TCD during CEA.

Methods Patients Inclusion criteria were: 1) indication for CEA according to the recommendations of the Asymptomatic Carotid Atherosclerosis Study and Asymptomatic Carotid Surgery Trial studies for asymptomatic patients and the North American Symptomatic Carotid Endarterectomy Trial and European Carotid Surgery Trial studies for symptomatic patients,19-21 2) an accessible temporal skull window for TCD registration. The institutional review board approved the study (NL 33061.041.10), which was conducted according to the declaration of Helsinki and all patients provided written informed consent. Patient characteristics, preoperative physical status according to the American Society of Anesthesiologists (ASA) classification, comorbidities and clinical follow up until the end of this study were collected by patient records. Carotid endarterectomy Patients underwent standard CEA with a longitudinal arteriotomy under general anaesthesia with standard neuromonitoring with electro-encephalography (EEG) and TCD measurements. 22 Surgery was executed by an experienced vascular surgeon or a vascular trainee under supervision. A shunt or patch was selectively used. In all patients, heparin (5 000 IU) was administered three minutes before cross-clamping. Trans cranial Doppler recordings A Delica UMS-9UA system equipped with a 1.6-MHz probe in a special head frame (SMT Medical, Wurzburg, Germany) was used to allow hands-off monitoring of the middle cerebral artery ipsilateral to the carotid artery that was operated on. TCD monitoring was performed to detect solid microemboli. The perioperative TCD monitoring started when the patients arrived in the operating room (prior induction) and ended when patients were fully awake from anaesthesia. Depth settings were between 48 and 56 mm, depending on pre-operative TCD measurement. Sample volume was maintained at 10 mm. Blood collection Arterial blood was collected prior induction of anaesthesia. Blood for FC and VerifyNow was collected via the arterial line, placed for standard intraoperative blood pressure monitoring. Blood for VerifyNow measurements was collected in 0.109 M tri-sodium

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Effect of clopidogrel on platelet reactivity and MES

citrate tubes (Greiner Bio-One, Alphen a/d Rijn, the Netherlands) and for the FC -ADP and -TRAP-6 in 0.105 M tri-sodium citrate (Becton Dickinson, Temse, Belgium) vacuum tubes. Blood samples were transported to the laboratory for platelet reactivity testing and quantification of the platelets with the CELL-DYN 1700 (Abbott Diagnostics, Wiesbaden, Germany). Platelet reactivity testing: Flow cytometric (FC) platelet activation assay 5 µL citrated whole blood was added to 50 µL HEPES-buffered saline (10 mM HEPES, 150 mM NaCl, 1 mM MgSO4, 5 mM KCl, pH 7.4) containing platelet agonist and FITCconjugated anti-fibrinogen antibodies (1:50; Dako, Glostrop, Denmark). Platelets were allowed to react for 20 minutes after which the reaction was stopped by addition of 500 µL fixative solution (0.2% formaldehyde, 150 mM NaCl). Samples were measured on a FACS Canto II flow cytometer (BD biosciences, San Jose, CA, USA). Platelets were identified with forward and sideward scatter. Fluorescence intensity in the FITC channel was used to determine fibrinogen binding, which indicates αIIbβ3 activation. Dose response curves were obtained for platelet agonists ADP (0.01-166 µM) (Sigma-Aldrich, Zwijndrecht, the Netherlands) and the protease activated receptor (PAR-1) agonist SFLLRN (TRAP-6; 0.05-833 µM) (Bachem, Weil am Rhein, Germany). Data are expressed as the sum of the median fluorescent signal for each agonist. High platelet reactivity based on FC was defined as the 75th percentile of the response of all included patients (P2Y12 inhibitor users and non-users), for ADP- and TRAP-6induced platelet reactivity separately. Platelet reactivity testing: VerifyNow VerifyNow (Accumetrics®, San Diego, CA, USA) is a point-of-care, aggregation-based test and supplies two different assay for either aspirin or P2Y12 inhibitor testing. The VerifyNow manual advices a cut-off value of respectively <550 aspirin reaction units (ARU) 23 or <208 P2Y12 reaction units (PRU) as good responders to antiplatelet therapy. Primary outcome: detection of solid emboli from TCD recordings The primary outcome was defined as number of solid micro emboli recorded during the CEA by the TCD. All recordings were performed by experienced Clinical neurophysiology technicians and the amplitude intensity threshold for recording events was 2.5 decibel (dB). From the audio output of the Delica UMS-9UA, the signal was sampled into the Embolic Detection System (EDS, SMT Medical, Würzburg, Germany) and stored. The EDS allows off-line analysis of the intensity, duration and zero crossing index of all recorded micro embolic signals (MES).24 TCD recordings were analysed by two human experts (HE), blinded to a patients identity. International Consensus criteria 25 were used by HE to identify solid emboli. Intra- and intraobserver agreement was previously assessed 26 and showed a good agreement, thus classification of solid emboli was performed only a single time for each patient. The HE did not only listen to the actual sound of the recorded events but was also supplied with zero crossing index and fast fourier transform (duration, intensity, and velocity display).

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Secondary outcome: clinical follow up Secondary outcomes were clinical thromboembolic events as stroke/ TIA (clinical diagnosis) either direct postoperative or within 1 days postoperative after a symptom-free period, postoperative troponin elevation (min. 1 measurement >0.06mcg/L within 3 days postoperative), postoperative bleeding and re-intervention, ipsilateral restenosis (based on Duplex or CT angiography where available), MACE (defined as stroke, TIA of AF, myocardial infarction and peripheral intervention) and mortality. Clinical outcomes are presented for 3 months follow up and complete follow-up. Statistical Analysis The analysis was performed with the use of SPSS (IBM SPSS Statistics 21 for Windows). First clinical characteristics and postoperative clinical events for all patients were displayed as percentage or mean with standard deviation. The correlation between platelet reactivity testing (FC and VerifyNow) was assessed per assay for number of solid micro embolic signals by the spearmans rho and linear regression analysis, reported as beta with 95% confidence intervals (CI). The predictive value of the platelet reactivity test for patients being at risk or not at risk (displaying >20 solid micro emboli) was assessed with logistic regression analysis, reporting the Odds Ratio and 95% CI. Next, the mean number of solid micro emboli was calculated for status of responder/ non- responder according to both Verify Now and flow cytometry results. Differences were calculated by non-parametric Wilcoxon signed-Rank test. Last, the effect of clopidogrel on platelet reactivity and number of solid micro emboli was calculated with the non-parametric Wilcoxon signed-Rank test and on occurrence of major adverse cardiovascular events (MACE) with Fisherâ&#x20AC;&#x2122;s exact test.

Results Baseline characteristics All patients were included between 2012-2015 in the University Medical Center Utrecht, the Netherlands. We included 197 patients (mean age 70.4 (SD 9.6)), of whom 148 were male (75.1%) (table 1). The majority was treated with a combination of aspirin and dipyridamole (39.6%) or monotherapy clopidogrel (34.5%). Symptomatic carotid stenosis was present in 88.3% of the patients with a stenosis grade of 0-50% (1.5%), 50-70% (8.6%) or 70-99% (89.8%). Platelet reactivity measurements for FC were available for all included patients (n=197). The type of VerifyNow assay performed per patient depended on the utilised APT for each individual patient, resulting in 78 P2Y12 assays and 106 aspirin assays. The mean follow up duration was 13.4 months (SD 10.9).

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Effect of clopidogrel on platelet reactivity and MES

Table 1. Patients characteristics. Baseline characteristics

N=197

% or SD

Male, n, (%) Female

148 49

75.1 24.9

Age (mean, SD)

70,4

9.6

History, n, (%) CAD, MI and or coronary interventions Peripheral vascular intervention (including carotid and upper limb) Renal Failure (GFR<50 ml/min)

68 38 30 /179

34.5 19.3 15.2

Cardiovascular risk factors, n, (%) Current smoking (year of operation) Diabetes Mellitus Hypertension Hypercholesterolemia, Body Mass Index, (mean. SD) eGFR based on MDRD, (mean, SD)

64 46 149 124/164 26,8 64.6

32.5 23.4 75.6 83.2 4.8 31.1

ASA Classification, n, (%) ASA 1 ASA 2 ASA 3 ASA 4

6 109 80 2

3.0 55.3 40.6 1.0

Medication, n, (%) Beta blocker Statin

72 171

36.5 86.8

Antiplatelet therapy, n, (%) Aspirin monotherapy with dipyridamole with P2Y12 inhibitor with oral anticoagulant P2Y12 inhibitor monotherapy Oral anticoagulant monotherapy

13 78 15 2 68 23

6.6 39.6 7.6 1.0 34.5 11.7

3 17 177

1.5 8.6 89.8

23 71 64 39

11.7 36.0 32.5 19.8

118/187 68 1

63.1 36.4 0.5

Type of closure, n, (%) Primary closure Venous patch Bovine patch

7/192 19 165

3.6 9.9 85.9

Duration of surgery, minutes (mean, SD)

Ipsilateral stenosis grade, n, (%) 0-50% 50-70% 70-99% Symptoms of presentation, n, (%) Asymptomatic Transient ischemic attack Stroke Amaurosis fugax Contralateral stenosis, n, (%) 0-50% 50-99% Occlusion

103.5

39.0

Indication of operation, n, (%) Restenosis De novo lesion

14 183

7.1 92.9

Preoperative hemoglobin (n= 196) Total cholesterol (n=97)

8,6 4,9

1.0 1.2

ASA American society of anesthesiologists, CAD coronary artery disease, GFR glomerular filtration rate, SD standard deviation, MI myocardial infarction

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Primary outcome: solid micro emboli A median number of 1 (IQR: 5) solid micro embolus was observed during CEA (Figure 1). Almost half of the patients of patients (n=87, 44.2%) did not display solid micro emboli. Eight patients had ≥ 20 solid micro emboli, classifying these patients as at high risk for perioperative stroke 12,26. These patients used aspirin monotherapy (n=1), aspirin and dipyridamole (n=5), clopidogrel (n=1) or aspirin and oral anticoagulant (n=1). Platelet reactivity assessed by FC was weakly correlated with the number of micro emboli, with ρ= 0.218 (p=0.003) for the response to ADP and ρ=0.195 (p=0.007) for the response to TRAP-6 (Figure 2 and table 2). Only FC- ADP could predict micro emboli frequency with β< 0.001 (95%CI: 0.000 to 0.000, p= 0.021).

Figure 1. Prevalence of solid micro emboli.

There was no correlation between the Verifynow- P2Y12 and -aspirin assay with number of micro embolic signals (ρ= -0.046 p= 0.689 and ρ= -0.068, p= 0.499). VerifyNow-P2Y12 could not predict the number of solid micro emboli (β=0.008 (-0.005 to 0.021, p=0.248). Remarkably, the slope of the Verifynow- aspirin results on micro emboli frequency was negative (β= - 0.028 (-0.074 to 0.018, p=0.232), reflecting that a higher number of VerifyNow ARU was related to less micro emboli measured with TCD. Utilizing the ‘’high platelet reactivity cut-off value’’ of the 75th percentile of platelet reactivity by FC analysis, 48 patients were identified with high platelet reactivity after stimulation with ADP. Equal analysis was performed after TRAP-6 stimulation. The type of VerifyNow assay performed per patient depended on the utilised APT for each individual patient, resulting in 29/78 (37.2%) patients with high platelet reactivity (PRU ≥ 208) according VN-P2Y12 and 7/106 (6.6%) patients according to VN- aspirin (ARU ≥ 550). Due to logistic reasons 2 FC- ADP and 5 VerifyNow P2Y12 assays were not performed for P2Y12 inhibitor users and 2 VerifyNow Aspirin assays were not performed for aspirin users.

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Effect of clopidogrel on platelet reactivity and MES

Table 2. The association between number of solid micro emboli with FC platelet activation test and VerifyNow N

Correlation coefficient (spearmans rho)

P- value

FC-ADP

‡

188

0.218

0.003*

FC-TRAP-6

188‡

0.195

0.007*

* VN P2Y12 assay

77‡

-0.046

0.689

** VN Aspirin assay

102

-0.068

0.499

‡

ADP adenosine diphosphate, CI confidence interval, FC flow cytometry, TRAP-6 thrombin receptor activation peptide, VN VerifyNow * Only patients with P2Y12 inhibitor treatment ** Only patients with aspirin treatment ‡ Invalid TCD registration for 9 patients in total

Figure 2. Scatterplots of platelet reactivity tests and micro emboli frequency.

To further investigate the clinical predictive value of both platelet function tests, we investigated the association of high platelet reactivity defined by either FC or VerifyNow with being at ‘high risk for stroke’ (displaying ≥ 20 solid micro emboli). Only high platelet reactivity determined by FC-ADP showed a significant association: OR 5.732 (95% CI: 1.314 - 25.003, p=0.020) with displaying ≥ 20 solid micro emboli (table 3). The FC-TRAP-6 and both VerifyNow assays did not show an association with ‘high risk for stroke’’ status. Next we compared the mean number of displayed micro emboli for patients with normal vs high platelet reactivity defined by FC and VerifyNow (Figure 3A.) The number of solid

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micro emboli differed between normal vs high platelet reactivity based on FC-ADP: resp. 3.6 vs. 8.6, p=0.043. This was not true for FC- TRAP-6: 4.4 vs. 6.0, p=0.347. Both VerifyNow -P2Y12 as -aspirin assay did now show a difference in displayed solid micro emboli by normal of high platelet reactivity status (2.2 vs 2.4, p=0.888 and 6.8 vs 0.71, p=0.203 for patients using respectively clopidogrel or aspirin (Figure 3B). To correct for the effect of clopidogrel usage in FC results, we consequently analysed the mean number of solid emboli in patients with normal vs. high platelet reactivity after ADP stimulation, only for patients who were actually treated with clopidogrel (fig 3B). No differences in solid micro emboli were found for normal vs. high platelet reactivity (2.2 vs. 2.3, p= 0.974) suggesting that the initial difference in solid micro emboli might be solemnly due to the P2Y12 inhibitor treatment.

Figure 3. Prevalence of solid micro emboli per responder status, classified by the FC for all patients (A) and classified by the FC and VerifyNow for P2Y12 inhibitor- or aspirin users (B). * 2 FC- ADP, 5 VerifyNow P2Y12 and 2 VerifyNow Aspirin assays were not performed due to logistic reasons.

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Table 3. Logistic regression analysis for patient at risk for stroke and MACE, as predicted by high platelet reactivity status. Platelet reactivity test: HPR status defined by

20 solid micro emboli (N=8)

Major adverse cardiovascular events (n=42)

Univariate OR (95% CI)

p-value

FC- ADP

5.732 (1.314 to 25.003)

0.020*

FC- TRAP-6

1.045 (0.204 to 5.367)

0.958

VerifyNow P2Y12 assay

0.442 (0.133 to 1.471)

0.183

VerifyNow Aspirin assay

1.731 (0.754 to 3.975)

0.196

FC- ADP

2.346 (1.126 to 4.890)

0.023*

FC- TRAP-6

1.765 (0.837 to 3.721)

0.136

VerifyNow P2Y12 assay

1.646 (0.706 to 3.840)

0.249

VerifyNow Aspirin assay

1.640 (0.780 to 3.448)

0.192

ADP adenosine diphosphate, ARU aspirin reactive units, AUC area under the curve, CI confidence interval, MFI median fluorescence intensity, FC flow cytometry, HPR high platelet reactivity PRU P2Y12 reaction units

7 Secondary outcome: clinical follow up Four patients showed direct postoperative neurological impairment: right-sided muscle weakness, central right-sided n. facialis paralysis and hemianopia (table 4.) Another 5 patients developed neurological symptoms (transient muscle weakness, right hand deficit, right sided mouth droop, hemiparesis and dysphasia) after a symptom-free period, within 1 day postoperative. The symptoms dissolved <24 hours in 4 patients. Fourteen patients (7.2%) had postoperative troponin elevation. Four patients (2.0%) showed an intervention related bleeding < 2 days post CEA with need for reintervention. A single patient died 12 days after CEA due to direct postoperative CVA, intervention related bleeding requiring reintervention with consequent palliative medical care. The overall mortality was 6.1% (n=12). Totally 42 major adverse events occurred: 14 cardiac (7.1%), 19 cerebral (stroke, TIA or AF) (9.7%) and 9 peripheral (4.6%) events. Some patients developed a recurrent ipsilateral stenosis of 51-70% (4.1%, n=8), 71-99% (3.6%, n=7) or total occlusion (1.0%, n=2) resulting in a re-CEA in 5 patients. The mean number of micro emboli in patients who developed MACE was significantly higher compared to patients without MACE (4.11 vs. 7.17, p=0.018). MACE developed in 37.5% of the patients who displayed â&#x2030;Ľ 20 micro emboli compared to 23.8%, p=0.238. A single patient with direct postoperative CVA showed 62 micro emboli during CEA. Other patients did not show increased embolization. Again, only the non-responder status determined by PACT-ADP showed an increased risk for MACE during follow up (OR 2.346, 95% CI: 1.126 to 4.890, p=0.023) (table 4). Other platelet reactivity tests did not show increased risk between test outcome and the occurrence of MACE during follow up.

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Table 4. Postoperative clinical events. Clinical events

N=197

% or SD

4 0

2.0 0.0

Stroke Transient ischemic attack

1 4

0.5 2.0

Hyper perfusion syndrome BP based BD and symptomatology

23 5

11.7 2.5

Direct postoperative Stroke Transient ischemic attack After symptom-free period, <1 day

Troponin elevation Bleeding Re-intervention bleeding direct stenosis

14/194

7.2

2.0

5 -4 -1

2.5 - 2 .0 - 0.5

0.5

3/193 13 6 5 1

1.6 6.7 3.1 2.6 0.5

3 months follow up Mortality Ipsilateral restenosis -0-30% 31-50% 51-70% 71-99% occlusion Total follow-up Duration (months), mean (SD)

13.4

10.9

Mortality Cardiovascular related death

12/196 4

6.1 2.0

Major adverse events Cardiac Cerebral (Stroke, TIA, AF) Peripheral

14/196 19 9

7.1 9.7 4.6

Ipsilateral restenosis 0-30% 31-50% 51-70% 71-99% occlusion

6/193 16 8 7 2

3.1 8.3 4.1 3.6 1.0

Reoperation due to stenosis < 1 year â&#x2030;Ľ 1 year

3/195 2

1.5 1.0

BP Blood pressure, TIA transient ischemic attack, AF amaurosis fugax

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Figure 4. Effect of clopidogrel on platelet reactivity, prevalence of solid micro emboli and major adverse events during follow-up.

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Effect of clopidogrel usage We observed a difference in solid micro emboli frequency by responder status of FC-ADP regarding all patients or only patients treated with clopidogrel. The high number of solid micro emboli in patients with ‘’high platelet reactivity’’ (mean 8.6) when analysing all patients, might be caused by those patients not using clopidogrel. Therefore we subsequently studied the effect of clopidogrel treatment on platelet reactivity measured with FC- ADP and -TRAP-6, number of micro embolic signals and occurrence of MACE and death during follow up (Figure 4): Clopidogrel usage resulted in decreased platelet reactivity after ADP and TRAP-6 stimulation (5 083 vs. 18 939 MFI, p< 0.001 and 6 850 vs. 20 479 MFI, p<0.001 ), less solid micro emboli (2.3 vs. 6.6, p= 0.001) and less MACE (14.6% vs 26.3%, p=0.049). P2Y12 inhibitor usage correlates to the amount of solid micro emboli displayed during CEA (ρ = -0.254, p<0.001). Clopidogrel usage lowers the amount of solid micro emboli frequency, β= -0.215 (95%CI: -7.161 to -1.500, p=0.003). Lastly, clopidogrel usage was not associated to the occurrence of MACE during follow up (OR= 0.480, 95%CI: 0.229 to 1.007 p=0.052). VerifyNow P2Y12 results were only available for patients using P2Y12 inhibitors, therefore previous analysis was not applicable to the VerifyNow results.

Discussion This study determined the correlation of a flow cytometry (FC)- and aggregation based (VerifyNow) platelet reactivity test with the number of solid micro emboli measured by the TCD during CEA. Platelet reactivity measured with flow cytometry after stimulation with ADP or TRAP-6 was correlated to solid micro emboli frequency, with an increased risk of being at risk (displaying ≥20 micro emboli) for patients with high platelet reactivity, based on FC- ADP. The VerifyNow-P2Y12 and -aspirin were not correlated to micro emboli frequency and the aspirin assay even showed a negative slope for prediction of micro emboli number. However, perioperative clopidogrel treatment showed the strongest effect on reducing the micro emboli frequency and thereby exceeded the effect of high platelet reactivity assessed by FC- ADP. Although the VerifyNow is the most commonly used test in monitoring APT, it shows divergent results regarding the correlation with clinical outcomes. High on clopidogrel platelet reactivity according to the VerifyNow P2Y12 assay was strongly related to stent thrombosis, MI or cardiovascular death in large trials with more than 2500 patients undergoing PCI. 27,28 Other trials show no significant predicting value 29,30, conclude the association only for high risk patients with diabetes and chronic kidney failure 31, or fail to show a benefit of adjusting APT based on VerifyNow test results. 6,7 Studies combining multiple platelet reactivity tests showed a poor correlation between the VerifyNow, Multiplate and/or Platelet Function Analyser- 100.32,33

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Studies comparing aggregometry and platelet activation by flow cytrometry regarding on treatment platelet reactivity are scarce; a single study compared platelet aggregation, measured by light transmission aggregometry, VerifyNow assays, and multiple electrode aggregometry in response to arachidonic acid (AA) and ADP in 316 patients receiving aspirin and clopidogrel after angioplasty with stent implantation. 34 AA- and ADP-induced P-selectin expression and activated glycoprotein (GP) IIb/IIIa were also measured with flow cytometry. The best correlation was seen between the results of the VerifyNow P2Y12 assay and activated GPIIb/IIIa (rho = 0.68). In line with our study results, AAinduced platelet reactivity showed poor outcome: no correlation between all different platelet aggregation tests, and between aggregometry and flow cytometry results. The utilized flow cytometry assay has been used before to determine platelet function in patients with thrombocytopenia after platelet transfusion 35,36 and with chronic kidney disease.37 A single previous study used flow cytometric analysis of platelet function to compare platelet reactivity in patients with chronic limb ischemia treated with aspirin or OAC with platelet reactivity in healthy controls without APT. 38 The current study is the first correlating FC measurements to thrombotic events. Although the FC has been used for other research aims, there are still important improvements to be made for the monitoring of APT: 1) the cut-off values for patients on different classes of APT need to be determined from a larger cohort of patients on APT, 2) the test should be performed under 37â&#x2014;ŚC to mimic the physiological temperature of blood and 3) all tests kits should be prepared at the same time, to minimize batch differences. The presence of MES has extensively been related to the occurrence of cerebral symptoms. 39 During CEA, occurrence of MES could be explained by manipulation of the atherosclerotic plaque (during dissection) and activation of platelets by the exposed collagen of the injured endothelium of the endarterectomy- and clamp site after restoration of the blood flow. MES can be classified in either gaseous or solid micro emboli, with different clinical consequences. Classifying exactly the right MES as solid micro emboli is a mentally strenuous procedure, potentially leading to less reliable results. Even more, stronger APT as clopidogrel treatment has been proven to decreased the number of MES 12,40 potentially causing the low mean micro emboli frequency in this study. In the current study clopidogrel treatment was the strongest predictor for a decrease in platelet reactivity measured with FC- ADP and -TRAP-6, solid micro emboli and MACE. Previous studies on P2Y12 inhibition on micro emboli frequency have initially demonstrated that platelet response to ADP was significantly higher in patients with > 25 micro emboli, measured with aggregometry and flow cytometry. 41 Next, a small cohort of 100 patients on aspirin treatment were randomized into clopidogrel (n=46) or placebo (n=54) prior surgery. Clopidogrel treatment resulted in a small reduction in platelet response to ADP, with a tenfold reduction in relative risk of displaying > 20 microemboli postoperatively. 12 This strong protective effect of clopidogrel during CEA was confirmed by a retrospective cohort study, investigating a cohort of 270 patients on aspirin and single tablet 75 mg clopidogrel compared to a historical control cohort. 40

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High embolization occurred more frequent in the historical cohort group than in the study group (3.2% vs. 0.4%, p= 0.0062). However, significant influence of different antiplatelet regimens on TCD detected postoperative embolization could not be found for either 1) Asasantin (Dipyridamole 200mg/Aspirin 25mg) twice daily, 2) Asasantin plus 75 mg Clopidogrel once daily or 3) Asasantin plus Rheomacrodex (Dextran 40) 100g/L iv; 500 ml.18 Despite this protective effect of clopidogrel, only 31% of the centres participating in the Asymptomatic Carotid Surgery Trial (ACST)-2 study treated patients undergoing CEA with dual antiplatelet therapy prior intervention and 24% post intervention. 42 For symptomatic patients no data is available. The current study holds some limitations; unfortunately, we were not able to investigate the influence of high on clopidogrel platelet reactivity on the micro emboli frequency, since only 4 patients for FC- ADP and 29 patients for VerifyNow P2Y12 were identified as non- responders to P2Y12 inhibitors. For the TCD registration only peroperative measurements were available, therefore we cannot make a statement concerning the postoperative embolization due to platelet activation on the operated endothelium site. Concluding, perioperative clopidogrel treatment showed the strongest effect on reducing platelet reactivity measured by flow cytometry and micro emboli frequency during carotid endarterectomy. High platelet reactivity assessed by FC- ADP showed an increased risk for displaying â&#x2030;Ľ20 micro emboli during CEA. To determine if clopidogrel treatment results in a reduction of stroke requires a future large randomised controlled trial.

Acknowledgements We would like to thank the all clinical neurophysiology technicians of the UMCU for their effort in registration of the TCD recordings.

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12.

Siddiqui TI, Kumar KSA, Dikshit DK. Platelets and atherothrombosis: causes, targets and treatments for thrombosis. Current medicinal chemistry. 2013 Ruggeri ZM, Mendolicchio GL. Adhesion mechanisms in platelet function. Circulation research. Jun 2007 Peeters Weem SM, van Haelst ST, den Ruijter HM, Moll FL, de Borst GJ. Lack of Evidence for Dual Antiplatelet Therapy after Endovascular Arterial Procedures: A Meta-analysis. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Aug 2016 Aradi D, Kirtane A, Bonello L, et al. Bleeding and stent thrombosis on P2Y12-inhibitors: collaborative analysis on the role of platelet reactivity for risk stratification after percutaneous coronary intervention. European heart journal. Jul 2015 Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA : the journal of the American Medical Association. Mar 2011 Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 Breet NJ, van Werkum JW, Bouman HJ, et al. Comparison of platelet function tests in predicting clinical outcome in patients undergoing coronary stent implantation. JAMA : the journal of the American Medical Association. Feb 2010 Leunissen TC, Peeters Weem SM, Urbanus RT, et al. High On-Treatment Platelet Reactivity in Peripheral Arterial Disease: A Pilot Study to Find the Optimal Test and Cut Off Values. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. May 2016 T. Leunissen PW, T. van Holten, P. de Groot, S. Korporaal, A. Koekman, F. Moll, M. Teraa, M. Verhaar, GJ. de Borst , R. Urbanus and M. Roest. The effect of P2Y12 inhibition on platelet activation assessed by aggregation- and flow cytometry based assays Platelets, in press. 2016 Goodall AH, Appleby J. Flow-cytometric analysis of platelet-membrane glycoprotein expression and platelet activation. Methods Mol Biol. 2004 Payne DA, Jones CI, Hayes PD, et al. Beneficial

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effects of clopidogrel combined with aspirin in reducing cerebral emboli in patients undergoing carotid endarterectomy. Circulation. Mar 2004 Ritter MA, Jurk K, Schriek C, et al. Microembolic signals on transcranial Doppler ultrasound are correlated with platelet activation markers, but not with platelet-leukocyte associates: a study in patients with acute stroke and in patients with asymptomatic carotid stenosis. Neurological research. Feb 2009 Kinsella JA, Tobin WO, Tierney S, et al. Increased platelet activation in early symptomatic vs. asymptomatic carotid stenosis and relationship with microembolic status: results from the Platelets and Carotid Stenosis Study. Journal of thrombosis and haemostasis : JTH. Jul 2013 Ackerstaff RG, Moons KG, van de Vlasakker CJ, et al. Association of intraoperative transcranial doppler monitoring variables with stroke from carotid endarterectomy. Stroke; a journal of cerebral circulation. Aug 2000 Skjelland M, Krohg-Sorensen K, Tennoe B, Bakke SJ, Brucher R, Russell D. Cerebral microemboli and brain injury during carotid artery endarterectomy and stenting. Stroke; a journal of cerebral circulation. Jan 2009 Timaran CH, Mantese VA, Malas M, et al. Differential outcomes of carotid stenting and endarterectomy performed exclusively by vascular surgeons in the Carotid Revascularization Endarterectomy versus Stenting Trial (CREST). Journal of vascular surgery. Feb 2013 de Borst GJ, Hilgevoord AA, de Vries JP, et al. Influence of antiplatelet therapy on cerebral micro-emboli after carotid endarterectomy using postoperative transcranial Doppler monitoring. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Aug 2007 Naylor AR, Rothwell PM, Bell PR. Overview of the principal results and secondary analyses from the European and North American randomised trials of endarterectomy for symptomatic carotid stenosis. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Aug 2003 Mayberg MR, Wilson SE, Yatsu F, et al. Carotid endarterectomy and prevention of cerebral ischemia in symptomatic carotid stenosis. Veterans Affairs Cooperative Studies Program 309 Trialist Group. JAMA : the journal of the American Medical Association. Dec 1991 Randomised trial of endarterectomy for recently symptomatic carotid stenosis: final results of the MRC European Carotid Surgery Trial (ECST). Lancet. May 1998 Pennekamp CW, Moll FL, de Borst GJ. The potential benefits and the role of cerebral monitoring in carotid endarterectomy. Current opinion in anaesthesiology. Dec 2011

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23. Accumetrics. Pocket Guide VerifyNow. San Diego, California, USA, 2013. 24. Keunen RW, Hoogenboezem R, Wijnands R, Van den Hengel AC, Ackerstaff RG. Introduction of an embolus detection system based on analysis of the transcranial Doppler audio-signal. Journal of medical engineering & technology. Jul 2008 25. Ringelstein EB, Droste DW, Babikian VL, et al. Consensus on microembolus detection by TCD. International Consensus Group on Microembolus Detection. Stroke; a journal of cerebral circulation. Mar 1998 26. T. Leunissen DvV, H. de Ruijter, F. Moll, W.H. Mess, GJ. de Borst. Validation of the automated electronic micro emboli detection system (EDS) in patients undergoing carotid endarterectomy. Conditionally accepted in the European Journal of Utrasound. 2016 27. Stone GW, Witzenbichler B, Weisz G, et al. Platelet reactivity and clinical outcomes after coronary artery implantation of drug-eluting stents (ADAPT-DES): a prospective multicentre registry study. Lancet. Aug 2013 28. Price MJ, Angiolillo DJ, Teirstein PS, et al. Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: a timedependent analysis of the Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety (GRAVITAS) trial. Circulation. Sep 2011 29. Paulu P, Osmancik P, Tousek P, et al. Lack of association between clopidogrel responsiveness tested using point-of-care assay and prognosis of patients with coronary artery disease. Journal of thrombosis and thrombolysis. Jul 2013 30. Legrand V, Cuisset T, Chenu P, et al. Platelet reactivity and cardiovascular events after percutaneous coronary intervention in patients with stable coronary artery disease: the Stent Thrombosis In Belgium (STIB) trial. EuroIntervention : journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology. Jun 2014 31. Viviani Anselmi C, Briguori C, Roncarati R, et al. Routine assessment of on-clopidogrel platelet reactivity and gene polymorphisms in predicting clinical outcome following drug-eluting stent implantation in patients with stable coronary artery disease. JACC. Cardiovascular interventions. Nov 2013 32. von Beckerath N, Sibbing D, Jawansky S, et al. Assessment of platelet response to clopidogrel with multiple electrode aggregometry, the VerifyNow P2Y12 analyzer and platelet Vasodilator-Stimulated Phosphoprotein flow cytometry. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis. Jan 2010

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33. Paniccia R, Antonucci E, Gori AM, et al. Comparison of different methods to evaluate the effect of aspirin on platelet function in high-risk patients with ischemic heart disease receiving dual antiplatelet treatment. American journal of clinical pathology. Jul 2007 34. Gremmel T, Koppensteiner R, Panzer S. Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists -Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy. PloS one. 2015 35. Bikker A, Bouman E, Sebastian S, et al. Functional recovery of stored platelets after transfusion. Transfusion. May 2016 36. Roest M, van Holten TC, Fleurke GJ, Remijn JA. Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients. Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie. Apr 2013 37. van Bladel ER, de Jager RL, Walter D, et al. Platelets of patients with chronic kidney disease demonstrate deficient platelet reactivity in vitro. BMC nephrology. 2012 38. P.P. Wisman MT, GJ de Borst, M. Verhaar, M. Roest, F. Moll. Increased baseline platelet activation, but reduced platelet reactivity in patients with critical limb ischemia. Submitted: PLoS One. 2015 39. King A, Markus HS. Doppler embolic signals in cerebrovascular disease and prediction of stroke risk: a systematic review and meta-analysis. Stroke; a journal of cerebral circulation. Dec 2009 40. Sharpe RY, Dennis MJ, Nasim A, et al. Dual antiplatelet therapy prior to carotid endarterectomy reduces post-operative embolisation and thromboembolic events: postoperative transcranial Doppler monitoring is now unnecessary. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Aug 2010 41. Hayes PD, Box H, Tull S, Bell PR, Goodall A, Naylor AR. Patients' thromboembolic potential after carotid endarterectomy is related to the platelets' sensitivity to adenosine diphosphate. Journal of vascular surgery. Dec 2003 42. Huibers A, Halliday A, Bulbulia R, Coppi G, de Borst GJ. Antiplatelet Therapy in Carotid Artery Stenting and Carotid Endarterectomy in the Asymptomatic Carotid Surgery Trial-2. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Mar 2016

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Chapter 8 Clinical risk factors and plaque characteristics associated with new development of contralateral stenosis in patients undergoing carotid endarterectomy Cerebrovascular Diseases, 2016 April; Volume 42, p 122-130

S. Merckelbach, T. Leunissen, J. Vrijenhoek, F. Moll, G. Pasterkamp, GJ. de Borst

Chapter 8

Abstract Background Following Carotid EndArterectomy (CEA), cerebrovascular haemodynamic may be hampered by ipsilateral restenosis or development of contralateral stenosis. It remains to be clarified if these patients need follow-up for identifying development of contralateral stenosis. Identification of risk factors contributing to development of contralateral stenosis could allow more specific follow-up. In this current study, we assessed clinical risk factors and plaque characteristics of patients undergoing CEA with development of new contralateral stenosis during mid-term follow-up. Methods 760 patients undergoing CEA between 2003 and 2011 at UMC Utrecht were included. Atherosclerotic plaques were excised and analysed for smooth muscle cells, collagen, macrophages, lipid core, plaque haemorrhage and vessel density. Patients underwent clinical and duplex ultrasound follow-up at 3 and 12 months and yearly thereafter. Association between plaque- and patient characteristics with development of contralateral stenosis â&#x2030;Ľ 50% was assessed with univariate and multivariate analysis. Clinical outcome during follow-up was associated with development of new contralateral stenosis. Results After a median follow-up time of 2.5 years, development of contralateral stenosis was observed in 108 patients (20%). Presence of high collagen (p=0.025) and high smooth muscle cells (p=0.027) was associated with development of new contralateral stenosis, whereas large lipid core was negatively associated with new development (p=0.034). The same plaque characteristics were related to contralateral occlusion. History of coronary artery disease (p=0.031) and asymptomatic presentation (p=0.000) were univariate associated with development of contralateral stenosis. Multiple regression analysis indicated that asymptomatic status was independently associated with contralateral stenosis (p=0.001). Patients with new development of contralateral stenosis showed more often symptoms during follow-up (p=0.049). Conclusion Dissection of a lipid-poor, collagen-rich or smooth muscle cell-rich plaque yielded an association with new development of contralateral stenosis during mid-term follow-up after CEA. Asymptomatic patients had a significantly higher risk for development of contralateral stenosis. New contralateral stenosis was related the presence of new cerebral symptoms. These findings may help to develop individual treatment algorithms for patient with cerebrovascular atherosclerotic burden.

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Introduction Carotid endarterectomy (CEA) reduces the risk of stroke for both symptomatic and subgroups of asymptomatic patients with high-grade carotid artery stenosis. 1 Nevertheless, this advantage may be attenuated by the development of restenosis. Restenosis is associated with a higher risk of recurrent symptoms, re-interventions and stroke and therefore hampers the benefit of CEA.2 In addition, following CEA patients are prone to develop atherosclerosis at other sites. Development of contralateral stenosis is relatively common after CEA,exceeds the rate of ipsilateral restenosis, reduces the contralateral cerebrovascular reserve and may also affect clinical outcome.3-7 It remains to be clarified if patients at risk for development of contralateral stenosis need follow-up and if so, for what period of time. Risk factors that might contribute to the development of contralateral stenosis remain to be established.1,8 Identifying patients at risk for developing contralateral stenosis would allow selective individualized follow-up after CEA. Next to clinical and demographic factors it could be useful to establish which plaque characteristics are predictive for contralateral stenosis development. Potentially, information derived from the removed carotid atherosclerotic plaque individualizes treatment decisions by using non-invasive plaque imaging. Our objectives were to establish the incidence and risk factors â&#x20AC;&#x201C;both clinically and based on plaque characteristics- for development of new contralateral stenosis or occlusion in mid-term follow-up after CEA.

Methods Patient population All patients included in this study are part of the Athero-Express study. The design of this study has been described earlier.10 In summary, this is a longitudinal bio bank study comprising carotid plaques of patients undergoing a primary CEA and long term followup. The plaques are collected and subjected to histological examination. All patients undergoing CEA at the University Medical Center Utrecht, the Netherlands were asked to participate. For the present study, we examined available data from all carotid arteries operated in patients included in the Athero-Express biobank from June 2003 until September 2011. There were no exclusion criteria. Indications for CEA were reviewed by a multidisciplinary vascular team and were based on recommendations of the Asymptomatic Carotid Atherosclerosis Study and Asymptomatic Carotid Surgery Trial studies for asymptomatic patients and the North American Symptomatic Carotid Endarterectomy Trial and European Carotid Surgery Trial studies for symptomatic patients. 3,11,12 This study was approved by the Institutional Review boards of the UMCU hospital. All patients provided informed consent. At baseline patients completed a questionnaire regarding medication use, cardiovascular risk factors and medical history.

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Atherosclerotic plaque examination CEA was performed by experienced vascular surgeons. Method of arterial closure was chosen by the surgeon and could be either primary or patch closure with Dacron, bovine or vein patch. The procedure remained the same during the inclusion period. The atherosclerotic plaque was immediately brought to the laboratory after removal during CEA. Plaque characteristics were scored by two independent observers blinded for clinical outcome with a good intra-observer and inter-observer reproducibility.9 Scoring was analysed in two categories (no/minor or moderate/ heavy staining) for macrophages, smooth muscle cells, calcification, collagen and lipid core. Plaque haemorrhage and fibrin staining were defined as absent or present. Vessel density was binned in two groups (low and high), split by the median of this density. More extensive information about tissue collection, tissue processing, and histological examination is described in earlier articles.13,14 Duplex Follow-up A follow-up with duplex ultrasound took place at 3 and 12 months after CEA and yearly thereafter. An IU22 ultrasound device from Philips Medical Systems (Eindhoven, the Netherlands) with linear transducer 9-3 mHz was used. The technicians who performed the duplexes were blinded for any data concerning this study. All the data regarding the duplex measurements were collected retrospectively and any lost to follow-up was defined as missing data on all follow-up moments thereafter. Clinical outcome Clinical follow-up of patients was performed using annual questionnaires, from inclusion date until lost to follow-up (or latest September 2013). Records of the patients in the electronic hospital database were reviewed for cardiovascular endpoints. Stroke (both ipsilateral and contralateral) and death were recorded for this study. Outcomes were verified by two independent researchers. Definition of Endpoints The primary endpoint of follow-up in this study was new development of contralateral stenosis at any time point. Contralateral stenosis is defined as occurrence of stenosis ≥ 50% or occlusion. In our database stenosis was classified in the following categories, adopted from the MRC European Carotid Surgery Trial: no stenosis (0-29%), mild stenosis (30-49%), moderate stenosis (50-69%), severe stenosis (70-99%) and occlusion. 3,15,16 This corresponds with the following peak systolic values (PSV): no stenosis (≤ 150 cm/s), mild stenosis (150 > PSV ≤ 210 cm/s ), moderate stenosis (210 > PSV ≤ 270 cm/s ), severe stenosis (PSV > 270 cm/s), subtotal occlusion (PSV < 40 cm/s and severe plaque) and occlusion (no flow).

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Data analysis SPSS version 20.0 (SPSS Inc, Chicago, Illinois) was used for all statistical analyses. Cross tables (2x2) were constructed to calculate the percentage of the development of contralateral stenosis for each baseline characteristic. The p-value was calculated with the Chi-square statistic. Continuous variables were analyzed using independent samples T-tests. To adjust for potential confounders, we performed a multivariable logistic regression analysis. We assumed a potential association for the clinical risk factors sex, age, hypertension, current smoking, diabetes, body mass index, acetylsalicylic acid and statin use, symptom status, type of patch used and year of operation. 14,17 Additional, a factor was assumed to be a potential cofounder if a P-value of <0.25 was found in univariate analysis with development of contralateral stenosis as outcome. The survival free-rate of development of contralateral stenosis was analyzed using Kaplan-Meier curves.

Results From 2003 until 2011 in total 811 primary CEAs were performed, of which 760 patients were included in this study. In total 51 patients were excluded because no follow-up was available (see flowchart; Figure 1). The mean age of the included patients was 69 years and 30% were woman. At the moment of inclusion smoking was reported in 33%, diabetes mellitus in 23% and hypertension in 74%. Clinical presentation was in 87% of the patients symptomatic, comprising mostly a transient ischemic attack (TIA). A majority of the patients used preoperative acetylsalicylic acid (82%) and statins (82%). Bilateral stenosis was found in 34% (138/401) of the patients, since they revealed a contralateral carotid stenosis of >50% at baseline DUS. Appendix 1 comprises the full baseline characteristics of all included patients. Clinical and plaque characteristics after follow-up The median follow- up of the total cohort was 2.5 years (IQR 1.7-4.3). During this followup 108 (20.3%) patients developed a new contralateral carotid stenosis of more than 50%. In 51 of these patients occlusion was detected. Of the total cohort, 424 patients did not develop contralateral stenosis, 138 were excluded because of contralateral stenosis at baseline and data were missing in 90 patients. In the flowchart those numbers are stated more clearly. Patients with a history of coronary artery disease (CAD) showed significantly more often development of contralateral stenosis (p=0.031) (table 1). Development of contralateral stenosis was also found significantly more often in patients who were asymptomatic at time of inclusion, compared to patients that were symptomatic (with TIA, amaurosis fugax or stroke). Other patient characteristics did not showed a significant association with the development of contralateral stenosis in our study population.

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Patients with an atherosclerotic plaque with a large lipid core showed significantly less development of contralateral stenosis (18% of the patients with development of contralateral stenosis had a large lipid core vs. 28% of the patients with no stenosis, p=0.034). High collagen and high smooth muscle cells in the plaque were associated with more development of contralateral stenosis (p=0.025 and p=0.027). Other plaque characteristics showed no correlation with development of contralateral stenosis in follow-up (Figure 2). As previously mentioned, in half of the patients with new contralateral atherosclerotic development a contralateral occlusion developed. Predicting clinical risk factors for development of a new contralateral occlusion were current smoking (47% in group with occlusion vs. 31 % in group without, p=0.023) and Body Mass Index (p=0.046). Besides that, asymptomatic patients significantly more often developed contralateral occlusion during follow-up (p=0.000). Same plaque characteristics that were predictable for the development of contralateral stenosis were associated with the development of contralateral occlusion. In table 1 clinical risk factors- and plaque-characteristics for both patient groups with and without development of contralateral stenosis and occlusion are reported.

Figure 1. Flowchart of patients undergoing carotid endarterectomy.

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Development of contralateral stenosis after CEA

Table 1. Characteristics of patients with and without contralateral stenosis or occlusion in follow-up. Patient characteristics

Contralateral No stenosis stenosis

P-value

Occlusion in FU

No occlusion in FU

P-value

Female gender

27/108 (25)

142/424 (34)

0.091

11/51 (22)

158/481 (33)

0.100

Mean age

69 (9)

68 (10)

0.444

66 (9)

69 (10)

0.423

Current smoker

41/108 (38)

132/418 (32)

0.208

24/51 (47)

149/475 (31)

0.023*

Diabetes mellitus

25/108 (23)

88/424 (21)

0.587

14/51 (28)

99/481 (21)

0.254

Hypertension

79/106 (74)

307/420 (73)

0.765

38/51(75)

348/475 (73)

0.848

Mean BMI

26.5 (4)

26.4 (4)

0.285

27 (4)

26 (4)

0.046*

Hypercholesterolemia

70/96 (73)

278/395 (70)

0.624

34/46 (74)

314/445 (71)

0.634

History of CAD

42/108 (39)

119/422(28)

0.031*

19/51 (37)

142/479 (30)

0.261

History of PI

15/108 (14)

80/421 (19)

0.217

10/51 (20)

85/478 (18)

0.747

Acetylsalicylic acid

88/108 (82)

350/421 (83)

0.685

43/51 (84)

395/478 (83)

0.763

Antiplatelet therapy

91/108 (84)

373/421 (87)

0.220

45/51 (88)

419/478 (88)

0.905

Statin use

97/109 (90)

347/422 (82)

0.056

47/51 (92)

397/479 (83)

0.088

Mean total cholesterol

4.2 (1.1)

4.6 (1.2)

0.339

4.2 (1.2)

4.6 (1.2)

0.787

High density lipoprotein

1.1 (0.36)

1.2 (0.51)

0.289

1.0 (0.27)

1.2 (0.51)

0.133

Low density lipoprotein

2.4 (0.89)

2.7 (0.99)

0.389

2.3 (0.99)

2.6 (0.98)

0.765

Triglycerides

1.6 (0.82)

1.6 (0.99)

0.378

1.7 (0.75)

1.6 (0.99)

0.198

Mean GFR, CG

72 (2.9)

73 (1.3)

0.114

81(32)

72 (25)

0.104

Asymptomatic

24/108 (22)

40/420 (10)

0.000*

19/51 (37)

45/477 (9)

0.000*

Amourosis fugax

16/108 (15)

64/420 (15)

0.133

2/51 (4)

78/477 (16)

0.455

TIA

45/108 (42)

209/420 (50)

0.368

22/51 (43)

232/477 (49)

0.119

Stroke

23/108 (21)

107/420 (26)

0.913

8/51 (16)

122/477 (26)

0.019*

Clinical presentation

Type of closure No patch, primary closure

5/10 7(5)

28/418 (7)

0.441

1/50 (2)

32/475 (7)

0.189

Venous

69/107 (65)

273/418 (65)

0.873

35/50 (70)

307/475 (65)

0.449

Bovine

22/107 (21)

85/418 (20)

0.959

8/50 (16)

99/475 (21)

0.419

Dacron

11/107 (10)

32/418 (8)

0.377

6/50 (12)

37/475 (8)

0.302

Moderate/ high marcrophages

47/104 (45)

209/412 (51)

0.313

28/49 (57)

232/467 (50)

0.320

Large lipid core

18/103 (18)

114/412 (28)

0.034*

6/49 (12)

126/466 (27)

0.024*

Moderate/ high collagen

90/104 (87)

314/411 (76)

0.025*

44/49 (90)

360/466 (77)

0.042*

Plaque haemorrhage

57/103 (55)

221/412 (54)

0.757

27/49 (55)

251/466 (54)

0.868

High vessel density

47/89 (53)

180/376 (48)

0.402

23/40 (58)

204/425 (48)

0.250

Moderate/ high SMC

82/104 (79)

279/412 (68)

0.027*

45/49 (92)

316/467 (68)

0.000*

Plaque characteristics

* P<0.05 Continuous values are expressed as mean with standard deviation, unless specified otherwise. Categorical values are expressed as n/total (%). Abbreviations: CAD: coronary artery disease; PI: peripheral intervention; GFR: Glomerular Filtration Rate ; CG: CockroftGauld; TIA: Transient ischemic attack; SMC: smooth muscle cells

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Figure 2. Contralateral stenosis at mid-term follow-up by different plaque characteristics.

A multi-regression analysis to estimate the relation between development of contralateral stenosis and potential factors, as mentioned earlier plus the potential cofounders found in univariate analysis, was performed until only variables with P-value <0.01 in the model were left. The potential cofounders found in univariate analysis were history of CAD, history of peripheral intervention, glomerular filtration rate, large lipid core, high collagen and high smooth muscle cells. After this analysis only asymptomatic status before CEA turned out to be associated with development of contralateral stenosis (p=0.001), values for significance are showed in table 2. Progression of baseline stenosis 138 patients showed a contralateral stenosis at baseline and were therefore excluded from the initial study analysis, in which we only looked at new development of contralateral stenosis. Of these 138 patients, nine (34.6%) with a baseline moderate contralateral stenosis progressed during follow-up into a severe stenosis. Eight (14.5%) patients with baseline severe contralateral stenosis developed an (sub)total occlusion. Other patients already displayed a (sub)total occlusion at baseline. No plaque characteristics were correlated with progression of contralateral stenosis except for the presence of a high amount of smooth muscle cells for patients with 50-69% stenosis at baseline (p=0.046). Clinical outcome Clinical outcome was scored by looking at symptoms of TIA, stroke or Amaurosis Fugax patients showed during follow-up. Patients who developed new contralateral stenosis during follow-up showed significantly more often symptoms than patients who did not developed a contralateral stenosis (14.3% vs 6.6%, p=0.049). 17.4% of the patients with contralateral stenosis at baseline showed symptoms during follow-up.

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Development of contralateral stenosis after CEA

Table 2. Multiregression analysis of potential cofounders. Adjusted R-square

0.040

F-test significance

0.009

Patient characteristics

Significance, p-value

Female gender

0.311

Mean age

0.163

Current smoker

0.080

Diabetes mellitus

0.507

Hypertension

0.888

Mean BMI

0.675

History of CAD

0.014

History of PI

0.135

Acetylsalicylic acid

0.255

Statin

0.090

Mean GFR, CG

0.871

Year of operation

0.930

Clinical presentation Asymptomatic

0.001*

Stroke

0.455

Type of closure No patch, primary closure

0.508

Bovine

0.704

Dacron

0.383

Plaque characteristics Large lipid core

0.628

Moderate/ high collagen

0.650

Moderate/ high SMC

0.628

*P-value <0.01 shows significance. Abbreviations: CAD: coronary artery disease; PI: peripheral intervention; GFR: Glomerular Filtration Rate ; CG: Cockroft-Gauld; TIA: Transient ischemic attack; SMC: smooth muscle cells

During follow-up 25.3% of the patients with baseline contralateral stenosis and 6.1% of patients with new developed contralateral stenosis underwent contralateral CEA. Survival rates Life table analysis showed a contralateral stenosis-free survival rate of respectively 97% (n-471), 89% (n=408) and 79% (n=202) at 1, 2 and 3 years of follow-up. After 8 years of follow-up 34% of the patients did not had contralateral stenosis (n=12). Kaplan-Meier survival analysis showed the same survival rates (Figure 3). The greatest risk for development of contralateral stenosis was between 6 and 9 years of follow-up (Hazard

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Ratio 0.24) and median survival free time was 6 years. When patients were grouped in asymptomatic and symptomatic at inclusion, life table analysis demonstrated that asymptomatic patients display earlier and more often development of contralateral stenosis. The Hazard Ratio was 0.27 after 3 years in asymptomatic patients, and 0.26 after 6 years in symptomatic patients. Cumulative stroke-free survival for all included patients after 3 years follow-up was 93% and after 9 years 59%, with a median follow-up time of 8.2 years (95% CI: 7.8-8.5 years). Overall survival was 91% after both 3 and 9 years, with and median follow-up time of 8.4 (95% CI: 8.1-8.6 years).

Figure 3. Kaplan-Meier curve for contralateral stenosis-free survival rate

Discussion In this study we aimed to explore the relation of clinical risk factors and plaque characteristics with the new development of contralateral stenosis in the follow-up after CEA. We reported new development of contralateral stenosis after CEA in 1 of every 5 patients. This finding corresponds with findings in other studies.8,18,19 New development of contralateral stenosis appeared after a median follow-up time of 2.5 years and approximately half of the developed stenosis was an occlusion. Further analysis showed that patients with a history of CAD are more likely to develop contralateral stenosis in follow-up. Patients who were asymptomatic at inclusion showed a significant correlation with the development of contralateral stenosis and occlusion.

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Development of contralateral stenosis after CEA

The association of asymptomatic presentation and new development of stenosis has previously been described for ipsilateral restenosis. 14,17 Next, we showed that a small lipid core is significantly associated with new development of contralateral stenosis. Previous studies have reported a correlation between small lipid core and development of ipsilateral restenosis after CEA. 9,10 The results of our study could therefore underline that atherosclerosis is a generated disease of the vascular system, 20,21 by showing risk factors for development of stenosis of the carotid artery that are similar to risk factors for ipsilateral stenosis, as found in previous studies. However, preceding studies of our research group could not find an association of large lipid core in local plaques and the occurrence of systemic cardiovascular outcomes. In contrast, local plaque hemorrhage was shown to be the single independent predictor of outcome.13 In this study, we found that plaques with high collagen or high smooth muscle cells were associated with more development of contralateral stenosis. Especially smooth muscle cells are components of a stable plaque phenotype, characterized by less inflammation and lower lipid content.22 Earlier studies reported that removal of a stable atherosclerotic plaque gives more restenosis 1 year after follow-up.9,14 In our study, removal of a stable plaque gives more contralateral stenosis, which suggests again that vascular disease is more extensive than only in the operated artery. Furthermore previous studies showed that lesions in asymptomatic patients have a more stable atherosclerotic plaque composition with smaller lipid cores. Symptomatic patients, who have had a TIA or stroke, show more unstable carotid plaques, with high macrophage contents and larger lipid cores.10 In line with these studies, we found that asymptomatic patients had more often a stable plaque with a small lipid core and high smooth muscle cells and also developed more often contralateral stenosis (data not shown). Summarizing, dissection of a stable atherosclerotic plaque with small lipid core and high content of smooth muscle cells increases the risk of new contralateral stenosis, as previously described for the development of ipsilateral restenosis. In this study grade of stenosis was measured using Duplex ultrasound derived PSV values. It could therefore be possible that restenosis in the ipsilateral artery also leads to higher PSV values in the contralateral artery and is thus measured as contralateral stenosis. 23 However, in half of the cases of development contralateral stenosis an occlusion is reported, which is measured as no PSV flow. Therefore, our findings cannot completely be declared with this assumption. Finally, based on the results of this study it can be considered to include asymptomatic patients and patients with a stable atherosclerotic plaque in a more intensive and longterm follow-up scheme. It would be useful to construct a prediction model that predicts the risk for restenosis and contralateral stenosis for each individual patient, since we showed that risk factors for contralateral stenosis are comparable to risk factors for ipsilateral restenosis indicated in earlier studies. This would allow more tailored treatment and selective follow-up. Our study has several limitations; firstly, duplex ultrasound scanning is a technique with drawbacks. Measurements can vary between different types of equipment, laboratories

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and observers. Equipment and laboratory have not changed during this study, but different technicians have performed the measurements. This may result in intra-observer variability, however we expect that this influence is negligible. Secondly, we defined contralateral stenosis as >50% with a PSV >210 cm/s, but cut-off values for a stenosis grade differ across studies and vascular laboratories.14 This could lead to outcomes that would be assessed different somewhere else.

Conclusion One in five patients develops new contralateral stenosis after CEA. Asymptomatic patients prior to CEA had a higher risk for developing contralateral stenosis. An atherosclerotic plaque removed during CEA with a small lipid core, a high amount of smooth muscle cells and/or a high amount of collagen increases the risk of new development of contralateral stenosis after CEA. New contralateral stenosis was related to the presence of new cerebral symptoms. Our study results may help to develop individual treatment algorithms based on clinical presentation and plaque characteristics for patients undergoing CEA. Acknowledgements The authors would like to thank Evelyn Velema and Sander van de Weg for their (continuing) technical and administrative work for the Athero-Express study.

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Development of contralateral stenosis after CEA

References 1.

10.

11.

12.

Reina-Gutierrez T, Serrano-Hernando FJ, Sanchez-Hervas L, Ponce A, Vega de Ceniga M, Martin A. Recurrent carotid artery stenosis following endarterectomy: natural history and risk factors. European journal of vascular and endovascular surgery : Apr 2005 Frericks H, Kievit J, van Baalen JM, van Bockel JH. Carotid recurrent stenosis and risk of ipsilateral stroke: a systematic review of the literature. Stroke; Jan 1998 Randomised trial of endarterectomy for recently symptomatic carotid stenosis: final results of the MRC European Carotid Surgery Trial (ECST). Lancet. May 1998 Fluri F, Engelter ST, Wasner M, Stierli P, Merlo A, Lyrer PA. The probability of restenosis, contralateral disease progression, and late neurologic events following carotid endarterectomy: a long-term follow-up study. Cerebrovasc Dis. 2008 Martin-Conejero A, Reina-Gutierrez T, SerranoHernando FJ, et al. Disease progression in the contralateral carotid artery after endarterectomy. Annals of vascular surgery. Sep 2005 Raman KG, Layne S, Makaroun MS, et al. Disease progression in contralateral carotid artery is common after endarterectomy. Journal of vascular surgery. Jan 2004 Sam K, Small E, Poublanc J, et al. Reduced contralateral cerebrovascular reserve in patients with unilateral steno-occlusive disease. Cerebrovasc Dis. 2014 Iafrati MD, Salamipour H, Young C, Mackey WC, Oâ&#x20AC;&#x2122;Donnell TF, Jr. Who needs surveillance of the contralateral carotid artery? American journal of surgery. Aug 1996 Hellings WE, Moll FL, De Vries JP, et al. Atherosclerotic plaque composition and occurrence of restenosis after carotid endarterectomy. JAMA : Feb 2008 Verhoeven B, Hellings WE, Moll FL, et al. Carotid atherosclerotic plaques in patients with transient ischemic attacks and stroke have unstable characteristics compared with plaques in asymptomatic and amaurosis fugax patients. Journal of vascular surgery. Dec 2005 Ferguson GG, Eliasziw M, Barr HW, et al. The North American Symptomatic Carotid Endarterectomy Trial : surgical results in 1415 patients. Stroke; Sep 1999 Mayberg MR, Wilson SE, Yatsu F, et al. Carotid endarterectomy and prevention of cerebral

13.

14.

15.

16.

17.

18.

19.

20.

21. 22.

23.

ischemia in symptomatic carotid stenosis. Veterans Affairs Cooperative Studies Program 309 Trialist Group. JAMA . Dec 1991 Hellings WE, Peeters W, Moll FL, et al. Composition of carotid atherosclerotic plaque is associated with cardiovascular outcome: a prognostic study. Circulation. May 4 2010 Vrijenhoek JE, de Borst GJ, den Ruijter HM, et al. A lipid-poor plaque and asymptomatic status in women are associated with higher peak systolic velocity on duplex ultrasound after carotid endarterectomy. Atherosclerosis. Dec 2014 Rothwell PM, Eliasziw M, Gutnikov SA, et al. Analysis of pooled data from the randomised controlled trials of endarterectomy for symptomatic carotid stenosis. Lancet. Jan 11 2003 MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (7099%) or with mild (0-29%) carotid stenosis. European Carotid Surgery Trialistsâ&#x20AC;&#x2122; Collaborative Group. Lancet. May 1991 van Lammeren GW, Peeters W, de Vries JP, et al. Restenosis after carotid surgery: the importance of clinical presentation and preoperative timing. Stroke; Apr 2011 AbuRahma AF, Cook CC, Metz MJ, Wulu JT, Jr., Bartolucci A. Natural history of carotid artery stenosis contralateral to endarterectomy: results from two randomized prospective trials. Journal of vascular surgery. Dec 2003 Ballotta E, Da Giau G, Meneghetti G, Barbon B, Militello C, Baracchini C. Progression of atherosclerosis in asymptomatic carotid arteries after contralateral endarterectomy: a 10-year prospective study. Journal of vascular surgery. Mar 2007 Arora R, Rai F. Atherothrombosis and the management of the vulnerable vascular patient. American journal of therapeutics. Jan-Feb 2009 Drouet L. Atherothrombosis as a systemic disease. Cerebrovasc Dis. 2002 Davies MJ, Richardson PD, Woolf N, Katz DR, Mann J. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage, and smooth muscle cell content. British heart journal. May 1993 AbuRahma AF, Richmond BK, Robinson PA, Khan S, Pollack JA, Alberts S. Effect of contralateral severe stenosis or carotid occlusion on duplex criteria of ipsilateral stenoses: comparative study of various duplex parameters. Journal of vascular surgery. Dec 1995

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Supplemental Appendix 1. Baseline characteristics of the study population. Characteristics

Â Total Cohort

No. of patients

760

Female gender

230/760 (30.3)

Mean age

69 (9.6)

Current smoker

247/751 (32.9)

Diabetes mellitus

171/760 (22.5)

Hypertension

558/750 (74.4)

Mean BMI

26 (3.7)

Hypercholesterolemia

496/695 (71.4)

History of CAD

138/756 (18.3)

History of PI

239/757 (31.6)

Acetylsalicylic acid use

623/757 (82.3)

Antiplatelet use

656/757 (86.3)

Statin use

624/758 (82.3)

Mean total cholesterol

4.5 (1.3)

High density lipoprotein

1.2 (0.46)

Low density lipoprotein

2.6 (1.0)

Triglycerides

1.6 (1.0)

Mean GFR, CG

72 (25.6)

Clinical presentation Asymptomatic

95/755 (12.6)

Amaurosis fugax

118/755 (15.6)

TIA

360/755 (47.7)

Stroke

182/755 (24.1)

Type of closure No patch, primary closure

62/748 (8.3)

Venous

478/748 (63.3)

Bovine

150/758 (20.1)

Dacron

58/748 (7.8)

Plaque characteristics Moderate/ high macrophages

383/743 (51.1)

Large lipid core

194/ 743 (26.1)

Moderate/ high collagen

586/740 (79.1)

Plaque haemorrhage

413/741 (55.7)

High vessel density

329/677 (48.6)

Moderate/ high SMC

516/742 (69.5)

Continuous values are expressed as mean with standard deviation, unless specified otherwise. Categorical values are expressed as n/total (%). Abbreviations: CAD: coronary artery disease; PI: peripheral intervention; GFR: Glomerular Filtration Rate ; CG: Cockroft-Gauld; TIA: Transient ischemic attack; SMC: smooth muscle cells

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Development of contralateral stenosis after CEA

141

PART IV Platelet reactivity in cardiac disease

Chapter 9 The use of platelet reactivity testing in patients on antiplatelet therapy for prediction of bleeding events after cardiac surgery Vasc Pharmacology, 2016 Feb; Volume 77, p 19-27

T. Leunissen, P. Janssen, J. ten Berg, F. Moll, S. Korporaal, GJ. de Borst, G. Pasterkamp and R. Urbanus

Chapter 9

Abstract Many patients are treated with platelet inhibitors such as aspirin and clopidogrel for prevention of thrombotic cardiovascular events. However, the inhibitory effect of antiplatelet therapy is variable between patients; in some, the platelets are hardly inhibited, while in others, the platelets are excessively inhibited. The newer and more potent platelet inhibitors, prasugrel and ticagrelor, often lead to low platelet reactivity, which potentially leads to bleeding events. Preoperative measurement of platelet reactivity in patients receiving platelet inhibitors who undergo cardiac surgery, could be useful to identify those with low platelet reactivity and thus have an increased risk of bleeding during or after surgery. In this review, we discuss the most commonly used platelet inhibitors and platelet function tests. Furthermore, we will provide an overview of the evidence for the prediction of post-operative bleeding at the operation site with preoperative platelet reactivity testing in patients undergoing cardiac surgery.

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Platelet reactivity as prediction of bleeding

Introduction Platelets are anucleated blood cells that play a key role in the maintenance of vascular integrity. They continuously monitor the vascular wall for breaches and are able to respond rapidly when defects are encountered. Upon exposure to the unique features of an injured vessel wall, they adhere to the site of injury despite the shear forces of the circulating blood, and immediately aggregate with other platelets. This results in the formation of a primary haemostatic plug that prevents blood loss and remains in place until it is reinforced with a strong fibrin network.1 Failure to form an adequate platelet plug due to low platelet reactivity (LPR) or low platelet count will lead to bleeding complications, while high platelet reactivity (HPR) is associated with an increased risk of thrombosis, mainly in the arteries. Although arterial thrombosis is a multifactorial disease with diverse genetic and acquired predisposing risk factors, the importance of platelets in this process is widely recognized. As a result, many people are treated with antiplatelet therapies for secondary prevention of thrombotic cardiovascular events (CVE), such as myocardial infarction, cerebrovascular accident and transient ischemic attack. Since the 1970s, the effect of aspirin as a platelet inhibitor has been studied extensively.2 Aspirin has been very efficient in the prevention of secondary CVE; a meta- analysis of randomized studies of various antiplatelet drugs showed a 25% decrease in thrombotic vascular outcomes in patients with pre-existing conditions on aspirin.3 With the addition of alternative and more potent P2Y12 inhibitors such as clopidogrel, prasugrel and ticagrelor, the prevention of secondary CVEs has become even more effective.4,5 The current recommended antiplatelet treatment for patients who undergo invasive cardiac surgery, such as coronary artery bypass graft (CABG), is aspirin monotherapy, with a recommended dose of 100-325 mg daily. Therapy should be initiated preoperatively, or at least within 6 hours post procedure and should be continued indefinitely to reduce the occurrence of vein graft closure and adverse cardiovascular events.6,7 However, a recently published review showed that HPR despite aspirin treatment (HAPR) occurs in some patients.8 The prevalence of aspirin resistance after CABG varies greatly though, with incidence rates ranging from 10% up to >90% in different studies, and seems to depend on both the assay used and the timing of measurements. 9 Possible causes of HAPR could be inadequate dosage, drug interactions, genetic polymorphisms of COX-1 and other genes involved in thromboxane biosynthesis, upregulation of nonplatelet sources of thromboxane biosynthesis and increased platelet turnover.10,11 To circumvent HAPR, alternative antiplatelet therapy as dipyridamole, warfarin or clopidogrel have been explored in multiple trials.12-15 So far, no other treatment regimen has been proven to be superior to aspirin, but multiple studies, including trials with the newer agents prasugrel and ticagrelor, are still underway (POPular CABG [NCT02352402] and Prasugrel for Prevention of Early Saphenous Vein Graft Thrombosis [NCT01560780]). However, the newer and more potent P2Y12 inhibitors have a potential downside: excessive inhibition of the platelets may cause LPR, potentially leading to bleeding events.

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Preoperative usage of clopidogrel on top of aspirin has already been associated with an increased risk of bleeding and mortality.16,17 In general, postoperative bleeding is a common complication after invasive cardiac surgery. The need for administration of blood products after cardiac surgery due to major blood loss is associated with an 8.1fold (95% CI, 3.9-17.0) increased risk of in-hospital mortality.18 Moreover, blood transfusion is associated with an increased risk for postoperative adverse events such as mortality, renal failure, extended ventilatory support, major infection, cardiac complications and neurologic events. 19 Besides extensive inhibition of the platelets by platelet inhibitors, multiple other mechanisms may contribute to LPR during cardiac surgery, including dysfunction of alpha-granule release, decreased glycoprotein Ib expression on the platelet membrane, prolonged circulation of activated, “exhausted” platelets, and impaired platelet aggregation.20,21 The association of HPR with increased risk of thrombotic events 22,23 and the relation of LPR with bleeding risk 24,25, suggest that there is a therapeutic window for platelet reactivity. If this is the case, assessment of PR in patients receiving antiplatelet therapy could identify those who are ‘overtreated’ and have an increased risk of bleeding during or after surgery. The timing of the operation can then be optimized in this group of patients and the risk of bleeding and transfusion requirement can be reduced. In this issue of vascular pharmacology, Polzin and colleagues have analysed the relationship between platelet reactivity and thrombotic or bleeding events in patients undergoing cardiac surgery. This review provides an overview of the evidence of preoperative platelet reactivity testing as predictor of post-operative bleeding complications in patients receiving aspirin or P2Y12 during invasive cardiac surgery. Since most of the available data are derived from studies involving CABG, we have used CABG as a model procedure. Antiplatelet therapy regimens Many antiplatelet regimens have been investigated, interfering with one or more stages in platelet activation. A brief overview of the antiplatelet regimens available and the activation pathways with which they interfere is provided below (figure 1). Aspirin, or acetylsalicylic acid (ASA) is an irreversible cyclooxygenase (COX)-1 inhibitor. When platelets are activated, arachidonic acid (AA) is liberated from the internal plasma membrane of the platelet by phospholipase A2 and converted into prostaglandin H2 by the enzyme COX-1. Prostaglandin H2 is subsequently converted into thromboxane A2, which will diffuse from the platelet interior and bind the thromboxane prostanoid receptor (TP). Signalling downstream from this G-protein coupled receptor leads to a rise in the intracellular Ca2+ concentration, as well as cytoskeletal rearrangement, causing shape change and enhanced platelet activation. Because platelets are anucleate, de novo synthesis of COX-1 by platelets is absent. As a result the effect of aspirin lasts for the remainder of the platelet lifetime (8-10 days). Although some studies have shown that high-dose aspirin also inhibits thrombin generation26 and erythrocyte-mediated platelet activation,27 aspirin is regarded as a weak platelet inhibitor. Because aspirin only targets the thromboxane-dependent platelet activation pathways, strong platelet agonists (e.g.

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Platelet reactivity as prediction of bleeding

Figure 1. Schematic representation of the major targets of antiplatelet therapy and the assays to measure their effects. When platelets are activated, an intracellular signaling cascade is initiated. This will lead to activation of the fibrinogen receptor, the integrin ÎąIIbÎ˛3. Aside from activation of the fibrinogen receptor, stimulation of platelets will lead to the initiation of two major auxiliary activation processes. First, platelets will secrete the content of their intracellular granules. Platelet dense granules contain ADP, which stimulate the purinergic receptors P2Y1 and P2Y12. Out of these two receptors, P2Y12 contributes most to the propagation of the prothrombotic response. Several antiplatelet agents target this receptor, rendering platelets less susceptible to activation. Aside from granule release, platelet activation will lead to activation of phospholipase A2, which will liberate the fatty acid arachidonic acid (AA). AA will subsequently be converted by cyclooxygenase (COX)-1 into prostaglandin H2, which will then be converted into thromboxane A2 (TxA2). TxA2 will pass through the platelet membrane and bind to the prostanoid receptor (TP). This will further augment platelet activation. The conversion of AA to prostaglandin H2 by COX-1 is inhibited by acetylsalicylic acid. By far the most platelet reactivity tests measure the final stages of platelet activation, i.e. platelet aggregation, platelet adhesion or clot retraction. The vasodilator stimulated phosphoprotein (VASP) assay is the exception, as it measures changes in signaling molecules within the platelet. LTA light transmission aggregometry, MEA multiple electrode aggregometry, PFA-100 platelet function analyzer 100, TEG thromboelastography,

thrombin or collagen) are able to bypass the blockade in thromboxane-production and still activate platelets after treatment with aspirin. During the process of platelet activation, platelets secrete the content of their dense granules, which includes adenosine diphosphate (ADP). Secreted ADP will subsequently bind to the purinergic receptors on the platelet, including P2Y12. Signalling downstream from P2Y12 leads to a decrease in intracellular cyclic adenosine monophosphate (cAMP), which inhibits the release of Ca2+ from intracellular stores. Binding of ADP to P2Y12 greatly enhances thrombus growth and stability by potentiating the effects of other agonists on dense granule secretion 28 and fibrinogen-receptor activation.29,30 This makes the ADP

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receptor P2Y12 a popular target for platelet inhibition. Several different P2Y12 inhibitors are available on the market, including thienopyridines (ticlopidine, clopidogrel, and prasugrel) and nucleoside analogues (ticagrelor and cangrelor). Thienopyridines, such as clopidogrel, inhibit platelet activation by blocking the ADPbinding site of the P2Y12 receptor. Metabolic activation into the active metabolite is required, since thienopyridines are prodrugs. Intestinal absorption of clopidogrel is limited by P-glycoprotein, also known as multidrug resistance protein 1. Over 85% of the absorbed prodrug (clopidogrel) is metabolized by omnipresent esterases into inactive metabolites. The other 15% is metabolized by the liver by the cytochrome P-450 (CYP) enzymatic pathway into an active metabolite. Clopidogrel activation requires a two-step oxidative process conversion into, first the inactive metabolite, 2-oxo-clopidogrel, secondly the active thiol metabolite. Both steps involve hepatic CYP isoenzymes such as CYP2C19, CYP3A4/5, CYP2C9, CYPP1A2 and CYP2B6.31 Inter-individual variability in the antiplatelet response to thienopyridines is observed, partially caused by genetic mutations in the CYP isoenzymes.32 Prasugrel is a newer thienopyridine, with a faster and stronger antiplatelet effect compared with clopidogrel due to its rapid metabolic activation. Like clopidogrel, prasugrel is a prodrug that first undergoes intestinal metabolisation into its intermediate thiolactone, which is then converted in a single-step reaction to its active metabolite by CYP3A, CYP2B6, CYP2C9 and CYP2C19 in the liver.33 Ticagrelor is a nucleoside analogue that, unlike thienopyridines, does not bind to the ADP-binding site but to a separate site of the P2Y12 receptor.34 Ticagrelor is not a prodrug and platelet inhibition is mediated by both the parent drug and its metabolite. Compared with thienopyridines, ticagrelor shows little inter-individual variability in the antiplatelet response. In addition to its effects on P2Y12, ticagrelor blocks the equilibrative nucleoside transporter, which results in increased plasma concentrations of adenosine.35 Adenosine mediates coronary vasodilation, reduction of ischemia and reperfusion injury, inhibition of inflammatory responses to stress and negative dromotropic and chronotropic effects on the heart. These effects may also contribute to the reduced mortality observed in trials amongst patients treated with ticagrelor. 36 A final step of platelet activation is the activation of the platelet fibrinogen receptor, integrin αIIbβ3. The αIIbβ3 is receptor is found exclusively on the surface of platelets.37 Upon activation of the platelet, the receptor undergoes conformational changes and binding sites for fibrinogen are exposed. Binding of fibrinogen subsequently leads to platelet aggregation. The αIIbβ3 receptor can be blocked with several αIIbβ3 inhibitors, such as abciximab, eptifibatide and tirofiban. Since fibrinogen binding to αIIbβ3 is the final step of platelet activation, inhibition of the receptor by the αIIbβ3 inhibitors is independent of the type of agonist. A disadvantage of αIIbβ3 inhibitors is that they require parenteral administration, which strongly limit their use in daily practice. Concluding, many different antiplatelet regimens are available, with all their specific site of action in the platelet activation. The different regimens differ in the extend of inhibition and the inter-individual variability.

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Platelet reactivity testing The extent of platelet inhibition in patients receiving antiplatelet therapy can be assessed by measuring the remaining platelet reactivity. There are multiple laboratory and pointof-care tests that measure diverse platelet responses and the reproducibility and applicability vary strongly between the tests. So far, no single test has been proven superior in the prediction of postoperative bleeding.38 Most of the available platelet reactivity tests are based on the platelet capacity to aggregate when exposed to a stimulus. The archetypical assay to measure platelet aggregation is Light Transmission Aggregometry (LTA), or Born aggregometry. The test has remained virtually unaltered since its introduction in the 1960s 39 and is traditionally considered the gold standard in platelet function testing. The test uses optical detection of the formation of aggregates in platelet-rich-plasma stimulated with manually added agonists. As platelet aggregation is influenced by many analytical and operator-dependent variables, the test is difficult to standardize. Several attempts have been made to improve standardization and reproducibility, with the most recent guideline published in 2013.40 A more modern approach to platelet aggregation is Multiple Electrode Aggregometry (MEA), which uses the changes in electrical impedance on electrodes rather than light transmission to measure platelet aggregation. This allows the assessment of platelet aggregation in whole blood, making the procedure faster and easier to perform. MEA devices such as the MultiplateÂŽ Analyser detect the increase of electrical impedance resulting from the adhesion and aggregation of platelets on two metal sensor wires. Platelets adhere to the sensor wires when activated and the electrical resistance between the wires increases. MEA can be used for monitoring of both aspirin and P2Y12 inhibitors; it has not only been used to identify cardiovascular patients not responding to antiplatelet therapy and at high risk of cardiovascular events, but also to distinguish patients with high platelet inhibition at risk of bleeding.41,42 The VerifyNow is a point-of-care test based on the agglutination of fibrinogen-coated beads in the presence of activated platelets. The rate of agglutination is directly proportional to the presence of the activated fibrinogen receptor and can therefore be used to estimate platelet reactivity.43 Multiple standardized cartridges with different agonists can be used to monitor platelet inhibition in response to aspirin, P2Y12 inhibitors, and ÎąIIbÎ˛3 inhibitors. The results are congruent with LTA38 but the assessment of sensitivity, specificity, and the definition of a cut-off value for patients at risk of cardiovascular event or bleeding differ greatly between studies.44-46 An even simpler approach to platelet aggregation is used in the Plateletworks assay. The principle of this test is based on the decrease in platelet count measured by cell counters when platelets aggregate in whole blood. By comparing an EDTA blood sample, in which platelets are resting, with a citrated blood sample in which platelets are stimulated with an agonist, the percentage of platelet aggregation after stimulation with a relevant agonist can be easily determined. 47 An alternative method for analysis of platelet function is used in the Platelet Function Analyser (PFA)-100. Here, whole blood is aspirated at high shear stress through a capillary

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tube towards an aperture in a collagen-coated membrane, which contains either ADP or epinephrine. The time it takes to occlude this opening is defined as closure time, a measure of overall platelet-related haemostasis.48 Although it is sensitive to aspirin treatment, 49 the PFA-100 appears to be insensitive towards P2Y12 inhibitors.50 The PFA100 is therefore considered to be mainly suitable as a screening tool for platelet disorders, integrated in a panel of other platelet function tests.51 Another method to analyse platelet function that does not depend on platelet aggregation is ThromboElastoGraphy (TEG). This whole blood clotting assay is based on plateletmediated clot contraction. Coagulation is initiated in whole blood in an oscillating cup. A stationary pin attached to an electrode measures changes in torque that occur when the pin is linked to the wall of the cup by fibrin strands. When activated platelets cause clot contraction, torque increases accordingly. The TEG platelet mapping (TEG PM) test for assessment of platelet inhibition is based on a comparison between the TEG results in fully activated blood in which thrombin causes full platelet activation and TEG results obtained in blood that is activated with a combination of a snake venom that specifically converts fibrinogen to fibrin and a weak platelet agonist such as ADP or arachidonic acid. Under these conditions, TEG results are sensitive to platelet inhibitors.52 Aside from looking at the functional consequences of platelet inhibition â&#x20AC;&#x201C; i.e. adhesion, aggregation, or clot contraction, there are tests that look at changes in platelet phenotype. One such example is the Vasodilator-Stimulated Phosphoprotein (VASP) assay. VASP is an intracellular platelet protein that is not phosphorylated when the P2Y12 receptors are active. When cAMP or cGMP levels are high, VASP is phosphorylated and dampens the platelet response to agonists.53 Persistent VASP phosphorylation, as measured with flow cytometry, correlates with P2Y12 receptor inhibition, reflecting the effect of the medical treatment. As aspirin induced platelet inhibition is not associated with changes in cGMP or cAMP levels, the VASP assay is only applicable for monitoring of P2Y12 inhibitors. Although the test has a standardized diagnostic assay kit, it is time consuming and requires experienced laboratory personnel. Different methodologies for the assessment of platelet reactivity to monitor the effectivity of antiplatelet therapy are approved for clinical use. No superiority has been proven of one test, although VerifyNow, PFA-100 and VASP are commonly used. Clinical trials regarding prediction of bleeding in patients undergoing CABG Several of the above mentioned tests were used to assess platelet reactivity in clinical trials amongst patients undergoing CABG. Here, we provide an overview of the available data. We wish to emphasize that this overview is intended as a summary of the available data and our assessment should not be considered as a systematic review or metaanalysis of all available literature on the utility of platelet reactivity testing regarding the prediction of postoperative bleeding. The sixteen relevant articles regarding bleeding prediction are summarized in table 1. Two studies evaluated the association between LTA results and bleeding.54,55 Both showed a good predictive value of LTA: one study demonstrated that LTA identified 12/45 patients

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on clopidogrel with a platelet response <40%, of whom 11 patients (92%) required platelet and red blood cell transfusion.54 In the other study, LTA with ADP as platelet agonist predicted postoperative bleeding and re-thoracotomy in patients on dual antiplatelet therapy when a cut-off value <50% platelet response was used.55 MEA was evaluated in three studies. 56-58 All reported a correlation between MEA and bleeding outcome. This depended strongly on the cut-off values used, which varied considerably between studies. In a study among 196 patients, preoperative MEA results were correlated to bleeding risk.56 Similar results were obtained in a retrospective analysis amongst 87 patients receiving thienopyridine treatment, in which MEA results were associated with postoperative bleeding and transfusions.57 The same group confirmed these results in a retrospective study amongst 361 patients.58 The ability of the VerifyNow P2Y12 assay to predict bleeding risk has been evaluated in five studies amongst patients receiving either dual antiplatelet therapy (aspirin and clopidogrel) or clopidogrel monotherapy. 59-63 The cut-off values used in these studies varied considerably and results were more ambiguous. One study demonstrated good correlation between the VerifyNow P2Y12 assay and both the VASP-assay and flow cytometric analysis of platelet reactivity and found that it was the only platelet reactivity test that correlated with clinical outcome (total blood loss and red blood cell transfusion).59 Another study showed that the VerifyNow could be used to discriminate between patients with and without major bleeding during surgery when a cut-off value of preoperative P-Reactive Units (PRU) â&#x2030;¤207 was used.60 A third study among 149 patients on dual antiplatelet therapy showed an increased risk of bleeding complications (defined as high chest tube output and the need for coagulation factor transfusion) in patients with PRU â&#x2030;¤290.61 In contrast, another study found no association between VerifyNow results and bleeding complications, although patients who discontinued clopidogrel â&#x2030;¤ 3 days prior to CABG received more platelet transfusion and had more chest tube output as compared with patients who had a longer washout period.62 One study amongst 238 patients reported that platelet reactivity below 200 PRU was associated with bleeding in patients with dual antiplatelet therapy only, and not in patients on ASA or clopidogrel monotherapy.63 The predictive value of Plateletworks for bleeding complications in patients receiving antiplatelet therapy was evaluated in three studies 64-66 and remains inconclusive. One study found that Plateletworks results correlated with postoperative chest drainage 5 hours and 12 hours after intervention in patients on dual antiplatelet therapy and that patients with Plateletworks results in the lowest tertile required more postoperative transfusion and produced a larger chest drain volume compared with other patients.64 However, these findings could not be confirmed in a comparable study by the same group with slightly more patients.65 In a study amongst 50 patients on monotherapy ASA, Plateletworks was not able to predict postoperative mediastinal blood loss and could not differentiate between patients who had ingested aspirin within the last 48 hours and those who had ingested aspirin more than 72 hours prior to blood sampling.66

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238

A: 45 B: 45

A: 33 B: 17

Study

Alström et al.59

Altheeb et al. 63

Chen et al.54

Dalen et al. 2012. 64

Dalen et al. 201465

ASA + Clopidogrel clopidogrel median withdrawal 48h prior surgery and ASA continued

A: ASA + clopidogrel clopidogrel mean withdrawal 98h prior surgery B: ASA ASA continued

A. ASA + Clopidogrel exposure <6 days- and stopped 2 days prior surgery B. ASA, no clopidogrel exposure ASA continued

Aspirin, clopidogrel or DAPT APT stopped 5 days prior surgery

ASA + clopidogrel: clopidogrel stopped ≤ 3 days prior surgery, ASA continued

APT regimen

Plateletworks: ADP

Plateletworks: ADP + collagen

LTA: ADP PFA-100

VerifyNow

Flow cytometry VASP VerifyNow P2Y12 assay Platelet- mapping

Platelet reactivity test

Postoperative blood loss Transfusion requirements

Postoperative chest drainage volume

RBCT, Transfusion of platelets

Major or minor bleeding

Total blood loss Total RBCT

Outcomes

No significant association ADP test and blood loss 12 h postoperative (estimate -7.51; 95%CI: -16.9-1.9, p=0.12) Significant association ADP test and platelet concentrates administered within 24h after surgery (IRR 0.95, 95%CI: 0.92-0.9, p<0.01)

Plateletworks ADP correlated to postoperative chest volume at 5 hours ρ =-0.83 and p<0.01, 12 hours ρ =- 0.55 and p<0.01 and in total ρ =-0.71 and p<0.01 Plateletworks collagen correlated weak to postoperative chest volume 12 hours ρ =- 0.33 and p=0.02

Preoperative platelet dysfunction measured by LTA-ADP (<40%) predicted all, but 1 case of severe coagulopathy requiring multiple transfusions Preoperative platelet dysfunction measured by PFA-100 (>170 s) did not predict coagulopathy requiring multiple transfusions

The range of 180-200 PRU suggested the likelihood of bleeding, p=0.004 for patients on DAPT, not for monotherapy ASA or clopidogrel

Correlation of VerifyNow P2Y12 assay with total blood loss ρ=0.29 (p=0.03) and RBCT = ρ=0.43 (p<0.01). Other PRTs showed no correlation with outcome

Summary of findings

Table 1. Summary of clinical trials that investigated the prediction of bleeding risk after CABG in patients receiving antiplatelet therapy.

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100

A: 16 B: 34

A: 103 B: 80 C: 13

A: 52 B: 50

A: 34 B: 25

Kwak et al. 68

Lennon et al. 66

Petricevic et al. 56

Plicner et al. 55

Preisman et al. 67

Ranucci et al. 2011 57

Ticlopidine or clopidogrel stopped 3.6 days (mean) prior surgery

A: ASA and clopidogrel B: ASA ASA and clopidogrel stopped 1 day (median) prior surgery

1. ASA + clopidogrel 2. ASA ASA continued until day of surgery clopidogrel stopped <5 days prior surgery

A: ASA B: ASA + clopidogrel C: Clopidogrel ASA continued, clopidogrel stopped (2-8 days prior surgery)

A: ASA <2 days before intervention B: no ASA >3 days before intervention

ASA+ Clopidogrel Clopidogrel stopped <5 days prior surgery and ASA continued

Volume of chest- and mediastinal drainage Transfusion requirements

Postoperative mediastinal blood loss

CTO RBCT

Peri- and postoperative bleeding

Re-exploration procedures CTO <24 h Consumption of blood products

Postoperative bleeding and platelet transfusion

TEG

Plateletworks LTA

Multiple electrode aggregometry: ASA (ASPI) and clopidogrel (ADP)

LTA: ADP

TEG ADP + AA

Multiple electrode aggregometry: ADP + TRAP

ADP test, cut off value 31 U, predicts postoperative bleeding: ROC AUC 0.71, p=0.013, sens. 72% and spec. 66%

Only ADP predicted bleeding tendency (ROC AUC: 0.81, p=0.004), with threshold defined at <42.5 mm (sens. 78% and spec. 84%) Multiplate and LTA detect bypass induced platelet dysfunction but not blood loss

LTA correlated with postoperative drainage, 6h and 12h postoperative, both ρ =-0.44 and p<0.05 Low platelet aggregation was only predictor of postoperative draining after 12 hours (R2=0.142, p,0.001) Low platelet aggregation was only predictor of rethoracotomy (OR=2.94, [1.12-7.75], p=0.029)

Significant difference in ‘’bleeders’’ among groups (p=0.039) ASPI and ADP test were correlated with 24h CTO (r=-0.170, p=0.014 and r=-0.206, p=0.003) Patients with PRBC administration has significant lower ASPI test results

Poor correlation plateletworks and outcome: ρ=0.07, p=0.58 Weak correlation LTA and outcome: ρ=-0.31, p=0.03

ROC of the percentage platelet inhibitory response to clopidogrel for postoperative transfusion requirement: AUC 0.771, 95%CI 0.647 to 0.868, P<0.001 Optimal cut off value: 70% platelet inhibitory response with sens. 77.8% and spec.75.0%

Platelet reactivity as prediction of bleeding

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149

A: 48 B: 11 C: 11

A: 36 B: 26

Reed et al. 2015 60

Rosengart et al. 61

Velik-Salchner et al.86

Yu et al.

A. Clopidogrel stopped ≤3 prior surgery B. Clopidogrel stopped >3 days prior surgery

A: No APT >7 days prior surgery B: ASA C: ASA + clopidogrel

ASA + clopidogrel no information of (dis) continuation of APT

ASA + clopidogrel MD or LD ≥3 days prior intervention ASA continued + clopidogrel stopped at operators discretion

A: ASA + ticlopidine B: ASA + clopidogrel C: ASA + prasugrel ASA continued, thienopyridines stopped 4 days (mean) prior surgery

VerifyNow P2Y12 assay

Multiple electrode aggregometry + LTA: collagen, ADP and AA

VerifyNow P2Y12 assay

Multiple electrode aggregometry: ADP + TRAP test

1. Chest tube output (CTO) 2. Platelet transfusion

Transfusion requirements

CTO Intra- and postoperative transfusions

Decrease in Hb, Hbg, HCT CTO Major and minor bleeding <24 hours after surgery

Postoperative bleeding Severe postoperative bleeding

Patients on clopidogrel ≤3 days prior surgery received more platelet transfusions and more CTO as compared with patients on clopidogrel who discontinued longer (p=0.01 and p=0.03) No difference in CTO or platelet transfusion between patients with PRU ≥ or < 250 LPR according VN, is not able to predict CTO or platelet transfusion

LTA Col 15 min and 3 h after protamine, discriminates between APT patients and controls Multiplate ADP 3 h after protamine, discriminates between patients on clopidogrel and controls Multiplate and LTA detect bypass induced platelet dysfunction but not blood loss

Lower-PRU patients were more likely to need coagulation factors (OR 2.82, p=0.0004), have high CTO or coagulation factor transfusion (OR 2.35, p=0.02)

Higher PRU was conversely correlated with every endpoint Preoperative PRU ≤207 predicts major bleeding during surgery (ROC AUC 0.76, 95%CI: 0.59-0.94, p=0.018)

Threshold of <22 U for ADP test: NPV 94%, PPV 20% Threshold of <75 U for TRAP test: NPV 95%, PPV 23% High risk subgroup (ADP <20U+TRAP <75U) was not associated with severe bleeding (NPV 100%,PPV 37%)

AA Arachidonic acid, APT antiplatelet therapy, ADP adenosine diphosphate, ASA acetylsalicylic acid, IRR Incidence rate ratio, PRU p-reactive units, CTO chest tube output, Hb haemoglobin, HCT Hematocrit, LTA Light transmittance aggregometry, PFA Platelet function analyser, PRBC Packed red blood cell concentrate, PRT Platelet reactivity test, RBCT Red blood cell transfusion, Sens. Sensitivity, Spec.Specificity, TEG Tromboelastography, TRAP Thrombin receptor-activating peptide, VN VerifyNow

A: 53 B: 298 C: 10

Ranucci et al. 2014 85

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Platelet reactivity as prediction of bleeding

TEG, evaluated in three studies 59,67,68, showed varying ability to predict blood loss and transfusion requirements. One study with 60 patients investigated preoperative platelet response in patients on dual antiplatelet therapy with the VASP assay, VerifyNow, and Plateletmapping (TEG). No agreement between TEG and the other platelet reactivity tests was found and TEG results did not correlate with bleeding.59 However, another study among 59 patients indicated that TEG was able to predict bleeding tendency (chest tube output and transfusion requirements) with good sensitivity and specificity.67 Similar results were obtained by Kwak et al, 68 who found that TEG could predict transfusion requirement and increased blood loss, regardless of the timing of clopidogrel discontinuation (1 or 3 days prior intervention). Future clinical trials regarding prediction of bleeding events should standardize the time between ingestion of antiplatelet therapy and platelet reactivity testing. 69 In most trials patients are treated with monotherapy aspirin or dual antiplatelet therapy (including aspirin). The number of platelets exposed to aspirin differs within the 24-hours dosing interval,70 which explains the time-dependent efficacy of aspirin.71 The variable use of anti-fibrinolytics and heparins during surgery should be taken into account as well, as these agents strongly influence the clinical outcomes. 72,73 Lastly, integrating genotyping, measurement of platelet biomarkers, fibrinogen levels, von WIllebrand factor or coagulations tests with platelet reactivity testing might improve the prediction of bleeding events. 74 In conclusion, the studies outlined above indicate that platelet reactivity tests can be used to predict peri- and postoperative bleeding in patients undergoing CABG. However, due to the variation in diagnostic tests used in these studies, differences in study design and differences in data analysis, the comparability of these studies is limited. No superiority of one platelet reactivity test over the other for risk stratification of bleeding can be established. The Society of Thoracic Surgeons has recommended platelet reactivity testing as a tool to manage perioperative antiplatelet therapy in their guidelines. 7 In patients on dual antiplatelet therapy in need of urgent operations, bleeding risk estimates based on platelet reactivity testing should be used to decide upon surgical delay rather than the arbitrary use of a specific period of time (class IIa recommendation). Next, preoperative platelet reactivity testing to assess bleeding risk may be useful in identifying patients with high residual platelet reactivity after usual dose of antiplatelet therapy and can therefore undergo operation without elevated bleeding risk (class IIb recommendation). Similarly, the use of perioperative platelet reactivity tests is recommended to limit blood transfusion (class IIb recommendation). After CABG, platelet reactivity testing may be considered to optimize the antiplatelet drug effect and minimize the thrombotic risk to vein grafts. Personalized antiplatelet therapy There is growing evidence that platelet reactivity tests can indeed predict perioperative blood loss in patients taking preoperative platelet inhibitors.75 The association of high PR with increased risk of thrombotic events and low PR with increased risk of bleeding

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events sparked the concept of tailoring antiplatelet therapy based on platelet reactivity test outcome. The basis of the concept is that adjusting antiplatelet therapy with the aim to achieve lower or higher PR, within a ‘’therapeutic window’’, will result in less thrombotic and/or bleeding events. 76 If platelet reactivity testing reveals that PR is below the lower boundary cut-off value, it seems reasonable to discontinue antiplatelet therapy and postpone surgery until platelet reactivity is restored. If platelet reactivity testing reveals that PR is above the upper bound cut off value, it is advised to (1) increase the antiplatelet therapy or administer different antiplatelet therapy and (2) provide platelet reactivity testing in the longitudinal follow up. The latter prevents that the risk of ischemic events switches towards an increased risk of bleeding and vice versa. Implementation of such a clinical decision rule would require established cut-off values for platelet reactivity. Thus far, however, the cut-off values for a therapeutic window for patients undergoing CABG have not been determined. Although there are various studies describing the variability in platelet inhibitory response to antiplatelet therapy, pooling of these results is not possible since different platelet function tests are used within different study settings. The effect of tailored antiplatelet therapy in patients undergoing CABG on perioperative bleeding events and timing of surgery has so far been evaluated in two studies. 77,78 Platelet reactivity was assessed in patients on background aspirin with (n=109) and without (n=95) clopidogrel prior to the surgery. CABG was performed 1 day, 3-5 days or >5 days after platelet reactivity testing, based on the TEG outcomes. The mean chest tube output in clopidogrel-patients was 93% of the amount, observed in clopidogrel- naïve patients and the total amount of red blood cell transfusions did not differ between the two groups. The mean waiting time was 2.7 days per patient, nearly a twofold reduction of the guideline recommended preoperative waiting period for clopidogrel- treated patients. Although utilized TEG cut-off value has not been validated yet, the results of this study seem promising regarding tailored antiplatelet therapy in patients undergoing CABG. The second study evaluated the impact of dual antiplatelet therapy among aspirinresistant patients who underwent CABG. 78 Four days after CABG, multiplate aggregometry diagnosed 219 aspirin-resistant patients (51% of the initially eligible patients), who were assigned to receive either dual antiplatelet therapy (aspirin and clopidogrel) or aspirinmonotherapy (300 mg). No differences were found between the primary clinical endpoints (all cause death, cardiovascular death, stroke, nonfatal MI and bleeding events) after six months. However in subgroup analysis, dual antiplatelet therapy led to lower rates of adverse events in patients with a body mass index >30 kg/m2 and those <65 years. The authors suggest the conduction of a larger scale study of a similar design, since this study was underpowered. In this respect, it is important to note that tailoring antiplatelet therapy based on platelet reactivity testing showed disappointing results thus far in patients undergoing percutaneous coronary intervention (PCI). Two large trials, GRAVITAS, (n=2,214) 79 and ARCTIC (n=2,240) 80 showed no difference in their primary composite endpoint (death of cardiovascular causes, nonfatal acute myocardial infarction and ST-elevation) or

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Platelet reactivity as prediction of bleeding

bleeding complications after 6 months in GRAVITAS and 1 year in ARCTIC, between patients receiving standard antiplatelet therapy and patients with tailored antiplatelet therapy. However few smaller studies showed a beneficial effect of tailored vs. standard antiplatelet therapy on a composite endpoint (e.g. cardiac death, stent thrombosis, recurrent ACS, re PCI< 1 year), but the effect on bleeding events remains doubtful.81-84 Future studies on personalized antiplatelet therapy in patients undergoing cardiac surgery Although platelet reactivity tests can be used to predict per- and postoperative bleeding in patients undergoing cardiac surgery, it has not yet been demonstrated that preoperative adjustment of antiplatelet therapy based on platelet reactivity measurements leads to a reduction in perioperative bleeding events. However, before this concept can be thoroughly studied, other important questions need to be answered: 1. What is the incidence of low on aspirin platelet reactivity (LAPR) and low on clopidogrel platelet reactivity (LCPR) in patients undergoing cardiac surgery? 2. What are the optimal cut-off values to predict perioperative bleeding events for each platelet reactivity test? 3. Do LAPR or LCPR change over time and can we accurately predict the occurrence of bleeding events with a single platelet reactivity test measurement? 4. When is the appropriate timing for platelet reactivity testing? 5. What is the optimal treatment strategy for patients undergoing cardiac surgery with LAPR or LCPR? 6. Should surgery be postponed in patients with low platelet reactivity, and if so, for what period of time? In patients undergoing PCI, the potential benefit of tailored antiplatelet therapy is currently studied in large trials (NCT01959451, NCT01538446) using validated cut-off values, focusing on high-risk cohorts of patients and implementing the therapeutic window of platelet inhibition to assess the potential clinical benefit on thrombotic and bleeding events. Tailored antiplatelet therapy based on genotyping is being studied in the TAILORED-PCI (NCT01742117). After PCI the conventional therapy arm will receive 75 mg and the prospective CYP2C19 genotype-based therapy arm will receive either 90 mg ticagrelor (CYP2C19 *2 or *3 reduced function allele patients) or clopidogrel 75 mg in non-*2 or -*3 CYP2C19 patients. We believe that the concept of personalized antiplatelet therapy should also be investigated in future clinical trials as a means to reduce post-operative bleeding after cardiac surgery. Preventing perioperative bleeding and administration of blood products might decrease the risk of in-hospital mortality, renal failure, infection and others.18,19

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Conclusion Platelet reactivity testing can be used to predict per- and postoperative bleeding in patients undergoing cardiac surgery. However, future studies towards the potential benefit of personalized antiplatelet therapy, based on platelet reactivity testing, are warranted in this specific patient population.

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de Groot PG, Urbanus RT, Roest M. Platelet interaction with the vessel wall. Handbook of experimental pharmacology. 2012 2. Evans G, Packham MA, Nishizawa EE, Mustard JF, Murphy EA. The effect of acetylsalicyclic acid on platelet function. The Journal of experimental medicine. Nov 1968 3. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. Jan 2002 4. Tang XF, Fan JY, Meng J, Jin C, Yuan JQ, Yang YJ. Impact of new oral or intravenous P2Y12 inhibitors and clopidogrel on major ischemic and bleeding events in patients with coronary artery disease: a meta-analysis of randomized trials. Atherosclerosis. Apr 2014 5. Gouya G, Arrich J, Wolzt M, et al. Antiplatelet treatment for prevention of cerebrovascular events in patients with vascular diseases: a systematic review and meta-analysis. Stroke; Feb 2014 6. Hillis LD, Smith PK, Anderson JL, et al. 2011 ACCF/ AHA guideline for coronary artery bypass graft surgery: executive summary: a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines. The Journal of thoracic and cardiovascular surgery. Jan 2012 7. Ferraris VA, Saha SP, Oestreich JH, et al. 2012 update to the Society of Thoracic Surgeons guideline on use of antiplatelet drugs in patients having cardiac and noncardiac operations. The Annals of thoracic surgery. Nov 2012 8. Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 9. Zimmermann N, Gams E, Hohlfeld T. Aspirin in coronary artery bypass surgery: new aspects of and alternatives for an old antithrombotic agent. European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. Jul 2008 10. Wurtz M, Grove EL. Interindividual variability in the efficacy of oral antiplatelet drugs: definitions, mechanisms and clinical importance. Current pharmaceutical design. 2012 11. Floyd CN, Ferro A. Antiplatelet drug resistance: Molecular insights and clinical implications. Prostaglandins & other lipid mediators. Jul 2015 12. Mulder BJ, van der Doef RM, van der Wall EE, et al. Effect of various antithrombotic regimens (aspirin, aspirin plus dipyridamole, anticoagulants) on the functional status of patients and grafts one year after coronary artery bypass grafting. European heart journal. Aug 1994

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reactivity for risk stratification after percutaneous coronary intervention. European heart journal. Jul 2015 25. Stone GW, Witzenbichler B, Weisz G, et al. Platelet reactivity and clinical outcomes after coronary artery implantation of drug-eluting stents (ADAPT-DES): a prospective multicentre registry study. Lancet. Aug 2013 26. Szczeklik A, Krzanowski M, Gora P, Radwan J. Antiplatelet drugs and generation of thrombin in clotting blood. Blood. Oct 1992 27. Santos MT, Valles J, Aznar J, Marcus AJ, Broekman MJ, Safier LB. Prothrombotic effects of erythrocytes on platelet reactivity. Reduction by aspirin. Circulation. Jan 1997 28. Dangelmaier C, Jin J, Smith JB, Kunapuli SP. Potentiation of thromboxane A2-induced platelet secretion by Gi signaling through the phosphoinositide-3 kinase pathway. Thrombosis and haemostasis. Feb 2001 29. Kauffenstein G, Bergmeier W, Eckly A, et al. The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism. FEBS letters. Sep 2001 30. Nieswandt B, Schulte V, Zywietz A, Gratacap MP, Offermanns S. Costimulation of Gi- and G12/G13mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets. The Journal of biological chemistry. Oct 2002 31. Mega JL, Simon T. Pharmacology of antithrombotic drugs: an assessment of oral antiplatelet and anticoagulant treatments. Lancet. Jul 2015 32. Wurtz M, Lordkipanidze M, Grove EL. Pharmacogenomics in cardiovascular disease: focus on aspirin and ADP receptor antagonists. Journal of thrombosis and haemostasis : JTH. Sep 2013 33. Rehmel JL, Eckstein JA, Farid NA, et al. Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent, with the cytochromes P450. Drug metabolism and disposition: the biological fate of chemicals. Apr 2006 34. Husted S, van Giezen JJ. Ticagrelor: the first reversibly binding oral P2Y12 receptor antagonist. Cardiovascular therapeutics. 2009 35. Cattaneo M, Schulz R, Nylander S. Adenosinemediated effects of ticagrelor: evidence and potential clinical relevance. Journal of the American College of Cardiology. Jun 2014 36. Wallentin L, Becker RC, Budaj A, et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. The New England journal of medicine. Sep 2009 37. Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood. Aug 1996

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38. Breet NJ, van Werkum JW, Bouman HJ, et al. Comparison of platelet function tests in predicting clinical outcome in patients undergoing coronary stent implantation. JAMA Feb 2010 39. Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. Jun1962 40. Cattaneo M, Cerletti C, Harrison P, et al. Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH. Journal of thrombosis and haemostasis : JTH. Apr 2013 41. Sibbing D, Schulz S, Braun S, et al. Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. Journal of thrombosis and haemostasis : JTH. Feb 2010 42. Hazarbasanov D, Velchev V, Finkov B, et al. Tailoring clopidogrel dose according to multiple electrode aggregometry decreases the rate of ischemic complications after percutaneous coronary intervention. Journal of thrombosis and thrombolysis. Jul 2012 43. Coller BS, Lang D, Scudder LE. Rapid and simple platelet function assay to assess glycoprotein IIb/ IIIa receptor blockade. Circulation. Feb 1997 44. Breet NJ, van Werkum JW, Bouman HJ, et al. High on-treatment platelet reactivity to both aspirin and clopidogrel is associated with the highest risk of adverse events following percutaneous coronary intervention. Heart. Jun 2011 45. Bouman HJ, Parlak E, van Werkum JW, et al. Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel. Journal of thrombosis and haemostasis : JTH. Mar 2010 46. Kim IS, Jeong YH, Kang MK, et al. Correlation of high post-treatment platelet reactivity assessed by light transmittance aggregometry and the VerifyNow P2Y12 assay. Journal of thrombosis and thrombolysis. Nov 2010 47. Carville DG, Schleckser PA, Guyer KE, Corsello M, Walsh MM. Whole blood platelet function assay on the ICHOR point-of-care hematology analyzer. The Journal of extra-corporeal technology. Dec 1998 48. Mammen EF, Comp PC, Gosselin R, et al. PFA-100 system: a new method for assessment of platelet dysfunction. Seminars in thrombosis and hemostasis. 1998 49. Harrison P, Segal H, Blasbery K, Furtado C, Silver L, Rothwell PM. Screening for aspirin responsiveness after transient ischemic attack and stroke: comparison of 2 point-of-care platelet function tests with optical aggregometry. Stroke; May 2005 50. Golanski J, Pluta J, Baraniak J, Watala C. Limited usefulness of the PFA-100 for the monitoring of ADP receptor antagonists--in vitro experience.

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Kent DM, Lau J. Testing of CYP2C19 Variants and Platelet Reactivity for Guiding Antiplatelet Treatment. Rockville (MD)2013. Janssen PW, ten Berg JM, Hackeng CM. The use of platelet function testing in PCI and CABG patients. Blood reviews. May 2014 Janssen PW, ten Berg JM. Platelet function testing and tailored antiplatelet therapy. Journal of cardiovascular translational research. Jun 2013 Mahla E, Suarez TA, Bliden KP, et al. Platelet function measurement-based strategy to reduce bleeding and waiting time in clopidogrel-treated patients undergoing coronary artery bypass graft surgery: the timing based on platelet function strategy to reduce clopidogrel-associated bleeding related to CABG (TARGET-CABG) study. Circulation. Cardiovascular interventions. Apr 2012 Gasparovic H, Petricevic M, Kopjar T, Djuric Z, Svetina L, Biocina B. Impact of dual antiplatelet therapy on outcomes among aspirin-resistant patients following coronary artery bypass grafting. The American journal of cardiology. May 2014 Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA Mar 2011 Montalescot G, Range G, Silvain J, et al. High on-treatment platelet reactivity as a risk factor for secondary prevention after coronary stent revascularization: A landmark analysis of the ARCTIC study. Circulation. May 2014 Siller-Matula JM, Francesconi M, Dechant C, et al. Personalized antiplatelet treatment after percutaneous coronary intervention: the

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Chapter 10 Lower platelet reactivity is associated with presentation of unstable coronary artery disease Accepted for publication in the International Journal of Angiologyâ&#x20AC;&#x192;

T. Leunissen*, C. Gijsberts*, PP. Wisman, A. Huisman, M. Ten Berg, F. Asselbergs, I. Hoefer, G. Pasterkamp, F. Moll, GJ. de Borst and M. Roest * Authors contributed equally tot his manuscript

Chapter 10

Abstract Background In patients with acute coronary syndrome, high platelet reactivity (PR) is associated with an increased risk of secondary thrombotic events. However, in patients undergoing elective percutaneous coronary intervention (PCI), no association between high PR and outcome has been demonstrated. At present, the relation of PR and clinical symptoms is unknown. Objectives To examine the association of PR with clinical indication for diagnostic angiography (stable or unstable coronary artery disease [CAD]bub), taking into account the influence of P2Y12 inhibitors. Methods A platelet function score (PFS) was determined in 195 patients by quantifying fibrinogen binding and P-selectin expression with flow cytometry. We evaluated the PFS with clinical presentation of stable or unstable CAD, angiographic severity of CAD, and the incidence of cardiovascular events during 2 years of follow-up. All data were analyzed stratified by P2Y12 inhibitor use (long-term and preprocedural vs. none). Results Surprisingly, among non-P2Y12 inhibitor users, the PFS was lower in patients with unstable CAD compared with stable CAD (5.6Âą1.8 vs. 7.4Âą1.6; p=0.001). Angiographic CAD severity showed no relation with PFS. The SYNTAX score tended to be inversely related with PFS: low PFS, 13.2 (IQR, 11.9-19.1); median PFS, 10.0 (IQR, 5.0-14.0); and high PFS, 8.0 (IQR, 5.0-13.0), without significance (p=0.304). Patients with low PFSs required more re-PCIs than patients with median and high PFSs (11.1% vs. 4.7% vs. 0.0%, p=0.004). This association was modified for patients using P2Y12 inhibitors. Conclusion Among patients without P2Y12 inhibitors undergoing coronary angiography, presentation of unstable CAD is independently associated with lower PR.

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Introduction Platelets are crucial for adequate regulation of hemostasis. Low platelet numbers (thrombocytopenia) or platelet dysfunction (thrombocytopathy) will lead to bleeding complications, whereas increased platelet reactivity leads to thrombosis, mainly in the arteries. Upon vascular endothelial injury, platelets bind to the exposed collagen via glycoprotein (GP)VI and integrin α2β1. This leads to αIIbβ3 activation and granule release.1 Secondary platelet activation is triggered by adenosine diphosphate (ADP) and thromboxane release, which bind to, respectively, P2Y12 and thromboxane receptors on the platelets and thereby reinforce platelet activation. These interconnected platelet activation pathways give multiple opportunities to inhibit platelet activation with antiplatelet therapy. Currently acetylsalicylic acid, P2Y12 inhibitors, and GPIIb/IIIa inhibitors belong to the standard medical treatment of patients with coronary artery disease (CAD), during coronary interventions, and as secondary prevention after myocardial infarction.2-4 The effectiveness of antiplatelet therapy has been measured by several commercially available platelet function tests, including the VerifyNow®, Platelet Function Analyser, Multiplate analyser, and the vasodilatorstimulated phosphoprotein-phosphorylation assay.5 Although some of these tests may identify patients at risk of atherothrombotic events, no benefit has been observed in adjusting the antiplatelet therapy regimen.5,6 Research on the role of platelet reactivity in CAD has thus far been dominated by clinical trials evaluating the potential protective effects of antiplatelet therapy, being mainly acetylsalicylic acid7 and P2Y12-receptor inhibitors.8 High on-treatment platelet reactivity has been associated with an increased risk of secondary cardiovascular events,9 especially among patients with acute coronary syndrome.10 Among patients with stable CAD undergoing elective percutaneous coronary intervention (PCI), this association could not be demonstrated in a recent meta-regression analysis.11 This discrepancy may be because a large proportion of stable CAD patients tolerate high levels of platelet reactivity without any adverse event. However, what the influence of the P2Y12 inhibitors is on the occurrence of secondary thrombotic events remains unclear in this meta-analysis. Hence, evidence is lacking on the influence of platelet reactivity in the development of clinical phenotypes, such as stable angina pectoris or acute myocardial infarction, between non-P2Y12 (NPIU) and P2Y12 inhibitor users (PIU). We therefore examined the association of platelet reactivity with clinical indication for diagnostic angiography (stable or unstable CAD), taking into account the influence of P2Y12 inhibitors. Because high on-treatment platelet reactivity is a risk factor for secondary thrombotic events, we hypothesized that high platelet reactivity would be more common in patients with unstable CAD.

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Methods Clinicaltrials.gov ID NCT02304744. Ethics statement This study was approved by the ethics committees of the University Medical Center in Utrecht the Netherlands, and conforms to the Declaration of Helsinki. All participants provided written informed consent before participation. Study population In this cross-sectional study we analyzed data from the UCORBIO cohort (clinicaltrials. gov identifier: NCT02304744), a biobank of patients undergoing coronary angiography, with or without PCI, in the University Medical Centre in Utrecht. From October 2011 to November 2012. We prospectively and consecutively enrolled 220 patients from the catheterization laboratories in whom platelet reactivity testing was performed. Valid platelet reactivity measurements were not obtained in 25 patients; thus leaving 195 valid platelet reactivity measurements for assessment. Age <18 years was the only exclusion criterion. Cardiovascular risk factors data and medical history were collected at baseline. We grouped the indication for coronary angiography into stable CAD (stable chest pain, dyspnea on exertion, or silent ischemia) and unstable CAD (unstable angina, non–STelevation myocardial infarction, and ST-elevation myocardial infarction) according to international guidelines.3,12 Administration of platelet inhibitors before the intervention and the consecutive treatment based on the diagnostic angiography were recorded. Treatment and periprocedural medication with aspirin, P2Y12 inhibitors, and/or GPIIb/IIIa inhibitors were left at the discretion of the operator. The interventional cardiologists were blinded for the platelet function score (PFS), and those who performed the platelet function tests were blinded for the indication for angiography and angiographic CAD severity. Blood collection Before coronary angiography, blood was drawn into a 4.5-mL Vacutainer® sodium citrate tube from the arterial sheath that is routinely inserted for the angiography procedure. Blood samples were transported to the laboratory for platelet reactivity testing and quantification of the platelets with the CELL-DYN Sapphire (Abbott Diagnostics, Wiesbaden, Germany). Platelet activation test (PACT) Material The PACT reaction mix was prepared in advance and contained 4.5 µmol/L ADP (01897; Zwijndrecht, the Netherlands), 6 µmol/L SFLLRN (TRAP-6) (H-2936; Bachem, Weil am Rhein, Germany), or 40 ng/mL cross-linked collagen-related peptide (xl-CRP, a generous

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gift from Professor Richard Farndale) in a HEPES-buffered saline mixture that contains a fixed concentration of R-phycoerythrin (RPE)-conjugated anti–P-selectin (1:25; 55524, BD Pharmingen™, Franklin Lakes, NJ, USA) and fluorescein isothiocyanate (FITC)conjugated antifibrinogen (1:100; F0111, Dako, Glostrup, Denmark). Methods The PACT was performed as previously described.13 In short, the agonist wells were filled with a 50 µL assay mixture into which 5 µL whole blood was pipetted. The mix was homogenized and incubated 8 minutes at room temperature. The reaction was stopped by pipetting 10 µL reaction mix into 190 µL fixative solution (0.2% formaldehyde/0.9% NaCl). Analysis of the samples was performed after a minimum of 30 minutes and maximum of 48 hours on the FACS Canto flow cytometer (BD Biosciences, San Jose, CA, USA). Single platelets were gated based on forward- and side-scatter properties. Fluorescence intensity in the RPE channel was used to determine P-selectin surface expression, and fluorescence intensity in the FITC channel was used to determine fibrinogen binding, which indicates αIIbβ3 activation. Platelet reactivity was quantified by the maximal expression of P-selectin and αIIbβ3 activation after stimulation. We normalized the maximum fluorescence intensity value per batch per agonist to the overall mean value per agonist (for P-selectin expression and fibrinogen binding separately) to reduce a possible batch effect. Platelet function score We designed a straightforward PFS based on the maximum fluorescence intensity measurements of the PACT. For each agonist (ADP, TRAP-6, and xl-CRP), we divided the platelet reactivity measurements into low, medium, and high tertiles and assigned a score of 1, 2, and 3, respectively (Figure 1). For each patient, we combined the tertile scores of the three agonists, leading to a PFS of 3 to 9. A score of 3 or 4 represents the lowest platelet reactivity (LPR), 5 to 7 corresponds to medium platelet reactivity (MPR), and a score of 8 or 9 is the highest platelet reactivity (HPR). This score was computed for fibrinogen binding and for P-selectin expression. Blood cell counts Data from blood cell counts were extracted from the Utrecht Patient Oriented Database (UPOD). UPOD is an infrastructure of relational databases comprising data on patient characteristics, hospital discharge diagnoses, medical procedures, medication orders, and laboratory tests for all patients treated at the UMC Utrecht since 2004. The structure and content of UPOD have been described in more detail elsewhere.14 UPOD data acquisition and data management are in line with current regulations in the Netherlands concerning privacy and ethics. Data used for this study were collected for patient care purposes and were used retrospectively. The automated blood cell analyses were performed with the Abbott Cell-Dyn Sapphire automated hematology analyser (Abbott Diagnostics, Santa Clara, CA, USA).

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Figure 1. Design of platelet function score (PFS) for fibrinogen. The PFS was based on the maximum fluorescence intensity measurements of the PACT: the results of each agonist (ADP, TRAP-6, and xl-CRP) were divided into tertiles and assigned a score of 1, 2, and 3 respectively. The tertile scores of the three agonists were combined, leading to a PFS of 3 to 9.

Angiographic CAD severity Angiographic data were collected and categorized into two categories: nonsignificant CAD (no stenosis, wall irregularities, <50% stenosis) and significant CAD (at least one epicardial vessel with >50% stenosis) based on the standard reporting of the clinical interventional cardiologists. SYNTAX, score of CAD complexity Two independent observers, using SYNTAX score calculator 2.11 software, measured the SYNTAX scores. The evaluation took place in the central core laboratory facility at the Utrecht University Medical Centre Department of Cardiology. The SYNTAX score allows for the characterization of coronary vasculature with respect to the number of lesions involved and the location and complexity of the lesions. Lesions are scored if they meet the required criteria (>50% stenosis and vessel diameter >1.5 mm).15 Higher scores are allocated to more complex lesions. The observers were blinded to the patientâ&#x20AC;&#x2122;s PFS. The two observers had unlimited access to quantitative coronary angiography software (CAAS, Siemens)16 to measure the percentage of stenosis or the dimension of the vessel if they were unsure about the significance of a lesion by visual estimation. The average of the SYNTAX scores of the two observers was used for the current analysis. Follow-up Questionnaires were used to collect follow-up data at 1 and 2 years to assess the occurrence of cardiovascular events (myocardial infarction, coronary revascularization, revascularization of other arteries, cerebrovascular events, hemorrhage, and death). The final occurrence of events was adjudicated by an independent event committee consisting of 3 cardiologists, 2 whom were interventional cardiologists.

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Statistical analysis Data were analyzed using the R statistical software package.17 We compared patient characteristics at baseline across the tertiles of the PFS. Data are presented as means ± standard deviations, medians with interquartile ranges (IQR), or as percentages (depending on normality). Categoric data are presented as percentages. Continuous data were compared using ANOVA (parametric) or Kruskal-Wallis (nonparametric) testing. Categorical variables were compared with χ2 testing. We performed multivariable ordinal regression to determine significant predictors of the PFS. Covariates in this analysis were P2Y12 inhibitor use, sex, age, diabetes, hypertension, hypercholesterolemia, smoking, indication for angiography, angiographic CAD severity, and CAD treatment (conservative, PCI, or coronary artery bypass graft [CABG]). All variables were entered in the model. This analysis was performed for the entire cohort and stratified by P2Y12 inhibitor usage. Follow-up events were collected, but no further analyses were performed because of the low event rate. At baseline, only PFS based on fibrinogen binding showed significant results and not PFS based on P-selectin expression; therefore, all following analyses were only performed for PFS based on fibrinogen binding.

Results Baseline characteristics The joint and stratified baseline patient characteristics are presented in Table 1. Of the 220 patients who were studied, platelet reactivity measurements were incomplete in 25 due to logistic problems. Thus, complete data were available from 195 patients. The median follow-up time was 662 days, during which 16 deaths and 12 re-PCIs occurred. Overall the participants (69.5% male) were an average age of 64.9 ± 11.0 years and showed a high prevalence of risk factors, consisting of diabetes (23.3%), hypertension (57.7%), hypercholesterolemia (50.0%), smoking (54.6%), kidney failure (3.2%), and overweight (mean body mass index, 27.1 ± 4.2 kg/m2). Approximately one-third of the patients had a history of CAD (acute coronary syndrome, 34.1%; previous PCI, 31.4%; and previous CABG, 15.9%). Coronary artery disease Antiplatelet therapy included aspirin in 77.4% of the participants, clopidogrel in 33.8%, prasugrel in 1.5%, and ticagrelor in 5.1% (Table 2). P2Y12 inhibitors were used by 74.4% of the patients with unstable CAD and by 33.1% of the patients with stable CAD. Surprisingly, among NPIU, the PFS was significantly lower in patients with unstable CAD (5.6 ± 1.8) than in those with stable CAD (7.4 ± 1.6; p<0.001; Figure 2). Consequently for NPIU, the presentation of unstable CAD was more common in patients with LPR (37.5%) compared with MPR (11.9%) or HPR (3.5%; p=0.006). The difference in the PFS was not significant among PIU, at 5.6 ± 2.1 for stable vs. 5.0 ± 1.9 for unstable (p=0.192).

173

174

106 (54.6)

Hypercholesterolemia (n, %)

Smoking (n, %)

75 (34.1)

69 (31.4)

35 (15.9)

History of ACS (n,%)

History of PCI (n,%)

History of CABG (n,%)

2 (22.2)

1 (11.1)

2 (22.2)

27.1 ±3.3

0 (0.0)

4 (50.0)

7 (77.8)

1 (11.1)

6 (66.7)

58.7 ±9.3

Low PFS

5 (11.6)

13 (30.2)

12 (27.9)

28.5 ±4.9

2 (4.7)

21 (61.8)

22 (52.4)

28 (65.1)

11 (25.6)

24 (55.8)

67.9 ±10.2

Medium PFS

High PFS

12 (19.0)

14 (22.2)

17 (27.0)

25.9 ±4.1

2 (3.2)

26 (48.1)

32 (50.8)

35 (55.6)

16 (25.4)

44 (69.8)

65.2 ±11.5

No P2Y12 inhibitor

0.535

0.403

5 (16.1)

14 (45.2)

13 (41.9)

26.8 ±3.6

0.016 0.941

1 (3.2)

16 (57.1)

14 (45.2)

13 (41.9)

6 (20.0)

24 (77.4)

64.9 ±9.4

0.772

0.452

0.984

0.341

0.628

0.330

0.066

p-value

Low PFS

3 (8.3)

13 (36.1)

17 (47.2)

26.7 ±2.7

1 (2.8)

18 (51.4)

14 (43.8)

21 (58.3)

10 (27.8)

28 (77.8)

62.2 ±11.7

Medium PFS

P2Y12 inhibitor

2 (15.4)

6 (46.2)

4 (30.8)

29.4 ±5.8

0 (0.0)

8 (66.7)

4 (36.4)

8 (61.5)

3 (23.1)

7 (53.8)

69.4 ±9.3

High PFS

0.593

0.699

0.587

0.084

0.814

0.649

0.877

0.316

0.760

0.204

0.106

p-value

0.425

0.050

0.040

0.928

1.000

0.569

0.675

0.470

0.920

0.101

0.150

p-value no P2Y12 vs. P2Y12 inhibitors

Data are shown as number of participants and percentage or mean with standard deviation. When data was incomplete, the percentage of participants that was accessible was calculated. Platelet function score (PFS) was assessed by stratifying the platelet reactivity per agonist in tertiles (1,2 and 3) for fibrinogen binding.

27.1 ±4.2

BMI (mean ± SD))

7 (3.2)

106 (50.0)

Hypertension (n, %)

Kidney Failure (n, %)

51 (23.3)

127 (57.7)

Diabetes (n, %)

153 (69.5)

Sex (n, % males)

220

64.9 ±11.0

Age (mean ± SD))

All

Table 1. Baseline clinical and demographical patients characteristics.

Chapter 10

39 (20.0)

15 (7.7)

103 (52.8)

74 (37.9)

2 (22.2)

4 (44.4)

3 (33.3)

(11.9, 19.1)

13.2

6 (66.7)

3 (33.3)

3 (37.5)

5 (62.5)

1 (11.1)

19 (30.6) 5 (8.1)

4 (9.5)

33 (52.4)

31 (72.1)

20 (47.6)

30 (47.6)

12 (27.9)

38 (61.3)

2 (3.5)

5 (11.9)

18 (42.9)

8.5

55 (96.5)

37 (88.1)

8.0

8 (12.7)

6 (14.0)

(5.0, 13.0)

10.0

(5.0, 14.0)

28 (90.3)

0.205

0.304

0.116

16 (53.3)

0.006

2 (6.7)

22 (73.3)

6 (20.0)

(5.1, 13.1)

3 (9.7)

14 (46.7)

(9.7)

0.960

7 (22.6)

2 (6.5)

23 (74.2)

28 (90.3)

39 (61.9)

33 (76.7)

0.275

2 (5.6)

28 (77.8)

6 (16.7)

(9.6, 20.0)

13.5

32 (88.9)

4 (11.1)

12 (35.3)

22 (64.7)

9 (25.0)

2 (5.6)

1 (2.8)

33 (91.7)

32 (88.9)

Medium PFS

P-value lowmediumhigh

P2Y12 inhibitor Low PFS

Medium PFS

High PFS

No P2Y12 inhibitor

6 (66.7)

Low PFS

* CAD coronary artery disease † PCI percutaneous coronary intervention ‡ CABG coronary artery bypass graft Data are shown as percentage, or median with interquartile range (IQR).

CABG ‡ (n, %)

†

PCI (n, %)

Conservative (n, %)

Treatment CAD

10.0

(5.0,16.0)

(median, IQR)

Significant (n, %)

SYNTAX score

54 (27.7)

141 (72.3)

Non-significant (n, %)

Angiographic CAD severity:

145 (74.4)

Unstable CAD (n, %)

Stable CAD* (n, %)

Indication for angiography:

Vit K antagonists (n, %)

3 (1.5)

10 (5.1)

Ticagrelor (n, %)

66 (33.8)

Prasugrel (n, %)

151 (77.4)

Clopidogrel (n, %)

195

Aspirin (n, %)

Medical therapy:

All

Table 2. Patient characteristics: antiplatelet therapy and coronary artery disease severity.

0 (0.0)

10 (76.9)

3 (23.1)

(5.0, 11.5)

7.0

11 (84.6)

2 (15.4)

3 (23.1)

10 (76.9)

1 (7.7)

0 (0.0)

10 (76.9)

13 (100.0)

High PFS

0.893

0.032

0.861

0.317

0.157

0.093

0.541

0.145

0.465

P-value lowmediumhigh

<0.001

0.261

<0.001

0.870

<0.001

0.142

<0.001

p-value no P2Y12 vs. P2Y12

Association of Platelet Reactivity with Coronary Artery Disease

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Chapter 10

Also, angiographic CAD severity showed no significant differences in the PFS. After diagnostic coronary angiography, 52.8% of patients underwent subsequent PCI. There was no association of the subsequent procedure and the PFS; however, more PCIs were performed within the PIU group than in the NPIU group (p<0.001).

Figure 2. Platelet function score (fibrinogen) by clinical presentation of coronary artery disease. Means and standard deviation platelet function score (fibrinogen) stratified by indication for coronary angiography and P2Y12 inhibitor usage. The p-values for the difference between stable and unstable CAD was p<0.001 among non-P2Y12 inhibitor users and p=0.192 for P2Y12 inhibitor users.

Blood cell counts Among NPIU, patients with low PFS displayed a significant lower platelet count compared with median PFS and high PFS (respectively, 247.3 [IQR, 201.9-279.9] vs. 223.9 [IQR, 186.2-264.7] vs. 193.7 [IQR, 161.0-248.8]; p=0.042; Table 3). No differences were seen in mean platelet volume. Within the same patient group, the white blood cell, neutrophil, and monocyte counts were increased in patients with LPR compared with median and high PFS. This resulted in, respectively, a white blood cell count of 9.3 × 109/L (IQR, 8.3-12.5 × 109/L), 6.6 × 109/L (IQR, 5.7-8.1 × 109/L), and 6.0 × 109/L (IQR, 5.0-7.2 × 109/L; p=0.001); a neutrophil count of 5.7 × 109/L (IQR, 5.4-9.4 × 109/L), 4.1 × 109/L (IQR, 3.1-5.2 × 109/L), and 3.5 × 109/L (IQR, 3.0-4.3 × 109/L; p=0.001); and a monocyte count of 0.75 × 109/L (IQR 0.66-0.94 × 109/L), 0.56 × 109/L (IQR 0.42-0.72 × 109/L), and 0.50 × 109/L (IQR 0.42-0.63 × 109/L; p=0.021). All medians were within the normal range of blood cell counts. The differences were not observed among PIU. SYNTAX score The SYNTAX score tended to be inversely related with PFS (fibrinogen) for NPIU: low PFS, 13.2 (IQR, 11.9-19.1); median PFS, 10.0 (IQR, 5.0-14.0); and high PFS, 8.0 (IQR, 5.0-

176

Association of Platelet Reactivity with Coronary Artery Disease

13.0), although this did not reach significance (p=0.304; Table 2 and supporting information 1). The SYNTAX score for PIU was significantly higher in the medium PFS group (p=0.032).

Table 3. Blood cell count in patients without P2Y12 inhibitor, stratified by platelet reactivity score (fibrinogen). Low PFS

Medium PFS

p-value

Leukocyte count (109 cells/L)

9.3 (8.3-12.5)

6.6 (5.7-8.1)

6.0 (5.0-7.2)

0.001

Neutrophil count (109 cells/L)

5.7 (5.4-9.4)

4.1 (3.1-5.2)

3.5 (3.0-4.3)

0.001

Monocyte count (10 cells/L)

0.75 (0.66-0.94)

0.56 (0.42-0.72)

0.50 (0.42-0.63)

0.021

2.1 (1.6-2.7)

1.6 (1.2-2.1)

1.5 (1.3-1.9)

0.314

0.08 (0.06-0.10)

0.13 (0.08-0.22)

0.13 (0.08-0.21)

0.224

Lymphocyte count (109 cells/L) Eosinophil count (109 cells/L) Basophil count (109 cells/L) Platelet count (109 cells/L) Mean platelet volume (fL)

High PFS

0.03 (0.03-0.04)

0.03 (0.02-0.05)

0.03 (0.01-0.04)

0.667

247.3 (201.9-279.9)

223.9 (186.2-264.7)

193.7 (161.0-248.8)

0.042

8.2 (7.8- 8.9)

7.7 (7.2-8.4)

8.2 (7.6-8.8)

0.096

All presented values are medians with interquartile ranges. Only values for non-P2Y12 users are shown.

Prediction of PFS A multivariable ordinal regression analysis was performed for the outcome PFS for fibrinogen (range, 3-9) with cardiovascular risk factors and coronary angiography characteristics as covariates (Table 4). As expected, P2Y12- inhibitor usage was a strong predictor of lower platelet reactivity (odds ratio: 0.22 (95% confidence interval (CI): 0.110.44, p<0.001). Again, the association of unstable CAD and PFS was confirmed among NPIU: the multivariable adjusted odds ratio of unstable CAD was 0.23 (95% CI: 0.06-0.83; p=0.026) among NPIU. Within all patients the multivariable odds ratio of unstable CAD for PFS was 0.34 (95% CI: 0.15-0.074; p=0.007). No association was found among PIU. These results may indicate an independent association of clinical presentation of CAD with decreased platelet reactivity. Follow-up In total, 16 patients died during a median follow-up duration of 662 days; causes of death were cardiovascular in 8 patients and noncardiovascular in 8 patients. This number was too low to perform reliable analyses. Also, other end points, such as myocardial infarction (n=6), cerebrovascular accident or transient ischemic attack (n=5), vascular intervention (n=8), and bleeding (n=4) were rare, and thus, we could not examine the relationship of PFS and clinical outcome in the 2-year follow-up interval. Repeat PCI was performed in 12 patients in the presence of residual symptoms and angiographic significant restenosis in the initially placed stent (n=3) or other location in

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the coronary arteries (n=4), both in-stent restenosis and stenosis at another location (n=4), and dissection after initial PCI (n=1). Within NPIU, significantly more repeat PCIs were performed in the low PFS than in median and high PFS (11.1% vs. 4.7% vs. 0.0%, respectively; p=0.004).

Table 4. Predictors of platelet function score (fibrinogen). All patients OR (95% CI)

p-value

P2Y12 usage (yes vs. no)

0.22 (0.11-0.44

<0.001

Aspirin usage (yes vs. no)

1.17 (0.58 - 2.36) 0.666

Sex (male vs. female)

1.01 (0.53-1.92)

Age (per year increase)

No P2Y12 inhibitor OR (95% CI)

p-value

P2Y12 inhibitor OR (95% CI)

p-value

0.91 (0.39 - 2.12) 0.829

3.37 (0.71 - 17.62)

0.133

0.969

1.34 (0.58-3.09)

0.492

0.55 (0.17-1.71)

0.302

1.01 (0.98-1.04)

0.464

1.02 (0.98-1.06)

0.357

1.00 (0.95-1.04)

0.914

Diabetes (yes vs. no)

1.10 (0.55-2.22)

0.791

0.85 (0.33-2.24)

0.744

1.45 (0.48-4.32)

0.507

Hypertension (yes vs. no)

0.87 (0.46-1.64)

0.677

0.57 (0.23-1.38)

0.211

1.63 (0.61-4.38)

0.332

Hypercholesterolemia 1.21 (0.67-2.21) (yes vs. no)

0.528

1.67 (0.73-3.87)

0.223

0.66 (0.25-1.77)

0.412

Smoking (yes vs. no)

1.08 (0.60-1.96)

0.790

1.02 (0.45-2.35)

0.955

0.94 (0.38-2.30)

0.887

Indication (unstable CAD* vs. stable CAD)

0.34 (0.15-0.74)

0.007

0.23 (0.06-0.83)

0.026

0.40 (0.14-1.07)

0.071

Angiographic CAD (significant vs. non-significant)

0.89 (0.31-2.62)

0.836

1.26 (0.35-4.71)

0.723

0.42 (0.05-3.45)

0.418

PCI† (vs. conservative)

0.85 (0.30-2.36)

0.761

0.62 (0.16-2.22)

0.461

1.29 (0.23-7.29)

0.768

CABG‡ (vs. conservative)

0.69 (0.18-2.58)

0.576

0.51 (0.09-2.71)

0.426

1.12 (0.10-2.71)

0.927

The presented odds ratios (OR) with 95% confidence intervals as the result from a multivariable ordinal regression analysis for the outcome Platelet Function Score (ranging from 3 to 9). All reported variables were entered in the model. * CAD coronary artery disease † PCI percutaneous coronary intervention ‡CABG coronary artery bypass graft

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Association of Platelet Reactivity with Coronary Artery Disease

Discussion Our study demonstrates that patients with unstable CAD had a lower PFS than those with stable CAD but that this association was modified by the use of P2Y12 inhibitors. Among NPIU, unstable CAD is more prevalent in patients with a low PFS than with a median or high PFS. The relationship between unstable CAD and PFS (fibrinogen) was independent of other baseline differences. No relationship was found between prevalence of unstable CAD and PFS in patients using P2Y12 inhibitors. P2Y12 inhibitor usage was a strong predictor for lower platelet reactivity. Although this correlation was contrary to our hypothesis, several previous studies have found similar results. In patients with stable or unstable AP, mostly using P2Y12 inhibitors, higher platelet reactivity was observed in response to mental stress. However in patients with cardiac syndrome x, not using P2Y12 inhibitors, a decrease in PR was found after mental stress, suggesting potential myocardial release of adenosine, a powerful antiplatelet agent. 18 Milovanovic et al. found an inverse relationship between platelet reactivity and severity of coronary blood flow obstruction in patients with stable angina pectoris after stimulation of the platelets with TRAP-6 and ADP.19 Another study in patients with critical limb ischemia and tissue loss showed lower platelet reactivity after in vitro stimulation compared with patients with intermittent calf claudication.20 A possible explanation for a relationship between LPR and a high prevalence of unstable CAD could be that platelets have a protective role in development of CAD. Unstable atherosclerotic plaques are characterized by the increased formation of neomicrovessels. These neomicrovessels have weak integrity and are leaky, which leads to recurrent intraplaque bleeding.21 This intraplaque bleeding could enable inflammatory cells (mostly monocytes and macrophages) to infiltrate the adventitia and to secrete proteases and inflammatory proteins, weakening the fibrous cap of the atherosclerotic plaque and hence increasing infarction risk. Platelets might stabilize this intraplaque bleeding and simultaneously prevent further atherosclerotic plaque development. Our finding that a low PFS is associated with unstable CAD in patients without P2Y12 inhibitors could be explained by increased intraplaque bleeding in patients with low PFS. Reduced platelet reactivity by ischemic preconditioning has also been reported. Brief episodes of myocardial ischemia paradoxically provide resistance of cardiomyocytes to a later sustained ischemic insult.22,23 The resistance is presumably due to preconditioninginduced attenuation of platelet-mediated thrombosis because it is accompanied by a significant decline in platelet-fibrinogen binding, a decrease in the formation of neutrophil-platelet aggregates, and a trend toward a reduction in platelet P-selectin expression.24 The mechanisms responsible for this attenuation in platelet activation and aggregation are still unknown. Ischemic preconditioning supports our finding of lower platelet reactivity in patients with unstable CAD, assuming that these patients were previously more often exposed to brief episodes of myocardial ischemia than patients with stable CAD.

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The current study found that a lower PFS was also related to a lower platelet count, potentially caused by platelet exhaustion and a high turnover of activated platelets. A low PFS in patients with unstable CAD could be caused by overstimulation of the platelets due to the irregularities of the atherosclerotic plaque, causing extinguishment of remaining platelet reactivity. Highly reactive platelets that have formed platelet-platelet and platelet-leukocyte aggregates are rapidly removed from the circulation, leaving the less responsive and the preactivated platelets that are no longer susceptible to further stimulation behind. This phenomenon has been described previously in different subsets of patients.25-27 Low platelet reactivity would not be the cause of more severe CAD in this case but rather the result of platelet prestimulation by more severe atherosclerosis. It can be debated whether high PR is a reflection of the patients overall cardiovascular risk rather than representing an independent modifiable parameter associated with clinical prognosis. Platelet reactivity in patients on P2Y12 inhibitors undergoing PCI was strongly influenced by clinical risk variables such as age, Body Mass Index, Diabetes Mellitus, serum creatinine and left ventricular function. 28 An inverse trend for the correlation between platelet reactivity and markers for renal function, creatinine, and urea was also found in patients with critical limb ischemia.29 Even more, increased PR was associated with impaired arterial stiffness in patients undergoing PCI and loading dose clopidogrel, suggesting a potential predicting clinical factor for high PR despite antiplatelet therapy.30 Risk factor assessment together with platelet function tests might improve future personalized antiplatelet therapy. Finally, most studies investigating platelet reactivity in patients with CAD are performed in patients who have been prescribed extensive antiplatelet therapy, such as P2Y12 inhibitors. Currently there is little knowledge about “unaffected platelet reactivity” in patients with CAD. In our opinion, the current consensus that high platelet reactivity is a prognostic risk factor for cardiovascular events in P2Y12 users is therefore not directly extractable to patients who do not use P2Y12 inhibitors. A clear distinction should be made between the term “high on-treatment platelet reactivity” and “high platelet reactivity.” Limitations An important limitation of our study was that it was underpowered, foremost in the follow-up events. Although there seems to be a difference in the occurrence of re-PCI in the low PFS compared with the median and high PFS, we could not thoroughly examine confounding factors because only 12 repeat PCIs occurred. Therefore, the follow-up results should be interpreted with care due to the limitation in statistical power. Furthermore, an unstable indication was more prevalent in patients with LPR, which could also be the cause of a higher number of re-PCIs in this group. However, more indicators of CAD severity, such as the SYNTAX score and angiographic CAD severity, tended toward the same results (the association of lower PFS in patients with more severe CAD), but no significant differences could be demonstrated (Appendix 1, SYNTAX score) because of the low numbers of participants.

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Association of Platelet Reactivity with Coronary Artery Disease

Conclusion Among patients undergoing coronary angiography, without P2Y12 inhibitors, presentation of unstable CAD is independently associated with lower platelet reactivity. These findings are perpendicular to results of studies reporting outcomes of patients with P2Y12 inhibitors.

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References 1.

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Suplemental

Figure 1. SYNTAX score in patients with and without P2Y12 inhibitor according to tertiles of Platelet function score (low, medium and high). Medians and interquartile ranges of the SYNTAX score stratified by platelet function score and P2Y12 inhibitor usage. The difference in SYNTAX score among non-P2Y12 inhibitors was not significant (p=0.304) but was significant among patients with P2Y12 inhibitor (p=0.032).

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Chapter 11 Reticulated platelets as predictor of myocardial injury and 30-day mortality after noncardiac surgery Submitted to the British Journal of Surgery

T. Leunissen, J. van Waes, W. van Klei, A. Huisman, F. Moll and GJ. de Borst

Chapter 11

Abstract Background A potential preoperative marker for identification of patients at risk for perioperative adverse events and 30-day mortality might be the percentage young, reticulated platelets (pRP). We aimed to determine the predictive value of preoperative pRP on postoperative myocardial injury (PMI) and 30-day mortality, in patients aged ≥60 years undergoing moderate-to high risk noncardiac surgery. Methods The incidence of PMI (Troponin I >0.06 µg/L) and 30-day mortality was compared for patients with normal- and high pRP (≥2.82%). The predictive pRP value was assessed using logistic regression. A prediction model for PMI or 30-day mortality with known risk factors was compared to a model including increased pRP using the area under the receiving operator characteristics curve (AUROC). Results In total, 26.5% (607/2289) patients showed preoperative increased pRP. Increased pRP was associated with higher rate of PMI and 30-day mortality as compared to normal pRP (36.1% vs. 28.3%, p<0.001 and 8.6% vs. 3.6%, p<0.001). Median pRP was higher in patients displaying PMI and 30-day mortality as compared to no (2.21 [IQR:1.57-3.11] vs. 2.07 [IQR:1.52-1.78], p=0.002 and 2.63 [IQR:1.76-4.15] vs 2.09 [IQR:1.52-3.98] p<0.001). pRP was independently related to PMI (OR:1.28 (95%CI:1.04-1.59) p=0.023) and 30-day mortality (OR:2.35 (95%CI:1.56-3.55), p<0.001). Adding increased pRP to the predictive model of PMI or 30-day mortality didn’t increase the AUROC (0.71 (95%CI:0.67-0.76) vs 0.72 (95%CI:0.67-0.76), p=0.30 and 0.80 (95%CI:0.78-0.82) vs. 0.81 (95%CI:0.67-0.76), p=0.07). Conclusion In patients undergoing major noncardiac surgery, preoperative increased percentage of reticulated platelets is independently related to 30-day mortality and to postoperative myocardial injury.

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Reticulated platelets as predictor

Introduction Recovery from noncardiac surgery is hampered by the occurrence of adverse events, including myocardial infarction and mortality. Myocardial infarction according to the latest universal definition1 occurs in 3-6% 2-5, while postoperative myocardial injury (PMI) defined as troponin elevation above the clinical cut-off level of 0.06 Îźg/L with or without clinical symptoms, occurs in approximately 20% of the patients. 6,7 Increased peak troponin values as measured in the first three days after noncardiac surgery are strongly associated with 30-day and one year mortality. 5-8 The first step in preventing PMI is to identify patients at risk, so that preventive measures can be taken. Current guidelines 9,10 recommend the revised cardiac risk index (RCRI) 11 and the National Surgery Quality Improvement Program (NSQIP) Myocardial Infarction and Cardiac Arrest (MICA) risk- prediction rules 12 as tools to identify patients at risk for postoperative cardiac complications. These models are constructed from a patients history, preoperative creatinine and procedure characteristics. The RCRI shows a moderate predictive value in discriminating patients at high risk for postoperative cardiac complications.13 In vascular surgery patients its performance is limited.13 Monitoring of troponin levels during the first three days after surgery substantially improved 30-day mortality risk stratification compared to stratification before surgery based on clinical risk factors only.5,7 Although implementing standard troponin surveillance improved prediction of all-cause mortality, it could not realise cardiovascular optimization by preventing further PMI, postoperative myocardial infarction and long term cardiovascular mortality. 6 Ideally, one should be able to better predict PMI and 30-day mortality prior surgery, in order to undertake perioperative precautions. A potential preoperative marker might be the newly released platelets, also termed reticulated platelets. Atherosclerosis reduces platelet survival and thereby increases the percentage of younger platelets in the circulation. Elevated levels of circulating reticulated platelets have been extensively associated with a prothrombotic phenotype and participate most actively in thrombosis. 14-17 Moreover, young platelets are more prone to participate in thrombus formation compared to older platelets.18 They tend to be larger in size, contain more dense granules and display a greater ex-vivo reactivity profile in response to agonists compared to mature platelets. 15,19 Elevated levels of reticulated platelets have been observed in the setting of acute coronary syndromes and stroke. 20,21 Therefore, we hypothesized that a high percentage of reticulated platelets (pRP) might be predictive for PMI and 30-day mortality. The purpose of this study was to determine the predictive value of the preoperative pRP on PMI and 30-day mortality, in patients aged â&#x2030;Ľ60 years undergoing moderate-to high risk noncardiac surgery.

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Methods Patients This observational cohort study included consecutive patients undergoing major noncardiac surgery between January 1st 2011 and December 31st 2012 at the University Medical Center Utrecht, the Netherlands.6 Patients were eligible if they were aged â&#x2030;Ľ60 years, were undergoing moderate- to high-risk noncardiac surgery with an expected postoperative hospital stay of â&#x2030;Ľ24 hours and if haematological measurements within 1 month prior surgery were available. For patients who underwent surgery more than once during this period, only the first operation was included in the analysis. Medical ethical approval The local medical ethics committee waived the need for informed consent since only routinely collected patient data were used and data was anonymised before analysis (UMC Utrecht Medical Research Ethics committee 14-189/C). The Utrecht Patient Orientated Database (UPOD) 22 containing haematology data of automated blood cell analysis, was set up in accordance with guidance of the Institutional Review Board (IRB) and privacy board of the UMC Utrecht, which allows the use of clinical data from patients who did not object to the use of their data for scientific purposes, as long as the patients cannot be identified directly from the data. Data collection All pre- and postoperative data was collected by electronical medical and administrative records. Data collected for all patients were patient characteristics, preoperative physical status for the American society of Anaesthesiologists (ASA) classification, comorbidities, postoperative troponin I measurements and death within 30 days after surgery. Mortality data were obtained from the municipal personal records database. Platelet measurements were obtained from UPOD, which contains haematology data of all automated blood cell analyses at the UMC Utrecht. Reticulated platelets The blood cell analyses were performed with the Cell- Dyn Sapphire (Abbott Diagnostics, Illinois, USA). A feature of this type of blood cell analyser is that it not only reports the parameters requested by the physician, but all haematological parameters that it is capable of measuring. 23 All data captured by the blood cell analysers were manually downloaded into the UPOD and anonymised for further research. From this database, the pRP, mean platelet volume and platelet count were obtained in blood samples that were ordered for preoperative blood typing and cross matching, maximum 28 days prior surgery. The cut-off for a high pRP was defined at 2.82% as to standard hospital protocol.24 This utilised threshold has been determined on the 2.5th and 97.5th percentile of repeated measurements in 151 healthy volunteers, according to the CLSI guidelines.25

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Aims The aim of the current study was to evaluate differences in baseline characteristics and occurrence of PMI and 30-day mortality between patients with normal vs. high preoperative pRP. Secondly we aimed to study the predictive value of pRP on the occurrence of PMI and 30-day mortality. Outcomes The primary outcome was PMI, defined as a troponin I >0.06 µg/L. This is the lowest value measurable with a 10% coefficient of variation above the 99th percentile of 0.04 µg/l of the assay used. Troponin was analysed with the use of the third-generation enhanced AccuTnl assay (Beckman Coulter, Brea, CA). 26 According to the hospital protocol, cardiac troponin I measurements were ordered for the first three days after surgery. For each patient the highest troponin value was used in the analysis. Mortality within 30 days was used as a secondary outcome. Statistical Analysis The analysis was performed with the use of SPSS (IBM SPSS Statistics 21 for Windows). In advance of data analysis we were aware that the pRP would not be available in all patients since not all patients had preoperative blood typing and crossmatching. Patients with missing platelet data <1 month prior intervention were not included. Next, patients with registered violation alarms from the data obtained by the Abbott Cell- Dyn Sapphire were excluded to ensure the data’s reliability. To check if the missing data and alarms could be considered ‘at random’, we compared the baseline characteristics for included (all data available) and excluded (incomplete data) patients. Statistics To investigate the abovementioned aims, we studied: 1. Differences in baseline characteristics using the chi-square or Fisher’s Exact test for categorical variables; and t test or Mann–Whitney U test for normally distributed or non-normally distributed continuous variables, respectively. A double sided p- value of less than 0.05 was considered statistically significant. 2. The incidence of PMI and 30-day mortality for patients with and without increased pRP using the Fisher’s Exact test. 3. The median pRP for patients with or without PMI and 30-day mortality by Kruskal– Wallis- and Mann-Whitney U test. 4. The predictive value of pRP on PMI and 30-day mortality using univariable and multivariable logistic regression analysis with clinical baseline and procedural characteristics as predictor variables. Results of the logistic regression were displayed as odds ratios (OR) with 95% confidence intervals (CI). 5. The survival of patients with and without increased pRP using Kaplan-Meier Curves (differences with log-rank analysis). 6. The predictive value of a model with known predictors of PMI and 30-day mortality, to a model in which the pRP was added using the area under the receiving operator characteristics curve.

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Results In total 2971 patients fulfilled the inclusion criteria. Of these patients, 682 patients were excluded because of missing troponin values (N=570) due to violation of the protocol for troponin monitoring or violation alarms of the Cell- Dyn Sapphire (N=112). In total, 2 289 (77%) patients were included in the analysis (Figure 1). Baseline characteristics of included and excluded patients due to violation alarms and missing troponin values are given in appendix 1. The urgency and duration of the procedure, and surgical specialty differed for in- and excluded patients: 27.5% vs. 35.5% emergency surgery (p<0.001), 199 vs. 153 minutes (p<0.001) respectively. More general and vascular surgery was reported in the included patients. PMI and 30 day mortality did not differ between the in-and excluded patients (30.4% vs. 25.6%, p=0.393 and 4.9% vs. 4.9%, p=1.00 respectively). The overall myocardial injury and 30 day mortality was 30.1% (716/2371) and 4.9%(145/2943). Baseline characteristics Of the 2 289 patients, 607 patients (26.5%) showed preoperative high pRP. Patients with high pRP were more frequently male, had a higher ASA classification, more diabetes type II and renal failure, underwent more emergency surgery and a longer hospital stay, as compared to patients with normal pRP levels (table 1). The mean platelet volume was smaller in patients with increased pRP (7.52 vs. 7.68 p=<0.001). Postoperative myocardial injury Patients with high pRP more often developed PMI as compared to patients with normal pRP (36.1% vs. 28.3%, p<0.001). The median pRP was higher in patients with PMI, 2.21% (IQR 1.57-3.11) compared to no PMI, 2.07% (IQR 1.52-2.78) p=0.002 (Figure 3, appendix table 2). In the univariable analysis, age, male sex, ASA classification, history of myocardial infarction and coronary intervention, renal failure, hypertension, chronic heart failure, peripheral arterial disease, increased pRP, emergency surgery and surgical specialty were significantly associated with PMI (table 2A). After adjustment for other variables, an increased pRP was an independent predictor of PMI (OR= 1.28 (95%CI: 1.04-1.59), p= 0.023). The strongest association was found for emergency surgery (OR= 2.59 (95%CI: 2.07-3.25), p<0.001), ASA IV (OR= 2.32 (95%CI: 0.97-5.55), p=0.060), ASA III (OR= 1.28 (95%CI: 0.85-1.92), p=0.232) and renal failure (OR= 1.70 (95%CI: 1.28-2.25), p<0.001). The area under the receiver operating characteristics curve (AUC) of the model with only the known preoperative predictors was 0.71 (95% CI 0.67- 0.76) with a sensitivity of 59.4% and specificity of 73.4%. Adding pRP to the model resulted in a AUC of 0.72 (95% CI 0.67- 0.76) with a sensitivity of 63.6% and specificity of 68.5% (p=0.30). 30-day mortality Patients with high pRP died more often within 30 days as compared to patients with a normal percentage pRP (8.6 vs. 3.6%, p<0.001). During the first 5 days postoperative the

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Figure 1. Flowchart for patients undergoing major noncardiac surgery, aged â&#x2030;Ľ 60 years.

survival of patients with and without elevated pRP was equal. After 10 days the survival decreased for patients with preoperative elevated pRP, resulting in the significant different 30-day survival (Figure 4). Equally, patients with PMI died more often within 30 days as compared to patients without PMI (9.6% vs 3.0%, p<0.001). Patients who died within 30-days more often displayed high pRP and PMI (46.4% vs 25.6%, p<0.001 and 58.9% vs. 30.7%, p<0.001). Of the high risk patients, who displayed an increased pRP and PMI, 12.0% (26/217) died within 30 days, as compared to 8.5% (40/471) patients with normal pRP but elevated postoperative troponin (p= 0.164).

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Table 1. Baseline characteristics of patients with and without increased young platelets. Reticulated platelets <2.82% N= 1 682

Reticulated platelets ≥ 2.82% N= 607

p-value

Age (yr) mean (SD)

71.2

7.8

71.5

7.9

0.247

Male gender, n (%)

906

53.9

357

58.8

0.036

-I

200

11.9

10.1

- II

1 052

62.5

357

58.8

- III

411

24.4

176

29.0

- IV

1.1

2.1

- MI, n (%)

169

10.0

9.4

- Coronary revascularisation, n (%)

217

12.9

10.2

0.027

ASA classification, n (%)

History of

Diabetes, n (%)

0.057 0.807

1376

81.8

494

81.4

Yes

306

18.2

113

18.6

Smoking, n (%) - Current

0.623

0.605 300/1 520

19.7

98/524

18.7

- Ever smoker

458

30.1

150

28.6

- Never smoker

762

50.1

276

52.7

892

53.0

299

49.3

0.118

3.5

3.6

0.798

Hypertension, n (%) Chronic heart failure, n (%) Peripheral arterial disease, n (%)

184

10.9

12.9

0.207

Hypertension, n (%)

892

53.0

299

49.3

0.118

Renal failure, n (%) (GFR<50ml/min)

210

12.5

16.0

0.026

Emergency surgery, n (%)

429

25.5

200

33.0

0.001

1253

74.5

407

67.1

General surgery, n (%)

542

32.2

240

39.5

Vascular surgery, n (%)

292

17.4

110

18.1

Gynaecology/Urology, n (%)

181

10.8

8.6

Elective surgery, n (%)

Other surgery, n (%) Surgery duration (minutes) [mean (SD)] Time in Hospital (days) [mean (SD) MPV (fL) [mean (SD)]

0.004

666

39.6

205

33.8

199.2

131.2

198.23

130.3

0.823

10.9

12.8

15.37

19.1

<0.001 <0.001

7.7

1.0

7.5

1.02

Platelet count (x 109/L) [mean (SD)]

268.6

94.7

271.2

117.29

0.699

Reticulated platelets (%)[mean (SD)]

1.8

0.5

4.3

1.59

<0.001

476

28.3

219

36.1

<0.001

60/1 666

3.6

52/605

8.6

<0.001

Myocardial injury (Tropinin I ≥ 0.06µg/l), n (%) 30 day mortality, n (%)

ASA American society of anaesthesiologists, MI myocardial infarction

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Reticulated platelets as predictor

The median pRP was higher in patients with 30-days mortality, 2.63% (IQR 1.76-4.15) as compared to no 30-day mortality, 2.09% (IQR 1.52-2.89), p<0.001 (Figure 3, appendix table 2). In the univariable analysis age, ASA classification, renal failure, platelet count, mean platelet volume, increased pRP, surgery -duration, -indication (elective/ emergency) and -type and PMI were significantly associated with 30-day mortality (table 2B). After adjustment for other predictor variables, increased pRP was associated to 30-day mortality: OR = 2.35 (95% CI: 1.56- 3.55, p<0.001). As expected, myocardial injury was strongly related to 30-day mortality (OR = 2.33 (95%CI: 1.52-3.57, p<0.001). The strongest correlation was found for ASA IV classification (OR = 4.60 (95% CI: 1.23-17.16), p=0.23) and emergency surgery (OR = 3.39 (95% CI: 2.14-5.37), p<0.001) The AUC of the model with only the known preoperative predictors was 0.80 (95% CI 0.78- 0.82) with a sensitivity of 74.8% and specificity of 75.5%. Adding pRP to the model resulted in a AUC of 0.81 (95% CI 0.67- 0.76) with a sensitivity of 67.6% and specificity of 84.7%. This was no significant increase (p=0.07).

11 Figure 2. Incidence of myocardial injury and 30-day mortality, stratified by percentage reticulated platelets.

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Table 2A. Univariable and multivariable associations with postoperative myocardial injury. Univariable

Multivariable

OR (95% CI)

p-value

OR (95% CI)

p-value

Elevation pRP (%)

1.42 (1.17-1.73)

0.000

1.28 (1.04-1.59)

0.023

Platelet count (x 109/L))

1.00(1.00-1.00)

0.834

Mean platelet volume (fL)

1.03 (0.95-1.13)

0.447

Age (year)

1.04 (1.03-1.05)

<0.001

1.04 (1.02-1.05)

<0.001

Male sex

1.38 (1.16-1.66)

<0.001

1.22 (1.00-1.50)

0.051

ASA classification ASA I

Ref

ASA II

1.35(0.98-1.85)

0.066

Ref -

ASA III

2.53 (1.81-3.55)

<0.001

1.28 (0.85-1.92)

0.232

ASA IV

0.060

6.10 (2.81-13.23)

<0.001

2.32 (0.97-5.55)

History myocardial infarction

2.17 (1.64-2.87)

<0.001

0.65 (0.41-1.02)

History coronary intervention

1.88 (1.46-2.43)

<0.001

Renal failure (GFR < 50 ml/min)

2.50 (1.96-3.19)

<0.001

Hypertension

1.32 (1.10-7.58)

0.002

Diabetes

1.25 (1.00-1.57)

0.049

Chronic heart failure

2.03 (1.30-3.18)

0.002

1.41 (0.84-2.45)

0.192

Peripheral arterial disease

1.57 (1.20-2.05)

0.001

1.12 (0.81-1.55)

0.482

- Current

1.07 (0.83-1.37)

0.616

- Ever

0.061 0.204

1.70 (1.28-2.25)

<0.001 0.236

Smoking

0.85 (0.68-1.07)

0.168

Emergency surgery

2.40 (1.99-2.92)

<0.001

2.59 (2.07-3.25)

<0.001

Surgery duration (minutes)

1.00 (1.00-1.00)

<0.001

1.00 (1.00-1.01)

<0.001

Surgical specialty - Other surgery

Ref

- General

1.90 (1.54-2.34)

<0.001

1.84 (1.47-2.31)

<0.001

- Gynaecology/Urology

0.44 (0.29-0.66)

<0.001

0.63 (0.41-0.99)

0.044

1.52 (1.18-1.96)

0.001

1.37 (1.01-1.85)

0.046

- Vascular

ASA American society of anaesthesiologists. Bold values indicate significant association with outcome. fL femtolitre

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Reticulated platelets as predictor

Table 2B. Univariate and multivariate associations with 30-day mortality. Univariate

Elevation pRP (%) Platelet count (x 109/L)) Mean platelet volume (fL) Age (year) Male sex

Multivariate

OR (95% CI)

p-value

OR (95% CI)

p-value

2.54 (1.73-3.73)

<0.001

2.35 (1.56-3.55)

<0.001

1.00 (1.00-1.000)

0.020

1.00 (1.00-1.00)

0.093

1.20 (1.01-1.43)

0.043

1.08 (0.88-1.33)

0.452

1.05 (1.030-1.08)

<0.001

1.02 (1.00-1.05)

0.109

1.15 (0.79-1.69)

0.466

ASA classification ASA I

Ref

ASA II

1.49 (0.63-3.53)

0.001

1.28 (0.53-3.11)

Ref 0.558

ASA III

4.10 (1.74-9.66)

<0.001

2.10 (0.82-5.34)

0.120

ASA IV

10.10 (3.02-33.60)

<0.001

4.60 (1.23-17.16)

0.023

History myocardial infarction

1.67 (0.98-2.87)

0.059

History coronary intervention

1.02 (0.57-1.81)

0.945

Renal failure (GFR < 50 ml/min)

2.50 (1.61-3.86)

<0.001

1.49 (0.88-2.52)

0.136

Hypertension

1.14 (0.78-1.67)

0.496

Diabetes

1.60 (1.03-2.48)

0.035

Chronic heart failure

1.93 (0.87-4.30)

0.107

Peripheral arterial disease

0.92 (0.50-1.70)

0.780

0.93 (0.51-1.69)

0.926

Smoking - Current

0.72 (0.41-1.27)

0.724

Emergency surgery

- Ever

6.03 (4.02-9.05)

<0.001

3.39 (2.14-5.37)

<0.001

Surgery duration (minutes)

1.00 (1.00-1.00)

0.035

1.00 (1.00-1.00)

0.778

Time in Hospital (days)

1.01 (1.00-1.02)

0.189

0.94 (0.63-1.41)

0.772

0.60 (0.38-0.94)

0.025

Surgical specialty Other surgery General

0.13 (0.03-0.53)

0.005

0.23 (0.06-1.00)

0.050

Gynaecology/Urology

0.38 (0.19-0.76)

0.006

0.26 (0.12-0.56)

0.001

3.55 (2.41-5.23)

<0.001

2.33 (1.52-3.57)

<0.001

Vascular Postoperative myocardial injury

ASA American society of anaesthesiologists. Bold values indicate significant association with outcome. fL femtolitre

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Figure 3. Distribution of percentage reticulated platelets over severity of myocardial injury and 30-day mortality

Figure 4. The area under the receiver operating characteristics curve for myocardial injury and 30-day mortality.

Discussion This study shows that an increased percentage of RP before surgery is independently related to 30-day mortality and is associated with postoperative myocardial injury for patients undergoing major noncardiac surgery, aged â&#x2030;Ľ60 year. Patients with an increased pRP have more cardiovascular risk factors (male sex, diabetes and renal failure) as compared to patients with normal pRP. This study confirms the presence of an increased pRP in patients with multiple cardiovascular risk factors as high age, male sex, renal failure, diabetes and higher ASA classification. A loss of glomerular filtration rate, as in chronic kidney disease,

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Reticulated platelets as predictor

Figure 5. Survival plot: Kaplan Meier Survival plot for patients with and without increased percentage reticulated platelets.

independently predicts mortality and accelerates the overall progression of cardiovascular disease. 27 This suggest that elevation of immature platelets may be utilized as marker in patients with an extensive cardiovascular profile and consequently increased risk of PMI and 30-day mortality. Measuring the pRP prior surgery is a very simple and quick method to identify patients who require careful perioperative cardiovascular risk management. The percentage of circulating young platelets reflects thrombopoesis, increasing with increased synthesis and decreased with decreased production. Measuring pRP has already been used for many years as a diagnostic tool for patients with thrombocytopenia to differentiate between decreased production of thrombocytes or peripheral destruction and to monitor the thrombocytopenic phase after chemotherapy and transplantation of haematological malignancies. 28 It has been proven that preferentially immature, reticulated platelets are recruited into arterial thrombosis compared to mature platelets 18 and elevated pRP is associated with a prothrombotic fenotype. 15,29 Younger platelets also tend to be larger in size, contain more dense granules and display a greater ex-vivo reactivity profile in response to agonists compared to mature platelets. 15,19 Consequently, researchers in the field of cardiovascular disease have also gained interest in the measuring the pRP. So far, increased pRP has been associated in nonsurgical patients with acute coronary syndrome 20,30 and cardioembolic stroke 21,31 and non-response to antiplatelet therapy in patients with coronary artery disease.19,29,32 Enhanced platelet turnover, as seen in patients with diabetes or acute coronary syndrome, may interfere with the anti- aggregatory effect of antiplatelet therapy. Even more, patients

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with enhanced platelet turnover might even represent the subgroup of patients that are not sufficiently protected by standard antiplatelet therapy.33 A single study compared specific agonist- dependent platelet function with platelet turnover in patients with antiplatelet drugs, regarding outcome. Measuring platelet turnover, by mean platelet volume, resulted in a higher predictive value for cardiac death than platelet function testing with VerifyNow.34 Platelet turnover might thus represent a major predictive factor for cardiovascular risk, while platelet reactivity testing is only allows specific identification of non-responders to APT.35 The pRP can be assessed on two commercially available, haematology analysers; Sysmex (XE- and XN- series) and CELL-DYN Sapphire, applying different reference ranges. Direct comparison between the two analysers showed low- to moderate correlation, with a higher sensitivity of the CELL-DYN for identification of patients with high platelet turnover. 36,37 The CELL-DYN Sapphire is an easy-to-use, standardized, flow-cytometry based analyser that is available is most laboratories and therefore suitable for utilization in standard care. Multiple preoperative biomarkers have been studies as predictors in cardiovascular risk stratification including N-terminal pro-brain natriuretic peptide (NT-BNP), cystatin C, C-reactive protein (CRP) and coronary artery calcium (CAC). 38 NT-BNP shows a 75-88% sensitivity and 62-100% specificity, studied in vascular surgery patients. 39 It is well known that CRP is related to the inflammatory mechanism of atherosclerotic disease, however no sensitivity and specificity have been determined. 40,41 The evidence concerning these biomarkers is modest, therefore routine preoperative measurement is not recommended. Some study limitations for the present study have to be recognized. First, although the reference range of 2.82% for reticulated platelets is used for standard clinical care in our hospital, different reference ranges are applied in studies, rising up to 6.0%. 42 The usage of a more strict cut-off value for elevation of pRP, may have resulted in different outcomes. Second, although we have found an association between preoperative pRP with PMI and 30-day mortality, we cannot conclude a causal connection since the study holds a retrospective study design. Multiple other (cardiovascular) factors may contribute to PMI and 30-day mortality. Finally, the overall incidence of PMI is relatively high in this cohort of patients (30.4%) compared to previous studies, which can be explained by the exclusion of patients without preoperative blood testing <1 month prior surgery. 7 43 Low- risk, healthy patients had no indication for preoperative blood sampling and could therefore not be included in this study, leaving a high risk population for inclusion. The applicability of reticulated platelets as preoperative marker in low-mediate risk patients should be determined in future studies. Concluding, preoperative increased pRP is an independent predictor of postoperative myocardial injury and 30-day mortality in patients undergoing major noncardiac surgery, aged â&#x2030;Ľ60 year. Increased pRP may reflect extensive cardiovascular disease. Future, prospective studies with consequent adjustment of cardiovascular risk management after pRP measurement, should determine the clinical relevance.

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Reticulated platelets as predictor

References 1.

Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD. Third universal definition of myocardial infarction. Global heart. Dec 2012 2. Mauermann E, Puelacher C, Buse GL. Myocardial injury after noncardiac surgery: an underappreciated problem and current challenges. Current opinion in anaesthesiology. Mar 2016 3. Landesberg G, Beattie WS, Mosseri M, Jaffe AS, Alpert JS. Perioperative myocardial infarction. Circulation. Jun 2009 4. Botto F, Alonso-Coello P, Chan MT, et al. Myocardial injury after noncardiac surgery: a large, international, prospective cohort study establishing diagnostic criteria, characteristics, predictors, and 30-day outcomes. Anesthesiology. Mar 2014 5. Devereaux PJ, Chan MT, Alonso-Coello P, et al. Association between postoperative troponin levels and 30-day mortality among patients undergoing noncardiac surgery. JAMA : the journal of the American Medical Association. Jun 2012 6. van Waes JA, Grobben RB, Nathoe HM, et al. OneYear Mortality, Causes of Death, and Cardiac Interventions in Patients with Postoperative Myocardial Injury. Anesthesia and analgesia. Apr 2016 7. van Waes JA, Nathoe HM, de Graaff JC, et al. Myocardial injury after noncardiac surgery and its association with short-term mortality. Circulation. Jun 2013 8. van Klei WA, Grobbee DE, Grobben RB, van Waes JA, Nathoe HM. Detection and management of asymptomatic myocardial injury after noncardiac surgery. European journal of preventive cardiology. Dec 2013 9. Fleisher LA, Fleischmann KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. Journal of the American College of Cardiology. Dec 2014 10. Kristensen SD, Knuuti J, Saraste A, et al. 2014 ESC/ ESA Guidelines on non-cardiac surgery: cardiovascular assessment and management: The Joint Task Force on non-cardiac surgery: cardiovascular assessment and management of the European Society of Cardiology (ESC) and the European Society of Anaesthesiology (ESA). European journal of anaesthesiology. Oct 2014 11. Lee TH, Marcantonio ER, Mangione CM, et al. Derivation and prospective validation of a simple index for prediction of cardiac risk of major noncardiac surgery. Circulation. Sep 1999 12. Gupta PK, Gupta H, Sundaram A, et al.

13.

14.

15.

16.

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18.

19.

20.

21.

22.

23.

24.

Development and validation of a risk calculator for prediction of cardiac risk after surgery. Circulation. Jul 2011 Ford MK, Beattie WS, Wijeysundera DN. Systematic review: prediction of perioperative cardiac complications and mortality by the revised cardiac risk index. Annals of internal medicine. Jan 2010 Richards EM, Baglin TP. Quantitation of reticulated platelets: methodology and clinical application. British journal of haematology. Oct 1995 Guthikonda S, Lev EI, Patel R, et al. Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin. Journal of thrombosis and haemostasis : JTH. Mar 2007 Cesari F, Marcucci R, Caporale R, et al. Relationship between high platelet turnover and platelet function in high-risk patients with coronary artery disease on dual antiplatelet therapy. Thrombosis and haemostasis. May 2008 Fager AM, Wood JP, Bouchard BA, Feng P, Tracy PB. Properties of procoagulant platelets: defining and characterizing the subpopulation binding a functional prothrombinase. Arteriosclerosis, thrombosis, and vascular biology. Dec 2010 McBane RD, 2nd, Gonzalez C, Hodge DO, Wysokinski WE. Propensity for young reticulated platelet recruitment into arterial thrombi. Journal of thrombosis and thrombolysis. Feb 2014 Ibrahim H, Nadipalli S, DeLao T, Guthikonda S, Kleiman NS. Immature platelet fraction (IPF) determined with an automated method predicts clopidogrel hyporesponsiveness. Journal of thrombosis and thrombolysis. Feb 2012 Lakkis N, Dokainish H, Abuzahra M, et al. Reticulated platelets in acute coronary syndrome: a marker of platelet activity. Journal of the American College of Cardiology. Nov 2004 Nakamura T, Uchiyama S, Yamazaki M, Okubo K, Takakuwa Y, Iwata M. Flow cytometric analysis of reticulated platelets in patients with ischemic stroke. Thrombosis research. May 2002 ten Berg MJ, Huisman A, van den Bemt PM, Schobben AF, Egberts AC, van Solinge WW. Linking laboratory and medication data: new opportunities for pharmacoepidemiological research. Clinical chemistry and laboratory medicine : CCLM / FESCC. 2007 Muller R, Mellors I, Johannessen B, et al. European multi-center evaluation of the Abbott Cell-Dyn sapphire hematology analyzer. Laboratory hematology : official publication of the International Society for Laboratory Hematology. 2006 Costa O, Van Moer G, Jochmans K, Jonckheer J, Damiaens S, De Waele M. Reference values for new red blood cell and platelet parameters on the Abbott Diagnostics Cell-Dyn Sapphire. Clinical chemistry and laboratory medicine : CCLM / FESCC. May 2012

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25. Horowitz A, Boyd, Ceriotti, Garg, Horn, Pesce, Sine and Zakowski. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guidelineâ&#x20AC;&#x201D;Third Edition. Clinical and laboratory standard institute. 2010 26. Thygesen K, Alpert JS, White HD, et al. Universal definition of myocardial infarction. Circulation. Nov 2007 27. Schefold JC, Filippatos G, Hasenfuss G, Anker SD, von Haehling S. Heart failure and kidney dysfunction: epidemiology, mechanisms and management. Nature reviews. Nephrology. Aug 2016 28. Hoffmann JJ. Reticulated platelets: analytical aspects and clinical utility. Clinical chemistry and laboratory medicine : CCLM / FESCC. Aug 2014 29. Guthikonda S, Alviar CL, Vaduganathan M, et al. Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease. Journal of the American College of Cardiology. Aug 2008 30. Grove EL, Hvas AM, Kristensen SD. Immature platelets in patients with acute coronary syndromes. Thrombosis and haemostasis. Jan 2009 31. McCabe DJ, Harrison P, Sidhu PS, Brown MM, Machin SJ. Circulating reticulated platelets in the early and late phases after ischaemic stroke and transient ischaemic attack. British journal of haematology. Sep 2004 32. Perl L, Lerman-Shivek H, Rechavia E, et al. Response to prasugrel and levels of circulating reticulated platelets in patients with ST-segment elevation myocardial infarction. Journal of the American College of Cardiology. Feb 2014 33. Martin JF, Kristensen SD, Mathur A, Grove EL, Choudry FA. The causal role of megakaryocyteplatelet hyperactivity in acute coronary syndromes. Nature reviews. Cardiology. Nov 2012 34. Choi SW, Choi DH, Kim HW, Ku YH, Ha SI, Park G. Clinical outcome prediction from mean platelet volume in patients undergoing percutaneous coronary intervention in Korean cohort: Implications of more simple and useful test than platelet function testing. Platelets. 2014

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35. Freynhofer MK, Gruber SC, Grove EL, Weiss TW, Wojta J, Huber K. Antiplatelet drugs in patients with enhanced platelet turnover: biomarkers versus platelet function testing. Thrombosis and haemostasis. Aug 2015 36. Bruegel M, Nagel D, Funk M, Fuhrmann P, Zander J, Teupser D. Comparison of five automated hematology analyzers in a university hospital setting: Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000. Clinical chemistry and laboratory medicine : CCLM / FESCC. Jun 2015 37. Meintker L, Haimerl M, Ringwald J, Krause SW. Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients. Clinical chemistry and laboratory medicine : CCLM / FESCC. Nov 2013 38. Zethelius B, Berglund L, Sundstrom J, et al. Use of multiple biomarkers to improve the prediction of death from cardiovascular causes. The New England journal of medicine. May 2008 39. Zarinsefat A, Henke P. Update in preoperative risk assessment in vascular surgery patients. Journal of vascular surgery. Aug 2015 40. de Ferranti S, Rifai N. C-reactive protein and cardiovascular disease: a review of risk prediction and interventions. Clinica chimica acta; international journal of clinical chemistry. Mar 2002 41. Shankar A, Li J, Nieto FJ, Klein BE, Klein R. Association between C-reactive protein level and peripheral arterial disease among US adults without cardiovascular disease, diabetes, or hypertension. American heart journal. Sep 2007 42. Dusse LM, Freitas LG. Clinical applicability of reticulated platelets. Clinica chimica acta; international journal of clinical chemistry. Jan 2015 43. Grobben RB, Vrijenhoek JE, Nathoe HM, et al. Clinical Relevance of Cardiac Troponin Assessment in Patients Undergoing Carotid Endarterectomy. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. Apr 2016

Reticulated platelets as predictor

Supplemental Appendix Table 1. Baseline characteristics of in-and excluded patients. Included patients (N=2 289) Age [mean (SD)]

Excluded patients (N=682)

p-value

71.2

7.8

70.7

7.5

0.108

1263

55.2

351

52.5

0.088

-I

261

11.4

12.0

0.633

- II

1 409

61.6

406

59.5

- III

587

25.6

187

27.4

- IV

1.4

1.0

Male gender, n (%) ASA classification, n (%)

Diabetes, n (%) No

1 870

81.7

557

81.7

Yes

419

18.3

125

18.3

Renal failure, n (%)

308

13.5

100

14.7

0.447

Emergency surgery, n (%)

629

27,5

242

35.5

<0.001

Elective surgery, n (%)

1 660

72.5

440

64.5

General surgery, n (%)

531

23.2

145

21.3

Vascular surgery, n (%)

402

17.6

13.2

Gynaecology/Urology, n (%)

146

6.4

1210

52.9

401

58.8

Other surgery, n (%) MPV (fL), [mean (SD)]

1.000

0.015

7.67

1.02

7.71

1.14

0.920

Platelet count (x 109/L) , [mean (SD)]

269.44

101.52

268.11

113.07

0.262

Reticulated platelets (%), [mean (SD)]

2.46

1.44

4.50

7.41

0.057

Surgery duration (minutes) [mean (SD)]

199.2

130.9

152.79

97.46

<0.001

Time in Hospital (days) [mean (SD)]

12.06

14.90

12.16

16.35

0.829

Myocardial injury, n (%)

695/2 289

30.4

21/82

25.6

0.393

30 day mortality, n (%)

112/2 271

4.9

33/672

4.9

1.000

ASA indicates American Society of Anesthesiologists;

Appendix Table 2. Distribution of percentage reticulated platelets. Myocardial injury No (Troponin I) <0.06 Âľg/L) Yes (Troponin I 0.06-0.6 Âľg/L) 30-day mortality

Median reticulated platelets (%)

1594

2.07

695

2.21

Median reticulated platelets (%)

IQR (25-75%) 1.52-2.78 1.57-3.11 IQR (25-75%)

2159

2.09

1.52-3.98

Yes

112

2.63

1.76-4.15

203

PART V Summary and discussion

Chapter 12 General discussion and summary

T. Leunissen

Chapter 12

Inhibition of platelet activation, aggregation and consequent thrombus formation is an essential target in primary and secondary prevention for secondary cardiovascular events (CVE). Although stronger platelet inhibitory therapy has been developed, there are still individual patients who develop a second CVE, such as stroke and myocardial infarction, while other patients develop bleeding events with the same dose of platelet inhibitors. This argues for tailored treatment strategies. Several platelet reactivity tests have been used to identify patients with high on treatment platelet reactivity, and therefore increased risk for CVE, however adjusting antiplatelet therapy based on these measurement has not yet been proven effective. This may be due to the validity of the specific tests, the unstandardized cut-off values, timing of testing, unknown optimal antiplatelet therapy in non- responders and more. The aim of this thesis was to evaluate the present state of platelet reactivity testing in patients with vascular disease as stepping stone towards tailored antiplatelet therapy. Platelet function testing in vascular disease 1. Currently many different platelet reactivity tests are used in trials and daily clinical practice. Although LTA, VerifyNow, PFA-100 and VASP are widely available there is little agreement between the tests with regard to definition of high on treatment platelet reactivity.1 The LTA is seen as laboratory gold standard, but the test is timeconsuming with a sensitivity and specificity from respectively 54.6- 60.2% and 53.160.9% for prediction of atherothrombotic events. 2 The VerifyNow, PFA-100 and VASP are clinical point of care tests, all with their pros and cons. We have shown that measurement of platelet fibrinogen binding using flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response, identifies different patients as non-responders to P2Y12 inhibitors, compared to only ADP- induced aggregation-based assays such as the VerifyNow (Chapter 2). Multiple good responders to P2Y12 inhibitors, as identified with VerifyNow, show normal ÎąIIbÎ˛3 activation after stimulation with PAR-1 and CRP-xl, suggesting there might be a threshold in platelet aggregation after which full aggregation takes place, while flow cytometry can still detect quantification in platelet activation. Both flow cytometry and VerifyNow were used to study the association with solid micro embolic signals, as markers of platelet activation, during carotid endarterectomy (Chapter 7). Flow cytometry after ADP stimulation did show association with solid micro emboli frequency while VerifyNow -P2Y12 and -aspirin could not show an association with micro emboli frequency. High platelet reactivity, defined by flow cytometry after ADP stimulation, showed an increased risk for displaying â&#x2030;Ľ20 micro emboli during CEA. However, when adjusting for the usage of clopidogrel, no association between the platelet reactivity tests and micro emboli frequency could be shown. Clopidogrel treatment was thus the strongest predictor of solid micro emboli frequency and cardiovascular events. Another future approach to identify patients with an increased risk for cardiovascular events is by reticulated platelet (RP) measurements (Chapter 11). Preoperative

208

General discussion and summary

percentage of increased RP was independently related to postoperative myocardial injury and 30-day mortality in patients undergoing major noncardiac surgery, aged â&#x2030;Ľ 60 year. Increased percentage RP may reflect extensive cardiovascular disease in patients. Future prospective studies with consequent adjustment of cardiovascular risk management after RP measurement, should determine the clinical relevance. Potentially RP can complement or replace platelet reactivity testing in future tailored antiplatelet therapy. 2. Another challenge in accurate platelet reactivity testing is that, due to the heterogeneity in platelet reactivity tests, there is no clear definition of high platelet reactivity. This leads to difference in prevalence of high platelet reactivity between studies studying the same patient population. For patients with peripheral arterial disease undergoing PTA a large variance in proportion of high platelet reactivity existed between the VerifyNow, VASP- assay and flow cytometry (Chapter 4). Frequency of high platelet reactivity despite aspirin or clopidogrel treatment was also different in patients undergoing carotid endarterectomy, when measured with either flow cytometry or VerifyNow (Chapter 7). 3. To further optimize platelet reactivity testing a consensus should be made about the usage of either venous or arterial blood. Recently a study compared platelet reactivity measured with LTA, VerifyNow and PFA-100 in blood from the coronary artery-, femoral artery and femoral vein, showing wide differences between the utilized tests as well as between coronary and venous blood.3 The results of a study from the Antonius Ziekenhuis evaluating the difference in platelet reactivity, measured by VerifyNow, in venous or arterial blood can be expected soon. 4. Future research should also focus on potential variability in platelet reactivity and the consequent timing of testing; data have demonstrated that the mean platelet reactivity across a population is constant in different samples. 4 In contrast the same study shows when evaluating each patient individually, that 15.7% of patients taking clopidogrel 75 mg and 11.4% of patients taking 150 mg had a change in their responder status when tested at 2 different time points (p < 0.001). In this thesis we observed a large variance of high platelet reactivity in patients with PAD when testing <24 hours or > 24 hours after intervention in the same patient (Chapter 4). Studies on healthy subjects who are not on APT, have shown that platelet reactivity can vary significant over time. 5,6 These findings suggest that many physiological factors, other than medications, may affect platelet function even in normal individuals. For patients on clopidogrel treatment, one can only speculate which factors might contribute to intraindividual platelet reactivity; potential factors might include either true alterations in platelet reactivity due to fluctuations in platelet production and expression of theÂ P2Y12Â receptor, changes in hepatic metabolism (based on CYP2C19 mutated alleles) altering the level of clopidogrel bio activation, unrecognized noncompliance, or artefactual changes in measured platelet reactivity due to technical issues affecting the platelet reactivity tests.

209

Chapter 12

5. If indeed a variability of platelet reactivity despite antiplatelet therapy is found, the next hurdle will be the timing of platelet reactivity testing: after stent placement during percutaneous transluminal angioplasty (PTA), patients often receive loading dose clopidogrel and continue dual antiplatelet therapy for three months. We have shown that platelet reactivity is more inhibited when tested >24 hours after loading dose compared to the day postoperative (Chapter 4). Technological improvements in the endovascular armentarium have made increasingly complex atherosclerotic lesions eligible for endovascular interventions, which has led to a primarily endovascular first strategy in peripheral arterial disease (Chapter 3). However as restenosis mostly occurs within 3 to 6 months after PTA, adequate platelet inhibition during the initial period after endovascular intervention is of particular importance.7 Unfortunately it is probably not possible to prevent all mechanical complications related to the procedure with adequate platelet inhibition, as is suggested by the results from the ARCTIC- trial 8 in percutaneous coronary intervention (PCI) patients and the IMPACT- trial in carotid artery stenting patients.9 A study investigating the adequate timing of platelet reactivity measurement at 1, 3 and 5 days after initiation of loading dose clopidogrel (e.g. in patients undergoing percutaneous transluminal angioplasty (PTA) with stent placement) in association with restenosis and secondary cardiovascular events is recommended to answer this question. Tailored antiplatelet therapy in vascular disease Besides optimization of platelet reactivity testing, more obstacles need to be surmounted before tailored antiplatelet therapy can be studied. As previously mentioned the cut-off values for high platelet reactivity per platelet reactivity test should be defined. However, one can question if different types of vascular disease hold an equal definition for high on treatment platelet reactivity. For example, patients with or without diabetes have a different cut-off value for the VerifyNow P2Y12 (resp. PRU 256 and >229) for prediction of major adverse cardiovascular events (MACE).10 Vascular disease patients who require surgical intervention might be in need for a less stringent cut-off value than patients who do not require intervention, due to an increased bleeding risk. Furthermore, we have shown that despite the fact that high platelet reactivity is a prognostic risk factor for cardiovascular events in P2Y12 users, this conclusion is not directly extractable for patients who do not use P2Y12 inhibitors (Chapter 10). In 195 patients undergoing coronary angiography, for non-P2Y12 inhibitor users, platelet reactivity was lower in patients with unstable CAD compared with stable CAD (5.6Âą1.8 vs. 7.4Âą1.6; p=0.001). The SYNTAX score tended to be inversely related with platelet reactivity: low: 13.2 (IQR, 11.9-19.1); median: 10.0 (IQR, 5.0-14.0); and high: (IQR, 5.013.0), without significance (p=0.304). Patients with low platelet reactivity required more re-PCIs than patients with median and high platelet reactivity (11.1% vs. 4.7% vs. 0.0%, p=0.004). This association was modified for patients using P2Y12 inhibitors.

210

General discussion and summary

These results support our hypothesis that platelet reactivity is not a fixed value for all vascular patients with different classes of antiplatelet therapy, but requires an individualised approach for choosing the appropriate therapeutic window. Also a clear distinction should be made in literature between the term “high on-treatment platelet reactivity” and “high platelet reactivity” since the clinical consequences might be contrary. Lastly, future research might also focus on new antiplatelet therapy, such as the proteaseactivated receptor-1 (PAR-1) inhibitors (e.g. vorapaxar and atopaxar). Two clinical trials showed somewhat controversial results regarding the clinical benefits of vorapaxar; in patients with stable atherosclerotic disease voraxapar reduced the risk of cardiovascular death or ischemic events compared to standard therapy, however with increased risk of moderate to severe bleeding, including intracranial haemorrhage. 30, For patients with ACS, the addition of vorapaxar to standard therapy did not significantly reduce the primary composite end point but significantly increased the risk of major bleeding, including intracranial haemorrhage. 31 Thus PAR-1 antagonism coupled with existing dual oral antiplatelet therapy may potentially offer more comprehensive platelet inhibition although potentially related to increased bleeding especially patients with a history of transient ischemic attack or stroke.11 Tailored antiplatelet therapy in peripheral arterial disease The 2011 European Society for Vascular Surgery (ESVS) Guidelines for the management of critical limb ischemia (CLI) suggests that stronger platelet inhibitors such as thienopyridines (clopidogrel) or dual antiplatelet therapy might confer additional benefits to monotherapy aspirin following complex endovascular revascularization. 12 The CHARISMA trial concluded that for patients with peripheral arterial disease (PAD), dual therapy provided some benefit over aspirin alone for the rate of MI and hospitalization for ischaemic events, at the cost of an increase in minor bleeding. 13 The few studies investigating platelet reactivity in PAD, all showed a higher incidence of high platelet reactivity compared with patients with coronary artery disease, confirming the relevance of platelet reactivity testing in PAD patients. 14-16 Importantly, the continuing low patency rates after PTA leave room for randomized control trials to prove significant benefit of tailored compared to standard antiplatelet therapy after the abovementioned questions are answered. Since the risk of haemorrhagic complications might increase by the use of the stronger P2Y12 inhibitors and dual APT, the use of a combined clinical benefit endpoint, comprising both thrombotic and bleeding events, would be advised in potential future trials. Tailored antiplatelet therapy in carotid artery disease For patients undergoing CEA, monotherapy aspirin or dual antiplatelet therapy is used as perioperative medical treatment but this treatment strategy is not evidence based and still little consensus exists regarding the optimal combination of APT (Chapter 5). 17 In Chapter 7 we showed that clopidogrel usage is the strongest predictor of solid micro emboli frequency, which is in line with the study results of Naylor et all. 18-20 This suggests

211

Chapter 12

that addition of a P2Y12 inhibitor prior surgery is mandatory to minimize the risk of perioperative stroke. For carotid artery stenting (CAS) a combination of aspirin and clopidogrel 4 days prior to the procedure has been standardized. However, if high on clopidogrel platelet reactivity occurs, it remains undefined what the next step in changing antiplatelet should be for either CEA and CAS. The role of the relatively newer P2Y12inhibitors (prasugrel, ticagrelor and cangrelor), cilostazol and GPIIb/IIIa-antagonists has not yet been studied. Developing a randomized controlled trial to study tailored vs. standard antiplatelet therapy in CEA meets another challenge: perioperative stroke and death occurs only in up to 5% of carotid endarterectomies 21, leading to an unfeasible large sample size, if chosen as primary outcome. With the introduction of new antiplatelet drugs, we expect that the thrombotic event rates will become lower. Solid micro emboli frequency might therefore be an acceptable surrogate marker for perioperative stroke and thus for the efficacy of APT on platelet reactivity. 22 Quantifying solid micro emboli frequency however is a time consuming (consequently expensive) and mentally strenuous procedure. Ideally solid micro emboli detection should be automated but so far all attempts to develop an automatic detection system failed to match the accuracy of human experts (Chapter 6). In the future cut-off values for high platelet reactivity in patients undergoing CEA and CAS should be determined. However, we should also focus on low platelet reactivity especially in patients undergoing CEA, as this is a more invasive procedure with a higher bleeding risk. The therapeutic window could be much smaller in this population as compared to patients undergoing stenting procedures. Finally, we reported new development of contralateral stenosis after CEA in 1 of every 5 patients (Chapter 8). Contralateral stenosis occurred mainly in patients with asymptomatic presentation: dissection of a stable atherosclerotic plaque with small lipid core and high content of smooth muscle cells increased the risk of new contralateral stenosis, as previously described for the development of ipsilateral restenosis. These study results may help to develop individual treatment algorithms based on clinical presentation and plaque characteristics for patients undergoing CEA. Tailored antiplatelet therapy in cardiac disease For patients with acute coronary syndrome, platelet reactivity testing has a low-level of recommendation (class IIb) 23. This is due to the lack of adequately sized, positive, randomized, controlled studies showing an improvement in clinical outcomes by using platelet reactivity testing in patients undergoing PCI. Failure of these previous studies 8,24,25 Â demonstrated that future trials should 1) be large multi-centre studies that are realistically powered for ischaemic endpoints, 2) include patients at high risk for stent thrombosis (preferably acute myocardial infarction), 3) use strong P2Y12-inhibitors such as prasugrel/ ticagrelor instead of double-dose clopidogrel when high on treatment platelet reactivity is detected and 4) test the clinical value of other newer platelet function assays. 26 Current guidelines 23,27Â state that ACS patients are recommended to be treated

212

General discussion and summary

with prasugrel or ticagrelor: 22, 26 new trials with ACS patients using standard-dose clopidogrel group is therefore unethical and contradicts guidelines. Another focus for future research is preventing bleeding complications, especially in patients undergoing invasive cardiac surgery, such as coronary artery bypass graft (CABG). This is particularly important with novel P2Y12-inhibitors in low-risk ACS patients, in the elderly population and in patients in whom both antiplatelet drugs and oral anticoagulants are indicated. Preventing perioperative bleeding and administration of blood products might decrease the risk of in-hospital mortality, renal failure, infection and others.28,29 We believe that the concept of personalized antiplatelet therapy should also be investigated in future clinical trials as a means to reduce post-operative bleeding after cardiac surgery (Chapter 9).

Future studies To determine if intraindividual platelet reactivity is variable over time we are designing a study with patients undergoing carotid endarterectomy (CEA), treated with aspirin and clopidogrel, to measure platelet reactivity prior surgery, 1 day-, 2 weeks- and 3 months postoperative with different platelet reactivity tests. Tailored antiplatelet therapy is currently studied in large trials as the TROPICAL-ACS (NCT01959451) and ANTARCTIC (NCT01538446) using validated cut-off values, focusing on high-risk cohorts of patients and implementing the therapeutic window of platelet inhibition to assess the potential clinical benefit on thrombotic and bleeding events. Tailored antiplatelet therapy based on genotyping is being studied in the TAILORED-PCI (NCT01742117). After PCI the conventional therapy arm will receive 75 mg and the prospective CYP2C19 genotype-based therapy arm will receive either 90 mg ticagrelor or clopidogrel 75 mg depending on the mutation. STEMI patients are recruited for the POPULAR GENETICS study (NCT01761786), in which antiplatelet therapy is tailored to CYP2C19 metabolizer status. In the genotyping group of this randomized controlled trial, the patients with a CYP2C19 loss of function allele are treated with ticagrelor or prasugrel and non-carriers of these loss of function alleles are treated with clopidogrel. The control group is treated with ticagrelor or prasugrel as recommended by the ESC STEMI guidelines. The results of all above mentioned ongoing studies and new studies will broaden our knowledge about optimal platelet reactivity testing and contribute to the promising concept of tailoring antiplatelet therapy, consequently reducing thrombotic and bleeding events. Concluding, platelet reactivity testing in vascular patients to monitor and consequently tailor antiplatelet therapy will be pivotal to balance between a thrombotic and bleeding risk.

213

Chapter 12

References 1.

10.

11.

12.

13.

14.

214

Wisman PP, Roest M, Asselbergs FW, et al. Plateletreactivity tests identify patients at risk of secondary cardiovascular events: a systematic review and meta-analysis. Journal of thrombosis and haemostasis : JTH. May 2014 Breet NJ, van Werkum JW, Bouman HJ, et al. Comparison of platelet function tests in predicting clinical outcome in patients undergoing coronary stent implantation. JAMA : Feb 2010 Hu YF, Lu TM, Wu CH, et al. Differences in high on-treatment platelet reactivity between intracoronary and peripheral blood after dual antiplatelet agents in patients with coronary artery disease. Thrombosis and haemostasis. Jul 2013 Hochholzer W, Ruff CT, Mesa RA, et al. Variability of individual platelet reactivity over time in patients treated with clopidogrel: insights from the ELEVATE-TIMI 56 trial. Journal of the American College of Cardiology. Jul 2014 Hevelow ME, McKenzie SM, Siegel JE. Reproducibility of platelet function testing. Laboratory hematology : official publication of the International Society for Laboratory Hematology. 2007 Refaai MA, Frenkel E, Sarode R. Platelet aggregation responses vary over a period of time in healthy controls. Platelets. 2010 Thukkani AK, Kinlay S. Endovascular intervention for peripheral artery disease. Circulation research. Apr 2015 Collet JP, Cuisset T, Range G, et al. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. The New England journal of medicine. Nov 2012 Van Der Heyden J, Van Werkum J, Hackeng CM, et al. High versus standard clopidogrel loading in patients undergoing carotid artery stenting prior to cardiac surgery to assess the number of microemboli detected with transcranial Doppler: results of the randomized IMPACT trial. The Journal of cardiovascular surgery. Jun 2013 Mangiacapra F, Peace A, Barbato E, et al. Thresholds for platelet reactivity to predict clinical events after coronary intervention are different in patients with and without diabetes mellitus. Platelets. 2014 Angiolillo DJ, Capodanno D, Goto S. Platelet thrombin receptor antagonism and atherothrombosis. European heart journal. Jan 2010 Dick F, Ricco JB, Davies AH, et al. Chapter VI: Follow-up after revascularisation. European journal of vascular and endovascular surgery : Dec 2011 Cacoub PP, Bhatt DL, Steg PG, Topol EJ, Creager MA. Patients with peripheral arterial disease in the CHARISMA trial. European heart journal. Jan 2009 Spiliopoulos S, Pastromas G, Katsanos K, Kitrou P,

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Karnabatidis D, Siablis D. Platelet responsiveness to clopidogrel treatment after peripheral endovascular procedures: the PRECLOP study: clinical impact and optimal cutoff value of on-treatment high platelet reactivity. Journal of the American College of Cardiology. Jun 2013 Pastromas G, Spiliopoulos S, Katsanos K, et al. Clopidogrel responsiveness in patients undergoing peripheral angioplasty. Cardiovascular and interventional radiology. Dec 2013 Attubato CKABBSFFJSM. Dual antiplatelet therapy responsiveness in patients undergoing percutaneous revascularization for peripheral arterial occlusive disease. Journal of American college of cardiology. 2012 Hamish M, Gohel MS, Shepherd A, Howes NJ, Davies AH. Variations in the pharmacological management of patients treated with carotid endarterectomy: a survey of European vascular surgeons. European journal of vascular and endovascular surgery :. Oct 2009 Hayes PD, Box H, Tull S, Bell PR, Goodall A, Naylor AR. Patientsâ&#x20AC;&#x2122; thromboembolic potential after carotid endarterectomy is related to the plateletsâ&#x20AC;&#x2122; sensitivity to adenosine diphosphate. Journal of vascular surgery. Dec 2003 Payne DA, Jones CI, Hayes PD, et al. Beneficial effects of clopidogrel combined with aspirin in reducing cerebral emboli in patients undergoing carotid endarterectomy. Circulation. Mar 2004 Sharpe RY, Dennis MJ, Nasim A, et al. Dual antiplatelet therapy prior to carotid endarterectomy reduces post-operative embolisation and thromboembolic events: postoperative transcranial Doppler monitoring is now unnecessary. European journal of vascular and endovascular surgery Aug 2010 Timaran CH, Mantese VA, Malas M, et al. Differential outcomes of carotid stenting and endarterectomy performed exclusively by vascular surgeons in the Carotid Revascularization Endarterectomy versus Stenting Trial (CREST). Journal of vascular surgery. Feb 2013 de Borst GJ, Hilgevoord AA, de Vries JP, et al. Influence of antiplatelet therapy on cerebral micro-emboli after carotid endarterectomy using postoperative transcranial Doppler monitoring. European journal of vascular and endovascular surgery :. Aug 2007 Hamm CW, Bassand JP, Agewall S, et al. ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: The Task Force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). European heart journal. Dec 2011 Price MJ, Berger PB, Teirstein PS, et al. Standardvs high-dose clopidogrel based on platelet

General discussion and summary

function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA : Mar 2011 25. Trenk D, Stone GW, Gawaz M, et al. A randomized trial of prasugrel versus clopidogrel in patients with high platelet reactivity on clopidogrel after elective percutaneous coronary intervention with implantation of drug-eluting stents: results of the TRIGGER-PCI (Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy With Prasugrel) study. Journal of the American College of Cardiology. Jun 2012 26. Aradi D, Storey RF, Komocsi A, et al. Expert position paper on the role of platelet function testing in patients undergoing percutaneous coronary intervention. European heart journal.

Jan 2014 27. Steg PG, James SK, Atar D, et al. ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation. European heart journal. Oct 2012 28. Karkouti K, Wijeysundera DN, Yau TM, et al. The independent association of massive blood loss with mortality in cardiac surgery. Transfusion. Oct 2004 29. Koch CG, Li L, Duncan AI, et al. Morbidity and mortality risk associated with red blood cell and blood-component transfusion in isolated coronary artery bypass grafting. Critical care medicine. Jun 2006

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Chapter 13

Dit proefschrift betreft onderzoek naar de rol van gepersonaliseerde behandeling met bloedplaatjesremmers bij patiënten met vaatziekten in de benen, halsslagaders en kransslagaders van het hart. Slagaderverkalking (atherosclerose) Hart- en vaatziekten zijn nog steeds de belangrijkste oorzaak van sterfte en verantwoordelijk voor meer dan 25% van de wereldwijde mortaliteit. Slagaderverkalking, ook wel atherosclerose genoemd, is de belangrijkste oorzaak van hart- en vaatziekten. De grootste gevolgen van atherosclerose zijn coronaire hartziekten (hartinfarct en angina pectoris), beroerten en vaatlijden in de benen (perifeer arterieel vaatlijden). Atherosclerose is een aandoening van de middelgrote en grote slagaders (arteriën) en is bij iedereen in meer of mindere mate aanwezig. De vaatwand bestaat uit drie lagen, de binnenste laag: tunica intima, de middelste laag: tunica media en de buitenste laag: tunica adventitia. De tunica intima bestaat uit een aaneengesloten laag van gladde, platte en uitgestrekte cellen, het endotheel. Bij geboorte is de binnenzijde van de vaatwand nagenoeg glad. Echter al snel ontwikkelt het eerste stadium van atherosclerose, de ‘’fatty streak’’ waarbij ontstekingscellen (monocyten) de vaatwand binnen dringen en aldaar transformeren in macrofagen. Deze macrofagen eten het aanwezige cholesterol (om cholesterol ‘’op te ruimen’’) en zwellen op. De macrofagen geven groeifactoren af waardoor de omliggende gladde spiercellen zich gaan delen. Deze toename in spiercellen zorgt voor een toename van schade aan de vaatwand en toename van het onstekingsproces met het aantrekken van nieuwe ontstekingscellen. De atherosclerotische plaque ontstaat wanneer dit onstekingsproces wordt omkapseld met een nieuw gevormde fibreuze (uit bindweefsel bestaande) kap. Op een zeker moment dringt de plaque het lumen (holte) van de arterie binnen en reduceert het de bloedstroom. Ook kan de plaque scheuren, waardoor trombose ontstaat aan het oppervlak van de plaque of stukjes van de plaque het bloedvat inschieten (emboli) en leiden tot klinische symptomen als hersen- of hartinfarct. Hoewel iedereen een zekere mate van atherosclerose ontwikkelt, zijn er een aantal risicofactoren bekend die sterk gerelateerd zijn aan het krijgen van atherosclerose zoals roken, hoge bloeddruk, overgewicht, weinig lichaamsbeweging, diabetes mellitus, erfelijkheid en een hoog cholesterol. Bloedplaatjes Bloedplaatjes zijn de kleinste cellen in het bloed en spelen een essentiële rol in de bloedstolling. De levensduur van een bloedplaatje is 8- 10 dagen en de afbraak vindt plaats in de milt, lever en longen. Bloedstolling is een buitengewoon complex proces waarbij vooral bloedplaatjes en stollingsfactoren (eiwitten) betrokken zijn. Na het optreden van vaatwandschade treedt eerst vasoconstrictie op, het samentrekken van het beschadigde bloedvat. De bloedplaatjes reageren op onregelmatigheid door te hechten aan het vrijgekomen bindweefsel (collageenvezels). Zodra de bloedplaatjes aan collageen gehecht zijn,

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worden ze geactiveerd waardoor bloedplaatjes aan elkaar kunnen binden en verscheidene stoffen vrijgeven. De vrijgekomen stoffen zijn onder andere serotinine, een vasoconstrictor, en adenosine difosfaat (ADP) dat zelf weer nieuwe bloedplaatjes activeert. Door deze cascade ontstaat een stevige bloedprop die de beschadigde vaatwand afsluit en overmatig bloedverlies tegen gaat. Deze bloedprop vormt zelf weer een oppervlak voor het binden van stollingsfactoren en verdere stappen in de bloedstolling. Geactiveerde bloedplaatjes spelen een belangrijke rol in de vorming van bloedpropjes en kunnen worden geactiveerd door zowel vaatwandschade als plaqueonregelmatigheden waarbij collageen, trombine, ADP en/of thromboxaan is vrijgekomen. Bloedplaatjesremmers en tailored antiplatelet therapy Patiënten die een cardiovasculair event zoals hart- of herseninfarct hebben doorgemaakt, worden behandeld met bloedplaatjesremmers (o.a. aspirine en clopidogrel). Het doel van deze bloedplaatjesremmers is om bloedplaatjes, de cellen die stolling initiëren bij vaatwandschade, minder werkzaam te maken. Hierdoor reduceert de kans op stolsels in de vaten en dus het risico op volgende cardiovasculaire events. De komst van aspirine als secundaire profylaxe voor cardiovasculaire events heeft geresulteerd is een forse afname van recidief klachten in patiënten met bekende vaatziekten. Aspirine zorgt voor een blokkade van thromboxaan vorming, en dus vermindering van bloedplaatjesactivatie. Na de komst van sterkere bloedplaatjesremmers, zoals clopidogrel, prasugrel en ticagrelor, is het aantal recidiverende klachten nog verder afgenomen. Dit gaat echter ten koste van een toename in aantal bloedingen. Monotherapie met aspirine of duaal therapie met bijvoorbeeld aspirine én clopidogrel is inmiddels de gouden standaard geworden als primaire of secundaire preventie of gedurende ingrepen aan hart- en vaten. Uit voorgaand onderzoek is gebleken dat ongeveer 20% van de patiënten behandeld met aspirine en 40% van de patiënten behandeld met clopidogrel, nog steeds een verhoogde bloedplaatjes activiteit vertonen met een bijbehorend verhoogd risico op het ontwikkelen van een volgend cardiovasculair event. In sommige patiënten kom dit door een mutatie in de genen die coderen voor cytochroom P450 2C19 (CYP2C19) activiteit, een enzym die clopidogrel omzet naar het actieve metaboliet (eindproduct). Andere onafhankelijk factoren die zijn gerelateerd aan hoge restactiviteit van bloedplaatjes ondanks clopidogrel behandeling zijn therapie-ontrouw, diabetes mellitus, nierfalen, een snelle vervanging van de bloedplaatjes en niet- roken. Aan de andere kant van het spectrum resulteert een zeer verlaagde bloedplaatjes restreactiviteit in een toename in bloedingen. Het is mogelijk om de restactiviteit van bloedplaatjes te meten in het bloed van patiënten. Momenteel zijn er zeer veel verschillende bloedtesten beschikbaar om bloedplaatjesactiviteit te meten. Helaas zijn deze bloedtesten tot nu toe te arbeidsintensief of te weinig specifiek om in de kliniek individuele patiënten met een afwijkende restactiviteit van de bloedplaatjes aan te wijzen. Gezien het risico van trombose aan de ene kant van het spectrum en het risico van

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bloeding aan de andere kant van het spectrum, lijkt er sprake van een therapeutische breedte van de bloedplaatjesremmers. Het concept tailored antiplatelet therapy past op basis van de uitslagen van de bloedtesten de medicamenteuze therapie aan door aanpassingen in dosering of vervanging door een ander type bloedplaatjesremmers. Het doel is om hiermee zowel het aantal trombotische events als het aantal bloedingen te reduceren. Doelen van dit proefschrift Dit proefschrift bevat studies naar de restactiviteit metingen van de bloedplaatjes in patiënten met (on)stabiele angina pectoris, perifeer vaatlijden en halsslagaderverkalking ter voorbereiding op het concept tailored antiplatelet therapy. We hebben een bloedplaatjesreactiviteitstest ontwikkeld die verschillende activatieroutes en activatiemarkers van bloedplaatjes kwantificeert, de prognostische waarde van deze test onderzocht in verschillende typen vaatpatiënten en we hebben deze test vergeleken met de huidige klinische gouden standaard, de VerifyNow. Daarnaast hebben we gezocht naar de optimale manier van bloedplaatjesactiviteit metingen, het tijdstip en de rol van de leeftijd van bloedplaatjes op hun restactiviteit. De belangrijkste bevindingen van het proefschrift zijn hieronder beschreven. Conclusie en toepassingen Het proefschrift bestaat uit zes delen. Deel I geeft een algemene introductie over vaatziekten en de doelstellingen van dit proefschrift. Achtereenvolgend worden studies beschreven over bloedplaatjesreactiviteit bij patiënten met vaatziekten in de benen (deel II), vaatziekten in de halsslagaders (deel III) en vaatziekten in het hart (deel IV). In deel V worden alle bevindingen samengevat in een Engelstalig en Nederlandstalig overzicht. Deel VI omvat de appendix met een overzicht van de leescommissie, het dankwoord, de publicatielijst en het curriculum vitae van de auteur. Deel I begint met hoofdstuk 1 met een introductie over vaatziekten, de rol van bloedplaatjes bij vaatziekten, bloedplaatjesremmers als therapie bij vaatziekten en uitleg over het concept tailored antiplatelet therapy. Het hoofdstuk besluit met de doelstellingen van dit proefschrift. In hoofstuk 2 worden de resultaten beschreven van een studie naar het effect van P2Y12 inhibitie (bijvoorbeeld door clopidogrel) op αIIbβ3-activatie, P-selectin en CD63expressie, aggregaat vorming en uitstoot van vrijgekomen stoffen uit de alpha en dense granules. P2Y12 inhibitors hadden een sterk effect op dense granule secretie na PAR- en CRP geïnduceerde plaatjesactivatie, maar niet op alpha granule secretie. Een deel van de patiënten die door de VerifyNow als ‘goede responders’ op bloedplaatjesremmers werden geïdentificeerd, vertoonde normale fibrinogeen binding gemeten met flow cytometrie na stimulatie met PAR- agonisten of CRP, ondanks volledige remming van fibrinogeen na ADP stimulatie. Dit wijst op een suboptimale bloedplaatjesremming ondanks medicamenteuze behandeling, geclassificeerd door flow cytometrie.

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Geconcludeerd identificeren flow cytometrie en VerifyNow andere patiënten als ‘nonresponders’ op bloedplaatjesremmers. Klinische vervolgstudies zullen moeten uitwijzen welke van de testen de beste voorspellende waarde heeft op klinische trombotische eindpunten. Deel II van het proefschrift richt zich op bloedplaatjesreactiviteit in patiënten met perifeer arterieel vaatlijden. In hoofdstuk 3 wordt de casus van een 73-jarige patiënt beschreven met acute verergering van zijn vaatlijden door een occlusie van zijn beenslagader, de arterie femoralis superficialis. Hij is behandeld met trombosuctie en vervolgens een dotter (percutane transluminale angioplastiek [PTA]) met stentplaatsing. Hierna werd de patiënt behandeld met duale bloedplaatjesremmers echter uit de VerifyNow bleek dat de remming van zijn bloedplaatjes hierop 0% was. Er is een genetische test verricht en er bleek sprake te zijn van een CYP2C19 mutatie. Om deze reden is prasugrel gestart welke bij testen wel effectief bleek te zijn (remming 41%). Bovenstaande casus is een voorbeeld van tailored antiplatelet therapy. Echter in perifeer vaatlijden zijn er weinig studies gedaan naar de optimale bloedplaatjestest en bijbehorende referentiewaarden, het ideale tijdstip voor testen en de beste volgende medicamenteuze optie indien de intiele bloedplaatjesremmer niet voldoende blijkt te werken. In hoofdstuk 4 beschrijven wij de resultaten van een kleine pilot studie in 30 patiënten met perifeer vaatlijden die een PTA met stentplaatsing hebben ondergaan en behandeld zijn met duale plaatjesremmers. Een dag na de ingreep zijn drie typen bloedplaatjestesten en een DNA test afgenomen. Het belangrijkste resultaat van de studie was een buitengewoon hoog percentage non-responders op bloedplaatjesremmers één dag na de ingreep. Bij enkele patiënten is de bloedplaatjesreactiviteit op latere momenten gemeten waarbij wé een goede remming gezien werd. Dit suggereert dat meten één dag na de ingreep (en dus starten van de duaal therapie) te vroeg is. Daarnaast werd er weinig overeenkomst gezien van de resultaten tussen de verschillende bloedplaatjestesten. Deel III van het proefschrift richt zich op bloedpaatjesreactiviteit in patiënten met een vernauwing in de halsslagader. Hoofdstuk 5 geeft een overzicht van de literatuur over bloedplaatjesremmers en bloedplaatjestesten in patiënten die carotis revascularisatie ondergaan en het postoperatieve traject. Het hoofdstuk concludeert dat er te weinig studies zijn vervaardigd om een uitspraak te doen over het juiste type bloedplaatjesremmers en het nut van bloedplaatjestesten in deze patiëntengroep. Er lijkt een voordeel te bestaan voor het gebruik van duale remming met clopidogrel en aspirine maar mogelijk verdwijnt dit voordeel met de komst van sterkere plaatjesremmers zoals prasugrel, ticagrelor maar ook cilostazol en GPIIb/IIIa- antagonisten. Patienten met een halsslagadervernauwing kunnen micro embolieën schieten in de cerebrale circulatie. Deze micro embolische signalen (MES) kunnen gedetecteerd worden met de transcraniële doppler (TCD) in de arteria cerebri media. De micro embolieën bestaan voornamelijk uit klonters van bloedplaatjes. Een toename van aantal geregistreerde MES is dan ook gerelateerd aan een verhoogd risico op herseninfarct.

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Goede diagnostiek van het aantal MES is dan ook noodzakelijk om patiënten met een verhoogd risico te identificeren. In hoofdstuk 6 wordt automatisch micro embolie detectie software gevalideerd ten opzichte van de gouden standaard, de human expert. Geregistreerde signalen door de TCD zijn tweemaal door twee onafhankelijke human experts geclassificeerd als zijnde artefact, vaste- of gasembolie. De overeenkomst met de classificatie van de software werd berekend. Helaas was de overeenkomst tussen de human experts en de software laag in het classificeren van MES als vaste embolieën. De human expert en software identificeren andere patiënten als zijnde ‘’at risk’’ voor herseninfarct (≥20 vaste micro embolieën). Derhalve kan de automatische software de human experts op dit moment nog niet vervangen. Hoofdstuk 7 beschrijft een studie naar de associatie van bloedplaatjesreactiviteit gemeten met flow cytrometrie en de VerifyNow (aggregatie test) met als primaire uitkomstmaat het aantal MES gemeten door de TCD tijdens carotis endarterectomie en als secundaire uitkomstmaat klinische cardiovasculaire events. De resultaten toonden dat alleen hoge bloedplaatjes reactiviteit gemeten door flow cytometrie na stimulatie met ADP een verhoogd risico op ≥20 micro embolieën tijdens carotis endarterectomie identificeerde. Behandeling met clopidogrel rondom de operatie bleekt het sterkste effect te hebben op een op lagere bloedplaatjesactiviteit en aantal micro embolieën. Of clopidogrel behandeling resulteert in een vermindering van aantal herseninfarcten, zal nog moeten worden onderzocht. Nadat een carotis endarterectomie kan de baat van de ingreep worden teniet gedaan door het optreden van een (nieuwe) ipsi- of contralaterale stenose. Hoofdstuk 8 toont de resultaten van een studie naar de identificatie van klinische risicofactoren en plaque karakteristieken die geassocieerd zijn met het ontwikkelen van een nieuwe contralaterale stenose. Van de 760 patiënten die een carotis endarterectomie ondergingen, ontwikkelde 108 patiënten (20%) een contralaterale stenose na een mediane follow- up van 2.5 jaar. Dissectie van een vetarme, collageenrijke of gladde spiercelrijke plaque was geassocieerd met het ontwikkelen van een nieuwe contralaterale stenose. Patiënten die zich initieel asymptomatisch presenteerden hadden een verhoogd risico op contralaterale stenose. Deel IV van het proefschrift richt zich op bloedplaatjesreactiviteit in patiënten met hartziekten. Hoofdstuk 9 richt zich op de andere kant van het spectrum van bloedplaatjesreactiviteit, namelijk het risico op bloeding. De nieuwere, sterkere plaatjesremmers leiden vaak tot lagere bloedplaatjesreactiviteit met mogelijk een toename in bloedingen. Preoperatieve bloedtesten in patiënten die hartoperaties ondergaan, zouden zinvol kunnen zijn om patiënten met een verhoogd risico op bloeding tijdens en na de operatie te identificeren. In dit hoofdstuk worden meerdere studies besproken die een positief resultaat laten zien van verschillende bloedtesten om hoogrisico patiënten te identificeren. Echter het nut van aanpassen van de bloedplaatjesremmers in hoogrisico patiënten moet nog worden onderzocht.

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In patiënten met een acuut coronair syndroom is hoge bloedplaatjesreactiviteit geassocieerd met een toegenomen risico op cardiovasculaire events. Voor patiënten die een electieve percutane coronaire interventie (dotter [PCI]) ondergaan is deze relatie echter nooit aangetoond. In hoofdstuk 10 worden de resultaten van een studie met patiënten die een diagnostische angiografie ondergaan op basis van stabiel of onstabiele coronair lijden beschreven. De associatie tussen de indicatie (stabiel en onstabiel coronair lijden) met de bloedplaatjesreactiviteit werd onderzocht bij 195 patiënten waarbij werd gecorrigeerd voor P2Y12 inhibitor gebruik. Tegen de verwachting in werd gezien dat voor patiënten zonder P2Y12 inhibitor, een lagere bloedplaatjesreactiviteit geassocieerd was met onstabiel coronairlijden. Ook was er een grotere noodzaak voor reïnterventie bij patiënten met lagere bloedplaatjesreactiviteit. Deze associaties werden niet gezien voor patiënten die wel P2Y12 inhibitors gebruikten. In het laatste hoofdstuk wordt een mogelijke nieuwe marker voor een verhoogd cardiovasculair risico onderzocht. Hoofstuk 11 toont de resultaten van een studie naar de preoperatief voorspellende waarde van de jonge, reticulated bloedplaatjes (rP) op het optreden van postoperatieve verhoogde troponine waarden en 30- dagen mortaliteit bij patiënten die major non-cardiac surgery ondergaan. In totaal had 26.5% (607/2289) preoperatief een verhoogd percentage rP. Een verhoogd percentage rP was onafhankelijk geassocieerd met postoperatief verhoogde troponine waarde en 30-dagen mortaliteit. Hoge rP reflecteert mogelijk uitgebreid cardiovasculair vaatlijden. Toekomstige studies naar het aanpassen van cardiovasculair risico management n.a.v. de rP meting zal de klinische relevantie van deze resultaten moeten aantonen. Dit proefschrift toont aan dat hoewel het concept ‘tailored antiplatelet therapy’ veelbelovend is er nog vele grootschalig klinische onderzoeken moeten plaats vinden naar o.a. de beste bloedplaatjestest, de afkapwaarden van deze bloedtesten per vaataandoening, de optimale standaard bloedplaatjesremmers per vaataandoening en de optimale volgende keuze van bloedplaatjesremmer bij patiënten met hoge plaatjesreactiviteit ondanks gebruik van standaard bloedplaatjesremmers. Tot slot, bloedplaatjesmetingen in patiënten met vaatziekten ter monitoring en in de toekomst gepersonaliseerd aanpassen van de bloedplaatjesremmers zal essentieel worden om een balans te houden tussen het trombotische- en bloedingsrisico.

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PART VI Appendix

Review committee Dankwoord Authorâ&#x20AC;&#x2122;s list of publications Curriculum Vitae

Appendix

Review committee Prof. dr. S.A.J. Chamuleau Department of Cardiology University Medical Center Utrecht, the Netherlands Prof. dr. J.F. Hamming Department of Vascular Surgery Leiden University Medical Center, the Netherlands Prof. dr. L.J. Kappelle Department of Neurology University Medical Center Utrecht, the Netherlands Prof. dr. W.A. van Klei Department of Anesthesiology University Medical Center Utrecht, the Netherlands Prof. dr. F.L.J. Visseren Department of Vascular Medicine University Medical Center Utrecht, the Netherlands

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Dankwoord Eind 2013 mocht ik op gesprek komen bij Prof. Moll en (toen nog) Dr. de Borst om de mogelijkheden van onderzoek binnen de vaatchirurgie te bespreken. Ik werd warm welkom geheten binnen het UMCU met een prodent- smile en grootste plannen. Hoe had ik toen ooit kunnen bedenken dat dit gesprek de start zou zijn van een fantastische periode waarin ik niet alleen op wetenschappelijk gebied, maar ook op persoonlijk vlak enorm veel van mijn begeleiders heb geleerd. Beste Prof. dr. F.L. Moll, allereerst dank voor de mogelijkheid die u mij heeft gegeven om onderzoek te komen doen binnen uw vakgroep. Vanaf het eerste moment heeft u me steun en vertrouwen gegeven en ik wil u bedanken voor alle wijze lessen, die overigens vaker níet dan wél over wetenschap gingen. Met uw motiverende woorden ging ik altijd vol vertrouwen weer de kamer uit om vervolgens de lat nog hoger te leggen. Bedankt voor de wereldwijze blik die u uw promovendi mee geeft. Deze ‘’bitch’’ (het zijn uw woorden) is enorm trots dat ze onder uw begeleiding mag promoveren! Beste Prof. dr. GJ. de Borst, beste Gert Jan, ik had me geen betere promotor kunnen wensen. Jij had eerder vertrouwen in mij dan dat ik het zelf had en je hebt me verder gebracht dan ik had durven denken. Ik heb enorm veel waardering voor de manier waarop jij de scepter zwaait binnen de vaatchirurgie: oprecht, vol kennis en kunde, recht door zee, altijd vriendelijk, geduldig en fanatiek. Jij bent mijn mentor en een groot voorbeeld: niets is onmogelijk. Door jouw enthousiasme en vertrouwen heb ik mijn weg gevonden binnen de chirurgie. Bedankt voor alle mooie onderzoekservaringen in binnenén buitenland. Ik hoop nog veel van je te mogen leren in de toekomst en kijk uit naar jouw officiële oratie. Beste Dr. Urbanus, beste Rolf, jij bent de motor van de bloedplaatjesgroep. Ik wil je enorm bedanken voor jouw opvang binnen het LKCH toen mijn directe begeleiding wegviel. Ik bewonder je gedrevenheid, oog voor detail, bedachtzaamheid en jouw daadkracht. Jij hebt mijn onderzoek naar een hoger niveau gebracht met jouw kritisch blik, indrukwekkende bloedplaatjeskennis, statistische vaardigheden en taalgevoel. Daarnaast waardeer ik jouw rust en aandacht voor persoonlijke zaken. Na een gesprek met jou verliet ik altijd gerustgesteld en met strakke planning weer de kamer. Ik ben blij dat je mijn copromotor bent. Beste Dr. Roest, beste Mark, wat heb ik genoten van onze samenwerking! Jij bent een uniek persoon: excentriek en ongeremd, eerlijk en oprecht en heerlijk impulsief. Ondanks onze korte samenwerking in het UMCU ben je altijd nauw betrokken gebleven bij mijn promotietraject. Je hebt een verfrissende blik op de manuscripten en altijd goede ideeën voor nieuwe onderzoeksprojecten. Dank voor de leuke tripjes naar Chicago, Toronto,

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de NVTH en niet te vergeten de Dam-tot-Dam loop. Ik wens je veel succes toe in Maastricht en volgend jaar september loop ik je er wél uit (!) Geachte leden van de leescommissie: Prof. dr. Visseren, Prof. dr. Chamuleau, Prof. dr. Hamming, Prof. dr. Kappelle en Prof. dr. van Klei, graag wil ik jullie bedanken voor de kritische beoordeling van mijn proefschrift en de bereidheid plaats te nemen in de leescommissie. Prof. dr. Mess en Dr. Scheltinga, bedankt dat jullie zitting willen nemen in de oppositie. Dr. Scheltinga, dank voor het allereerste zetje in de juiste richting. Naast de hulp van mijn (co-)promotoren, is dit proefschrift tot stand gekomen met de hulp van vele anderen. Hoewel de lijst onuitputtelijk is, wil ik graag in het bijzonder nog de volgende mensen bedanken: Beste Prof. dr. Pasterkamp, beste Gerard, hoogleraar experimentele cardiologie, afdelingshoofd experimentele cardiologie & LKCH, wetenschapper, epidemioloog, clinicus, zakenman en sportman in één met, zoals je dat wellicht normaal juist van de chirurgie gewend bent, perfecte verpakking. Bedankt voor jouw interesse in zowel mijn onderzoek als in mij als persoon. Jouw reactie na het vertrek van mijn copromotor was goud waard, dank hiervoor. Beste Dr. Korporaal, beste Suus, ik heb bewondering voor de manier waarop jij je, als enige dame in de bloedplaatjesgroep, al jaren weet te profileren. Wat fijn dat je er was! Ik heb enorm genoten van zowel jouw (zeer kritische) wetenschappelijke bijdragen, als jouw zorgzame persoonlijkheid: T&H barbecues in de achtertuin, koffieleuten, samen winkelen in Toronto en jouw nachtelijke strijkpartij na de GT’s. Dank! Beste Peter Paul Wisman, als ik aan iemand veel te danken heb dan is dat aan jou. Dank voor je hulp, uitleg en kritische blik op de studies. Ik hoop dat het geworden is zoals jij het ooit voor ogen had. Nooit had ik verwacht dat ik het zo naar mijn zin zou hebben op een laboratorium en dat zal dan ook grotendeels door de collega’s komen: LKCH, dank voor jullie open houding naar weer zo’n witte jas, alle uitleg en begeleiding maar vooral voor de prettige (humoristische) werksfeer. De volgende mensen wil ik in het bijzonder bedanken: Ivar van Asten, van student naar PhD- student, ontzettend goed gedaan! Veel succes met je eigen onderzoek en je carrière als klinisch chemicus. Steven de Maat, dank voor alle (on) zin op en buiten het lab. Wees zuinig op die leuke vriendin van je. Tesy Merkx, dank voor het vele pipeteerwerk waarmee je me hebt geholpen. Arnold en Arjan, de vaste steunpilaren in het lab, dank voor al jullie hulp met proeven, het repareren van de flow cytometer en vanzelfsprekende aanwezigheid bij het maken van filmpjes en borrels. Sander Kooijmans, Susan van Dommelen, Jessica Molhoek, Rick Huisjes, Marco van der Stoep, Mirjam Mebius, dank voor het delen van de promotiefrustraties, veel succes nog

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in jullie verdere carrières! Ray Schiffelers, Coen Maas, Harry Heijnen, Pieter Vader, Marcel Fens en Roy van der Meel, dank voor jullie motiverende enthousiasme voor de wetenschap! Secretaresses van de Heelkunde en in het bijzonder Susan en Cobie, dank voor de hulp bij het plannen én cancelen van al die (on)mogelijke afspraken. Maar bovenal, bedankt voor al de tijd die ik bij jullie kwam volkletsen als ik op de professoren aan het wachten was. Ik ga jullie missen! Dank aan alle auteurs die hebben meegewerkt aan de manuscripten: Prof. dr. de Groot, Prof. dr. Asselbergs, Prof. dr. Mess, Prof. dr. Verhaar, Dr. Voncken, Hester den Ruijter, Martin Teraa, Albert Huisman, Maarten ten Berg, Imo Hoefer, Judith van Waes, Crystel Gijsberts, Thijs van Holten, Arnold Koekman, Daniel van Vriesland en Sophie Merckelbach. Jurriën ten Berg en Paul Janssen, hopelijk volgen er nog vele succesvolle samenwerkingen. Tamara Marees en Marianne van Es, dank voor het wegwijs maken in het doolhof van regels en formulieren rondom het opstarten van onderzoek. Wat fijn dat jullie er zijn voor alle bleue nieuwe onderzoekers. Beste laboranten van de klinische neurofysiologie, beste Daniel, Barbara, Elise, Anja en William, dank voor alle uren TCD-metingen die jullie hebben verricht. Daniel, dank voor je enthousiasme en vooral geduld met het scoren van de embolieën! Heel veel dank ook aan alle vaatchirurgen en PA’s in het UMCU voor de hulp bij het includeren van de studiepatiënten en de feedback bij besprekingen: Joost van Herwaarden, Raechel Toorop, Stijn Hazenberg, Bart-Jeroen Petri, Boudewijn Reichmann, Paul van Schaik, Olaf Bakker, Anouk Jansze, Marye Rutten en Trijntje Tuin. Alle stafleden en arts-assistenten vanuit het UMCU: dank voor het warme welkom en de gezelligheid zowel in het ziekenhuis als bij de wetenschapsdag- en diner, skireizen, chirurgencup en overige activiteiten. Wat een heerlijke club bij elkaar! Lieve Toren 5.0: Stefanie Peters-Weem, Laurien Waaijer, Anne Huibers, Sjoerd Nell, Steven van Haelst, Jakob Kist en Kevin Parry. Promoveren was heel wat zwaarder geweest zonder jullie! Dag in, dag uit samen werken in de toren, gezamenlijk sporten, congressen bezoeken, internationale tripjes, een fietsweekend bij mijn ouders en ontelbare feestjes… Volgens mij valt dit buiten de term ‘’collega’’. Dank voor de fantastische tijd! De enorme club (nieuwe) vaatonderzoekers: Aarent Brand, Armelle van Meershoek, Djurre de Waard, Evelien de Vries, Joep Wijnands, Leonie Fassaert en Vanessa Pourier. Veel succes met het vervolgonderzoek, jullie weten het ongetwijfeld tot een mooi einde te brengen.

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Mede-chirurgie onderzoekers van het UMCU: samen is alles leuker. Lieve Kari Trumpi, Mark Haverkort, Lucas Goense en Lutske Lodewijk, hoewel wij op onderzoeksgebied volstrekt niets met elkaar te maken hebben gehad, wil ik jullie toch graag bedanken voor de bijzondere momenten die zijn geweest en hopelijk nog gaan komen! Veel dank gaat verder uit naar: Amy Gunning, Diederik Smeeing, Dorothee van Trier, Els Visser, Hylke Brenkman, Ian van Koevorden, Inge Ubink, Janine Simons, Jasmijn Smits, Leonie Haverkamp, Linde van Veenendaal, Maarten Burbach, Maarten Seesing, Marieke Walma, Morsal Samim, Nicola Frenkel, Peter van Rossum, Pien Hellebrekers, Pieter Leliefeld, Quirina de Ruijter, Steffi Rombouts en Thomas Vellinga. Stafleden en assistenten van het Diakonessenhuis, dank voor jullie geduld, wijze adviezen, gezelligheid en met name aanstekelijke enthousiasme. Vanaf nu kan ik me volledig richten op de mooie dingen in het Diakonessenhuis. Ik voel me in deze paar maanden al enorm bij jullie thuis en hoop nog lang met jullie samen te werken! Dr. Weits, dank voor de mooie afbeelding op de omslag van dit boekje! Lieve dames van Bon’ Aparte, speciaal lieve Josyne, Liza, Maran, Juliette, Josje, Barbara, Annemarie en Eveline, ik prijs me gelukkig met jullie om me heen. Jullie weten als geen ander dat de boog niet altijd gespannen kan zijn en hebben me dan ook met overtuiging afgeleid van mijn promotie. Dank daarvoor! 030, dat iedereen maar voor altijd in Utrecht blijft wonen! Lieve boekenclub (Voes, Layla, Lotte, Anna, Michelle en Marjolein), onze boekenclub staat nu officieel op papier dus we kunnen het niet meer ontkennen. De meest veelzijdige boekenclub die ik ken; ADE, internationaal naar Oxford, gay bar, 90’s party, heerlijke diners en eindeloze discussies. Met pijn in mijn hart even terug getrokken maar nu dit boek af is (als eerste op de lijst voor 2017) graag weer present! Zuid Afrika 2011: Lieve Jessica, Diane, Myrna en Mark. Het meest onwaarschijnlijke clubje bij elkaar maar toch hebben we inmiddels ons 5-jarig jubileum gevierd. Dank dat jullie me eraan herinneren dat er nog een wereld is buiten de medische wereld. Dank voor jullie inzichten en wereldwijsheid. Bovenal, dank dat jullie er altijd voor me zijn en begrip tonen. Lieve Wim, Simone, Suze en Michelle, ik prijs me gelukkig dat ik jullie er zomaar bij heb gekregen. Wat ben ik trots deze dag tegemoet te gaan met mijn paranimfen aan mijn zijde: Tes, je was er, je bent er en je blijft altijd. Eén ding is zeker, wat de toekomst ook brengt: samen gaan we er iets moois van maken! Jennifer, Chirurgencup 2014 in combinatie met een paar La Chouffe bleek de basis voor een nieuwe vriendschap. Ik heb de afgelopen jaren

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fantastische hoogtepunten met je beleefd en ik hoop dat er dit in de toekomst alleen maar meer worden! Lieve Rik, mijn grote broer. Jij bent altijd mijn voorbeeld geweest en ik wilde alles doen wat jij ook deed. Inmiddels zijn we beiden een geheel eigen kant op gegaan en vullen we elkaar perfect aan. Dank voor jouw steun! Lieve Nanet, wat fijn dat jij ook bij onze familie hoort! Lieve papa en mama, ik vrees dat ik het nooit zo heb gezegd, maar ik had me geen betere ouders kunnen wensen. Dank voor de onvoorwaardelijke liefde en de ruimte die jullie me hebben gegeven. Hoewel ook kleine meisjes groot worden, maak ik nog steeds graag gebruik van jullie (toch wel wijze) adviezen en hoop ik dit nog heel lang te kunnen doen! Lieve Hinke, zo hetzelfde en zo verschillend... Je begrijpt me als geen ander, je daagt me uit, stimuleert me en laat zien dat niets onmogelijk is. Ik ben ontzettend trots op dat gekke vriendje van me en je maakt me zielsgelukkig. Ik wil niets liever dan samen met jou een prachtige toekomst aangaan. Heb ik trouwens wel eens gezegd dat ik je leuk vind?!

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Author’s list of publications T. Leunissen, PP. Wisman, D. van Vriesland, L. Schropp, S. Korporaal, F. Moll, G. Pasterkamp, M. Roest, R. Urbanus and GJ. de Borst. Clopidogrel is the strongest suppressor of platelet reactivity and perioperative solid micro emboli during carotid endarterectomy. Manuscript in preparation. T. Leunissen, J. van Waes, W. van Klei, A. Huisman, F. Moll and GJ. de Borst. Reticulated platelets as predictor of myocardial injury and 30-day mortality after noncardiac surgery. Submitted to the British Journal of Surgery. T. Leunissen, D. van Vriesland, H. de Ruijter, F. Moll, W.H. Mess and GJ. de Borst. Validation of the automated electronic microemboli detection system in patients undergoing carotid endarterectomy. European Journal of Ultrasound (accepted under conditions). T. Leunissen, PP. Wisman, T. van Holten, P. de Groot, S. Korporaal, A. Koekman, F. Moll, GJ. de Borst, M. Teraa, M. Verhaar , R. Urbanus, M. Roest; The effect of P2Y12 inhibition on platelet activation assessed by aggregation- and flow cytometry based assays. Platelets 2016 (in press). T. Leunissen*, C. Gijsberts*, PP. Wisman, F. Asselbergs, I. Hoefer, G. Pasterkamp, GJ. de Borst, F. Moll and M. Roest. Lower platelet reactivity is associated with unstable coronary artery disease. International Journal of Angiology 2016 (in press) T. Leunissen*, S. Peeters Weem*, R. Urbanus, H. den Ruijter, F. Moll, F. Asselbergs and GJ. de Borst. High on-treatment platelet reactivity in peripheral arterial disease – finding the optimal test and cut-off values. European Journal of Vascular and Endovascular surgery: August 2016; 52: 198-204. S. Merckelbach, T. Leunissen, J. Vrijenhoek, F. Moll, G. Pasterkamp and GJ. de Borst. The contralateral carotid artery in patients undergoing carotid endarterectomy: a mid-term follow up study. Cerebrovascular Diseases: April 2016; 42:122-130 T. Leunissen, M. de Boer, R. van der Hulst, J. Slatman. Exploring novel dimensions of body experience after breast reconstruction. Journal of Plastic, Reconstructive & Aesthetic Surgery Open, March 2016; 7: 32–41 T. Leunissen, P. Janssen, J. ten Berg, F. Moll, S. Korporaal, GJ. de Borst, G. Pasterkamp and R. Urbanus. The use of platelet reactivity testing in patients on antiplatelet therapy for prediction of bleeding events after cardiac surgery. Vascular Pharmacology: February 2016; 77: 19-27

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Authorâ&#x20AC;&#x2122;s list of publications

T. Leunissen*, S. Peeters Weem*, M. Teraa. E.J. Vonken, GJ. de Borst and F. Moll. Personalized antiplatelet therapy following endovascular revascularization in peripheral arterial disease: A novel concept. European journal of vascular and endovascular surgery, Short Reports, October 2015; 29: 11-17 T. Leunissen, GJ. de Borst, P. Janssen and J. ten Berg. The role of perioperative antiplatelet therapy and platelet reactivity testing in carotid revascularization; overview of the evidence. Journal of Cardiovascular surgery, April 2015; 56:165-175

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Curriculum Vitae Tesse Charlotte Leunissen was born on January the 19th, 1990 in Breda, the Netherlands. After graduating (cum laude) at the Stedelijk Gymnasium in Breda, she started her medical study at Maastricht University in September 2007. Her interest in surgery emerged during the very first internship at the Department of Surgery in the Maxima Medical Center in Veldhoven (the Netherlands). To confirm her initial interest in surgery, she completed her elective internship at the Burns Center, Rode Kruis Ziekenhuis in Beverwijk (the Netherlands). During her master, she worked on a collaborative qualitative research project of the department of plastic and reconstructive surgery and the department of health, ethics, and society. This project was focussed on the bodily experiences of women after breast reconstruction. Besides she worked on a study evaluating the effect of remodeling helmets in children with plagiocephaly and brachycephaly. She combined medical school with being an active sorority member (and board member), cycling through the hills of Limburg and discovering other countries: between her bachelor and master she travelled throughout Zambia, Malawi, Mozambique and South Africa. She followed her internship ophthalmology in Aarhus (Denmark) and pediatrics in Manipal (India). She continued this international medical work in 2016 during a mission to Nairobi (Kenia) with Medical Checks for Children. After finishing medical school in 2013, she was given the opportunity to work as a Ph.D.candidate at the Department of Vascular Surgery and Clinical Chemistry & Hematology of the University Medical Center Utrecht under the supervision of prof. dr. Moll, prof. dr. GJ. de Borst, dr. Roest and dr. Urbanus. The results of her research are presented in this thesis. This research project was made possible by the Prof. Heimburger Award Global Research Grant from CSL Behring for coagulation research. Parts of the work described in this thesis were presented and awarded on (inter)national congresses as the Charing Cross certificate of merit for excellent abstract presentation and trainee travel award at the International Symposium of Laboratory Hematology. Currently she is working as a surgical resident (not in training) at the Diakonessenhuis in Utrecht (the Netherlands) under the supervision of dr. van Dalen.

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Towards tailored antiplatelet therapy in vascular disease ÂŠ Tesse Charlotte Leunissen, 2016