Stakos

Platelet ac(va(on in Acute Coronary Syndromes Dimitrios A. Stakos, MD, FESC Assistant Professor of Cardiology Democritus University of Thrace, Alexandroupolis WWW.cardioalex.gr

The last act of an Acute Coronary Syndrome

How this started Optical coherence tomography (OCT)

Initial lesion (lipid accumulation, activated endothelium) .. platelet first contact (endothelium, WBCs) Pathology

In#mal  thickening

Plaque rupture – Acute coronary syndrome Pretreatment Cyclo-oxygenase inhibitors (ASA) GPIIb/IIIa inhibitors P2Y12 inhibitors Thrombin “inhibitor” (heparin)

Fibrous cap

Initial lesion transformed to more complex

Platelet activation in ACS Treatment

Antiplatelet agents Recovery Cyclo-oxygenase inhibitors (ASA) P2Y12 inhibitors Thrombin “inhibitor� (heparin)

Discharge on Cyclo-oxygenase inhibitors (ASA) P2Y12 inhibitors

Platelet activation in ACS prevention / stent thrombosis

Most PLTs never undergo firm adhesion to the endothelium GP Ib

---

vWFactor

collagen---

IIb P G

PGI2

aI I /I

PLTs

NO

cAMP and cGMP; CD39 ectoADPase, heparan sulfate, #ssue plasminogen ac#vator

This changes after plaque rupture or erosion..

Sub-endothelial layers are exposed

or in uncovered stent struts

uts

U

ve nco

re

t str n e t ds

Optical frequency domain imaging (OFDI),

Platelet activation and adhesion begins

Rolling at high shear

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

Collagen / vW / vitronectin/ laminin, fibronectin

GP Â Ib/V/IX

Collagen / vW / vitronectin/ laminin, fibronectin

Initial Adhesion / Rolling at High Shear (binding to vWF)

GPIbα

Promote more firm adhesion/ 1 2β Integrin activation α P

G

VI P G

vWF

Initial Adhesion (binding to collagen) / Rolling at High Shear

GPVI ITAM (immunoR Tyr Activation Motif)

Ia a I I / IIb

Insi d

mb

ra

ne

tiva tion

PLCγ

M ITA

me

t ac

n

M ITA

PL T

e-ou

tio a v i ct

α2β1

SY

K

SLP-76

GADS LAT

(ATVB, 2006)

Initial Adhesion (collagen) / Rolling at High Shear

ITAM (immunoR Tyr Activation Motif)

GPVI α2β1

Out si

de-

in a

ctiv

atio

a A I I I n IIb/

atio v i t c

n

SYK

PLpCγ

mb

ra

ne

M ITA

me

M ITA

PL T

SLP-76

GADS LAT

(ATVB, 2006)

SECRETION OF PLATELET AGONISTS

TF

ADP

TXA2

T sCD40L

vWF

THROMBIN AND PAR-1

PAR-1: Protease Activated Receptor

Fibrinogen

T

PA R

-1

Fibrin

TFmRNA

vWF TF exposure in atherosclerotic tissue

Tissue Factor mRNA in patients with Acute Coronary Syndromes

ATVB, 2008

THROMBIN AND PAR-1

GPIIb/IIIa inactive

T

GPIIb/I IIa active Shape change Ps

ele

TXA2 Ser ADP Epinephrine

ctin

TROMBOXANE A 2 AND TPα Receptor ADP Ca++

Gra n

Rel

eas

e

Shape change TPαR /IIIa b I I GP tion a activ

TXA2

Arach Acid

COX-1 TX synthase TXA2

Ser

Downstream Micro-vessel contraction

ADP RECEPTORS P2Y12R AND P2Y1R Dense granule release from adjacent PLTs

Y

1R

P2Y12R ADP

P2

cAMP Gi

Ca++ dense granule release alpha granule release (P-selectin expression) PLT recruitment

GPIIb/IIIa inactive

GPIIb/I IIa active

Pro-Coagulation TXA2 generation Enhancement of TXA2/ TH PLT activation

Current knowledge supports that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury.

There is more than collagen during platelet adhesion and aggrega#on in ACS The Local Shear Micro-­‐Gradient

Shear Micro-gradient induced PLT aggregation in ACS

g ou

e dh

re

h

Ib GP

Extrusion

r th

A

IIb/IIIa

Nature Med, 2009

Platelet aggregation may be driven by changes in blood flow parameters (rheology), Shear Micro-gradient induced PLT aggregation in ACS

Plaque  Constric#on Thrombus

Nature Med, 2009

INTEGRIN ACTIVATION AS FINAL RESULT

Shear Micro-gradient

FINAL RESULT

INITIAL PLT RICH THROMBUS

ct) a ( IIa IIb/I

PLT-PLT interactions

Fi b

rin

og

en

Firm adhesion

vWF

Coag CASCADE Fibrin

Fibrinogen FIIa

FXIIIa

FXIII FXa

FXI

FII FX

Fi b

FVII

Fibrin

FXIa

rin

Fibrinogen

og

e FIXa n FVIIa FIX TF/VIIa

FVIIIa FIIa

FXa STEMI FX

FVIII FII

PLT MICROPARTICLES AND DOWNSTREAM VASOCONSTRICTION Contribute to ischemia in ACS

PLT MICROPARTICLES AND DOWNSTREAM VASOCONSTRICTION

Ischemia despite successful recanalization and normal epicardial blood flow

PLT MICROPARTICLES AND DOWNSTREAM VASOCONSTRICTION

Ischemia despite successful recanalization and normal epicardial blood flow

PLATELETS AND INFLAMMATION PLTs-IL release PLTs-WBCs interactions PLTs – inflammatory molecules PLTs-oxLDL interactions

PLATELETS AND INFLAMMATION (IL secretion) Thrombin – Integrins – Interleukin 1β Coagulation Inflammation IL1β

TCPs (Translational Control Proteins)

TCP TCP

TCP

Cell 2005; NEJM, 2007

PLATELETS AND INFLAMMATION (Neutrophil - PLT aggregates) In patients with ACS

Circulation, 1996

PLATELETS AND INFLAMMATION (Neutrophil - PLT aggregates) In patients with ACS

Circulation, 1996

PLATELETS AND INFLAMMATION (Monocyte - PLT aggregates) In patients with ACS

(Circulation, 2001)

PLATELETS AND INFLAMMATION (Leukocyte - PLT aggregates) In patients with ACS

J Thromb Haemost, 2007

PLATELETS AND INFLAMMATION (β-amyloid) es

as t e r

a β-

nd

ec s γ

Alzheimer Disease amyloid β (Aβ) peptides

Amyloid Precursor Protein (APP)

Ann N Y Acad Sci. 1993

PL Td

BAC

eri

ve d

Se

AP

P

cre

Em

RNA

tas

es

Aß1–40

Macrophage activation

MMPs De Meyer G, Circul Res, 2002

Electron micrograph shows cultured J774 macrophages incubated with human blood platelets (ratio 1:200) for 2 hours.

Colocalization of activated MC with AĂ&#x; and platelets in advanced human atherosclerotic plaques

Mcact PLTs

N

PLT induced MC activation through Aβ peptides in atherosclerosis No PLTs + PLTs

MC (APP pos) after incubation with PLTs

MC (APP neg)

MC (Aβ1-40 pos) after incubation with PLTs

Circulation Research. 2002

Abeta peptide 1-40 in ACS

ANOVA P<0.0001

140 P<0.0001

Aβ40 (pg/ml)

120 100

P=0.02

P=0.0003

80 60 40 20 00 CNTR

SAP

ACS Preliminary data

Abeta peptide 1-40 in ACS and association with PLT activation

Aβ42 / Aβ40 ratio (pg/ml)

R= 0.40 P=0.01

P selectin (MFI)

Preliminary data

PLATELETS AND INFLAMMATION (oxLDL induced PLT activation) LDL and oxLDL in ACS

CD36 scavenger receptor

P selectin

Plateled-bound-anti oxLDL (MFI)

Platelet-bound OxLDL is increased in patients with ACS

P < 0.05

200 150 100 50 0 SAP

ACS

(n= 182)

(n= 174)

Preliminary data

Log (oxLDL)

oxLDL correlates positively with platelet activation

2.4

2.4

2.0

2.0

1.6

1.6

n=356 r= 0.189 P< 0.05

1.2

n=356 r= 0.257 P< 0.05

1.2

1.0

1.5

2.0

Log (platelet-bound cd62p)

2.5

0.5 1.0 1.5 2.0 2.5 3.0 Log (GPIIb/IIIa)

Preliminary data

oxLDL bound PLTs Roll more on ECs or Collagen

Rolling

Rolling platelets per high powerfield

In vitro study 200

Resting Plts Ox-LDL activated Plts

*

150

*

100 50 0 ECs

Collagen

Preliminary data

oxLDL bound PLTs Adhere more on ECs or Collagen

Firm Adhesion Adherent platelets per high powerfield

In vitro study 60

Resting Plts Ox-LDL activated Plts

50 40

*

*

30 20 10 0 ECs

Collagen

Preliminary data

Most information about the role of platelets in Acute Coronary Syndromes & Thropmbus formation came from studies in animal models and in vitro studies

However, markers of PLT activation are found to be elevated in pts with ACS Antiplatelet therapy have shown to improve outcomes in pts with ACS

From pathophysiology to markers of PLT activation for the diagnosis or prognosis after an ACS

No ASA TXA2 metabolites in pts with ACS

NEJM, 1986

It is known that PLT activation is associated (In general) with poorer prognosis in ACS

Platelet aggregation and prognosis in Myocardial infarction (20 years ago..)

SPA nega#ve SPA intermediate

SPA posi#ve

SPA: Spontaneous Platelet Aggrega#on NEJM, 1990

P-selectin is increased in Acute Coronary Syndromes

Time frame

Blood. 1996

Over the first 48h

Circulation, 1996

Platelet activation in ACS (P-selectin)

P=0.002

Plt- CD62P FITC

16 12 8 4 0 SAP (n=348)

ACS (n=319)

J Thromb Haemost, 2010

P-selectin and infarct size

Plt-CD62P FITC

40 30 20 10 n= 139 r=0.316 P<0.001

0 0

200

400

600

800

1000

Maximum CK-MB (U/l) J Thromb Haemost, 2010

Markers of PLT activation in ACS (collagen receptor GPVI)

Eur Heart J, 2006

Prognosis in ACS according to sCD40L

NEJM, 2003

ANTIPLATELET THERAPY..

Thromboxane Inhibitors (ASPIRIN)

Inhibition of platelet function and clinical outcomes (CVD prevention by ASPIRIN)

Lancet, 2009

The P2Y12 antagonists The combination of aspirin and clopidogrel was superior to aspirin alone in preventing vascular events in cardiac patients with unstable angina

ASA +

+ ASA

NEJM, 2001

or in those requiring percutaneous coronary intervention..

JAMA, 2002

No risk reduction in non-ACS patients however

â&#x20AC;Śit showed no overall benefit in patients with stable vascular disease

Role of platelet inhibition in acute events

NEJM, 2006

â&#x20AC;ŚSTEMI Potent platelet inhibition prior to thrombolysis seems to be beneficial in patients with STEMI Aspirin Heparin Thrombolysis

NEJM, 2005

Newer P2Y12 antagonists (PRASUGREL vs CLOPIDOGREL)

cy a c ffi E

Safety

fit Net bene

fit e n be

t Ne

TRITON–TIMI 38, NEJM, 2007

The P2Y12 antagonists Newer antiplatelet agents (TICAGRELOL vs CLOPIDOGREL)

NEJM, 2009

The GPIIb/IIIa inhibitors in patients with ACS

(PURSUIT, NEJM, 1998)

The GPIIb/IIIa inhibitors in patients with ACS

lower incidence of ischemic events ACS pts than heparin + aspirin

(PRISM PLUS, NEJM, 1998)

Prognosis in ACS according to sCD40L levels and treatment with Abciximab

sCD40L

NEJM, 2003

Collaborative meta-analysis of RT on antiplatelet therapy (ASA, Clop, Dipyrid, IIb/IIIa)

BMJ, 2002

Inhibition of platelet function and Bleeding from ASPIRIN) Number of events (aspirin vs control) Primary Secondary preven#on (660 preven#on (43 000 person-­‐ 000 person-­‐ years) years) Major coronary event

Rate ra;o (95% CI) (aspirin vs control)

Primary preven#on

Secondary preven#on

Yearly absolute difference (% per year)

p value for heterogeneity

Primary preven#on

Secondary preven#on

934 vs 1115

995 vs 1214

0·∙82 (0·∙75–0·∙90) 0·∙80 (0·∙73–0·∙88)

0·∙7

−0·∙06

−1·∙00*

Non-­‐fatal MI

596 vs 756

357 vs 505

0·∙77 (0·∙69–0·∙86) 0·∙69 (0·∙60–0·∙80)

0·∙5

−0·∙05

−0·∙66

CHD mortality

372 vs 393

614 vs 696

0·∙95 (0·∙82–1·∙10) 0·∙87 (0·∙78–0·∙98)

0·∙4

−0·∙01

−0·∙34

655 vs 682

480 vs 580

0·∙95 (0·∙85–1·∙06) 0·∙81 (0·∙71–0·∙92)

0·∙1

−0·∙01

−0·∙46*

Haemorrhagic

116 vs 89

36 vs 19

1·∙32 (1·∙00–1·∙75) 1·∙67 (0·∙97–2·∙90)

0·∙4

0·∙01

..†

Ischaemic

317 vs 367

140 vs 176

0·∙86 (0·∙74–1·∙00) 0·∙78 (0·∙61–0·∙99)

0·∙5

−0·∙02

..†

Unknown cause

222 vs 226

304 vs 385

0·∙97 (0·∙80–1·∙18) 0·∙77 (0·∙66–0·∙91)

0·∙1

−0·∙001

..†

619 vs 637

825 vs 896

0·∙97 (0·∙87–1·∙09) 0·∙91 (0·∙82–1·∙00)

0·∙4

−0·∙01

−0·∙29

1671 vs 1883 1505 vs 1801 (0·∙51% vs 0·∙57% (6·∙69% vs 8·∙19% 0·∙88 (0·∙82–0·∙94) 0·∙81 (0·∙75–0·∙87) per year) per year)

0·∙1

−0·∙07

−1·∙49*

0·∙2

0·∙03

..†

Stroke

Vascular death

Any serious vascular event

Major extracranial bleed

335 vs 219

23 vs 6

1·∙54 (1·∙30–1·∙82) 2·∙69 (1·∙25–5·∙76)

Lancet, 2009

Inhibition of platelet function and Bleeding from Clopidogrel and Prasugrel

Newer antiPLT therapies on the basis of similar if not lower bleeding risk

TRITONâ&#x20AC;&#x201C;TIMI 38, NEJM, 2007

Inhibition of platelet function and Bleeding from Ticagrelol

Newer antiPLT therapies on the basis of similar if not lower bleeding risk

NEJM, 2009

Bleeding because they interfere with mechanisms of primary haemostasis

The need for more potent antiplatelet drugs with safer profile

RPR in patients on dual antiplatelet therapy and acute coronary events

High-on Platelet Reactivity (death, myocardial infarction, stent thrombosis, or ischemia requiring a hospital stay)

JACC, 2007

Triple antiplatelet therapy (+ cilostazol a type III phosphodiesterase inhibitor)

Circulation, 2009

New antiPLT drugs may emerge from molecules that can be targeted and functionally inactivated in-vivo ..

..finally representing novel target-proteins for effective prophylaxis and better clinical outcomes in pts with Acute Coronary Syndromes

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (Adhesion through collagen receptors): 1. Anti GPVI

GPVI-depleted

Scanning electron micrographs of carotid arteries 2 h after vascular injury

J Exp Med, 2003

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets:(Adhesion through collagen receptors): 1. Anti GPIbα (p0p/B antibody)

Control

GPIbα−/− mice

adhesion in arterioles from GPIba-/- mice is virtually absent for the entire observation period

PNAS, 2006

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (adhesion): 2. Phospholipase D1 (PLD1) Shear-dependent IIbIIIa activation and thrombus stability GPIIb/IIIa activation GPVI GPIb

T G-PrCRs

PhAcid

CHL

Ph/dyl/chol Pld1-/- mice display reduced IIb/IIIa activation after G-PrCR stimulation Sci Signal, 2010

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (activation): 3. C-type LECtin lilke type II trans-membrane receptor (CLEC-2)

GPIIb/IIIa

CLEC-2 G-PrCRs

PhdyloInosP2 IP3

kindilin3

DAG

talin1

Hydrolysis actin

PrKC Rap1b GTPase

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (PLT activation): 3. C-type LECtin lilke type II trans-membrane receptor (CLEC-2)

Defective aggregation Reduced surface coverage

INU1: anti CLEC-2

Blood, 2009

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (Ca++ signaling): 4. Stromal Interaction Molecule 1 (STIM1) & Orai1

GPIIb/IIIa

CLEC-2

Orai1

G-PrCRs Ca++ Ca++ ++ Ca Ca++

PIP2

Ca++

PLpCγ2

IP3

PLpCβ

Ca++

actin

IP3R Ca++

Ca++ Ca++

talin1

Sustained Ca++ influx

1 IM T S Ca++

kindilin3

Ca++DAG/GEF1

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (Ca++ signaling): 4. Stromal Interaction Molecule 1 (STIM1) STIM1-/- mice

Increased occlusion and tail bleeding times

J Exp Med, 2008

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (GPIIb/IIIa activation): 5. Ca++ & DiAcylGlycerol-regulated Guanine-nucleotide Exchange Factor I (Cal-DAG-GEF1)

GPIIb/IIIa

CLEC-2

Orai1

G-PrCRs Ca++ Ca++ ++ Ca Ca++ Ca++

kindilin3

1 IM T S Ca++ Ca++

actin

IP3R Ca++

Ca++ Ca++

talin1

Cal-DAG/GEF1

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (GPIIb/IIIa activation): 5. Diacylglycerol-regulated guaninenucleotide exchange factor I (CalDAG-GEF1)

No thrombus ajer 30 min

J Clin Invest, 2007

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (GPIIb/IIIa activation): 6. Talin1

GPIIb/IIIa activation GPVI GPIb

T G-PrCRs

kindilin3

PhAcid

CHL

Ph/dyl/chol

talin1

actin

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (GPIIb/IIIa activation): 6. Talin1

Talin-­‐/-­‐

platelets did not adhere to the vessel wall and thombus formation did not occur

J Exp Med, 2008

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (GPIIb/IIIa activation): 7. Kindilin3

GPIIb/IIIa activation GPVI GPIb

T G-PrCRs

kindilin3

PhAcid

CHL

Ph/dyl/chol

talin1

actin

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (GPIIb/IIIa activation): 7. Kindilin3

Nat Med, 2008

C1qTNFâ&#x20AC;&#x201C;related protein-1 (CTRP-1)

Other targets A protein that blocks VWF binding to collagen D

flow traces

re-establishes blood flow

Treatment with CTRP-1 in injured carotid artery

Control Blood flow Control 0.5mg/Kg 1.0 mg/Kg

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (thrombin-induced PLT activation): 8. PAR-1 R

Thrombin

PAR-1 active

PAR-1 inactive

Action •GPIIb/IIIa activation •Secretion •proCoagulant activity •Shape change

PLATELETS ACTIVATION in ACUTE CORONARY SYNDROMES New Targets (thrombin-induced PLT activation): 8. PAR-1 R

PLT inhibi#on of TRAP-­‐induced aggrega#on

Under investigation Lancet, 2009

PLATELET ACTIVATION IN ACUTE CORONARY SYNDROMES Conclusions • Platelets are central to the pathogenesis of cute coronary syndromes (ACS) with complex mechanisms similar of that of primary haemostasis • Some indices of platelet ac#va#on may be used as diagnos#c and prognos#c markers in ACS in the future • Current guidelines recommend combina#on an#platelet therapy for ACS •There remains a significant incidence of thrombo#c events in pts receiving available agents •Bleeding is also an important issue

•There is a need for newer agents that provide both • comprehensive platelet inhibi#on (effec#ve protec#on) •without interfering with hemostasis (no incremental risk of bleeding)

Newer methods may provide further insight into the mechanisms that underlie platelet activation and may help to identify novel pharmacologic targets Platelet Proteome analysis

K N A H T

U YO

Blood, 2010