TWO-FACED BEHAVIOR OF AMIDATED SINGLE-WALLED CARBON NANOTUBES IN TERMS OF PLATELET FUNCTION JÁNOS FENT, PÉTER BIHARI AND SUSAN LAKATOS Department of Pathophysiology, Laboratory Institute for Health Protection, Medical Centre of Hungarian Defense Forces H-1134, Róbert Károly krt. 44, Budapest, Hungary e-mail: latyakos24@gmail.com
BACKGROUNDS 80
* p<0.01
Various single-walled carbon nanotubes induce surface expression of CD62P activation marker on platelets in whole blood
* *
CD62P MFI
60 40 *
*
*
20
HOW TO EXPLAIN
0 Control
20
Ohm
15
ADP
CONH2- COOHpPEGSWCNT SWCNT SWCNT SWCNT
that in the presence of amidated SWCNTs the CD62P positive platelets
* p<0.01
Pristine, pegylated, carboxylated but not amidated single-walled carbon nanotubes induce platelet aggregation
* *
10 *
*
5
DO NOT AGGREGATE?
0 Collagen
pSWCNT
CONH2SWCNT
COOHSWCNT
PEGSWCNT
During aggregation platelets are bound to each other with fibrinogen. Fibrinogen
Activated fibrinogen receptor can be detected by PAC-1 antibody
RESULTS 40
* p<0.01
1000 Impedance (AU)
* PAC-1 MFI
30 * 20
*
* *
10
SWCNT-CONH2 Collagen
800 600 400
SWCNT-CONH2
200
Control
0
0 Control
ADP
CONH2- COOHpPEGSWCNT SWCNT SWCNT SWCNT
0
500
1000 Time (sec)
1500
2000
Amidated single-walled carbon nanotubes do not prevent collagen induced whole blood aggregation
In the presence of SWCNTs even in case of amidated ones platelets evolve PAC-1 antibody binding ability
A PUTATIVE EXPLANATION Amidated SWCNTs may induce a conformational change of fibrinogen receptor resulting in a „metastable” form of GPIIbIIIa fibrinogen receptor with - perfect epitopes for PAC-1 antibody binding but with - no fibrinogen binding ability.
However, the classical platelet agonist (collagen) can convert this „metastable” form of fibrinogen receptor into the perfect activated form.
MATERIALS AND METHODS SINGLE-WALLED CARBON NANOPARTICLES (SWCNT) SWCNT SES Research SWCNT-PEG Sigma-Aldrich SWCNT- CONH2 Sigma-Aldrich SWCNT-COOH NanoAmor
MEASUREMENT OF IN VITRO PLATELET ACTIVATION MARKERS BY FLOW CYTOMETRY citrated human whole blood +/- 0.1 mg/ml final concentration of the appropriate nanopartilcle P-selectin (CD62P) activation marker PAC- 1 (activated fibrinogen receptor)
PREPARATION OF NANOPARTICLE DISPERSIONS stock solutions in distilled water → sonication → addition of human serum albumin → sonication → addition 10× concentrated PBS solution → sonication
WHOLE BLOOD AGGREGOMETRY citrated human whole blood +/- 0.1 mg/ml final concentration of the appropriate nanopartilcle impedance change at 37 °C with continuous stirring (1000 rpm) After 20 min, 5 μM of collagen was added to check the aggregation ability of platelets