Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through protease-activated receptor-1.
OBJECTIVE
In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca2+ concentration ([Ca2+]i) and the various platelet receptors for thrombin.


RESULTS
We demonstrate that procoagulant activity is related to a sustained increased [Ca2+]i, which in turn depends on extracellular Ca2+ influx. Increased PtdSer exposure coincides with increased [Ca2+]i and was observed in a subpopulation (approximately 14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIbalpha made clear that this receptor did not signal for platelet procoagulant activity. Inhibition of protease-activated receptor 1 (PAR-1) and PAR-4 by selective intracellular inhibitors and selective desensitization of these receptors revealed that PAR-1, but not PAR-4, activation is a prerequisite for both sustained elevations in [Ca2+]i and procoagulant activity induced by collagen plus thrombin.


CONCLUSIONS
The interaction of thrombin with PAR-1 mediates a synergistic effect on collagen-induced procoagulant activity by inducing a sustained elevation in [Ca2+]i in a subpopulation of platelets.
Keuren
Jeffrey F W
JF
Sanquin, Blood Bank region South-East, Maastricht, The Netherlands.
Wielders
Simone J H
SJ
Ulrichts
Hans
H
Hackeng
Tilman
T
Heemskerk
Johan W M
JW
Deckmyn
Hans
H
Bevers
Edouard M
EM
Lindhout
Theo
T
D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000493:pharmacokinetics; D003094:Collagen / Q000378:metabolism / Q000494:pharmacology*; D004357:Drug Synergism; D006490:Hemostatics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D013925:Thromboplastin / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D006490:Hemostatics; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D044463:Receptor, PAR-1; C032193:glycoprotein receptor GPIb-IX; C101296:prostate apoptosis response-4 protein; D003094:Collagen; D013925:Thromboplastin; D013917:Thrombin; D002118:Calcium