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. Methods and 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 (approximate to 14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIb alpha 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.