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 Ca concentration ([Ca]i) and the various platelet receptors for thrombin. METHODS AND RESULTS—We demonstrate that procoagulant activity is related to a sustained increased [Ca]i, which in turn depends on extracellular Ca influx. Increased PtdSer exposure coincides with increased [Ca]i and was observed in a subpopulation (≈14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIbα 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 [Ca]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 [Ca]i in a subpopulation of platelets.