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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1499.)
© 2005 American Heart Association, Inc.

Thrombosis

Synergistic Effect of Thrombin on Collagen-Induced Platelet Procoagulant Activity Is Mediated Through Protease-Activated Receptor-1

Jeffrey F.W. Keuren; Simone J.H. Wielders; Hans Ulrichts; Tilman Hackeng; Johan W.M. Heemskerk; Hans Deckmyn; Edouard M. Bevers; Theo Lindhout

From the Sanquin (J.F.W.K.), Blood Bank region South-East, Maastricht, and the Department of Biochemistry (S.J.H.W., T.H., J.W.M.H., E.M.B., T.L.), Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands; and the Laboratory for Thrombosis Research (H.U., H.D.), K.U. Leuven, Campus Kortrijk, Belgium.

Correspondence to Dr T. Lindhout, Department of Biochemistry, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands. E-mail t.lindhout{at}bioch.unimaas.nl

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

Our study demonstrates that the interaction of thrombin with PAR-1 mediates a synergistic effect on collagen-induced platelet procoagulant activity by inducing a sustained elevation in [Ca²⁺]_i in a subpopulation of platelets.

Key Words: thrombin • collagen • platelets • procoagulant activity • protease-activated receptors

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