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Updated: Aug 18, 2026

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Published on: May 24, 2024
Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through
Jeffrey F W Keuren1, Simone J H Wielders, Hans Ulrichts
1Sanquin, Blood Bank region South-East, Maastricht, The Netherlands.
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 (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.
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