Rapid Ca2+-mediated activation of Rap1 in human platelets

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Abstract
Rap1 is a small, Ras‐like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP‐bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with α‐thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+‐chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin‐induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist‐induced as well as Ca2+‐induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist‐induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration.