Rapid Ca2+-mediated activation of Rap1 in human platelets

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 farm of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of Ra1GDS (RBD), Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RED in vitro, Binding to RED 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 I-2 (PGI(2)), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1, From our results, rye 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.