RAPID CA2-MEDIATED ACTIVATION OF RAP1 IN HUMAN PLATELETS()

Rap1 is a small, Ras-like GTPase whose function and regulation are sti ll largely unknown. We have developed a novel assay to monitor the act ive, GTP-bound farm of Rap1 based on the differential affinity of Rap1 GTP and Rap1GDP for the Rap binding domain of Ra1GDS (RBD), Stimulatio n of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RED i n vitro, Binding to RED increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation, An incr ease in the intracellular Ca2+ concentration is both necessary and suf ficient for Rap1 activation since it was induced by agents that increa se intracellular Ca2+ and inhibited by a Ca2+-chelating agent, Neither inhibition of translocation of Rap1, to the cytoskeleton nor inhibiti on of platelet aggregation affected thrombin-induced activation of Rap 1. In contrast, prostaglandin I-2 (PGI(2)), a strong negative regulato r of platelet function, inhibited agonist-induced as well as Ca2+-indu ced activation of Rap1, From our results, rye conclude that Rap1 activ ation in platelets is an important common event in early agonist-induc ed signalling, and that this activation is mediated by an increased in tracellular Ca2+ concentration.