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 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 alpha-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.
Franke
B
B
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Akkerman
J W
JW
Bos
J L
JL
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011464:Epoprostenol / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010974:Platelet Aggregation; D011493:Protein Kinase C / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins
D002614:Chelating Agents; D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D014157:Transcription Factors; D020726:ral Guanine Nucleotide Exchange Factor; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D011464:Epoprostenol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013917:Thrombin; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D002118:Calcium