CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets

Abstract

Second messenger-mediated inside-out activation of integrin αIIbβ3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent αllbβ3-mediated aggregation of CalDAG-GEFI-deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for αllbβ3 activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI-deficient platelets, however, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for αllbβ3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Gαi-coupled receptor for ADP. P2Y12. Independent roles for CalDAG-GEFI and PKC/Gαi signaling were also observed for PAR4p-induced activation of the small GT Pase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and αllbβ3 activation in mouse platelets activated through the PAR4 receptor. © 2008 by The American Society of Hematology.