Second messenger-mediated inside-out activation of integrin αIIbβ3 is a key step in platelet aggregation. We have recently shown strongly impaired aggregation in response to various agonists in CalDAG-GEFI-deficient platelets. Thrombin-induced aggregation, however, was affected only at low but not high doses of the agonist, suggesting CD-GEFI-dependent and -independent pathways for integrin activation in platelets. In this study, we further evaluated the roles of CalDAG-GEFI and PKC for integrin activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared to wild-type (WT) 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. In contrast, aggregation/αIIbβ3 activation was significantly reduced at any tested PAR4p concentration in CalDAG-GEFI-deficient platelets pre-treated with Ro31–8220. PAR4p-induced αIIbβ3 activation in the absence of CalDAG-GEFI required co-signaling through the Gi-coupled receptor for ADP, P2Y12, while this was not the case in WT platelets treated with Ro31–8220. Consistent with these results, release of ADP-containing dense granules was completely inhibited by Ro31–8220 at all PAR4p concentrations tested. In contrast, dense granule release was impaired at low but not high dose PAR4p in CD-GEFI-deficient platelets. Independent roles of CalDAG-GEFI- and PKC/Gi-signaling were also observed for PAR4p-induced activation of Rap1, with CalDAG-GEFI mediating the rapid activation of this small GTPase. In summary, our studies identify CalDAG-GEFI and PKC as independent pathways leading to Rap1 and αIIbβ3 activation in mouse platelets activated through the PAR4 receptor.
Disclosure: No relevant conflicts of interest to declare.