HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Differential requirements for calcium and Src family kinases in platelet GPIIb/IIIa activation and thromboxane generation downstream of different G-protein pathways

G12/13 or Gq signaling pathways activate platelet GPIIb/IIIa when combined with Gi signaling. We tested whether combined Gi and Gz pathways also cause GPIIb/IIIa activation and compared the signaling requirements of these events. Platelet aggregation occurred by combined stimulation of Gi and Gz pathways in human platelets and in P2Y1-deficient and G qdeficient mouse platelets, confirming that the combination of Gi and Gz signaling causes platelet aggregation. When Gi stimulation was combined with Gz stimulation, there was a small mobilization of intracellular calcium. Chelation of intracellular calcium decreased the extent of this platelet aggregation, whereas it abolished the Gq plus Gi-mediated platelet aggregation. Costimulation of Gi plus Gz pathways also caused thromboxane generation that was dependent on outside-in signaling and was inhibited by PP2, a Src family tyrosine kinase inhibitor. Src family tyrosine kinase inhibitors also inhibited platelet aggregation and decreased the PAC-1 binding caused by costimulation of Gi and Gz signaling pathways in aspirin-treated platelets. However, Src family kinase inhibitors did not affect Gq plus Gi-mediated platelet aggregation. We conclude that the combination of Gi plus Gz pathways have different requirements than Gq plus Gi pathways for calcium and Src family kinases in GPIIb/IIIa activation and thromboxane production. (Blood. 2005;105:2749-2756)