Src Family Kinases Contribute to G_I and G_q-Mediated Platelet Activation

Abstract 5263

The Src family kinases (SFKs) play an essential role in collagen- and von Willebrand factor (vWF)-mediated platelet activation. However, the role of SFKs in G protein-coupled receptor (GPCR)-mediated platelet activation is not fully understood, and little is known about the molecular mechanisms by which SFKs are activated by GPCR stimulation. Here we demonstrate that SFKs are activated by the G_q and G_i pathways, respectively and SFKs play important roles in G_q- and G_i-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and G_q^−/− platelets, and ADP-induced SFK phosphorylation was inhibited by the P2Y₁₂ antagonist AR-C69931MX or P2Y₁₂ knockout but was not affected by the P2Y₁ antagonist MRS-2179. Lyn and Fyn were co-immunoprecipitated with G_i2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn^−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the G_i pathway activated via its receptor P2Y₁₂. Furthermore, epinephrine also dose-dependently induced SFK phosphorylation in mouse platelets. A selective inhibitor of Src family kinase PP2 inhibited ADP-induced Akt phosphorylation, fibrinogen binding, and platelet aggregation. Thus, activation of G_i is sufficient to induce SFK activation, which plays an important role in Gi-dependent platelet activation. We further show that the thrombin receptor PAR4 peptide AYPGKF elicited SFK phosphorylation in P2Y₁₂^−/−, but not in G_q^−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that G_q-dependent SFK phosphorylation is downstream from the Ca²⁺ signaling. The calcium ionophore, A23187-induced TXA₂ synthesis, platelet aggregation and secretion were inhibited by pre-incubation of platelets with PP2. PAR4-induced TXA₂ synthesis was also abolished by PP2. Moreover, PAR4-mediated granule secretion, integrin a_IIbb₃ activation, and aggregation of P2Y₁₂ deficient platelets were partially inhibited by PP2 or a PKC inhibitor Ro-31-8220, but were completely abolished by Ro-31-8220 plus PP2 or dimethyl-BAPTA, suggesting that Ca²⁺/SFKs and PKC represent two parallel signaling pathways mediating G_q-dependent platelet activation. In summary, SFKs can be activated by G_q/Ca²⁺-dependent mechanisms and by G_i-dependent mechanisms, and SFKS play important roles in G_q- and G_i-dependent platelet activation.