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 Gq and Gi pathways, respectively and SFKs play important roles in Gq- and Gi-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and Gq−/− 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 Gi2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the Gi 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 Gi 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 Gq−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that Gq-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 aIIbb3 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 Gq-dependent platelet activation. In summary, SFKs can be activated by Gq/Ca2+-dependent mechanisms and by Gi-dependent mechanisms, and SFKS play important roles in Gq- and Gi-dependent platelet activation.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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