G12/13 Pathways Potentiate Akt Phosphorylation in Platelets Mediated by Gi/Gz Pathways in a Src Kinase-Dependent Manner.

Akt is a serine/threonine kinase that is activated by various agonists including thrombin and ADP in platelets, and activation of Akt in platelets is known to require Gi signaling pathways. Even though thrombin-induced Akt phosphorylation depends on secretion/Gi pathways, thrombin caused much stronger Akt phosphorylation than ADP and epinephrine. In this study, we investigated the contribution of G12/13 pathways to Akt phosphorylation mediated by Gi or Gz pathways. We used selective agonists to activate different G protein pathways. PAR4-activating peptide (AYPGKF) and thrombin failed to induce Akt phosphorylation in Gαq-deficient platelets, but Akt phosphorylation was restored to the levels achieved by AYPGKF and thrombin in wild-type platelets by selective supplement of either Gi or Gz signaling with 2-MeSADP and epinephrine, respectively. This phosphorylation of Akt was dramatically inhibited in the presence of PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src family tyrosine kinase, but not by PP3, an inactive structural analog. Importantly, AYPGKF and thrombin induced the activation of Src kinase in Gαq-deficient platelets, suggesting the involvement of Src-dependent pathways in the G12/13 signaling in potentiation of Akt phosphorylation. When human platelets were stimulated with low concentrations of YFLLRNP, a PAR1-specific partial agonist, to selectively activate the G12/13 signaling cascade, Akt phosphorylation did not occur. However, combined stimulation of YFLLRNP-mediated G12/13 signaling and selective activation of Gi pathways caused the Akt phosphorylation. This Akt phosphorylation was blocked by the P2Y12 receptor antagonist AR-C69931MX, or the PI 3-kinase inhibitor LY294002. Platelet aggregation induced by co-activation of both G12/13 and Gi signaling was dramatically inhibited by ML-9 (1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride), an Akt selective inhibitor, suggesting an important role of Akt in platelet aggregation stimulated by combined G12/13 and Gi pathways. These data demonstrate that G12/13 signaling alone does not cause Akt phosphorylation in platelets, but G12/13 signaling has a significant role in potentiating Akt phosphorylation mediated by selective Gi or Gz signaling in human as well as mouse platelets. Finally, we conclude that Src family kinases play an important role in this Akt phosphorylation in platelets.