G_12/13 Pathways Potentiate Akt Phosphorylation in Platelets Mediated by G_i/G_z 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 G_i signaling pathways. Even though thrombin-induced Akt phosphorylation depends on secretion/G_i pathways, thrombin caused much stronger Akt phosphorylation than ADP and epinephrine. In this study, we investigated the contribution of G_12/13 pathways to Akt phosphorylation mediated by G_i or G_z pathways. We used selective agonists to activate different G protein pathways. PAR4-activating peptide (AYPGKF) and thrombin failed to induce Akt phosphorylation in Gα_q-deficient platelets, but Akt phosphorylation was restored to the levels achieved by AYPGKF and thrombin in wild-type platelets by selective supplement of either G_i or G_z 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 Gα_q-deficient platelets, suggesting the involvement of Src-dependent pathways in the G_12/13 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 G_12/13 signaling cascade, Akt phosphorylation did not occur. However, combined stimulation of YFLLRNP-mediated G_12/13 signaling and selective activation of G_i 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 G_12/13 and G_i 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 G_12/13 and G_i pathways. These data demonstrate that G_12/13 signaling alone does not cause Akt phosphorylation in platelets, but G_12/13 signaling has a significant role in potentiating Akt phosphorylation mediated by selective G_i or G_z 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.