A novel P2Y₁₂-Independent Signaling Pathway Mediating Akt Phosphorylation In Response to Thrombin Receptors

Abstract 3191

The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on G_i signaling activated primarily by secreted ADP through its receptor P2Y₁₂. By using P2Y₁₂ knockout mice combined with recombinant CHO models, we demonstrate a G_i-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y₁₂ deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr³⁰⁸ and Ser⁴⁷³ in P2Y₁₂ deficient platelets, demonstrating that there are P2Y₁₂-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y₁₂ deficient platelets, suggesting that G_i activation by thrombin or AYPGKF is dependent on ADP receptor P2Y₁₂. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y₁₂ deficient platelets is G_i independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y₁₂ pathway, because CHO cells apparently do not express functional P2Y₁₂. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y₁₂ deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y₁₂ dependent. In addition, P2Y₁₂-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y₁₂-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y₁₂ deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y₁₂-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.