P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

In thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 3 1, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi(2)-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin alpha(IIb)beta(3) outside-in signaling with RGDS or S 1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA(2)-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA(2) receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA2 signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled alpha(2A)-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI3-K) activation. Elevating cAMP levels with PGl(2) or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADR as well as subsequent P2Y12 receptor-mediated Gi coupling.