Stimulating human platelets with thrombin induces the activation of the extracellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 microM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 microM) and MRS2179 (100 microM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 microM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 microM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via alpha(2A)-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthio-ADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2 -receptor with U46619 (10 microM), which leads to ADP secretion and P2Y12 receptor-dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.

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, 31, 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 S1197 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 TXA(2) 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 (PI 3-K) activation. Elevating cAMP levels with PGI(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 ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.