ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets

Abstract

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.

Protease-Activated Receptors Differentially Regulate Human Platelet Activation through a Phosphatidic Acid-Dependent Pathway

“…Although PLD metabolism of PC to PA and DAG influence secretion, effects on secretion are unable to explain the differential effects of PAR1 versus PAR4 signaling. Other reports have indicated that ADP released from the dense granule is important for augmenting a second phase of platelet activation (Falker et al, 2004;Hechler et al, 2005). Our data are consistent with the hypothesis that PAR4 signals in cooperation with ADP, and PAR1 signaling does not require ADP (Holinstat et al, 2006).…”

Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase

“…Similar observations have been made in the case of ERK activation. 16 p38 has been shown to mediate platelet adhesion in static as well as flow conditions on low-collagen-density surfaces, 17 and platelet aggregation induced by low collagen, U46619, 18 and thrombin 8 concentrations. However, these studies relied on the use of p38 inhibitors (SB202190, SB203580), but unfortunately did not use appropriate controls such as the inactive analog SB202474.…”

Involvement of the Mitogen-activated Protein Kinase c-Jun NH2-terminal Kinase 1 in Thrombus Formation

“…Recently, our group demonstrated a critical role for ERK2 in platelet adhesion, in blood flow conditions, and in platelet spreading (18,19). With other agonists, such as low doses of collagen (20) or thrombin (21), ERK2 is involved in platelet aggregation. In these conditions, ERK2 acts on platelet secretion and activation of myosin light chain kinase (20,22).…”