Fälker Knut, Lange Danica, Presek Peter
Martin-Luther-University Halle-Wittenberg, 06097 Halle, Germany.
Thromb Haemost. 2004 Jul;92(1):114-23. doi: 10.1160/TH03-12-0729.
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.
用凝血酶刺激人血小板可诱导细胞外信号调节激酶2（ERK2）的激活。我们证明这种效应高度依赖于ADP分泌和P2Y12受体信号传导。AR-C69931MX（10微摩尔），一种与Gi偶联的P2Y12 ADP受体的特异性拮抗剂，可抑制凝血酶诱导的ERK2激活。与Gq偶联的P2Y1 ADP受体的拮抗剂A3P5P（500微摩尔）和MRS2179（100微摩尔）没有作用。单独的ADP及其更强效的类似物2-甲硫基-ADP（两者浓度均高达100微摩尔）不会诱导ERK2激活。此外，我们表明AR-C69931MX对0.1 U/ml凝血酶诱导的ERK2激活以及血小板聚集的抑制作用可被肾上腺素（1和10微摩尔）绕过，而单独的肾上腺素没有作用。肾上腺素主要通过α(2A)-肾上腺素能受体作用于血小板，该受体与P2Y12受体一样，与抑制性G蛋白偶联。此外，2-甲硫基-ADP以及肾上腺素在单独无明显作用的凝血酶浓度（0.05 U/ml）下可引发ERK2激活。血栓素A2（TXA2）与ADP一样，由活化的血小板释放，起正反馈介质的作用。用U46619（10微摩尔）刺激与Gq偶联的TXA2受体，可导致ADP分泌和P2Y12受体依赖性血小板聚集，也可诱导与P2Y12相关的ERK2激活。AR-C69931MX对U46619诱导的ERK2激活和血小板聚集的抑制作用也可被肾上腺素挽救。用阿司匹林预处理可抑制0.1 U/ml凝血酶诱导的ERK2激活，但在高浓度凝血酶时没有作用。U46619和凝血酶在单独无作用的浓度下联合使用可引发ERK2激活，表明凝血酶和释放的TXA2起协同作用。我们的数据表明，ERK2激活既需要通过Gq的初级信号传导（引发ADP分泌），也需要随后通过P2Y12受体经Gi偶联。
