McNicol Archibald, Jackson Elke C G
Department of Oral Biology, University of Manitoba, 780 Bannatyne Avenue, Winnipeg, Man., Canada R3E 0W2.
Biochem Pharmacol. 2003 Apr 15;65(8):1243-50. doi: 10.1016/s0006-2952(03)00069-8.
The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.
多种激动剂激活人血小板的过程伴随着丝裂原活化蛋白（MAP）激酶的细胞外信号调节激酶（ERK）亚型的磷酸化。然而，这些酶在血小板功能中的作用以及作用底物仍不清楚。对血小板中ERK的研究依赖于药理学工具，包括ERK激活抑制剂U0126 [1,4 - 二氨基 - 2,3 - 二氰基 - 1,4 - 双（2 - 氨基苯硫基）丁二烯]。在本研究中，检测了U0126及其“无活性”类似物U0125 [1,4 - 二氨基 - 2,3 - 二氰基 - 1,4 - 双（苯硫基）丁二烯]对人血小板聚集和MAP激酶活性的影响。研究了几种具有多种信号通路的激动剂，包括凝血酶、血栓素类似物、花生四烯酸、胶原蛋白、钙离子载体以及佛波酯佛波醇肉豆蔻酸酯乙酸酯（PMA）。在与抑制分离酶一致的浓度下，U0126抑制了每种激动剂诱导的ERK磷酸化，进而抑制了MEK激活。在这种情况下，U0126不影响第二种MAP激酶p38（MAPK）的磷酸化；然而，血小板聚集也未受影响。更高浓度的U0126和U0125抑制了胶原蛋白和PMA诱导的血小板聚集，而对其他激动剂诱导的聚集没有影响。这些结果将ERK激活与血小板聚集分离，提示ERK在血小板功能中具有其他作用。此外，更高浓度的U0126的作用可能是由于对蛋白激酶C的作用，可能与ERK抑制无关，这表明抑制剂浓度对于此类研究的解释至关重要。
