Quinton T M, Kim S, Jin J, Kunapuli S P
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
J Thromb Haemost. 2005 May;3(5):1036-41. doi: 10.1111/j.1538-7836.2005.01325.x.
ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-beta-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Galpha(i) activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Galpha(z)-coupled agonist epinephrine. Finally, Galpha(i) preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Galpha(i) signaling downstream of P2Y12 activation, but not Galpha(q) or Galpha(z) signaling downstream of P2Y1 or alpha2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.
在响应其他激动剂时，活化血小板分泌的二磷酸腺苷（ADP）在促进止血过程中起着重要作用。脂筏是质膜内富含胆固醇和鞘脂的微结构域，与血小板激动剂的刺激机制有关。我们试图通过用甲基-β-环糊精消耗胆固醇来破坏脂筏，从而确定脂筏在经由G蛋白偶联的P2Y1和P2Y12受体介导的ADP诱导的血小板活化中的重要性。用ADP刺激胆固醇耗竭的血小板导致聚集程度降低，但形状改变程度或细胞内钙释放无差异。此外，向先前耗竭的膜补充胆固醇可恢复ADP介导的血小板聚集。此外，血小板胆固醇耗竭后，P2Y12介导的环磷酸腺苷（cAMP）形成抑制作用显著降低。用依赖Gαi激活才能完全激活的激动剂刺激胆固醇耗竭的血小板时，聚集和分泌显著丧失，但同时用Gαz偶联的激动剂肾上腺素刺激时则恢复。最后，通过蔗糖密度离心法确定，Gαi优先定位于脂筏。我们得出结论，P2Y12激活下游的Gαi信号传导，而非P2Y1或α2A激活下游的Gαq或Gαz信号传导，在ADP介导的血小板活化中发挥功能时需要脂筏。
