Crouch M F, Lapetina E G
Molecular Biology Department, Wellcome Research Laboratories, Research Triangle Park, NC 27709.
Biochem Biophys Res Commun. 1988 May 31;153(1):21-30. doi: 10.1016/s0006-291x(88)81184-7.
Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.
用凝血酶刺激人血小板时，磷脂酶C和A2都会被激活。这些被认为是相继发生的事件，磷脂酶A2的激活是由肌醇磷脂的预先水解和细胞内钙储存的动员引起的。然而，我们实验室和其他实验室最近对这一观点提出了质疑，现在我们提供进一步的证据表明，在凝血酶刺激过程中，这些酶可能通过不同的机制被激活。α-凝血酶诱导肌醇磷脂的快速水解，以及肌醇三磷酸和磷脂酸的形成。这与细胞内钙储存的动员同时发生。这些反应被前列环素阻断了约50%。相比之下，α-凝血酶诱导的花生四烯酸释放被前列环素完全抑制。效果较差的激动剂血小板活化因子（PAF）和γ-凝血酶也都刺激磷脂酶C，但PAF引起快速且短暂的反应，而γ-凝血酶的反应则延迟且更持久。这些激动剂诱导钙储存释放的能力与磷脂酶C的激活密切相关。然而，这两种试剂达到的最大细胞内钙浓度是相同的。尽管如此，γ-凝血酶而非PAF能够释放少量花生四烯酸。当在α-凝血酶刺激血小板之前给予肾上腺素时，磷脂酶C的激活、钙动员和聚集都会增强。γ-凝血酶和PAF也是如此。然而，与α-凝血酶不同，γ-凝血酶刺激的花生四烯酸释放并未被肾上腺素增强，反而有所减少。这些结果表明，在活化的血小板中，磷脂酶C和磷脂酶A2的激活是可分离的事件。α-凝血酶刺激磷脂酶A2的机制似乎不是通过抑制性GTP结合蛋白Gi的解离，因为γ-凝血酶降低百日咳毒素诱导的41 kDa蛋白的ADP核糖基化程度与α-凝血酶相同，但在诱导花生四烯酸释放方面比α-凝血酶的效果要差得多。
