Crouch M F, Lapetina E G
Molecular Biology Department, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
J Biol Chem. 1988 Mar 5;263(7):3363-71.
Stimulation of washed human platelets with alpha-thrombin was accompanied by aggregation, formation of inositol phosphates and phosphatidic acid, liberation of arachidonic acid, mobilization of intracellular Ca2+ stores, and influx of Ca2+ from the extracellular medium. Each of these responses was potentiated by a short pretreatment with epinephrine, although alone this agent was ineffective. A prolonged (5 min) stimulation with alpha-thrombin desensitized both phospholipase C and Ca2+ mobilization to a further thrombin challenge. Epinephrine added following thrombin desensitization restored both the ability of thrombin to release Ca2+ stores and stimulate inositol phospholipid hydrolysis. Resensitization was mediated by alpha 2-adrenergic receptors and lasted about 3 min, after which the Ca2+ levels returned again to basal levels. Pretreatment of platelets with phorbol dibutyrate at concentrations which specifically activate protein kinase C increased the rate of desensitization of the thrombin-induced release of Ca2+ stores and abolished the ability of epinephrine to restore the thrombin response. The protein kinase C inhibitor, staurosporine, blocked the inhibitory effect of phorbol ester and also reduced the rate of desensitization of thrombin and subsequent epinephrine action. These results suggest that thrombin activation of protein kinase C phosphorylates and inactivates a signaling protein which is common to both thrombin and alpha 2-adrenergic receptors. This protein is involved in thrombin stimulation of phospholipase C but is not directly stimulatory since epinephrine alone does not activate this enzyme. We searched for a known second messenger protein common to both thrombin and alpha 2-adrenergic receptors which was phosphorylated in intact platelets by protein kinase C in parallel with thrombin-induced desensitization. The alpha subunit of the inhibitory GTP-binding protein, Gi, was the only candidate which fulfilled all of these criteria as shown by immunoprecipitation. Therefore, we suggest that alpha i maintains the thrombin receptor in a state which can couple to phospholipase C when activated with thrombin. This permissive state of alpha i is blocked by phosphorylation by thrombin-activated protein kinase C.
用α-凝血酶刺激洗涤过的人血小板会伴随聚集、肌醇磷酸和磷脂酸的形成、花生四烯酸的释放、细胞内钙库的动员以及细胞外介质中钙离子的内流。尽管单独使用肾上腺素无效，但用其进行短暂预处理可增强上述每种反应。用α-凝血酶进行长时间（5分钟）刺激会使磷脂酶C和钙动员对进一步的凝血酶刺激产生脱敏作用。凝血酶脱敏后添加肾上腺素可恢复凝血酶释放钙库和刺激肌醇磷脂水解的能力。再敏化由α2-肾上腺素能受体介导，持续约3分钟，之后钙离子水平再次恢复到基础水平。用特异性激活蛋白激酶C的浓度的佛波醇二丁酸酯预处理血小板会增加凝血酶诱导的钙库释放的脱敏速率，并消除肾上腺素恢复凝血酶反应的能力。蛋白激酶C抑制剂星形孢菌素可阻断佛波醇酯的抑制作用，还可降低凝血酶和随后肾上腺素作用的脱敏速率。这些结果表明，凝血酶激活蛋白激酶C会使一种信号蛋白磷酸化并使其失活，该信号蛋白是凝血酶和α2-肾上腺素能受体共有的。这种蛋白参与凝血酶对磷脂酶C的刺激，但不是直接刺激性的，因为单独的肾上腺素不会激活这种酶。我们寻找了一种已知的凝血酶和α2-肾上腺素能受体共有的第二信使蛋白，该蛋白在完整血小板中被蛋白激酶C磷酸化，与凝血酶诱导的脱敏同时发生。抑制性GTP结合蛋白Gi的α亚基是唯一符合所有这些标准的候选蛋白，免疫沉淀结果表明了这一点。因此，我们认为αi使凝血酶受体维持在一种状态，当被凝血酶激活时可与磷脂酶C偶联。αi的这种允许状态被凝血酶激活的蛋白激酶C的磷酸化所阻断。
