Lyons R M, Shaw J O
J Clin Invest. 1980 Feb;65(2):242-55. doi: 10.1172/JCI109666.
Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction of the platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43--loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin- and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0 +/- 1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3 +/- 1.3 s SE (n = 3) for P7P and 3.7 +/- 0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P less than 0.015) by removal by Ca2+ from the medium at a time point before any thrombin- or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the rabbit platelet is an appropriate model in which to study the effects of Ca2+ on protein phosphorylation during the platelet release reaction.
钙离子流和蛋白质磷酸化被认为在血小板释放反应的诱导过程中发挥重要作用。然而，钙离子、蛋白质磷酸化与释放反应之间的相互作用一直难以研究，因为人血小板的分泌不依赖细胞外钙离子。因此，我们研究了兔血小板，与人类血小板不同，兔血小板的5-羟色胺释放需要细胞外钙离子。用凝血酶、嗜碱性粒细胞血小板激活因子（PAF）或离子载体A23187处理完整的、负载了32PO43-的兔血小板，分别导致P7P和P9P的磷酸化增加200%-400%。这些肽在所有方面都与凝血酶处理的人血小板中磷酸化的肽相似。当用乙二醇双四乙酸（EGTA）替代培养基中的钙离子时，（a）凝血酶和PAF诱导的兔血小板[3H]5-羟色胺释放被抑制60%-75%，而离子载体诱导的释放则被完全阻断；（b）凝血酶、PAF或离子载体诱导的P9P磷酸化被抑制60%；（c）离子载体诱导的P7P磷酸化降低60%，而凝血酶或PAF引起的P7P磷酸化仅降低20%。在0.25-0.5 U/ml的凝血酶浓度下，磷酸化先于[3H]5-羟色胺释放，P7P的最大释放时间的一半为26.0±1.3秒标准误（n=；3），P9P的最大磷酸化时间的一半为12.3±1.3秒标准误（n=3），P9P为3.7±0.17秒标准误（n=3）。在任何凝血酶或离子载体诱导的5-羟色胺释放可检测到之前的一个时间点，通过从培养基中去除钙离子，P9P磷酸化受到显著抑制（P<0.015）。因此，我们的数据表明钙离子流先于5-羟色胺分泌的开始，并且兔血小板是研究血小板释放反应过程中钙离子对蛋白质磷酸化影响的合适模型。
