Siess W, Siegel F L, Lapetina E G
J Biol Chem. 1983 Sep 25;258(18):11236-42.
Exogenous unlabeled arachidonic acid (AA) added to human platelets prelabeled with [3H]AA induces breakdown of [3H]phosphatidylinositol and the rapid and transient formation of [3H]1,2-diacylglycerol and [3H]phosphatidic acid (PA), indicating activation of phosphatidylinositol-specific phospholipase C. Formation of [3H]1,2-diacylglycerol and [3H]PA is inhibited by pretreatment of platelets with aspirin, which suggests that endoperoxides or thromboxane A2 are responsible for AA-induced stimulation of phospholipase C. Exogenous unlabeled AA also induces the formation of [32P]PA or [14C]PA in platelets that have been prelabeled with 32Pi or [14C]AA, respectively. Increased radioactivity in PA reflects increased content of PA as measured by the fatty acid composition of PA. The relation of PA production, which reflects stimulation of phospholipase C, to specific platelet responses was further investigated. Low concentrations of AA (0.05-0.2 microM) induces platelet shape change in parallel to formation of 50-100% PA and phosphorylation of a 40,000 molecular weight protein. Higher concentrations of AA (0.5-50 microM) stimulate the formation of a further amount of PA (200-250%), and phosphorylation of 40,000 molecular weight protein, platelet aggregation, and serotonin release. Indomethacin inhibits all these observed changes by inhibiting the conversion of AA by platelet cyclooxygenase. In contrast, prostacyclin blocks these responses without affecting conversion of AA by platelet cyclooxygenase and thromboxane synthetase. We conclude that formation of endoperoxides and thromboxane A2 is necessary but not sufficient for platelet activation by AA. Only if PA is formed are platelets activated. The results indicate a central role for the phospholipase C pathway in the process of platelet activation.
将外源性未标记的花生四烯酸（AA）添加到预先用[3H]AA标记的人血小板中，会诱导[3H]磷脂酰肌醇的分解以及[3H]1,2 - 二酰甘油和[3H]磷脂酸（PA）的快速短暂形成，这表明磷脂酰肌醇特异性磷脂酶C被激活。用阿司匹林预处理血小板可抑制[3H]1,2 - 二酰甘油和[3H]PA的形成，这表明内过氧化物或血栓素A2是AA诱导磷脂酶C激活的原因。外源性未标记的AA还分别在预先用32Pi或[14C]AA标记的血小板中诱导[32P]PA或[14C]PA的形成。PA中放射性的增加反映了通过PA的脂肪酸组成测量的PA含量的增加。进一步研究了反映磷脂酶C激活的PA产生与特定血小板反应之间的关系。低浓度的AA（0.05 - 0.2 microM）诱导血小板形状改变，同时形成50 - 100%的PA并使分子量为40,000的蛋白质磷酸化。较高浓度的AA（0.5 - 50 microM）刺激进一步形成一定量的PA（200 - 250%），使分子量为40,000的蛋白质磷酸化、血小板聚集和5 - 羟色胺释放。吲哚美辛通过抑制血小板环氧化酶对AA 的转化来抑制所有这些观察到的变化。相比之下，前列环素阻断这些反应，而不影响血小板环氧化酶和血栓素合成酶对AA的转化。我们得出结论，内过氧化物和血栓素A2的形成对于AA激活血小板是必要的，但不是充分的。只有当形成PA时血小板才被激活。结果表明磷脂酶C途径在血小板激活过程中起核心作用。
