Karniguian A, Grelac F, Levy-Toledano S, Legrand Y J, Rendu F
INSERM U150-CNRS URA 334, Hôpital Saint-Louis, Paris, France.
Biochem J. 1990 Jun 1;268(2):325-31. doi: 10.1042/bj2680325.
This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.
本研究分析了胶原诱导的血小板活化过程中的早期生化事件。在延迟期发生的一个早期代谢事件是磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）特异性磷脂酶C的活化。磷脂酸（PtdOH）的形成、P43和P20的磷酸化、血栓素B2（TXB2）的合成以及血小板分泌在延迟期后开始，并且除了延迟的TXB2合成外，它们在时间上具有相似的依赖性。胶原诱导广泛的P43磷酸化，而P20磷酸化较弱且始终低于凝血酶诱导的水平。P43磷酸化和颗粒分泌的剂量反应曲线相似，两者在7.5微克胶原/毫升时达到峰值，该剂量诱导了半最大PtdOH和TXB2的形成。假定抑制蛋白激酶C的鞘氨醇平行抑制P43磷酸化和分泌。然而，鞘氨醇对蛋白激酶C不具有特异性，因为15微摩尔浓度的鞘氨醇不抑制P43磷酸化，但却使TXB2合成受阻50%。即使在100微摩尔浓度下，鞘氨醇对PtdOH的形成也没有任何影响，这表明胶原自身诱导了这种PtdOH的形成，与TXB2的生成无关。外部Ca2+的缺失允许多磷酸肌醇的裂解和肌醇三磷酸（InsP3）的积累发生，但损害了P43磷酸化、PtdOH和TXB2的形成以及分泌；只有添加0.11微摩尔/升的Ca2+才能恢复这些过程。总之，对胶原的血小板膜受体的刺激引发了一种不依赖于外部Ca2+的PtdInsP2特异性磷脂酶C的活化，这可能是直接的受体相关反应。Ca2+内流对于触发随后的血小板反应是必不可少的。这种刺激主要涉及与分泌相关的蛋白激酶C途径，并且至少在其初始阶段似乎不是由TXB2介导的。
