Shattil S J, Brass L F
J Biol Chem. 1987 Jan 25;262(3):992-1000.
We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.
我们使用经皂角苷通透处理的血小板来研究血小板活化导致纤维蛋白原表面受体暴露的机制。使用125I - 纤维蛋白原和125I - PAC1（一种对纤维蛋白原受体活化形式具有特异性的单克隆抗体）检测受体暴露情况。所研究的潜在介质包括鸟苷 - 5'-基亚氨基二磷酸（Gpp(NH)p）和鸟苷5'O -（硫代三磷酸）（GTPγS），它们可导致血小板中G蛋白依赖性磷脂酶C活化；肌醇1,4,5 - 三磷酸（IP3），可使血小板致密管状系统释放Ca2+；以及二酰基甘油和佛波酯，它们可激活蛋白激酶C。这些分子中的每一种都能引起纤维蛋白原和PAC1结合。通过提高通透处理血小板中的胞质游离Ca2+浓度可模拟IP3的作用。然而，吲哚美辛或阿司匹林可消除IP3和Ca2+诱导的PAC1结合，而它们对Gpp(NH)p、佛波酯或二酰基甘油引起的PAC1结合没有影响。这表明对IP3和Ca2+的反应是由于花生四烯酸代谢产物的形成。一种这样的代谢产物，血栓素A2（TxA2），被认为通过刺激G蛋白依赖性磷酸肌醇水解来激活血小板。事实上，我们发现G蛋白抑制剂鸟苷 - 5'-基硫代磷酸（GDPβS）可抑制血栓素A2类似物（U46619）、IP3和Ca2+引起的PAC1结合，但对二酰基甘油或佛波酯诱导的PAC1结合没有影响。凝血酶诱导的PAC1结合和磷酸肌醇水解也受到GDPβS和百日咳毒素的抑制。增加凝血酶浓度可克服GDPβS对PAC1结合的抑制，但不能克服对磷酸肌醇水解的抑制。这些观察结果表明，纤维蛋白原受体暴露至少通过两条途径发生。其中一条途径，响应凝血酶和U46619等激动剂，由G蛋白依赖性磷酸肌醇水解引发，涉及IP3和二酰基甘油的形成。IP3似乎通过刺激Ca2+依赖性花生四烯酸代谢起作用，进而触发进一步的磷酸肌醇水解。二酰基甘油通过刺激蛋白激酶C起作用。第二条途径由高浓度凝血酶激活，且不依赖于磷酸肌醇水解。
