Kloprogge E, Akkerman J W
Biochem J. 1986 Dec 1;240(2):403-12. doi: 10.1042/bj2400403.
When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [( 3H]arachidonate mobilization and thromboxane synthesis) and secretion [( 14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.
在平衡条件下（60分钟，22摄氏度，非搅拌悬浮液），将人血小板与500 nM - PAF - 乙酰乙醚（血小板活化因子，1 - O - 烷基 - 2 - 乙酰 - sn - 甘油 - 3 - 磷酸胆碱）一起孵育时，会暴露两类纤维蛋白原结合位点：一类具有高亲和力[解离常数（Kd）（7.2±2.1）×10⁻⁸ M，2367±485个位点/血小板，n = 9]，另一类具有低亲和力[Kd（5.9±2.4）×10⁻⁷ M，26972±8267个位点/血小板]。用环氧化酶抑制剂（乙酰水杨酸、吲哚美辛）或血栓素合成酶抑制剂（UK 38.485）预孵育可完全消除高亲和力结合，而低亲和力结合不变。相反，ADP清除剂（磷酸肌酸/肌酸激酶或磷酸烯醇丙酮酸/丙酮酸激酶）可完全阻止低亲和力结合，而高亲和力结合不受影响。初始结合研究（孵育2 - 10分钟）证实了这些发现，大部分结合对ADP清除剂敏感，一小部分对吲哚美辛敏感，两种抑制剂联合使用则完全阻断结合。将温度升高到37摄氏度会使低亲和力结合位点的数量减少6倍，而高亲和力结合不变。以单个血小板消失速率衡量的聚集，在纤维蛋白原浓度为10 nM或更低时取决于高亲和力结合，而在纤维蛋白原浓度为100 nM或更高时低亲和力结合占主导。这些发现表明在结合实验过程中血小板有相当程度的活化。然而，花生四烯酸代谢[（³H）花生四烯酸动员和血栓素合成]和分泌[（¹⁴C）血清素和β - 血小板球蛋白]约为最佳条件下（5单位凝血酶/毫升，37摄氏度，搅拌）所发现量的10%或更少。我们得出结论，在结合条件下PAF - 乙酰乙醚诱导的血小板活化很少。然而，血栓素A2和分泌的ADP的量足以通过相互独立的机制启动高亲和力和低亲和力纤维蛋白原结合。
