Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.
Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.
复制标题
PAF-乙醚（1-O-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱）与完整人血小板的结合动力学。
作者：
E. Kloprogge;J. Akkerman
在 22 摄氏度下测量 [3H]PAF-乙醚（1-O-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱）与完整人凝胶过滤血小板的结合。在高剂量的 [3H]PAF-乙醚 (0.5-0.9 nM) 下，特异性结合在 15 分钟内达到饱和，而使用低剂量 (0.02-0.5 nM) 时需要约 90 分钟。超过 1 nM，[3H]PAF-乙醚非特异性结合逐渐增加，这与 3H 标记代谢物的证明一起表明 [3H]PAF-乙醚的摄取和代谢。平衡分析揭示了一类特异性受体，Ka 为 18.86 +/- 4.82 X 10(9) M-1，每个血小板有 242 +/- 64 个结合位点。非平衡结合显示出类似的 Ka (16.87 X 10(9) M-1)。长时间孵育后，特异性结合变得不可逆，这一过程随着[3H]PAF-乙醚浓度的增加而增强。通过预先与未标记的PAF-乙醚温育而对PAF-乙醚脱敏的血小板未能结合第二剂量的PAF-乙醚（3H-标记的），这表明脱敏是由于可用结合位点的丢失造成的。在结合研究的条件下，PAF-乙醚诱导纤维蛋白原受体的暴露、聚集（在搅拌的悬浮液中）和（聚）-磷脂酰肌醇的改变。这些结果表明 PAF-乙醚通过受体介导的过程启动血小板反应。
The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82 X 10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87 X 10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.