血小板活化过程中膜糖蛋白Ⅱb/Ⅲa构象变化的研究

Study on conformational changes of GP Ⅱb/Ⅲa complex during platelet activation

目的 对血小板活化过程中膜糖蛋白（ Ｇ Ｐ） Ⅱｂ／ Ⅲａ 构象变化进行初步探讨。方法 分别用供体荧光（ Ｆ Ｉ Ｔ Ｃ） 与受体荧光（ Ｔ Ｒ） 标记识别 Ｇ ＰⅡｂ／ Ⅲａ 上不同抗原决定簇的单抗。用流式细胞仪检测活化血小板在５３０ ｎｍ 处的荧光强度值，并计算荧光供受体间的荧光共振能量转移值（ Ｆ Ｒ Ｅ Ｔ Ｖ） 。结果 不论何种单抗作为荧光供体，静息态血小板均可被测得一低而稳定的 Ｆ Ｒ Ｅ Ｔ Ｖ（ 平均５ ．５ ％ ） 。血小板被激活时 Ｆ Ｒ Ｅ Ｔ Ｖ 会有显著升高，表明 Ｇ ＰⅡｂ／ Ⅲａ 内的亚单位间发生了位置或（ 和） 方向上的变化，该变化也可因胞外钙离子的清除而发生，但不依赖于纤维蛋白原与其受体的结合。结论 血小板活化时 Ｆ Ｒ Ｅ Ｔ Ｖ 的升高可定性反映出荧光标记单抗所结合的 Ｇ ＰⅡｂ／ Ⅲａ 内部亚单位间所发生的重新排列，这种构象的改变可最终导致纤维蛋白原受体的表达。

Objective To determine whether a conformational change in GPⅡb/Ⅲa occurs during platelet activation. Methods Epitopes on GPⅡb/Ⅲa complex were labeled with McAbs to GPⅡb (SZ 22) and GPⅢa (SZ 21) conjugated to either a donor fluorescein (FITC labeled) or an acceptor (TR labeled) chromophore. The FRET between platelet bound FITC and TR was measured and calculated by flow cytometry during platelet activation induced by some agonists. Results In unstimulated platelets, a small but consistent change of FRET (5.5%) was detected, regardless of which antibody served as FRET donor. Platelet activation resulted in an obvious increase in FRET, suggesting a significant change in the separation or orientation of the epitopes within GPⅡb/Ⅲa. This kind of changes could also occur on removing the extracellular calcium , and was independent of receptor occupancy. Conclusion The increase in FRET can reflect the rearrangement of the chromophore labeled McAbs bound to GPⅡb/Ⅲa during platelet activation, and this kind of structural rearrangement subunits of GPⅡb/Ⅲa complex subunits can induce fibrinogen receptor expression.