血小板活化过程中膜糖蛋白Ⅱb/Ⅲa构象变化的研究

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。

Study on conformationalchanges of GP

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.