Mátyus L, Bene L, Hársfalvi J, Alvarez M V, González-Rodríguez J, Jenei A, Muszbek L, Damjanovich S
Department of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt 98, H-4012 Debrecen, Hungary.
J Photochem Photobiol B. 2001 Dec 1;65(1):47-58. doi: 10.1016/s1011-1344(01)00255-x.
Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.
糖蛋白IIb/IIIa是糖蛋白IIb和IIIa的异源二聚体，在血小板表面作为纤维蛋白原和其他黏附蛋白的诱导型受体。尽管Calvete等人已在溶液中构建了GPIIb/IIIa分子的四级结构模型[《生物化学杂志》282 (1992) 523]，但完整血小板表面的相应模型仍然缺失。在本研究中，在生理条件下，以纳米分辨率研究了静息人血小板表面GPIIb/IIIa异源二聚体的构象和横向分布。实验基于荧光共振能量转移的双波长流式细胞术检测以及针对已明确描述的结合位点产生的一组单克隆抗体的应用。首次观察到GPIIb/IIIa异源二聚体的单分散分布，并在生理条件下在血小板膜上构建了抗体结合位点的详细三维邻近图谱。我们的数据支持GPIIb亚基处于弯曲构象的观点。还对一组单克隆抗体的K(d)值和结合位点数量进行了详细分析，为结合位点的拓扑结构提供了补充数据。我们的结果对GPIIb/IIIa异源二聚体的膜拓扑结构进行了优化。
