Electron microscopic analyses on platelet membrane glycoproteins using monoclonal antibodies

血小板粘附和聚集过程是由血小板表面特异性膜糖蛋白（GP）介导的。结合位点有两种；对于血管性血友病因子和凝血酶，在GPIb和GPIIb/IIIa复合体上的纤维原结合位点。我们使用免疫细胞化学方法检查了血小板活化过程中gp的分布。我们利用杂交瘤技术制备的TM60是一种抗GPIb的单克隆抗体。将戊二醛固定的血小板与TM60孵育后，观察到血小板表面膜上均匀分布代表GPIb定位的铁蛋白颗粒。颗粒以约100 nm的间隔分布。扫描电镜下单侧表面铁蛋白颗粒数为2070-4150个（2940<正负> - 790；平均<正负>SD, n = 10）。血小板活化后，颗粒分布紊乱，呈簇状结构，提示GPIb作为受体的作用。用山羊抗人纤维蛋白原抗体与金标记抗山羊IgG在电镜下反应，观察纤维蛋白原在adp和凝血酶诱导的血小板聚集中的关联。在静息的血小板中，金颗粒只出现在apha颗粒中，而不在血小板表面。当添加ADP时。在添加纤维蛋白原的情况下，在血小板表面检测到金颗粒。ADP引起正常血小板聚集，贴壁血小板之间和血小板表面有明显的金颗粒。当血小板分解时，金不再存在于表面。然而，在凝血酶诱导的聚集中，有几个聚集的团块在血小板表面没有纤维原结合。因此，纤维蛋白原可能不是形成凝血酶刺激的血小板之间联系的唯一原因

Processes of platelet adhesion and aggregation are mediated by specific membrane glycoproteins (GP) on the platelet surface.There are two kinds of binding sites; for von Willebrand factor and thrombin,on GPIb and fibriongen binding site on GPIIb/IIIa complex.We have examined distributions of GPs during platelet activation using immunocytochemical methods.TM60 which was produced by us using a hybridoma technique is a monoclonal antibody against GPIb. When glutaraldehyde-fixed platelets were incubated with TM60,a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm.The numbers of ferritin particles on the surface of one side were 2070-4150(2940<plus-minus>790;mean<plus-minus>SD, n = 10) under the scanning electron microscope. The distribution was disarranged and cluster-like structures of particles were observed after the activation of platelets suggestion the role of GPIb as a receptor. Association of fibrinogen in ADP-and thrombin-induced platelet aggregation was observed using a goat antihuman fibrinogen antibody which reacted with a gold-labeled anti-goat IgG under the electron microscope.In resting platelets, gold particles were appearent in only aplha-granules and not on the platelet surface.When ADP was added.gold particles were detected on the platelet surface in the presence of added fibrinogen. ADP caused aggregaion of normal platelets and gold particles were evident between the adherent platelets and on the platelet surface.When the platelets deaggregated, gold was no longer present on the surface.In thrombin-induced aggregation, however,there are several aggregated masses without fibringen binding on the platelet surface.Thus fibrinogen may not be solely responsible to forming the links between thrombin-stimulated plateleffe