Electron microscopic analyses on platelet membrane glycoproteins using monoclonal antibodies

使用单克隆抗体对血小板膜糖蛋白进行电镜分析

• 批准号: 61570597
• 负责人: YAMAZAKI Hiroh
• 金额: $ 1.41万
• 依托单位: The Tokyo Metropolitan Institute of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61570597/
• 关键词: Platelet membrane glycoproteins; Immunocytochemistry; Platelet membrane recoptors; thrombin aggregation; ADP aggregation; fibrinogen; mooclonal antibody; トロンビン凝集; 血小板無力症; 受容体刺激

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

血小板表面的膜糖蛋白（GP）介导了血小板的粘附和聚集过程。GPIb和GPIIb/IIIa复合物上有两种GP结合位点，分别是von Willebrand因子和凝血酶的结合位点和fibriongen的结合位点。我们用免疫细胞化学方法研究了GP在血小板活化过程中的分布。我们用杂交瘤技术制备了抗GPIb的单克隆抗体TM 60。当戊二醛固定的血小板与TM 60孵育时，在血小板表面膜上观察到代表GPIb定位的铁蛋白颗粒的均匀分布。颗粒间隔约100 nm分布，扫描电子显微镜下一侧表面铁蛋白颗粒数量为2070-4150个（2940 <plus-minus>790个;平均<plus-minus>SD，n = 10）。血小板活化后颗粒分布紊乱，呈簇状结构，提示GPIb作为受体发挥作用。用羊抗人纤维蛋白原抗体与金标记的抗羊IgG反应，在电镜下观察了ADP和凝血酶诱导的血小板聚集中纤维蛋白原的相关性。在静息血小板中，仅在血小板颗粒中出现金颗粒，而不在血小板表面。当ADP存在时，在加入纤维蛋白原的情况下，在血小板表面检测到added.gold颗粒。ADP引起正常血小板聚集，在血小板表面和粘附的血小板之间有明显的金颗粒。当血小板解聚时，血小板表面不再有金颗粒存在。然而，在凝血酶诱导的血小板聚集中，有几个聚集的团块，而没有凝血酶原结合在血小板表面。因此，纤维蛋白原可能不是形成凝血酶刺激的血小板与血小板之间连接的唯一原因。

项目成果

Suzuki,H,Kinlough-Rathbone,RL,Packham,MA,Tanoue,K,Tamazaki,H, Mustard,JF: "Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibringen during ADP-induced responses,and enhanced fibringen binding in a medium w

Suzuki,H,Kinlough-Rathbone,RL,Packham,MA,Tanoue,K,Tamazaki,H,Mustard,JF：“纤维蛋白原在洗涤的人血小板上的免疫细胞化学定位。ADP 诱导的反应期间缺乏对纤维蛋白原的需求，并且纤维蛋白原增强

Yamazaki H;Mustard JF: "Platelet Activation" Academic Press, 210 (1987)

Yamazaki H；Mustard JF：“血小板激活”学术出版社，210（1987）

Yamazaki H.and Mustard J.F.: "Platelet Activation" Academic Press, 210 (1987)

Yamazaki H.和 Mustard J.F.：“血小板激活”学术出版社，210（1987）

Yamamoto,K,Yamamoto,N,Kitagawa,H,Tanoue,K,Kosaki,G,Yamazaki,H: "Localization of a thrombin-binding site on human platelet membrane glycoprotein Ib determined by a nomoclonal antibody" Thrombosis and Haemostasis. 55. 162-167 (1986)

Yamamoto,K,Yamamoto,N,Kitakawa,H,Tanoue,K,Kosaki,G,Yamazaki,H：“通过单克隆抗体确定人血小板膜糖蛋白 Ib 上凝血酶结合位点的定位”血栓形成和止血。

Suzuki K;Kinlough-Rathbone R.L.;Packham M.A.;Tanoue K.;Yamazaki H.;Mustard J.F.: Blood.

铃木 K；Kinlough-Rathbone R.L.；Packham M.A.；Tanoue K.；山崎 H.；芥末 J.F.：血。