Coller B S, Peerschke E I, Scudder L E, Sullivan C A
J Clin Invest. 1983 Jul;72(1):325-38. doi: 10.1172/jci110973.
To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP- and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of approximately 40,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IIIa, and both glycoproteins bound to an affinity column of the antibody. These studies indicate that there is probably a single anatomic site that is crucial to the binding of all fibrinogen molecules and that this site is most likely on the glycoprotein IIb/IIIa complex. It also suggests that the thrombasthenic phenotype can be completely accounted for on the basis of the inhibition of fibrinogen binding to platelets.
为了更明确纤维蛋白原受体在血小板生理学中的作用并对其进行生化特性鉴定，借助功能筛选试验，通过杂交瘤技术制备了一种能完全阻断纤维蛋白原与血小板表面结合的鼠单克隆抗体。纯化的F(ab')2片段和/或完整抗体完全阻断了由ADP、凝血酶或肾上腺素诱导的聚集以及由ADP诱导的放射性标记纤维蛋白原与血小板的结合。该抗体不阻断瑞斯托菌素对甲醛固定血小板的凝集作用，也不阻断由ADP或凝血酶诱导的形态变化。该抗体完全抑制了由ADP和肾上腺素诱导的ATP释放，但对由胶原和凝血酶诱导的释放的抑制呈剂量依赖性且不完全。该抗体还显著抑制血小板在玻璃珠柱中的滞留、血小板与玻璃的黏附以及血块回缩。因此，该抗体诱导了一种类血小板无力状态。免疫荧光研究证实了该抗体对正常血小板和巨核细胞的特异性，并表明血小板无力症患者的血小板中可检测到的抗原明显减少。放射性标记抗体在正常血小板上平均结合约40,000个位点，但在血小板无力症患者的血小板上结合少于2,000个位点。该抗体免疫沉淀了糖蛋白IIb和IIIa，并且这两种糖蛋白都与该抗体的亲和柱结合。这些研究表明，可能存在一个对所有纤维蛋白原分子结合至关重要的单一解剖学位点，并且该位点很可能位于糖蛋白IIb/IIIa复合物上。这也表明，基于纤维蛋白原与血小板结合的抑制作用，可以完全解释血小板无力症的表型。
