Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody.
Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody.
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DOI：
10.1172/jci109983
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发表时间：
1980-12
期刊：
影响因子：
--
通讯作者：
R. McEver;N. Baenziger;P. Majerus
中科院分区：
文献类型：
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作者：
R. McEver;N. Baenziger;P. Majerus
我们采用杂交瘤技术进一步表征了Glanzmann血栓减少症的血小板糖蛋白异常。将人血小板免疫的Balb/c小鼠脾细胞与小鼠骨髓瘤细胞系Sp2/0-Ag14融合。采用次黄嘌呤-氨基蝶呤-胸苷选育法分离并克隆了产生多种抗血小板抗体的杂交瘤细胞系，并从6株杂交瘤细胞系中制备了纯化的单克隆IgG。其中一种细胞系8aB5-9产生一种抗体Tab，它与正常血小板上的一种蛋白质结合，而不是血栓性血小板。我们用Tab-Sepharose亲和层析法从Triton X-100溶解的正常血小板膜中分离到该蛋白。SDS聚丙烯酰胺凝胶电泳测定，分离的蛋白为糖蛋白IIb和IIIa的复合物，因为这两个亚基与整个血小板的糖蛋白IIb和IIIa是同源的，并且在二硫键还原后表现出相同的迁移率变化。我们制备了（125）I-Tab，通过直接结合法测定正常血小板和凝血血小板上糖蛋白IIb-IIIa复合物的数量。来自17个正常供体的血小板结合39,000+/-4,600 (SD) Tab分子/血小板。4例血栓减少患者的血小板缺乏Tab结合位点（<5%）。5个专性和4个假定的血栓性贫血杂合子结合24,500+/-5,800个Tab分子/血小板。血小板异体抗原Pl（Al）不能被Tab识别，因为来自三个Pl（Al）阴性受试者的血小板正常结合Tab。使用Tab抗体进行的研究(a)可以定量测定正常血小板膜上的糖蛋白IIb-IIIa复合物的数量，(b)证明血栓纯合子缺乏和杂合子部分缺乏这种复合物，(c)可以分离正常血小板聚集和凝块缩回所需的这种膜蛋白。
We used the hybridoma technique to characterize further the platelet glycoprotein abnormality in Glanzmann's thrombasthenia. Spleen cells from Balb/c mice immunized with human platelets were fused to mouse myeloma cell line Sp2/0-Ag14. Hybridoma lines producing a variety of antiplatelet antibodies were isolated by hypoxanthine-aminopterin-thymidine selection and cloned, and purified monoclonal IgG from six lines was prepared. One of these lines, 8aB5-9, produced an antibody, Tab, that binds to a protein on normal but not thrombasthenic platelets. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-Sepharose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein is a complex of glycoproteins IIb and IIIa, because the two subunits comigrate with glycoproteins IIb and IIIa of whole platelets and show identical changes in mobility after disulfide bond reduction. We prepared (125)I-Tab to determine the number of glycoprotein IIb-IIIa complexes on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal donors bound 39,000+/-4,600 (SD) Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%). Five obligate and four presumed heterozygotes for thrombasthenia bound 24,500+/-5,800 Tab molecules/platelet. The platelet alloantigen, Pl(Al), is not that recognized by Tab, because platelets from three Pl(Al)-negative subjects bound Tab normally. Studies with the Tab antibody have (a) enabled quantitation of the number of glycoprotein IIb-IIIa complexes on normal platelet membranes, (b) demonstrated that thrombasthenic homozygotes lack and heterozygotes have a partial deficiency of this complex, and (c) made possible the isolation of this membrane protein which may be required for normal platelet aggregation and clot retraction.