PURIFICATION AND PRELIMINARY CHARACTERIZATION OF THE GLYCOPROTEIN IB COMPLEX IN THE HUMAN-PLATELET MEMBRANE
PURIFICATION AND PRELIMINARY CHARACTERIZATION OF THE GLYCOPROTEIN IB COMPLEX IN THE HUMAN-PLATELET MEMBRANE
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DOI：
10.1111/j.1432-1033.1985.tb09152.x
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发表时间：
1985-01-01
期刊：
影响因子：
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通讯作者：
CASTALDI, PA
中科院分区：
文献类型：
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作者：
BERNDT, MC;GREGORY, C;CASTALDI, PA
人血小板糖蛋白Ib （GP Ib）是一种主要的整体膜蛋白，已被确定为血小板结合位点，介导血小板与血管内皮下层的因子VII/血管性血血病因子依赖性粘附。最近的证据表明GP Ib通常与另一种血小板膜蛋白GP IX络合。在本研究中，用含有0.1% (v/v) Triton X-100的缓冲液选择性地溶解人血小板质膜。GP Ib复合物（GP Ib + GP IX）在.apprxeq中纯化至均匀性。利用偶联琼脂糖的抗（糖蛋白Ib复合物）小鼠单克隆抗体WM 23对膜提取物进行免疫亲和层析，收率为30%；随后，利用偶联琼脂糖的第二抗（糖蛋白Ib复合物）单克隆抗体FMC 25对GP Ib复合物进行免疫亲和层析，分离出GP Ib和GP IX作为纯化组分。经十二烷基硫酸盐/聚丙烯酰胺凝胶电泳评估，纯化的GP Ib与完整血小板上的分子相同，在非还原条件下的表观相对分子质量为170,000，而在135,000 （. α）。亚单位)和25,000 (.beta。亚基)在还原条件下。GP IX在非还原和还原条件下的表观Mr均为22,000。纯化的Gb Ib复合体和GP Ib抑制利多司汀介导的、人因子VIII/冯氏血癌因子依赖的和牛因子VIII/冯氏血癌因子依赖的人血小板凝集，表明这些蛋白已经以功能活性形式分离出来。GP IB.alpha。与先前报道的蛋白水解降解产物糖钙素的氨基酸组成相似。GP b. β的氨基酸组成。与GP IX相似，但在谷氨酸、丙氨酸、组氨酸和精氨酸水平上存在显著差异。GP b. α的n端。GP IX被阻断；GP Ib.beta。具有n端序列Ile-Pro-Ala-Pro-。在交叉免疫电泳中，发现GP Ib和GP IX出现在相同的免疫沉淀区(s)，无论血小板在没有或存在钙依赖性蛋白酶抑制剂lepeptin的情况下被溶解。在富血小板血浆中的结合研究表明，有相似数量的结合位点(.hivin。x。+ -。SD)为3种抗（糖蛋白Ib复合物）单克隆抗体；a51, GP b. α表位。(22000 + -。2700, n = 3), wm23, GP b.alpha表位。(21000 + -。3400, n = 3), FMC 25， GP IX表位(20,100 .+-。2700, n = 3), FMC 25 (Fab ")2(27,100 .+-。800, n = 2)。综合证据表明GP Ib通常与GP IX结合，并且该配合物的化学计量比为1:1。
Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in .apprxeq. 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose, GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170,000 under nonreducing conditions and 135,000 (.alpha. subunit) and 25,000 (.beta. subunit) under reducing conditions. GP IX had an apparent Mr of 22,000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP IB.alpha. had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib.beta. and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib.alpha. and GP IX were blocked; GP Ib.beta. had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (.hivin.x .+-. SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies; AN 51, epitope on GP Ib.alpha. (22,000 .+-. 2700, n = 3), Wm 23, epitope on GP Ib.alpha. (21,000 .+-. 3400, n = 3), FMC 25, epitope on GP IX (20,100 .+-. 2700, n = 3), and FMC 25 (Fab'')2 (27,100 .+-. 800, n = 2). The combined evidence suggests that GP Ib is normally bound to GP IX and that the stoichiometry of this complex is 1:1.