Tomasini B R, Mosher D F
Department of Medicine, University of Wisconsin, Madison 53706.
Blood. 1988 Sep;72(3):903-12.
A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme-immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin-antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of urea treatment and of adsorption of vitronectin onto tissue culture plastic (as is done in cell adhesion assays). The change may be important for expression of vitronectin activity.
使用竞争性酶免疫吸附测定法和免疫亲和色谱法，研究了单克隆抗体8E6（Hayman EG等人，《美国国家科学院院刊》80:4003，1983年）对黏附糖蛋白玻连蛋白（也称为S蛋白、血清扩散因子和表皮调节素）的识别差异。血清中玻连蛋白的识别率比血浆中玻连蛋白的识别率高约50倍。与肝素、凝血酶-抗凝血酶III复合物或肝素与凝血酶-抗凝血酶III复合物一起孵育的玻连蛋白的识别率分别比单独的玻连蛋白高2.5倍、7倍或32倍。单独的凝血酶或抗凝血酶III不会诱导抗原变化。因子Xa-抗凝血酶III在诱导这种变化方面不如凝血酶-抗凝血酶III有效。硫酸葡聚糖和岩藻依聚糖在诱导抗原变化方面比肝素更有效，而硫酸皮肤素、透明质酸、硫酸乙酰肝素、硫酸软骨素或硫酸角质素则效果较差。对血清以及与凝血酶和抗凝血酶III一起孵育的玻连蛋白进行免疫印迹分析，结果表明存在由玻连蛋白和凝血酶-抗凝血酶III组成的复合物，这些复合物只能用还原剂解离。N-乙基马来酰亚胺完全阻断了可能由二硫键连接形成的复合物的形成，并部分阻断了抗原变化。非二硫键连接和二硫键连接的玻连蛋白都能从玻连蛋白和凝血酶-抗凝血酶III的混合物中与抗体-琼脂糖结合。用8摩尔/升尿素处理玻连蛋白会导致单克隆抗体对其识别增强。因此，8E6抗体与一个表位反应，该表位优先由非共价和共价连接的玻连蛋白/凝血酶-抗凝血酶III复合物以及经尿素处理的玻连蛋白表达。因此，凝血酶-抗凝血酶III诱导的玻连蛋白变化是尿素处理以及玻连蛋白吸附到组织培养塑料上（如细胞黏附试验中所做的那样）的生理相关物。这种变化可能对玻连蛋白活性的表达很重要。
