Karczewski J, Knudsen K A, Smith L, Murphy A, Rothman V L, Tuszynski G P
Department of Medicine, Medical College of Pennsylvania, Philadelphia 19129.
J Biol Chem. 1989 Dec 15;264(35):21322-6.
The interaction of human platelet thrombospondin (TSP) with human platelet glycoproteins GPIIb-IIIa was studied using a solid-phase binding assay. Polystyrene test tubes were coated with TSP, and 125I-labeled GPIIb-IIIa was added, allowed to bind, and the bound radioactivity was measured. After 90 min, the binding became time independent, and in most experiments, more than 10% of the exogenously added radioactivity was bound to the tube. Analysis of the bound radioactivity by polyacrylamide gel electrophoresis and autoradiography indicated that it was from labeled GPIIb-IIIa. Several lines of evidence indicate that the binding of GPIIb-IIIa to TSP was specific. (a) TSP immobilized on plastic or Sepharose bound 3-10-fold more GPIIb-IIIa than immobilized bovine serum albumin. (b) Addition of unlabeled excess GPIIb-IIIa reversed the binding of 125I-labeled GPIIb-IIIa to immobilized TSP. (c) Addition of EDTA inhibited the binding of GPIIb-IIIa to TSP by more than 90%, whereas addition of 1 mM CaCl2 and 1 mM MgCl2 potentiated the binding by more than 100%. (d) Monoclonal antibodies against TSP and GPIIb-IIIa inhibited the binding by 30-70% as compared with control and polyclonal anti-fibrinogen anti-serum. (e) A plot of GPIIb-IIIa bound versus GPIIb-IIIa added was best described as a rectangular hyperbola by regression analysis with half-saturation at 60 ng/ml GPIIb-IIIa. Similar results were obtained when labeled TSP was added to tubes coated with GPIIb-IIIa. These results show that TSP and GPIIb-IIIa can specifically interact in vitro and suggest that GPIIb-IIIa may function as a platelet TSP receptor during platelet aggregation.
采用固相结合试验研究了人血小板凝血酶敏感蛋白（TSP）与人血小板糖蛋白GPIIb-IIIa之间的相互作用。将凝血酶敏感蛋白包被在聚苯乙烯试管上，加入125I标记的GPIIb-IIIa，使其结合，然后测量结合的放射性。90分钟后，结合变得与时间无关，在大多数实验中，超过10%的外源添加放射性与试管结合。通过聚丙烯酰胺凝胶电泳和放射自显影分析结合的放射性表明，其来自标记的GPIIb-IIIa。几条证据表明，GPIIb-IIIa与凝血酶敏感蛋白的结合是特异性的。（a）固定在塑料或琼脂糖上的凝血酶敏感蛋白结合的GPIIb-IIIa比固定的牛血清白蛋白多3至10倍。（b）添加未标记的过量GPIIb-IIIa可逆转125I标记的GPIIb-IIIa与固定化凝血酶敏感蛋白的结合。（c）添加乙二胺四乙酸（EDTA）可使GPIIb-IIIa与凝血酶敏感蛋白的结合抑制超过90%，而添加1 mM氯化钙和1 mM氯化镁可使结合增强超过100%。（d）与对照和多克隆抗纤维蛋白原抗血清相比，针对凝血酶敏感蛋白和GPIIb-IIIa的单克隆抗体可使结合抑制30%至70%。（e）通过回归分析，结合的GPIIb-IIIa与添加的GPIIb-IIIa的关系图最好描述为矩形双曲线，在60 ng/ml GPIIb-IIIa时达到半饱和。当将标记的凝血酶敏感蛋白添加到包被有GPIIb-IIIa的试管中时，也获得了类似的结果。这些结果表明，凝血酶敏感蛋白和GPIIb-IIIa在体外可特异性相互作用，并提示GPIIb-IIIa在血小板聚集过程中可能作为血小板凝血酶敏感蛋白受体发挥作用。
