Ramsamooj P, Doellgast G J, Hantgan R R
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103.
Thromb Res. 1990 Jun 15;58(6):577-92. doi: 10.1016/0049-3848(90)90304-u.
We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a Kd in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP- and thrombin-stimulated gel filtered platelets caused a 2-3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.
我们通过扁豆凝集素亲和层析，随后进行凝胶过滤层析，从人血小板的膜组分中纯化了整合素GPIIb:IIIa。以纯化的GPIIb:IIIa作为抗原，我们制备了单克隆抗体CS-1，免疫印迹显示其对天然GPIIIa具有特异性；GPIIIa的二硫键还原导致免疫反应性丧失。放射性标记配体结合研究表明，CS-1识别每个血小板约55,000个位点，结合常数在纳摩尔范围内，且与血小板激活状态无关。CS-1或其Fab片段与ADP和凝血酶刺激的凝胶过滤血小板结合，导致可溶性配体纤维蛋白原和纤维蛋白原纤维的结合受到2至3倍的抑制。CS-1还分别使ADP和凝血酶刺激的血小板聚集受到2倍和4倍的抑制。由于CS-1抑制纤维蛋白（原）与GPIIb:IIIa的相互作用，我们提出CS-1识别的GPIIIa上构象依赖性表位构成了受体中参与纤维蛋白（原）结合的区域。
