Human platelet adhesion to fibrin is mediated by beta1-as well as beta3-integrins.

我们研究了由FXIII-交联型纤维蛋白组成的血小板与表面黏附的机制。将人纤维连接蛋白耗竭纤维蛋白原(Fbgen)(约2.0 mg/ml)、凝血酶(Lu/ml)和CaCl2(2 MM)在最终的100 MU1中混合在96-免疫板的孔中。然后用0.3%的BSA和凝血酶抑制肽PPACK封闭纤维蛋白表面。作为比较，还将Fbgen(30mug/ml)涂覆在孔上，以制备固定化Fbgen表面(ImFbgen)。用荧光染料BCECF-AM负载人血小板，洗涤后悬浮至50×10~(-3)/ml，将100×10~(-1)负载的血小板放置在纤维蛋白和imFbgen表面。间隔后取出非粘附性血小板，用Fluoloskan测定粘附性血小板。在37ﾟ中孵育1h后，血小板与纤维蛋白和imFbgen的粘附率分别为15.7±2.3%(M*SD，n=11)和12.8×2.3%(n=9)，凝集抑制单抗T74抑制了血小板与…的粘附性与imFbgen的粘附率增加了90%，但与纤维蛋白的粘附性仅被抑制了20%。在37ﾟC下，用4 mM EDTA在37ﾟC下处理1小时后，血小板与imFbgen和纤维蛋白的粘附率分别为对照组的28%和52%。RGD多肽呈剂量依赖性地抑制血小板与imFbgen和纤维蛋白的粘附性。纤维蛋白原-γ链C端十二肽抑制血小板与imFbgen的黏附，但不抑制与纤维蛋白的黏附。II型血小板对免疫纤维蛋白原和纤维蛋白的粘附率分别为正常血小板的26.7%和75.4%。扫描电子显微镜研究显示，血小板减少，与EDTA治疗的血小板一样，呈圆形，附着在纤维蛋白上，但不扩散。一些抗β1抗体如JBS5和LM534可抑制血小板与纤维蛋白的黏附，但不能抑制imFbgen的黏附。此外，C32TG细胞是一种黑色素瘤来源的细胞系，此前有报道称它可以收缩纤维蛋白凝块，它附着在纤维蛋白上，而纤维蛋白可被Beta1和Beta3抗体抑制。这些结果表明，血小板与纤维蛋白的黏附不仅由β3介导，也由β1整合素介导。他们还提出，纤维蛋白原向纤维蛋白的转化与一个新的粘附域的暴露和纤维蛋白原伽马链的功能C末端十二肽的丢失有关。较少

We studied mechanisms of platelet adhesion to surface consisting of FXIII-cross-linked fibrin. Human' fibronectin-depleted fibrinogen (Fbgen) (f. c. 2.0mg/ml) , thrombin (lU/ml) and CaCl_2 (2 mM) in a final 100 mu1 were mixed in wells of 96-immunoplates. Fibrin surface was then blocked with 0.3% BSA and PPACK, a thrombin-inhibitory peptide. For comparison, immobilized Fbgen surface (imFbgen) was also prepared by coating Fbgen(30 mug/ml) on the wells. Human platelets were loaded with BCECF-AM, an fluorescent dye, washed and finally suspended to a concentration of 50 x 10^3/ml. 100mu1 of the BCECF-loaded platelets were placed on the surfaces of fibrin and imFbgen. After intervals, non-adhesive platelets were removed and adhering platelets were measured with Fluoloskan. Platelet adhesion to fibrin and imFbgen were 15.7*2.3 % (M*SD, n=11) and 12.8*2.3 % (n=9), respectively, after 1 hr incubation at 37ﾟC.T74, an aggregation-inhibiting monoclonal anti-beta3 antibody, inhibited platelet adhes … More ion to imFbgen by 90%, but, in a sharp contrast, adhesion to fibrin was inhibited only by 20%. Adhesion to imFbgen and fibrin of the platelets previously treated with 4mM EDTA at pH 7.4 at 37ﾟC for 1 hr were 28 and 52 %, respectively, of control platelets treated with EDTA at 22ﾟC.RGD-peptides dose-dependently inhibited platelet adhesion to both imFbgen and fibrin. Dodecapeptide of C-terminus of fibrinogen gamma chain inhibited platelet adhesion to imFbgen but not to fibrin. Adhesion of Type II thrombasthenic platelets to imFbgen and fibrin were 26.7 and 75.4%, respectively, of normal platelets. Scannning electron microscopic studies revealed that thrombasthenic platelets, as likely as EDTA-treated platelets, as a round shape, adhered to but did not spread on fibrin. Some anti-beta1 antibodies such as JBS5 and LM534 inhibited platelet adhesion to fibrin but not to imFbgen. In addition, C32TG cells, a melanoma-derived cell line, and previously reported to retract fibrin clot, adhered to fibrin which was inhibited by beta1 as well as beta3 antibodies. These results indicate that platelet adhesion to fibrin is mediated by not only beta3 but also beta1 integrins. They also suggest that conversion of fibrinogen to fibrin is associated with exposure of a new adhesive domain and loss of functional C-terminal dodecapeptide of fibrinogen gamma chain. Less