Salatti J A, Anton P, Nemerson Y, Sakariassen K S
Biotechnology Centre of Oslo, University of Oslo, Norway.
Blood Coagul Fibrinolysis. 1993 Dec;4(6):881-90.
Fibrin forms on and binds to the extracellular matrix of endotoxin-stimulated endothelium when exposed to flowing non-anticoagulated blood. These processes have been investigated by employing a human ex vivo perfusion model, a synthetic peptide Arg-Gly-Asp-Val and a monoclonal anti-tissue factor antibody which inhibits tissue factor/FVIIa-induced coagulation. Procoagulant extracellular matrix on plastic cover-slips was prepared from cultures of endotoxin-stimulated human endothelium following brief exposure to 0.1 M NH4OH. Non-anticoagulated blood was drawn directly from an antecubital vein by a pump at venous (100/s) and arterial (650/s) wall shear rates over the matrix-coated cover-slips positioned in parallel-plate perfusion chambers. Deposition of fibrin and platelets on the matrix was quantified by morphometry. Preincubation of the matrix with Arg-Gly-Asp-Val inhibited fibrin deposition by 80-90% at both venous (P < 0.001) and arterial shear (P < 0.05). However, the peptide had no effect on the clotting time in a modified one-stage clotting assay where coagulation was initiated by lysed endotoxin-stimulated endothelial cells, indicating that the peptide interfered with the binding of fibrin to the matrix in the perfusion model. Preincubation of the matrix with the anti-tissue factor antibody, which blocked the coagulant activity ( > 95%, P < 0.01) in the modified coagulation assay, also inhibited fibrin deposition on the matrix by 90-95% (P < 0.01) at both shear rates. In the absence of either inhibitor, platelets adhered preferentially to the fibrin meshwork, and more so at arterial shear. Platelet thrombus formation on the fibrin coat was in particular pronounced at arterial shear. Thus, it appears that the extracellular matrix of endotoxin-stimulated endothelium initiates coagulation predominantly through tissue factor/FVIIa and that the resulting fibrin meshwork forming on the surface induces rapid platelet thrombus formation. The inhibitory effect of Arg-Gly-Asp-Val on the binding of fibrin to the matrix may indicate the presence of specific matrix fibrinogen/fibrin binding site(s) with a recognition sequence of Arg-Gly-Asp.
当暴露于流动的非抗凝血液时，纤维蛋白会在内毒素刺激的内皮细胞外基质上形成并与之结合。这些过程已通过使用人体离体灌注模型、合成肽Arg-Gly-Asp-Val和一种抑制组织因子/FVIIa诱导凝血的单克隆抗组织因子抗体进行了研究。塑料盖玻片上的促凝细胞外基质是由内毒素刺激的人内皮细胞培养物在短暂暴露于0.1 M NH4OH后制备的。通过泵以静脉（100/s）和动脉（650/s）壁剪切速率从肘前静脉直接抽取非抗凝血液，流过平行板灌注室中涂有基质的盖玻片。通过形态计量学对纤维蛋白和血小板在基质上的沉积进行定量。用Arg-Gly-Asp-Val预孵育基质，在静脉剪切（P < 0.001）和动脉剪切（P < 0.05）时，纤维蛋白沉积均受到80 - 90%的抑制。然而，在通过裂解的内毒素刺激内皮细胞启动凝血的改良一步凝血试验中，该肽对凝血时间没有影响，这表明该肽在灌注模型中干扰了纤维蛋白与基质的结合。用抗组织因子抗体预孵育基质，该抗体在改良凝血试验中阻断了促凝活性（> 95%，P < 0.01），在两种剪切速率下，也使纤维蛋白在基质上的沉积受到90 - 95%的抑制（P < 0.01）。在没有任何一种抑制剂的情况下，血小板优先黏附于纤维蛋白网络，在动脉剪切时更是如此。纤维蛋白涂层上的血小板血栓形成在动脉剪切时尤为明显。因此，似乎内毒素刺激的内皮细胞外基质主要通过组织因子/FVIIa启动凝血，并且在表面形成的纤维蛋白网络会诱导快速的血小板血栓形成。Arg-Gly-Asp-Val对纤维蛋白与基质结合的抑制作用可能表明存在具有Arg-Gly-Asp识别序列的特定基质纤维蛋白原/纤维蛋白结合位点。
