Modulation of procoagulant activity of extracellular endothelial matrix by anti-tissue factor antibody and the synthetic peptide Arg-Gly-Asp-Val. Experiments with flowing nonanticoagulated human blood

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Abstract

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 humanex vivoperfusion 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. Plateelet 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.

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