MODULATION OF PROCOAGULANT ACTIVITY OF EXTRACELLULAR ENDOTHELIAL MATRIX BY ANTITISSUE FACTOR ANTIBODY AND THE SYNTHETIC PEPTIDE ARG-GLY-ASP-VAL - EXPERIMENTS WITH FLOWING NONANTICOAGULATED HUMAN BLOOD

Fibrin forms on and binds to the extracellular matrix of endotoxin-sti mulated endothelium when exposed to flowing non-anticoagulated blood. These processes have been investigated by employing a human ex vivo pe rfusion model, a synthetic peptide Arg-Gly-Asp-Val and a monoclonal an ti-tissue factor antibody which inhibits tissue factor/FVIIa-induced c oagulation. Procoagulant extracellular matrix on plastic cover-slips w as prepared from cultures of endotoxin-stimulated human endothelium fo llowing brief exposure to 0.1 M NH4OH. Non-anticoagulated blood was dr awn 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 p ositioned in parallel-plate perfusion chambers. Deposition of fibrin a nd platelets on the matrix was quantified by morphometry. Preincubatio n 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-sta ge clotting assay where coagulation was initiated by lysed endotoxin-s timulated endothelial cells, indicating that the peptide interfered wi th the binding of fibrin to the matrix in the perfusion model. Preincu bation of the matrix with the anti-tissue factor antibody, which block ed the coagulant activity (> 95%, P < 0.01) in the modified coagulatio n assay, also inhibited fibrin deposition on the matrix by 90-95% (P < 0.01) at both shear rates. In the absence of either inhibitor, platel ets adhered preferentially to the fibrin meshwork, and more so at arte rial shear. Platelet thrombus formation on the fibrin coat was in part icular pronounced at arterial shear. Thus, it appears that the extrace llular matrix of endotoxin-stimulated endothelium initiates coagulatio n predominantly through tissue factor/FVIIa and that the resulting fib rin meshwork forming on the surface induces rapid platelet thrombus fo rmation. The inhibitory effect of Arg-Gly-Asp-Val on the binding of fi brin to the matrix may indicate the presence of specific matrix fibrin ogen/fibrin binding site(s) with a recognition sequence of Arg-Gly-Asp .