Active Site–Blocked Factors VIIa and IXa Differentially Inhibit Fibrin Formation in a Human Ex Vivo Thrombosis Model

Abstract

Abstract The role of tissue factor/factor VIIa (FVIIa) and factor VIIIa/factor IXa (FVIIIa/FIXa) complexes in thrombus formation was examined in a human ex vivo blood flow system by use of active site–blocked FVIIa (FVIIai) and FIXa (FIXai) as selective inhibitors. Blood was drawn directly from the veins of volunteers into a mixing device where FVIIai and FIXai were mixed with flowing blood. The blood then entered parallel-plate chambers containing coverslips coated with human fibrillar collagen or tissue factor–expressing cell layers of tumor necrosis factor–α–stimulated human endothelial cells, human smooth muscle cells, and J82 cells. Exposure of stimulated endothelial cells to blood flowing at a venous shear rate of 65/s led to fibrin deposition, which was inhibited by infusion of FVIIai (IC 50 , 3 nmol/L), as quantified by microdensitometry of fibrin-stained coverslips. Whereas FIXai (600 nmol/L) was only a weak inhibitor, FVIIai (60 nmol/L) reduced fibrinopeptide A (FPA) plasma levels from 504±79 to 171±27 ng/mL and concomitantly inhibited platelet thrombus deposition. Similarly, experiments with smooth muscle cells and J82 cells showed that FVIIai but not FIXai efficiently reduced FPA levels. Conversely, with tissue factor–free collagen, which induces platelet-dependent fibrin formation, infusion of FIXai but not of FVIIai inhibited fibrin deposition (IC 50 , 8 nmol/L) and reduced FPA levels from 55±8 to 9±5 ng/mL. However, FIXai did not affect the number of platelet thrombi deposited on collagen. The results suggest that fibrin formation on tissue factor–expressing cellular surfaces is initiated by tissue factor/FVIIa–dependent direct activation of factor X, while on the tissue factor–free collagen surface, factor X activation and subsequent fibrin formation is dependent on the platelet FVIIIa/FIXa complex.