ACTIVE SITE-BLOCKED FACTORS VIIA AND IXA DIFFERENTIALLY INHIBIT FIBRIN FORMATION IN A HUMAN EX-VIVO THROMBOSIS MODEL

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 hu man fibrillar collagen or tissue factor-expressing cell layers of tumor necrosis factor-alpha-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 (IC50, 3 nmol/L), as quantified by microdensitometry of fibrin-stained coverslips. Whereas FIXai (600 nmol/L) was only a weak inhibitor, FVIIai (GO 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 (IC50, 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.