Effects of Binding of Ligand (FVIIa) to Induced Tissue Factor in Human Endothelial Cells

Abstract

The tissue factor protein is structurally related to the cytokine receptors and ligand binding (factor VIIa) has been reported to give an intracellular calcium signal, thus indicating that tissue factor is a true receptor. In view of the attempts to use recombinant factor VIIa as a therapeutic agent in hemophilia, its binding effects may be of clinical interest. We have studied the effect of ligand binding to human endothelial cells that were stimulated with interleukin-1 to express tissue factor. Human umbilical cord vein endothelial cells produce and release a wide variety of proteins that participate in coagulation and fibrinolysis, and we have investigated whether binding of recombinant factor VIIa to tissue factor altered the release of some of these compounds. Three main findings are reported. (1) After an initial increase, the measurable tissue factor activity in endothelial cells decreased more rapidly in the presence of factor VIIa (half-life 3.7±0.7 hours) than in its absence (half-life 7.4±1.5 hours). This difference was not seen when tissue factor antigen was measured, indicating that ligand binding did not increase the degradation of the protein. (2) Tissue factor pathway inhibitor was detected on the cell surface, in cell homogenates, and in cell medium. When recombinant factor VIIa was added to the cells there was a significant decrease in the release of tissue factor pathway inhibitor to the medium. Four hours after recombinant factor VIIa was added, the levels were 7.5-fold higher in the medium of untreated cells compared to the medium of cells treated with recombinant factor VIIa. (3) We observed increased release of von Willebrand factor (vWF). After 1 and 6 hours with recombinant FVIIa the release was significantly greater than in controls without FVIIa. We did not detect significant differences in the release of tissue plasminogen activator or tissue factor pathway inhibitor.