Effects of binding of ligand (FVIIa) to induced tissue factor in human endothelial cells.

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.

Cellular effects of initiation of the extrinsic pathway of blood coagulation.

In the initial phase of scientific research into blood clotting around 50 years ago, most studies focused on investigating blood samples to find out what took place in the flowing blood. With the purification and cloning of Tissue Factor (TF) it was realized that TF was an integral membrane protein sitting in the cell surface membrane. This shifted the emphasis to investigations of what happened on the cell surface, and later to the cell biology of TF and its inducibility in monocytes/macrophages and endothelial cells. During the last 8 years, researchers have become increasingly interested in studying the processes going on inside the cells that carry TF when coagulation is initiated on their surface. Cells carrying TF have been incriminated in tumorigenesis, metastasis, angiogenesis, and a number of other cellular phenotypes. That binding of the plasma clotting Factor VIIa upregulates a number of genes involved in regulation of growth, transcription, and cellular motility, as well as cytokines, makes it possible to suggest a link between the formation of the TF/Factor VIIa complex and these cellular processes.

Induction of tissue factor synthesis in human umbilical vein endothelial cells involves protein kinase C.

Incubation of human umbilical vein endothelial cells with one of the following compounds: endotoxin, recombinant interleukin-1 beta, recombinant tumor necrosis factor alpha, allogenic lymphocyte subpopulations or phorbol ester resulted in significant induction of tissue factor synthesis. Diacylglycerol had the same effect and also enhanced synergistically the induction caused by endotoxin and interleukin-1 beta. Two different inhibitors of protein kinase C, H7 and sphingosine, inhibited tissue factor synthesis at concentrations which did not depress protein synthesis in general, suggesting that protein kinase C is involved in the processes leading to tissue factor synthesis. Cells down-regulated for the tissue factor response to TPA responded essentially normally to endotoxin and interleukin-1 with regard to tissue factor synthesis.