Human recombinant tissue factor pathway inhibitor induces apoptosis in cultured human endothelial cells

Tissue factor pathway inhibitor (TFPI) is mainly synthesized in vascular endothelial cells and exhibits a strong and specific inhibitory activity against tissue factor-mediated blood coagulation. In the present study, we demonstrate that human recombinant TFPI (h-rTFPI) inhibits the growth of cultured human umbilical vein endothelial cells (HUVECs) by inducing apoptosis. In a growth-rate assay of HUVECs, the growth of the cultured HUVECs is completely abolished by the addition of 1 microM h-rTFPI to the culture medium containing fetal bovine serum (FBS), basic fibroblast growth factor, and epidermal growth factor. In addition, h-rTFPI and h-rTFPI-C which lacks the carboxyl-terminal basic region prevent the survival of growth-arrested HUVECs which are starved in a medium containing 2%, FBS alone, suggesting that h-rTFPI directly induces the death of these HUVECs. This hypothesis is supported by the finding that h-rTFPI does not inhibit the synthesis of DNA in HUVECs during proliferation, as shown by a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Furthermore, Giemsa staining and a gel electrophoretic analysis of DNA fragmentation show that the HUVEC death mediated by h-rTFPI has the typical characteristics of apoptosis. However, the apoptosis in HUVECs is considerably inhibited in the presence of 1 microg/ml of the protein synthesis inhibitor, cycloheximide. Therefore, the process of apoptosis triggered by h-rTFPI is, at least in part, actively conducted by the cells.