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

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Abstract
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 μM 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 μg/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.

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