Co-Interaction and Increased Release of Tissue Factor Pathway Inhibitor by Heparanase.

Abstract

Background and objectives. Tissue factor pathway inhibitor (TFPI) is a potent direct inhibitor of factor Xa and factor VIIa-tissue factor complex. In addition, TFPI was shown to be an inhibitor of angiogenesis and metastasis. Heparanase is an endo-beta-D-glucuronidase of 65 kDa that cleaves heparan sulfate chains on cell surfaces and in the extra-cellular matrix an activity that closely correlates with cell invasion, angiogenesis and tumor growth. The study hypothesis was that heparanase may reduce the level of TFPI or release it from the cell surface in an attempt to increase heparanase prometastatic potential.Material and methods. The effect of exogenous heparanase on TFPI expression and release to the medium was studied in HUVEC by immunoblotting, real time RT-PCR, and flow-cytometry. Human cell lines (MDA-MB-435 breast carcinoma; U87 glioma; HEK-293 embryonic kidney) were transfected to over express heparanase and the effect on TFPI was studied. TFPI expression was explored in heparanase transgenic mice by immunoblotting and immunostaining. Transfections with various modified forms of heparanase were used to further explore the effect of heparanase. Interaction between TFPI and heparanase was studied by co-immunoprecipitation analysis.Results. Heparanase was found to increase the release of TFPI to the medium, reduce the level of TFPI at the cell surface, and to up-regulate its expression in the cells. These results were verified in HUVEC, tumor cell lines, and in the animal model. The effect was independent of heparanase activity or interaction with heparan sulfate, and dependent on heparanase secretion. A protein co-interaction between TFPI and heparanase was found.Conclusions. Overall, a cell surface interaction is suggested in which heparanase impose increased release of TFPI from the cell surface to the medium, providing a local procoagulant and a systemic anticoagulant environment.