טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNadir Yona
SubjectRegulatory Effect of Heparanase on Tissue Factor and Tissue
Factor Pathway Inhibitor
DepartmentDepartment of Medicine
Supervisors Professor Israel Vlodavsky
Professor Benjamin Brenner
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endo-β-D-glucuronidase that is capable of cleaving heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPG) on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Taking into account the pro-metastatic and pro-angiogenic functions of heparanase, its over-expression in human malignancies and abundance in platelets, we hypothesized that heparanase is also involved in blood coagulation.

Evidence is provided that heparanase over-expression in human leukemia, glioma and breast carcinoma cells results in a marked increase in tissue factor (TF) levels verified by immunoblot and realtime PCR analyses. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. TF induction was further confirmed in heparanase over-expressing transgenic mice and correlated with heparanase expression levels in leukemia patients. Notably, TF induction was independent of heparanase enzymatic activity, further expanding the multiple non-enzymatic effects of heparanase in normal and pathological processes. Interestingly, a direct interaction between TF and heparanase was shown using co-immunoprecipitation and far-western techniques.

We hypothesized that heparanase not only induces TF expression, but is also involved in the regulation of additional factors participating in the coagulation machinery. We examined the possible involvement of heparanase in the regulation of tissue factor pathway inhibitor (TFPI), a potent endogenous inhibitor of factor Xa and factor VIIa-TF complex. TFPI is expressed by endothelial cells and other cells of the vascular wall, contributing to the non-thrombogenic properties of the endothelial cell surface. It was shown that heparanase over-expression or exogenous addition induces 2-3 fold induction of TFPI expression, evident by immunoblotting and real-time PCR analyses. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. Extracellular accumulation exceeded, however, the observed increase in TFPI at the protein level, and appeared to be independent of HS and heparanase enzymatic activity. Instead, a physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local pro-thrombotic function of heparanase.