Summary. Background: Over-expression of tissue factor (TF) and activation of the coagulation system are common in cancer patients. Heparanase is an endo-b-D-glucuronidase that cleaves heparan sulfate chains on cell surfaces and in the extracellular matrix, activity that closely correlates with cell invasion, angiogenesis and tumor metastasis. The study was undertaken to investigate the involvement of heparanase in TF expression. Methods: Tumor-derived cell lines were transfected with heparanase cDNA and TF expression was examined. The effect of exogenous addition of active and inactive heparanase on TF expression and activity was studied in tumor cell lines and primary human umbilical vein endothelial cells. TF expression was also explored in heparanase over-expressing transgenic (Tg) mice. Blast cells were collected from acute leukemia patients and TF and heparanase expression levels were analyzed. Results: Over-expression of heparanase in tumor-derived cell lines resulted in a 2-fold increase in TF expression levels, and a similar trend was observed in heparanase Tg mice in vivo. Likewise, exogenous addition of heparanase to endothelial or tumor-derived cells resulted in enhanced TF expression and activity. Interestingly, TF expression was also induced in response to enzymatically inactive heparanase, suggesting that this effect was independent of heparanase enzymatic activity. The regulatory effect of heparanase on TF expression involved activation of the p38 signaling pathway. A positive correlation between TF expression levels and heparanase activity was found in blasts collected from 22 acute leukemia patients. Conclusions: Our results indicate that in addition to its well-known function as an enzyme paving a way for invading cells, heparanase also participates in the regulation of TF gene expression and its related coagulation pathways.

BackgroundHeparanase is an endo-β-D-glucuronidase dominantly involved in tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Our hypothesis was that heparanase is directly involved in activation of the coagulation cascade. Design and MethodsActivated factor X and thrombin were studied using chromogenic assays, immunoblotting and thromboelastography. Heparanase levels were measured by enzyme-linked immunosorbent assay. A potential direct interaction between tissue factor and heparanase was studied by coimmunoprecipitation and far-western assays. ResultsInterestingly, addition of heparanase to tissue factor and activated factor VII resulted in a 3-to 4-fold increase in activation of the coagulation cascade as shown by increased activated factor X and thrombin production. Culture medium of human embryonic kidney 293 cells overexpressing heparanase and its derivatives increased activated factor X levels in a non-enzymatic manner. When heparanase was added to pooled normal plasma, a 7-to 8-fold increase in activated factor X level was observed. Subsequently, we searched for clinical data supporting this newly identified role of heparanase. Plasma samples from 35 patients with acute leukemia at presentation and 20 healthy donors were studied for heparanase and activated factor X levels. A strong positive correlation was found between plasma heparanase and activated factor X levels (r=0.735, P=0.001). Unfractionated heparin and an inhibitor of activated factor X abolished the effect of heparanase, while tissue factor pathway inhibitor and tissue factor pathway inhibitor-2 only attenuated the procoagulant effect. Using co-immunoprecipitation and farwestern analyses it was shown that heparanase interacts directly with tissue factor. ConclusionsOverall, our results support the notion that heparanase is a potential modulator of blood hemostasis, and suggest a novel mechanism by which heparanase increases the generation of activated factor X in the presence of tissue factor and activated factor VII.Key words: heparanase, coagulation cascade, tissue factor, Xa level.Citation: Nadir Y, Brenner B, Fux L, Shafat I, Attias J, and Vlodavsky I. Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII. Haematologica 2010;95(11):1927-1934. doi:10.3324/haematol.2010 Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII

SummaryHeparanase activity is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we have reported that heparanase stimulates tissue factor (TF) expression in endothelial and cancer cells, resulting in elevation of coagulation activity. We hypothesized that heparanase regulates other coagulation modulators, and examined the expression and localization of tissue factor pathway inhibitor (TFPI) following heparanase over-expression or exogenous addition. Primary human umbilical vein endothelial cells (HUVEC) and human tumor-derived cell lines were incubated with heparanase, or were stably transfected with heparanase gene-constructs, and TFPI expression and secretion were examined. Heparanase over-expression or exogenous addition stimulated TFPI expression by 2–3 folds. TFPI accumulation in the cell culture medium exceeded in magnitude the observed induction ofTFPI gene transcription reaching 5– to 6-fold increase. Extracellular accumulation of TFPI was evident already 60 min following heparanase addition, prior toTFPI protein induction, and correlated with increased coagulation activity. This effect was found to be independent of heparanase enzymatic activity and interaction with heparan-sulfate, and correlated with reduced TFPI levels on the cell surface. Data were verified in heparanase transgenic mice tissues and plasma. Interaction between heparanase and TFPI was evident by co-immunoprecipitation. Interaction of heparanase with TFPI resulted in its displacement from the surface of the vascular endothelium and in increased pro-coagulant activity. Thus, heparanase facilitates blood coagulation on the cell surface by two independent mechanisms:dissociation ofTFPI from the vascular surface short after local elevation of heparanase levels, and subsequent induction of TF expression.

Heparanase is a mammalian endo-β-D-glucuronidase that cleaves heparan sulfate (HS) side chains at a limited number of sites. Heparanase enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix (ECM) and to facilitate cell invasion associated with tumor metastasis, angiogenesis and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase up regulation was detected in an increasing number of primary human tumors, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The present study was undertaken to develop a highly sensitive ELISA assay suitable for the determination and quantification of human heparanase in tissue extracts and body fluids. The assay preferentially detects the 8+50 kDa active heparanase heterodimer vs. the latent 65 kDa pro-enzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200 pg/ml, and is suitable for quantification of heparanase in tissue extracts and urine.

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