Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII

• Published in: Haematologica (Roma) Vol. 95; no. 11; pp. 1927 - 1934
• Main Authors: NADIR, Yona, BRENNER, Benjamin, FUX, Liat, SHAFAT, Itay, ATTIAS, Judith, VLODAVSKY, Israel
• Format: Journal Article
• Language: English
• Published: Pavia Ferrata Storti Foundation 01-11-2010
• Subjects: Adolescent; Adult; Aged; Anticoagulants - pharmacology; Biological and medical sciences; Blood Coagulation; Factor VIIa - genetics; Factor VIIa - metabolism; Factor Xa - genetics; Factor Xa - metabolism; Female; Glucuronidase - antagonists & inhibitors; Glucuronidase - genetics; Glucuronidase - metabolism; Glycoproteins - pharmacology; HEK293 Cells; Hematologic and hematopoietic diseases; Heparin - pharmacology; Humans; Leukemia, Myeloid, Acute - enzymology; Leukemia, Myeloid, Acute - genetics; Male; Medical sciences; Middle Aged; Original; Precursor Cell Lymphoblastic Leukemia-Lymphoma - enzymology; Precursor Cell Lymphoblastic Leukemia-Lymphoma - genetics; Serine Proteinase Inhibitors - pharmacology; Thromboplastin - genetics; Thromboplastin - metabolism; Blood coagulation; Serine endopeptidases; Hematology; Enzyme; Tissue factor; Coagulation Factor Xa; Xa level; Factor VII; Peptidases; coagulation cascade; Heparanase; Hydrolases; Coagulation Factor VIIa
• ISSN: 0390-6078; 1592-8721
• DOI: 10.3324/haematol.2010.023713

Heparanase 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.
Activated 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 co-immunoprecipitation and far-western assays.
Interestingly, 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 over-expressing 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 far-western analyses it was shown that heparanase interacts directly with tissue factor.
Overall, 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.