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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Nadir
Yona
Y
Thrombosis and Hemostasis Unit, Department of Hematology and Bone Marrow Transplantation, Rambam Medical Center, Haifa 31096, Israel. y_nadir@rambam.health.gov.il
Brenner
Benjamin
B
Fux
Liat
L
Shafat
Itay
I
Attias
Judith
J
Vlodavsky
Israel
I
D000293:Adolescent; D000328:Adult; D000368:Aged; D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation; D015942:Factor VIIa / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology; D057809:HEK293 Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology / Q000235:genetics; D008297:Male; D008875:Middle Aged; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism; D000099175:Heparanase
D000925:Anticoagulants; D015942:Factor VIIa; D015951:Factor Xa; D005966:Glucuronidase; D006023:Glycoproteins; D006493:Heparin; D015842:Serine Proteinase Inhibitors; D013925:Thromboplastin; D000099175:Heparanase; C021240:tissue-factor-pathway inhibitor 2