title: Thrombin enhances degradation of heparan sulfate in the extracellular matrix by tumor cell heparanase.
abstract: The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.
authors.affiliation: Department of Oncology, Hadassah University Hospital, Jerusalem, Israel.
authors.forename: M
authors.initials: M
authors.lastname: Benezra
authors.forename: I
authors.initials: I
authors.lastname: Vlodavsky
authors.forename: R
authors.initials: R
authors.lastname: Bar-Shavit
mesh_terms: D000818:Animals; D015551:Autoimmunity; D002417:Cattle; D002478:Cells, Cultured / Q000187:drug effects; D003315:Cornea / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D007249:Inflammation / Q000503:physiopathology; D009362:Neoplasm Metastasis / Q000503:physiopathology; D013917:Thrombin / Q000494:pharmacology*; D000099175:Heparanase
chemical_list: D005966:Glucuronidase; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D013917:Thrombin; D000099175:Heparanase