INACTIVATION OF THROMBIN BY A COMPLEX BETWEEN RAT MAST-CELL PROTEASE-1 AND HEPARIN PROTEOGLYCAN

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with (SO42-)-S-35, RMCP-1 was recovered in a macromolecular complex with [S-35]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombininactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [S-35]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [S-35]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.