Identification of the Proteolytic Thrombin Fragments Formed after Cleavage with Rat Mast Cell Protease 1

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Abstract
We have previously identified rat mast cell protease 1 (RMCP-1), a chymotrypsin-like secretory granule serine protease, as a potent inactivator of thrombin. The present study outlines the cleavage pattern obtained after degradation of thrombin by RMCP-1. The cleavage sites in thrombin were identified by N-terminal amino acid sequence analysis of recovered thrombin fragments. Incubation of thrombin with RMCP-1 resulted in the rapid formation of a 37-kDa fragment, due to cleavage of the Phe1G-Gly1F bond in the thrombin A chain (numbering of amino acid residues according to topological equivalencies with chymotrypsinogen). Further incubation resulted in cleavage of the Trp148-Thr149 bond in the B chain, along with the formation of fragments of 27 kDa and 15 kDa. When the RMCP-1/thrombin mixtures were incubated further, successive degradation of the 37-kDa, 27-kDa and 15-kDa fragments was observed, along with cleavage of the Tyr117-Ile118 bond in the B chain and the formation of fragments of 12, 9 and 6 kDa. No residual thrombin activity was detected after the degradation process had proceeded to this stage. Heparin was shown to markedly enhance the rate of thrombin degradation by RMCP-1.