The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

Abstract

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34 -55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the ␣-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptasespecific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the ␣-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.

The mouse mast cell–restricted tetramer‐forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis

“…Although their amino acid sequences are ϳ75% identical, mMCP-6 and mMCP-7 are functionally distinct tryptases (40,41). Because recombinant hTryptase ␤1 (42,43) has a substrate specificity more similar to that of recombinant mMCP-6 (41) than recombinant mMCP-7 (40), mMCP-6 is the mouse ortholog of hTryptase ␤1.…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…73) ( Figure 1). Tryptase can process prothrombin (74), generate C3a from complement C3 (75), and degrade ECM components including fibrinogen (76,77), denatured type I collagen (78), and fibronectin (79). Tryptase and chymase can further promote ECM degradation by activating proMMPs (80)(81)(82)(83)(84) and pro-uPA (85).…”

Suppressor of Cytokine Signaling 1 Regulates an Endogenous Inhibitor of a Mast Cell Protease

“…Scanning the primary amino acid sequence of murine p85 has revealed a few potential cleavage sites with Ser-Ser-Arg/Lys residues at P3, P2, and P1 positions specific for mMCP-7 (35), raising the possibility that mMCP-7 could be one of the mast cell proteases dysregulated in the absence of SOCS1. Direct examination of this possibility using the ␣-fibrinogen degradation assay (35), revealed that the dysregulated protease of SOCS1-deficient BMMCs resembles mMCP-7 in specifically cleaving ␣-fibrinogen. However, the protease regulated by SOCS1 is unlikely to be mMCP-7, because (i) it generates a proteolytic fragment that was not observed followed mMCP-7 digestion, and (ii) unlike mMCP-7, the dysregulated protease of SOCS1-deficient BMMCs was completely inhibited by mouse plasma.…”

“…Because the protease inhibitor experiments have indicated that the dysregulated protease is a tryptase-like serine protease, we further examined whether the dysregulated protease in SOCS1-deficient cells is mMCP-7 using a specific assay described by Stevens and colleagues. Recombinant mMCP-7 specifically cleaves the ␣-isomer of fibrinogen, and this activity is not blocked by the natural protease inhibitors present in mouse plasma (35). When incubated with fibrinogen, SOCS1-deficient mast cell lysate specifically cleaved the ␣-isomer (Fig.…”