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

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function ex pressed by a subpopulation of mast cells that reside in numerous conne ctive tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive s ystemic anaphylaxis, we used this in vivo model system to identify a p hysiologic substrate of the tryptase. Plasma samples taken from V3 mas tocytosis 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 i ts purification, The resulting 1 recombinant mMCP-7 exhibited potent a nticoagulant activity in the presence of normal plasma and selectively cleaved the a-chain of fibrinogen to fragments of similar size as tha t seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide lib rary revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the al pha-chain of rat fibrinogen. Because fibrinogen is a physiologic subst rate of mMCP-7, this tryptase can regulate clot formation and fibrinog en/integrin-dependent cellular responses during mast cell-mediated inf lammatory reactions.