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 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 alpha-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 tryptase-specific, 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 alpha-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.
Huang
C
C
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Wong
G W
GW
Ghildyal
N
N
Gurish
M F
MF
Sali
A
A
Matsumoto
R
R
Qiu
W T
WT
Stevens
R L
RL
D000707:Anaphylaxis / Q000097:blood; D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D053818:Chymases; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018836:Inflammation Mediators / Q000378:metabolism*; D008415:Mastocytosis / Q000097:blood; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000097:blood*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D053802:Tryptases
D000925:Anticoagulants; D001798:Blood Proteins; D018836:Inflammation Mediators; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C509190:Tpsb2 protein, mouse; D005340:Fibrinogen; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509222:Tpsab1 protein, mouse; C509223:Tpsab1 protein, rat; C509221:Tpsb2 protein, rat; D053802:Tryptases