Human mast cell tryptase fibrinogenolysis: kinetics, anticoagulation mechanism, and cell adhesion disruption.
Tryptase is a 31 kDa, glycosylated, trypsin-like enzyme stored in and released from mast cell granules. Human tryptase exists as a tetramer, binds heparin, and has a limited substrate specificity, yet it displays remarkable resistance to inhibition by blood plasma proteinase inhibitors. In this study we have examined the cleavage of human fibrinogen by tryptase. alpha chain cleavage was shown to occur in the carboxyl terminal region at Arg572 and beta chain cleavage was found to occur at Lys21. Kinetic analyses of these reactions yielded Km values of 0.2 microM for alpha chain cleavage and 0.26 microM for beta chain cleavage, as well as kcat/KM values of 7 x 10(5) and 4.6 x 10(5) M-1 s-1 for alpha and beta chain reactions, respectively. Proteolysis at Arg572 destroyed the Arg-Gly-Asp (RGD) sequence motif recognized by cell surface alphavbeta3 integrins, and endothelial cell binding to tryptase-modified fibrinogen was significantly reduced, consistent with loss of the RGD motif. Tryptase competed with thrombin in clotting assays using pure fibrinogen with heparin or blood plasma in the absence of heparin. Thrombin failed to initiate the clotting of fibrinogen following modification by tryptase, and fibrin clotting initiated with Ancrod was stopped and partially reversed by tryptase. These data provide insight concerning the mechanism by which tryptase renders fibrinogen unclottable by thrombin and suggests a novel role for tryptase in the modulation of cellular interactions with fibrin(ogen).
Thomas
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VA
Department of Biochemistry and Molecular Biology, J. H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614-0581, USA.
Wheeless
C J
CJ
Stack
M S
MS
Johnson
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DA
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D053818:Chymases; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D053802:Tryptases
D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D005337:Fibrin; D005340:Fibrinogen; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D013917:Thrombin; D053802:Tryptases