HUMAN MAST-CELL TRYPTASE FIBRINOGENOLYSIS - KINETICS, ANTICOAGULATIONMECHANISM, 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 displ ays remarkable resistance to inhibition by blood plasma proteinase inh ibitors. In this study we have examined the cleavage of human fibrinog en by tryptase. a chain cleavage was shown to occur in the carboxyl te rminal region at Arg(572),, beta chain cleavage was found to occur at Lys(21) of these reactions yielded K-m values of 0.2 mu M for alpha ch ain cleavage and 0.26 mu M for beta chain cleavage, as well as k(cat)/ K-M values of 7 x 10(5) and 4.6 x 10(5) M-1 s(-1) for alpha and beta c hain reactions, respectively. Proteolysis at Arp(572) destroyed the Ar g-Gly-Asp (RGD) sequence motif recognized by cell surface alpha(v) bet a(3) s integrins, and endothelial cell binding to tryptase-modified fi brinogen was significantly reduced, consistent with loss of the RGD mo tif, Tryptase competed with thrombin in clotting assays using pure fib rinogen with heparin or blood plasma in the absence of heparin. Thromb in failed to initiate the clotting of fibrinogen following modificatio n by tryptase, and fibrin clotting initiated with Ancrod was stepped a nd partially reversed by tryptase:. These data provide insight concern ing the mechanism by which tryptase renders fibrinogen unclottable by thrombin and suggests a novel role for tryptase in the modulation of c ellular interactions with fibrin(ogen).