Action of metalloproteinases mutalysin I and II on several-components of the hemostatic and fibrinolytic systems

The zinc endopeptidases mutalysin I (100 kDa) and mutalysin II (22.5 kDa) h ave been previously isolated from bushmaster (Lachesis muta muta) snake ven om, Hemorrhagic activity was observed with as little as 0.5 mu g (2000 unit s/mg) and 17.8 mu g (56.2 units/mg) for mutalysin I and II, respectively. A dditionally, the proteases hydrolyse the A alpha>B beta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimat ed as 5.25 and 16.3 mu mol fibrinogen/min/mu mol protein for mutalysin I an d II, respectively. In vitro, the enzymes act directly on fibrin and are no t inhibited by serine proteinase inhibitors (SERPINS), Analysis by SDS-PAGE of fibrin hydrolysis by both enzymes showed that mutalysin II (0.22 mu M) completely digested the alpha- and gamma-gamma chains and partially the bet a-chain (in 120 min incubation). In contrast, mutalysin I (three fold highe r concentration than mutalysin II) hydrolyzed selectively the alpha-chain o f fibrin leaving the beta and gamma-gamma chains unaffected. Unlike with th e plasminogen activator-based thrombolytic agents (e.g,, streptokinase), mu talysins do not activate plasminogen. Neither enzyme had an effect on prote in C activation. Mutalysin II does not inhibit platelet aggregation in huma n PRP induced by collagen or ADP. However, mutalysin I showed a selective i nhibitory effect on collagen-induced aggregation of human PRP; it did not a ffect platelet aggregation with ADP as the agonist. The present investigati on demonstrates that both native and EDTA-inactivated mutalysin I dose depe ndently blocked aggregation of human PRP elicited by 10 mu g/mL of collagen with an IC50 of 180 and 580 nM, respectively. These studies suggest that, in addition to the metalloprotease region of mutalysin I, the disintegrin-l ike domain also participates in the inhibitory effect. The proteolytic acti vity of mutalysin II against dimethylcasein and fibrin was completely aboli shed by alpha 2-macroglobulin (alpha 2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of alpha 2-M. In contrast, the proteolytic ef fect of mutalysin I against the same substrates was not significantly inhib ited by alpha 2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated wi th the observed pathological effects of the venom. (C) 2000 Elsevier Scienc e Ltd. All rights reserved.