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) have been previously isolated from bushmaster (Lachesis muta muta) snake venom, Hemorrhagic activity was observed with as little as 0.5 mu g (2000 units/mg) and 17.8 mu g (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the A alpha>B beta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5.25 and 16.3 mu mol fibrinogen/min/mu mol protein for mutalysin I and II, respectively. In vitro, the enzymes act directly on fibrin and are not 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 beta-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the alpha-chain of fibrin leaving the beta and gamma-gamma chains unaffected. Unlike with the plasminogen activator-based thrombolytic agents (e.g,, streptokinase), mutalysins do not activate plasminogen. Neither enzyme had an effect on protein C activation. Mutalysin II does not inhibit platelet aggregation in human PRP induced by collagen or ADP. However, mutalysin I showed a selective inhibitory effect on collagen-induced aggregation of human PRP; it did not affect platelet aggregation with ADP as the agonist. The present investigation demonstrates that both native and EDTA-inactivated mutalysin I dose dependently 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-like domain also participates in the inhibitory effect. The proteolytic activity of mutalysin II against dimethylcasein and fibrin was completely abolished 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 effect of mutalysin I against the same substrates was not significantly inhibited by alpha 2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated with the observed pathological effects of the venom. (C) 2000 Elsevier Science Ltd. All rights reserved.