Action of Metalloproteinases Mutalysin I and II on Several Components of the Hemostatic and Fibrinolytic Systems

Abstract

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 μg (2000 units/mg) and 17.8 μg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aα>Bβ chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5.25 and 16.3 μmol fibrinogen/min/μmol 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 μM) completely digested the α- and γ-γ chains and partially the β-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the α-chain of fibrin leaving the β and γ-γ 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 μg/mL of collagen with an IC 50 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 α2-macroglobulin (α2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of α2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by α2-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.