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 microg (2000 units/mg) and 17.8 microg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aalpha>Bbeta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5. 25 and 16.3 micromol fibrinogen/min/micromol 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 microM) 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 microg/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 alpha2-macroglobulin (alpha2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of alpha2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by alpha2-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.
Estêvão-Costa
M I
MI
Centro de Pesquisa e Desenvolvimento, Fundaçao Ezequiel Dias, 30510-010, Belo Horizonte, Brazil.
Diniz
C R
CR
Magalhães
A
A
Markland
F S
FS
Sanchez
E F
EF
D000818:Animals; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects*; D006470:Hemorrhage / Q000139:chemically induced; D006487:Hemostasis / Q000187:drug effects*; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010958:Plasminogen / Q000187:drug effects / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010979:Platelet Function Tests; D011486:Protein C / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014757:Viper Venoms / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000511:alpha-Macroglobulins / Q000378:metabolism / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005338:Fibrin Fibrinogen Degradation Products; D011486:Protein C; D014757:Viper Venoms; D000511:alpha-Macroglobulins; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D008666:Metalloendopeptidases; C068795:hemorrhagic factor LHFII; C414406:mutalysin I protein, Lachesis muta muta