title: Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothrops jararaca venom by its only plasma inhibitor, alpha 2-macroglobulin.
abstract: Observations that a haemorrhagic metalloproteinase (jararhagin) from Bothrops jararaca venom had less effect on platelets suspended in plasma than in washed platelet suspensions, suggested that plasma contains naturally occurring inhibitor(s) of this enzyme. By using radiolabelled jararhagin and crossed immunoelectrophoresis, we have demonstrated the binding of this enzyme to alpha 2-macroglobulin in plasma. SDS-PAGE analysis of this binding revealed the presence of radioactivity in four bands with relative molecular masses of 640, 570, 520 and 410 kDa; in addition a small amount of 47 kDa free enzyme was demonstrable. Reduced samples showed an additional non-complexed 90 kDa fragment of alpha 2-macroglobulin generated by jararhagin. These results are compatible with a model in which, upon multiple cleavages of alpha 2-macroglobulin, the enzyme becomes covalently bound to the inhibitor, and the two halves of the inhibitor become crosslinked. However, jararhagin activity was not completely inhibited even after long incubation (60 min) with a large (10-fold) molar excess of alpha 2-macroglobulin either in plasma or a purified alpha 2-macroglobulin preparation. Kinetic studies showed that inhibition was comparatively slow, although jararhagin readily cleaved alpha 2-macroglobulin in the bait region. Therefore, the ineffectiveness of the inhibition could have resulted from a low tendency of this proteinase to form covalent complexes with the inhibitor. We conclude that the pronounced haemorrhagic activity of jararhagin can be attributed to prolonged access of this enzyme to high molecular weight substrates, even in the presence of a large molar excess of alpha 2-macroglobulin.
authors.affiliation: University Department of Haematology, Royal Liverpool University Hospital, UK.
authors.forename: A S
authors.initials: AS
authors.lastname: Kamiguti
authors.forename: H P
authors.initials: HP
authors.lastname: Desmond
authors.forename: R D
authors.initials: RD
authors.lastname: Theakston
authors.forename: C R
authors.initials: CR
authors.lastname: Hay
authors.forename: M
authors.initials: M
authors.lastname: Zuzel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D017837:Bothrops; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007123:Immunoelectrophoresis, Two-Dimensional; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D009930:Organic Chemicals; D010975:Platelet Aggregation Inhibitors; D000511:alpha-Macroglobulins / Q000502:physiology*; D000097146:Bothrops jararaca Venom
chemical_list: D002863:Chromogenic Compounds; D003435:Crotalid Venoms; D005340:Fibrinogen; D008666:Metalloendopeptidases; D009930:Organic Chemicals; D010975:Platelet Aggregation Inhibitors; D000511:alpha-Macroglobulins; D000097146:Bothrops jararaca Venom; C043626:hide powder azure