Characterization of the reaction of plasmin with alpha 2-macroglobulin: effect of antifibrinolytic agents.
The reaction of several plasmin derivatives with alpha 2-macroglobulin (alpha 2M) has been investigated. Titration experiments measuring conformational changes in alpha 2M, changes in the number of sulfhydryl groups available for titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and changes in the ability of alpha 2M to protect bound plasmin from inhibition by soybean trypsin inhibitor all suggested that between 1.3 and 1.5 mol of plasmin was bound per mole of inhibitor. Under experimental conditions where [plasmin] greater than [alpha 2M], the conformational change occurring in the inhibitor and thiol group appearance displayed biphasic kinetics. Examination of the extent of subunit cleavage by plasmin revealed that the rapid phase was associated with cleavage of approximately two to three of the four alpha 2M subunits, while cleavage of the remaining subunits occurred during the slow phase of the reaction. Binary (1:1) alpha 2M-plasmin complexes were prepared by reacting a large excess of alpha 2M with plasmin and purifying the resultant complex by immunoaffinity chromatography using a monoclonal antibody specific for a neoantigen on alpha 2M that is generated when the inhibitor reacts with proteases or with methylamine. Characterization of the purified complex revealed that two of the four subunits were cleaved, and the conformational change, measured by alterations in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS), was approximately 50% of that measured for a 2:1 complex. Thus it appears that proteolysis and conformational alterations associated with the binding of 1 mol of plasmin to alpha 2M are limited to one of two functional units in the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
D000933:Antifibrinolytic Agents / Q000494:pharmacology*; D002846:Chromatography, Affinity; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D000511:alpha-Macroglobulins / Q000378:metabolism*
D000933:Antifibrinolytic Agents; D000511:alpha-Macroglobulins; D004228:Dithionitrobenzoic Acid; D005341:Fibrinolysin