Characterization of the reaction of plasmin with .alpha.2-macroglobulin: effect of antifibrinolytic agents

Abstract

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)

Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

“…The sites of proteolytic cleavage have been extensively mapped (1,38,39) and generally conform to the primary substrate specificity of the proteinase in question. Several proteinases including trypsin, chymotrypsin, and elastase react rapidly with a 2 -M, while restrictive proteinases such as thrombin and plasmin react slowly (1,34,40). Evidently, each bait region contains one or more peptide bonds which can be cleaved by a wide variety of proteinases.…”

Recent Advances on Plasmin Inhibitors for the Treatment of Fibrinolysis‐Related Disorders

“…The other structural feature of plasmin is the C ‐terminal light chain (Val562‐Asn791), which is the trypsin‐like catalytic domain having the triad of His603, Asp646, and Ser741 residues (His57, Asp102, and Ser195 in chymotrypsinogen numbering) . The catalytic domain appears to bind to α 2 ‐macroglobulin, a physiological inhibitor of plasmin …”

“…This helps localize plasmin's proteolytic activity . Fibrinolysis can also be regulated by thrombin‐activatable fibrinolysis inhibitor, plasminogen activator inhibitor‐1 and ‐2, or α 2 ‐macroglobulin . In a clinical setting, fibrinolysis can be enhanced by using thrombolytics such as the fibrin‐cleaving preparations of streptokinase, urokinase, or tissue plasminogen activator (tPA) …”

The plasmin–antiplasmin system: structural and functional aspects

“…Concomitantly, the internal thioester bond is cleaved, which mediates the covalent binding of a 2 M to the protease. In the case of Plm, it appears that proteolysis and conformational changes in a 2 M are limited to one of the two subunits and the association constant K a for Plm is 1.3 9 10 5 M -1 s -1 [138]. a 2 M seems to be associated with Alzheimer disease (MIM 103950) and a deletion in exon 18 seems to be the cause of an increased risk of Alzheimer disease [139].…”