Interaction of human plasmin with human .alpha.2-macroglobulin

Cummings, Helen S.

doi:10.1021/bi00296a017

Cited by 25 publications

(13 citation statements)

References 35 publications

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“…The model is also consistent with the observation that antibodies directed against plasmin, a large proteinase, react with the a2M-plasmin complexes (1,36) since these proteinases could easily be envisoned as protruding from the trap.…”

Section: Discussionsupporting

confidence: 86%

Model of alpha 2-macroglobulin structure and function.

Feldman¹,

Gonias²,

Pizzo³

1985

Proc. Natl. Acad. Sci. U.S.A.

139

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A model of a2-macroglobulin is presented that is compatible with previous structural, functional, and phylogenetic studies of the protein. The model of the molecule resembles a hollow cylinder and is comprised of two identical functional halves with three C2 axes ofsymmetry and no mirror planes. The "trap mechanism" of this proteinase inhibitor is effected by slight movement of two trap arms per "halfmolecule." Evidence for this model is obtained from the study of the structure and proteinase binding of the molecule. By using this model, predictions are made concerning proteinase binding ratios, receptor recognition, and "slow-to-fast" conformational change of the molecule.

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Section: Discussionsupporting

confidence: 86%

Model of alpha 2-macroglobulin structure and function.

Feldman¹,

Gonias²,

Pizzo³

1985

Proc. Natl. Acad. Sci. U.S.A.

139

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“…Two other antibodies appeared to decrease the association rate by approximately 4-fold. These studies found that the heavy chain of plasmin in the a2M-plasmin complex is accessible to the antibodies (Cummings & Castellino, 1984). Thus, binding of large molecules to the plasmin heavy chain, which contains the lysine binding regions, seems to only have a moderate effect on the association rate and certainly does not prevent complex formation with a2M or alter the stoichiometry of plasmin binding with a2M.…”

Section: Discussionmentioning

confidence: 99%

“…The interaction of plasmin with a2M has been investigated in a number of laboratories (Cummings & Castellino, 1984;Christensen & Sottrup-Jensen, 1984; Gonias & Pizzo, 1983a; Straight & McKee, 1982), and discrepancies exist concerning the extent of plasmin binding and number of a2M subunits cleaved when plasmin associates with the inhibitor. To date, the effects of antifibrinolytic agents on the reaction have not been explored.…”

mentioning

confidence: 99%

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

Steiner¹,

Migliorini²,

Strickland³

1987

Biochemistry

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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)

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“…Peaks from the two gel filtrations containing the putative plasmin-␣-macroglobulin (peak I) and plasmin-plasmin inhibitor complexes (peak II) were pooled and concentrated for further analysis. sented stable complexes between plasmin and one or more of the three ␣-macroglobulins in rat plasma that are analogous to human ␣ 2 -macroglobulin and belong to the superfamily of large proteins which can entrap plasmin and a diversity of other proteinases (33,34). The entrapped proteinase molecules are unable to cleave large substrates, but retain the ability to cleave small ones.…”

Section: Characterization Of Rat Plasmin-inhibitor Complexesmentioning

confidence: 99%

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

Bangert¹,

Thorsen²

2000

Thromb Haemost

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SummaryAn improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

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Interaction of human plasmin with human .alpha.2-macroglobulin

Cited by 25 publications

References 35 publications

Model of alpha 2-macroglobulin structure and function.

Model of alpha 2-macroglobulin structure and function.

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

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

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