Abstract
Although the plasminogen activating equimolar complex of staphylokinase (STA) with human plasmin is very rapidly inhibited by alpha 2-antiplasmin, STA is a potent fibrinolytic agent in a human plasma milieu which contains 1 microM alpha 2-antiplasmin. In the present study, it was found that the complex of plasmin with recombinant STA (STAR), after neutralization with alpha 2-antiplasmin, retained the full plasminogen activating potential of STAR when added to a plasminogen solution (93 +/- 5% residual activity). When added to human plasma containing a 125I-fibrin-labeled plasma clot, equi-effective concentrations (causing 50% lysis in 2 h) were 17 +/- 3.0, 13 +/- 1.0, and 20 +/- 1.0 nM for STAR, equimolar plasmin-STAR mixtures, and plasmin-STAR mixtures neutralized by alpha 2-antiplasmin, respectively. Gel filtration of mixtures of plasmin(ogen) and STAR revealed elution as plasmin-STAR complex (Mr approximately 100,000), whereas after addition of alpha 2-antiplasmin, STAR eluted with an apparent Mr of 20,000. When mixtures of plasmin and STAR were adsorbed to lysine-Sepharose, STAR adsorbed quantitatively (96 +/- 1%) to the gel, whereas it was nearly quantitatively recovered in the unbound fraction (92 +/- 4%) after addition of alpha 2-antiplasmin to the mixture. Scatchard analysis of the binding of STAR to plasmin-Sepharose yielded a dissociation constant of 55 nM, whereas no specific binding of STAR to plasmin-alpha 2-antiplasmin-Sepharose could be demonstrated. These findings indicate that, both in purified systems and in a human plasma milieu containing a 125I-fibrin-labeled plasma clot, neutralization of the plasmin-STAR complex by alpha 2-antiplasmin results in dissociation of functionally active STAR from the complex and recycling of STAR to other plasminogen molecules. This dissociation-recycling process may explain the high fibrinolytic potency of STAR in a plasma milieu in the presence of high concentrations of alpha 2-antiplasmin.
Highlights
The plasminogen activating equimocloamr - Staphylokinase (STA),’a 136-aminoacid protein produced plex of staphylokinase (STA) with human plasmin is by Staphylococcus aureus, was previously shown to have proveryrapidlyinhibitedbya,antiplasmin,STAis a fibrinolytic properties [1,2]
Inthepresent thenactivatesother plasminogen molecules following Mistudy, it was found that the complex of plasmin with chaelis-Menten kinetics [3, 4].In purified systems, a,antirecombinant STA (STAR), after neutralization with plasmin rapidly inhibits the complex of plasmin(ogen) with a,antiplasmin, retained the full plasminogen activat- recombinant STA (STAR)(5), althoughit does notinhibitthe solution (93f 5% residual activity)
Thereby its inhibition rate by a,antiplasmin is rewhereas no specific binding of STAR to plasmin-az- duced, allowing preferential plasminogen activationatthe antiplasmin-Sepharose could be demonstrated. These findings indicate that, both in purified systeImn sview of the high concentration of a,antiplasmin in and ina human plasmamilieu containing a lZ5I-fibrin- human plasma relative to fibrinolytically active labeledplasmaclot,neutralization of theplasmin- STAR concentrations
Summary
The abbreviations used are: STA, staphylokinase; STAR,recombinant STA; plasmin-STAR, preformed complex obtained by incubation of human native Glu-plasminogen withan equimolar amount of S T A R Plg,plasminogen;rPlg-Ala7“, recombinant plasminogen with the active site Ser7“ mutagenized to Ala; 6-AHA, 6-aminohexanoic acid; S-2251, D-valyl-leucyl-lysine-p-nitroanilideP;AGE, polyacrylamide gel electrophoresis; ELISA,enzyme-linkedimmunosorbant assay; cpm, counts/minute. Recombi- plasmin in plasma was monitored by addition of lZ'I-plasmin-STAR nant plasminogen with the active site Semr7u4ta1genized to Ala (rPlg- (final concentration 45 nM containing 4 X lo cpmltube) to300 p1 of. Dissociation of STAR from the Plasmin-STAR Complex after Insorptiononan unsolubilizedmonoclonal antibody (MA-34F7) di- hibition by a,-Antiplasmin-Themolecularform of STARinthe rected against a,-antiplasmin After depletion, this plasma contained plasmin-STAR complex beforeand after inhibitiobny as-antiplasmin less than 2% residual as-antiplasmin as determined by ELISA STAR mixtures neutralized with a2-antiplasmin(0.5 ml a t a concentration of p~ in 0.1 M phosphate buffer, pH 7.3) were adsorbed onto lysine-Sepharose (150 mg of dry gel) for 1h at 4 "C on a tilting table.
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