Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.7 (plasmin)
 9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Interaction between staphylokinase, plasmin(ogen), and alpha 2-antiplasmin. Recycling of staphylokinase after neutralization of the plasmin-staphylokinase complex by alpha 2-antiplasmin. 768 64

The effects of alpha 2-antiplasmin and fibrin on the activation of plasminogen by recombinant staphylokinase (STAR) were studied in an effort to elucidate further the molecular basis of the fibrin-specificity of this fibrinolytic agent. In purified systems consisting of 1.5 mumol/L intact or low-M(r) plasminogen and 3 mumol/L alpha 2-antiplasmin, at 37 degrees C and in the absence of fibrin, STAR did not induce plasminogen activation and plasmin-alpha 2-antiplasmin complex (PAP) formation. Addition of a purified fibrin clot (30% vol at a concentration of 3 mg/mL) to mixtures containing intact plasminogen caused approximately 40% plasminogen activation within 2 hours, whereas in mixtures containing low-M(r) plasminogen, no activation was observed. In contrast, 10 nmol/L streptokinase (SK) induced 74% to 100% plasminogen activation within 2 hours in mixtures containing either intact or low-M(r) plasminogen, in both the absence and the presence of fibrin. In citrated human plasma in the absence of fibrin, 30 nmol/L STAR did not induce measurable plasminogen activation and PAP formation (< 1.5% within 2 hours), whereas addition of a plasma clot (12% vol) resulted in complete clot lysis and conversion of 19% +/- 8% of the plasminogen to PAP within 2 hours. Addition of a second plasma clot produced 23% +/- 2% additional plasminogen activation. Equipotent concentrations for plasma clot lysis of SK (100 nmol/L) induced 54% +/- 11% plasminogen activation in the absence and 49% +/- 16% in the presence of fibrin. Addition of 50 mmol/L 6-aminohexanoic acid (6-AHA) abolished the effect of fibrin on plasminogen activation with STAR, but not on activation with SK. In alpha 2-antiplasmin-depleted human plasma in the absence of fibrin, 30 nmol/L STAR did not induce fibrinogen breakdown (> 90% residual fibrinogen after 6 hours), whereas 30 nmol/L preformed plasmin-STAR complex induced extensive fibrinogen degradation (70% within 20 minutes). Thus, in the absence of fibrin, alpha 2-antiplasmin inhibits the activation of plasminogen by STAR, by preventing generation of active plasmin-STAR complex. Fibrin stimulates plasminogen activation by STAR via mechanisms involving the lysine-binding sites of plasminogen, probably by facilitating the generation of plasmin-STAR complex and by delaying its inhibition at the clot surface.
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PMID:Regulation by alpha 2-antiplasmin and fibrin of the activation of plasminogen with recombinant staphylokinase in plasma. 768 90

The kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p'-anisate-HCl was 52 M-1 s-1 and its deacylation rate constant 1.2 x 10(-4) s-1 (t1/2 of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min. The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.
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PMID:Functional properties of p-anisoylated plasmin-staphylokinase complex. 823 43

A bio-immunoassay (BIA) for the determination of staphylokinase (STA) activity in a plasma milieu has been developed. MA-7H11, a murine monoclonal antibody raised against STA, which has a high affinity for STA but does not interfere with the complex formation between plasmin(ogen) and STA or with plasminogen activation by STA, was coated on microtiter plates at a concentration of 4 micrograms/ml. STA-containing samples were incubated overnight at 4 degrees C and, after extensive washing, bound STA was quantitated by incubation with plasminogen (final concentration 0.5 microM) for 1 h at 37 degrees C, followed by determination of generated plasmin from the absorbance at 405 nm 10 min after addition of the chromogenic substrate S-2403 (final concentration 0.3 mM). Calibration curves constructed with natural (STAN) or recombinant (STAR) STA were linear between approximately 1 and 10 nM, with a lower detection limit of < or = 1 nM in buffer and in plasma of the human, baboon or hamster. Following bolus injection of STAR in hamsters, the disposition rate of STAR activity from plasma, determined with the BIA correlated very well (r = 0.98) with that of STAR-related antigen determined by ELISA, indicating that STAR is cleared in a functionally active form. The initial half-life was about 2 min, as determined with both methods. Following continuous intravenous infusion over 1 h in baboons, the plasma clearance of STAR activity, determined from the infusion rate and the steady-state plasma level of STAR activity, ranged between 45 and 62 ml/min for doses of STAR between 0.063 and 0.250 mg/kg.
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PMID:Bio-immunoassay for staphylokinase in blood. 825 55

Recombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-delta 6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-delta 10 with NH2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma. In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-delta 10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with Km = 3.6 microM and k2 = 0.38 s-1. The specific clot lysis activities of HMW-STAR (122,000 +/- 8,000 units/mg) and LMW-STAR (129,000 +/- 8,000 units/mg) were indistinguishable. In an in vitro system consisting of a 60 microliters plasma clot submerged in 250 microliters plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular conversions of recombinant staphylokinase during plasminogen activation in purified systems and in human plasma. 825 56

The mechanism of activation of human plasminogen by recombinant staphylokinase (STAR) was studied using the active site titrant p-nitrophenyl-p'-guanidinobenzoate (NPGB). NPGB prevented active site exposure in equimolar mixtures of plasminogen and STAR but reacted stoichiometrically with mixtures preincubated in the absence of titrant. Active site generation occurred progressively, with a marked initial lag phase followed by an exponential growth phase, and was associated with the conversion of single-chain plasminogen to two-chain plasmin. Incubation of mixtures of plasminogen and STAR with catalytic amounts (< 0.2% molar ratio) of preformed plasmin.STAR complex or of urokinase shortened the lag hase, whereas catalytic amounts (5% molar ratio) of the plasmin inhibitor alpha 2-antiplasmin delayed active site generation. The following kinetic model for the activation of plasminogen (P) by STAR (S) fits the experimental data, [formula: see text] and is described by [formula: see text] or [formula: see text] In this model, plasminogen and STAR produce an inactive complex (P.S), in which active plasmin.STAR (p.S) is generated in a rate limiting step, which is accelerated by plasminogen activators and delayed by plasmin inhibitors. At room temperature in a 0.1 M Veronal buffer, pH 8.3, containing 0.1 M arginine, the data are adequately fitted by the integrated equation with k1 = 4.0 x 10(-7) s-1 and k2 = 1.3 x 10(-2) microM-1 s-1. The k1 value could be explained by contamination of the plasminogen preparation with 3 ppm plasmin, converted by S to p.S. It is concluded that STAR activates plasminogen via a mechanism which differs in several essential aspects from that of streptokinase.
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PMID:On the mechanism of the activation of human plasminogen by recombinant staphylokinase. 846 38