Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Matrix metalloproteinases (MMPs) can degrade a number of proteins that constitute the extracellular matrix. Previous studies have shown that atherosclerotic plaques contain substantial amounts of fibrin(ogen)-related antigen, and more recently, MMPs have been identified in such lesions. The hypothesis that MMPs play a role in the degradation of fibrinogen (Fg) and cross-linked fibrin (XL-Fb) was investigated. Fibrinogen became thrombin-unclottable when treated with matrix metalloproteinase 3 (MMP-3, stromelysin 1) but not with matrix metalloproteinase 2 (MMP-2, gelatinase A). Incubation of XL-Fb clots (made with 125I-Fg) with MMP-3 resulted in complete lysis after 24 h. A D monomer-like fragment was generated by MMP-3 degradation of fibrinogen, XL-Fb, and fragment DD. Immunoreactivity with monoclonal antibody (MoAb)/4-2 (anti-gamma 392-406) but not with MoAb/4A5 (anti-gamma 397-411) suggested that a major cleavage site was within the sequence participating in the cross-linking of two gamma-chains. NH2-terminal sequence analysis of they gamma-chain of the D monomer-like fragment and of a dipeptide isolated from the MMP-3 digest of XL-fibrin identified the hydrolysis of the gamma Gly 404-Ala 405 peptide bond. These data indicate that the degradation of Fg and XL-Fb by MMP-3 is specific and different from plasmin. This mechanism of fibrinolysis might be of relevance in wound healing, inflammation, atherosclerosis, and other pathophysiological processes.
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PMID:Degradation of cross-linked fibrin by matrix metalloproteinase 3 (stromelysin 1): hydrolysis of the gamma Gly 404-Ala 405 peptide bond. 885 41

Structure/function relationships in the activation of plasminogen with staphylokinase were studied using mutants of recombinant staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42D delta N10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,K11H) and Sak42D(K6H,K8H,K11H) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42 delta (K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42D delta C1, or of Lys135 and Lys136 in Sak42D delta C2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 x 10(5) M-1 and 1.4 x 10(7) M-1, respectively (as compared to a Ka of 1.1 x 10(8) M-1 for Sak42D). These results indicate that Lys11 and the COOH-terminal region of staphylokinase play a key role in the activation of plasminogen.
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PMID:Functional significance of NH2- and COOH-terminal regions of staphylokinase in plasminogen activation. 895 Jul 86

Serpin gene-1 from the tobacco hornworm, Manduca sexta, encodes, through alternative exon usage, 12 reactive site variants (Jiang, H., Wang, Y. and Kanost, M. R., (1994) J. Biol. Chem. 269, 55-58; Jiang, H., Wang, Y., Huang, Y., Mulnix, A. B., Kadel, J., Cole, K., and Kanost, M. R. (1996) J. Biol. Chem. 271, 28017-28023). These 43-kDa proteins differ from each other only in their COOH-terminal 39-46 residues, which include the reactive site. To test the hypothesis that these proteins are proteinase inhibitors of diverse selectivities and to begin to elucidate their physiological functions, we expressed the 12 serpin-1 variants in Escherichia coli. Seven of the variants inhibited mammalian serine proteinases, with association rate constants comparable with those of human serpins. Serpin-1A, with a P1 Arg residue, inhibited both trypsin and plasmin. Serpin-1B (P1 Ala) and serpin-1F (P1 Val) inhibited porcine pancreatic elastase and human neutrophil elastase. Serpin-1H, -1K, and -1Z, all with a Tyr residue at the P1 position, inhibited chymotrypsin and cathepsin G. Serpin-1I (P1 Leu) inhibited both elastase and chymotrypsin. Nine of the serpin variants were active as inhibitors of microbial serine proteinases, including subtilisin Carlsberg, proteinase K, and two proteinases secreted by an entomopathogenic fungus, Metarhizium anisopliae. In addition, one of the serpin variants, serpin-1J, strongly inhibited activation of M. sexta hemolymph phenoloxidase, a pathway involving a serine proteinase cascade. This pathway is a component of the defensive response of insects to microbial infection. These results suggest that the products of M. sexta serpin gene-1 may be important in regulating both exogenous and endogenous serine proteinases in hemolymph.
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PMID:Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta. 899 6

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.
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PMID:Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein. 926 Nov 79

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.
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PMID:Limited proteolysis of angiogenin by elastase is regulated by plasminogen. 933 Feb 25

Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCI's reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3' Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA-PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3' interact with PCI rather than uPA, consistent with P3' variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.
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PMID:Assessment of the interaction between urokinase and reactive site mutants of protein C inhibitor. 936 30

Staphylokinase (Sak), a 16-kDa bacterial protein, forms a 1:1 stoichiometric complex with the serine proteinase domain of human plasmin, which in turn converts other plasminogen molecules into plasmin. To identify amino acid residues critical for generating the Sak:plasmin activator complex, alanine-scanning mutagenesis was performed on phage-displayed micro-plasminogen (microPlg). Substitution of Arg719 with Ala [microPlg(R719A)] disrupted complex formation, although the sensitivity of phage-displayed microPlg(R719A) to activation by urokinase and the amidolytic activity of the micro-plasmin derivative [microPli(R719A)] remained unaffected. Likewise, the soluble microPlg(R719A) molecule did not generate a functional activator complex with Sak, whereas quantitative activation into plasmin was obtained upon incubation with either urokinase or the Sak:plasmin complex. Real-time biospecific affinity measurements revealed that the Arg --> Ala substitution at position 719 increased the equilibrium dissociation constant between microPlg(R719A) and Sak from 46 nM to 1 microM, primarily by reducing the association rate constant. Arg719 has recently also been implied in the functional complex formation between human plasmin and streptokinase [Dawson, K. M., Marshall, J. M., Raper, R. H., Gilbert, R. J., and Ponting, C. P. (1994) Biochemistry 33, 12042-12047.], suggesting that both bacterial cofactors may share common structural and/or mechanistic aspects for plasminogen activation.
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PMID:Arginine 719 in human plasminogen mediates formation of the staphylokinase:plasmin activator complex. 957 54

Changes in coagulation and fibrinolysis in the plasma (in vivo) and hepatocytes (ex vivo) were studied using hyperglycemic rats. Hyperglycemia was induced by intravenous injection of 50 mg/kg streptozotocin (STZ). Eight weeks after the injection, we observed increases in thrombin-antithrombin III complex and tissue type plasminogen activator activity, decreases in plasma levels of antithrombin III, plasminogen and alpha2-plasmin inhibitor, and significant shortening of activated partial thromboplastin time. In freshly isolated or cultured hepatocytes from STZ-induced hyperglycemic rats, concentrations of proteins related to coagulation were increased. An increase in alanine-aminotransferase leakage and decreases in the levels of amylase, triglycerides and phospholipids were observed in the culture medium of hepatocytes from STZ treated rats. In vivo study revealed that STZ-induced subchronic diabetes induced imbalance between coagulation and fibrinolysis, and ex vivo study in hepatocytes from STZ-treated rats showed membrane degeneration and reduction in amylase synthesis, while protein synthesis related to coagulation was not inhibited. These results suggest that, despite vulnerability of liver cells from STZ treated rats, coagulation activity in the liver is retained and rather enhanced in STZ-induced hyperglycemic rats, which may contribute to the promotion of atherosclerosis.
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PMID:Blood coagulability and fibrinolysis in streptozotocin-induced diabetic rats. 958 51

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.
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PMID:Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1. 969 48

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (</= 10%) Sak42DDeltaN11(M),G12K, active (>50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.
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PMID:NH2-terminal structural motifs in staphylokinase required for plasminogen activation. 971 54


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