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)

To determine whether glycoconjugates can be released into airways by surface epithelial cells that do not contain secretory granules and, if so, whether extracellular proteinases can affect this release, we studied dog tracheal epithelial cells after 8-10 days in culture. Ultrastructurally, these cells showed an extensive cell surface coat and no secretory granules. Cells were pulse labeled with radioactive sulfate (Na2 35SO4, 50 microCi/ml/24 h) and washed free of the unbound label. Release of sulfated products was then measured at 20-min intervals under basal conditions and again after 20 min of incubation with various extracellular proteinase. We found that these cells synthesized sulfated products and released them spontaneously and continuously into the medium. In addition, trypsin, Pseudomonas aeruginosa elastase, thermolysin, Staphylococcus aureus proteinase, mast cell chymase, plasmin, and kallikrein (each at 10(-7) M except plasmin, at 5 X 10(-6) M) increased the release of sulfated products to 77-667% over baseline release (p less than 0.01, n = 5 dogs for each); preliminary results showed that human neutrophil elastase was also very potent. The sulfated products released by trypsin had an apparent molecular weight of greater than or equal to 10(6) da as determined by gel filtration on Sepharose Cl-4B. Over 50% of these 35S-labeled products were digested to low-molecular-weight products (500-2000 da) upon incubation with endo-beta-galactosidase or with keratanase, suggesting that they are glycoconjugates containing poly(N-acetyllactosamine)-type carbohydrate chains. Decrease in cell staining by lectins specific for poly(N-acetyllactosamine), which accompanied the release of glycoconjugates, indicates that these sulfated glycoconjugates were released by proteinases from the apical cell surface. We conclude that cultured tracheal epithelial cells synthesize and transport sulfated macromolecular glycoconjugates to apical cell surfaces. These glycoconjugates are released from cell surfaces when exposed to extracellular proteinases. We therefore suggest that macromolecular glycoconjugates in airway secretions can originate not only from secretory granules but also from epithelial cell surfaces during airway inflammation.
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PMID:Dog tracheal epithelial cells in culture synthesize sulfated macromolecular glycoconjugates and release them from the cell surface upon exposure to extracellular proteinases. 331 21

Neutrophil elastase digests plasminogen to yield a fragment, mini-plasminogen, which is activatable to a mini-plasmin capable of escaping the action of the primary plasmin inhibitor. Such a molecule may play a role in joint destruction, either directly or by activation of procollagenase to collagenase. Synovial fluid samples from 34 acute joint effusions were examined by lysine-Sepharose chromatography and fibrinolytic assay of the fall-through (non-lysine-binding) fractions in presence of urokinase. Fragments similar to mini-plasminogen were found in 20 of 23 inflammatory effusions (cell count greater than 0.5 X 10(3)/microliter) and in none of 11 non-inflammatory (traumatic and osteoarthritic) effusions (cell count less than 0.5 X 10(3)/microliter) (p less than 0.001). Analysis of four inflammatory fluids by gel filtration on Bio-Gel P 100 and enzyme-linked immunoassay for plasminogen antigen revealed plasminogen fragments with molecular weight similar to mini-plasminogen (34,000 daltons) in three, and larger plasminogen fragments (or complexes of mini-plasminogen with other synovial fluid macromolecules) in all four. Fibrinolytic activity was demonstrable in fractions containing plasminogen fragments after treatment with tissue type plasminogen activator. In contrast with non-inflammatory effusions, inflammatory joint fluids contain plasminogen fragments with the properties of mini-plasminogen, suggesting their possible role in inflammatory joint destruction.
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PMID:Mini-plasminogen-like fragments of plasminogen in synovial fluid in acute inflammatory arthritis. 363 68

Human neutrophil elastase from normal donors has been purified using an isolation procedure which included sequential sodium chloride extraction, Aprotinin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil elastase and resulted in a higher specific activity of the final preparation. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of the reduced purified protein demonstrated three polypeptides of Mr 31,000, 28,000, and 27,500. Four polypeptides were resolved on acid gel electrophoresis; each of the four possessed amidolytic activity. Furthermore, peptide analysis of Staphylococcus aureus V8 protease digests indicated that these polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The apparent isoelectric points of these four forms as determined by two-dimensional electrophoresis range from 6.1 to 6.7. By utilizing microsequencing techniques, the first 40 residues of neutrophil elastase have been determined and compared with the reported sequence of elastase isolated from leukemic myeloid cells. In addition, a high degree of homology was found within the amino-terminal regions of neutrophil elastase and the serine proteinases porcine elastase, bovine chymotrypsin, human factor D, and the beta chain of plasmin.
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PMID:Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil elastase from normal donors. 385 49

The present study was undertaken to provide a highly sensitive detection system for the identification and characterisation of serine proteinases separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Biotinylated aprotinin of high specific activity (88-92% active) was prepared (i) by reaction of aprotinin directly with N-hydroxy sulfosuccinimidyl-6-(biotinamido) hexanoate, and (ii) by reaction of aprotinin-trypsin complex with N-hydroxy succinimidobiotin. Both biotinylated aprotinin samples were suitable as probes for the detection of the serine proteinases, neutrophil elastase and cathepsin G, pancreatic trypsin and chymotrypsin and plasmin on nitrocellulose blots. Specific irreversible chloromethyl ketone proteinase inhibitors used in combination with this detection system enabled respective proteinases to be selectively inactivated and thus positively identified. The biotinylated aprotinin detection system was highly sensitive and could detect as little as 0.2 ng (8.5 fmol) of active proteinase (trypsin). In summary, a method has been developed for the sensitive detection of serine proteinases separated by SDS-PAGE. The method is more sensitive and convenient to perform than conventional zymography and significantly, when used in conjunction with specific serine proteinase inhibitors or specific antibodies can yield appreciable information on the identity of the respective serine proteinases being examined. Furthermore the molecular mass of the serine proteinase may be reliably obtained by this method. This method should find application in identifying the role that serine proteinases play in the etiopathogenesis of connective tissue disorders.
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PMID:Biotinylated aprotinin: a versatile probe for the detection of serine proteinases on western blots. 758 24

The precursor of matrix metalloproteinase 9 (pro-MMP-9) forms a complex with the tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule, and the N-terminal domain of TIMP-1 in the complex interacts and inhibits active MMPs. We have reported that a catalytic amount of MMP-3 (stromelysin 1) activates pro-MMP-9 (Ogata, Y., Enghild, J. J., and Nagase, H. (1992) J. Biol. Chem. 267, 3581-3584). To activate pro-MMP-9 in the complex, however, an excess molar amount of MMP-3 is required to saturate the TIMP-1 in the complex. The aim of this study was to test the hypothesis that the requirement for excess MMP-3 can be circumvented by specific destruction of TIMP-1 by non-target proteinases. We have tested trypsin, plasmin, cathepsin G, neutrophil elastase, and chymotrypsin as possible inactivators of TIMP-1 and found that neutrophil elastase inactivates TIMP-1 in the complex without significant destruction of pro-MMP-9. Once TIMP-1 is inactivated, pro-MMP-9 can be readily activated by a catalytic amount of MMP-3. These results suggest that neutrophil elastase may participate in the connective tissue destruction at the inflammatory sites not only by its direct action on matrix macromolecules but also by rendering pro-MMP-9 in the pro-MMP-9.TIMP-1 complex activable by MMP-3 as well as activating pro-MMP-3.
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PMID:Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. 762 55

Aprotinin reduces blood loss after cardiac operations and decreases the bleeding time. The mechanism of action of aprotinin that produces these effects is not clear. During simulated extracorporeal circulation the contact and complement systems, platelets, and neutrophils are activated. We investigated the effect of aprotinin on kallikrein-C1-inhibitor complex and C1-C1-inhibitor complex formation, neutrophil degranulation, and platelet release and aggregation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated at 37 degrees C for 2 hours in a spiral coil membrane oxygenator-roller pump perfusion circuit. Changes in platelet count, leukocyte count, platelet response to adenosine diphosphate, and plasma levels of beta-thromboglobulin, kallikrein-C1-inhibitor complexes, C1-C1-inhibitor complexes, and neutrophil elastase were measured before and at 5, 30, 60, and 120 minutes of recirculation at 0, 0.015, 0.03, 0.06, and 0.12 mg/ml doses of aprotinin. Platelet counts decreased to 36% +/- 12% of control values at 5 minutes and increased to 56% +/- 13% at 120 minutes without aprotinin. Aprotinin did not affect platelet counts, but it did prevent the decrease in sensitivity of platelets to adenosine diphosphate and it attenuated beta-thromboglobulin release. In the absence of aprotinin, kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively to 0.53 +/- 0.14 U/ml and 2.38 +/- 0.33 U/ml, respectively, at 120 minutes. Kallikrein-C1-inhibitor complexes were completely inhibited and C1-C1-inhibitor complexes were partially inhibited at aprotinin concentrations of 0.03 mg/ml or greater. Release of neutrophil elastase was partially but not completely inhibited at the highest dose of aprotinin and was 50% inhibited at a dose of 0.03 mg/ml. Because activation of the fibrinolytic system does not occur in this system, the changes were independent of the inhibition of plasmin. We conclude that aprotinin in high doses completely inhibited kallikrein-induced activation of neutrophils and partially inhibited complement-induced activation. Aprotinin did not directly affect platelet adhesion or aggregation, but it indirectly preserved platelet sensitivity to agonists and also attenuated release of alpha-granule contents. The data indicate that in the presence of aprotinin platelet function was partially preserved, kallikrein production was totally inhibited, complement activation was partially inhibited, and neutrophil release was partially inhibited, thus attenuating the "whole body inflammatory response" associated with cardiopulmonary bypass.
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PMID:Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporeal perfusion. 768 93

A novel trypsin inhibitor, tentatively named countertrypin, was isolated from mouse plasma in an apparently homogeneous state. Countertrypin is a 53-kDa glycoprotein having about 30% carbohydrate, and did not cross-react immunologically with either mouse alpha 1-antiproteinase (also called alpha 1-proteinase inhibitor or alpha 1-antitrypsin) or contrapsin. Countertrypin had no inhibitory activity against chymotrypsin, pancreatic elastase, neutrophil elastase, thrombin, plasmin, plasma kallikrein, pancreatic kallikrein, clotting factor Xa, or papain. This inhibitory spectrum does not correspond to any of the known plasma proteinase inhibitors that have been well characterized in human or other mammals. NH2-terminal amino acid sequence analysis of the intact molecule and three peptides obtained by CNBr digestion revealed that a total of 93 amino acid residues could be aligned with stretches in human alpha 2-HS glycoprotein, bovine fetuin, and rat pp63 (rat fetuin). Human alpha 2-HS glycoprotein and bovine fetuin prepared without use of ethanol inhibited trypsin and pancreatic and neutrophil elastases. These results indicate that mouse countertrypin is a new member of the mammalian fetuin family, which possibly has the trypsin-inhibiting activity in common.
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PMID:Isolation and characterization of mouse countertrypin, a new trypsin inhibitor belonging to the mammalian fetuin family. 768 30

Both plasmin and elastase, a protease released from neutrophil granulocytes, are known to degrade fibrin(ogen). This raises the possibility that elevated plasma levels of split products such as D-dimer may in part result from elastase action. After incubation in vitro of fibrinogen and fibrin clots with elastase, a clearcut increase of D-dimer immunoreactivity was demonstrated by two commercial ELISA kits. In the plasma of 79 patients with inflammatory bowel disease, D-dimer values measured by one of the ELISA kits were correlated significantly not only with markers of thrombin and plasmin activation, but also with elastase-alpha 1-antitrypsin complexes (r = 0.3555; P = 0.014). Thus, the findings of this study suggest that indeed the D-dimer levels in patients with inflammatory disorders are influenced by neutrophil elastase. New tests discriminating effects of activated haemostasis from proteolysis by neutrophil enzymes might be helpful in differential diagnosis and monitoring of therapy.
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PMID:D-dimer tests detect both plasmin and neutrophil elastase derived split products. 778 49

Heparin and heparan sulfate, exhibiting wide biological interactions, are constituted of block structures. A defined pentasaccharide motif was found responsible for the enhancement of the rate of inactivation of factor Xa by antithrombin III. Heparin also interacts with other serine proteinase inhibitors as protease nexin I, and thus possibly modulates extracellular matrix proteolysis by serine proteinases in the pericellular environment. Human neutrophil elastase (HNE) activity is inhibited by heparin with Ki = 75 pM. This strong interaction is electrostatic, involving HNE/arginine residues disposed in a "cluster shoe" arrangement on the surface of the molecule and mainly OSO3- groups of heparin. HNE-heparin interactions also interfere with HNE associations with its natural inhibitors: it decreases the rate of association of HNE with alpha 1 proteinase inhibitor (alpha 1 P(i)) by 3 orders of magnitude, while increasing kass between HNE and mucus bronchial inhibitor (MBI) by > 10 fold. In vivo experiments demonstrated that heparin fragments lacking anticoagulant activity were able to nearly completely abolish emphysematous lesions induced in mice by a single intratracheal administration of 200 micrograms HNE. Long chain unsaturated fatty acids peptide conjugates were described as competitive HNE inhibitors (Hornebeck W. et al. 1985). We synthesized N-oleoyl heparin derivative (3 oleoyl groups/one molecule of heparin); such a lipophilic glycosaminoglycan (LipoGAG), although acting as an elastin protecting agent, possessed lower HNE inhibitory capacity as compared with heparin. In contrast, however, it was able to inhibit other serine proteinases such as urokinase, plasmin, porcine pancreatic apha-chymotrypsin and elastase. Such Lipo GAG's can be therefore useful to control matrix metalloproteinases (MMPs) during tissue remodeling or tumor invasion.
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PMID:Heparin and its derivatives modulate serine proteinases (SERPS) serine proteinase inhibitors (SERPINS) balance. Physiopathological relevance. 789 38

Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2':6',2"-terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.
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PMID:Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate. 836 3


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