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Query: EC:3.4.21.37 (neutrophil elastase)
 4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of brefeldin A, monensin, and the weak base NH4Cl on the biosynthesis and processing of cathepsin G and neutrophil elastase of myeloid cells were investigated. Monoblast-like U-937 cells were biosynthetically labeled with [35S]methionine, followed by subcellular fractionation, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Brefeldin A inhibited proteolytic processing, intracellular transport, and secretion. The effects were reversible inasmuch as removal of brefeldin A resulted in a normal pattern of processing and transfer to high-density organelles, corresponding to lysosomes, and restitution of constitutive secretion of precursor forms. Both cathepsin G and neutrophil elastase acquired resistance to endoglycosidase-H, suggesting that conversion to complex oligosaccharide side chains also occurs in the presence of brefeldin A. Monensin and NH4Cl inhibited final proteolytic processing, indicating either that acidification is necessary for directing cathepsin G and neutrophil elastase to lysosomal-like organelles or that the protease(s) responsible for processing requires an acid pH. We conclude that pH-dependent proteolytic processing of cathepsin G and neutrophil elastase occurs in post-Golgi structures and that transfer to lysosomes or an immediately prelysosomal compartment is mandatory for complete processing.
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PMID:Processing and intracellular transport of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937-modulation by brefeldin A, ammonium chloride, and monensin. 828 40

The biocompatibility and solute permeability characteristics of a high-permeability modified cellulose membrane (Hemophan-HP) (He-HP) were compared with those of two synthetic membranes (poly(ethylene-co-vinyl alcohol) (EVAL) and poly(acrylonitrile-co-sodium methallyl sulphonate) (AN69)) and Cuprophan in a multicentre, four-way cross-over clinical trial. Cuprophan membranes caused significant complement activation, leukopenia, and granulocyte elastase release. He-HP membranes demonstrated a lesser effect, which was similar to that observed for the EVAL membrane, although less than that seen with the AN69 membrane. A similar order for the four membranes was seen for their effect on platelets. Cuprophan membranes provided superior small-molecule removal to the other three membranes. In contrast, Cuprophan was essentially impermeable to beta 2-microglobulin, whereas He-HP, EVAL, and AN69 allowed the removal of 60-90 mg of beta 2-microglobulin per treatment. However, a decrease in the plasma concentration of beta 2-microglobulin was observed only with the AN69 membrane, most probably as a result of the ability of that membrane to adsorb proteins. Our results demonstrate that high-permeability membranes of comparable biocompatibility to some synthetic membranes can be fabricated from cellulose derivatives.
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PMID:Biocompatibility of a new high-permeability modified cellulose membrane for haemodialysis. 838 35

Cytokine activation of cultured human vascular endothelial cells renders them hyperadhesive for blood leukocytes. Co-incubation of freshly isolated, unstimulated human blood neutrophils with confluent cytokine-activated human endothelial monolayers for 90 minutes results in extensive endothelial detachment and destruction of monolayer integrity. In contrast, unactivated endothelial monolayers remain intact. Using this in vitro model, we have explored the neutrophil-effector mechanisms involved in this injury. Coincubation in the presence of a serine protease inhibitor (phenylmethylsulfonyl fluoride) or specific elastase inhibitors (Ala-Ala-Pro-Val-chloromethyl ketone or alpha-1-protease inhibitor) markedly diminished injury. In contrast, scavengers or inhibitors of oxygen-derived free radicals (superoxide dismutase, catalase, mannitol, or sodium azide) were not protective. Purified human neutrophil elastase mimicked the effect of the neutrophils suggesting a key role for elastase in the neutrophil-mediated injury in this model. Interfering with direct neutrophil-endothelial cell contact by interposing a microporous barrier insert prevented endothelial cell detachment. Furthermore, this neutrophil-mediated detachment could be inhibited with interleukin-8, an action correlated with a decrease in neutrophil adhesion to activated endothelial monolayers. By defining the role of endothelial activation in neutrophil-mediated injury, this in vitro model may provide useful insights into potential therapeutic interventions designed to prevent disruption of the endothelial barrier function.
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PMID:Neutrophil-mediated damage to human vascular endothelium. Role of cytokine activation. 842 50

Complement activation and leukocyte stimulation were prospectively studied during and after cardiopulmonary bypass in 16 children receiving sodium nitroprusside--a nitrovasodilator releasing nitric oxide--for vasodilation during the cooling and rewarming periods of extracorporeal circulation. Results were compared with those in 29 patients who were not treated with sodium nitroprusside during the operation. Patients treated with sodium nitroprusside had significantly less C3 conversion during cardiopulmonary bypass as measured by the ratio C3d/C3 (p <0.05) and significantly less C5a liberation immediately after cardiopulmonary bypass (p < 0.005) than patients not treated with sodium nitroprusside. C4 was not overtly consumed in our series. Leukocyte count during the rewarming period of cardiopulmonary bypass, but not leukocyte elastase release during cardiopulmonary bypass, was significantly reduced in patients treated with sodium nitroprusside (p <0.05). In vitro experiments were conducted to analyze the effect of sodium nitroprusside on complement hemolytic activity initiated by the classic and the alternate pathways and on zymosan-induced C3 conversion by the activation of the alternate pathway. The in vitro experiments clearly demonstrate inhibition of complement hemolytic activity by sodium nitroprusside in the sera tested. The 50% inhibitory concentration of sodium nitroprusside on the available complement hemolytic activity was less through the alternate pathway than through the classic one (4.2 +/- 0.8 mmol/L and 14.0 +/- 2.88 mmol/L, respectively). The decrease of complement hemolytic activity measured was dose-dependent and was enhanced by the sodium nitroprusside preincubation of the sera tested. This effect was related to the duration of preincubation. Sodium nitroprusside photodegradation (enhancing nitric oxide release) increased the anticomplementary effect of the drug, reducing the 50% inhibitory concentration on complement hemolytic activity to 0.24 to 0.02 mmol/L for the alternate pathway and 2.74 o 0.3 mmol/L for the classic pathway. the zymosan-induced C3 conversion was inhibited by sodium nitroprusside. Nitroglycerin and isosorbide dinitrate (other nitric oxide donors) had in vitro effects on complement hemolytic activity similar to those of nonphotodegraded sodium nitroprusside at similar concentrations (1 mmol/L). Our results suggest that sodium nitroprusside, both in vitro and in vivo, has an inhibiting effect on complement activation initiated by both classic and alternate pathways and that this effect is mediated by nitric oxide release from sodium nitroprusside. This is the first report on the anticomplementary effect of sodium nitroprusside by nitric oxide release.
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PMID:Effect of sodium nitroprusside on complement activation induced by cardiopulmonary bypass: a clinical and experimental study. 861 50

We purified a novel serine proteinase inhibitor (serpin)-like protein from the bovine brain and named it B-43 from its molecular mass, 43 kDa. A cleaved peptide from B-43 was copurified with the native B-43. Partial amino acid sequencing of the purified B-43 showed that this protein was homologous to glia-derived nexin/protease nexin-1 (GDN/PN-1), plasminogen activator inhibitor 2, leukocyte elastase inhibitor (LEI) and placental thrombin inhibitor (PTI) among the serpins. Although B-43 had a similar amino acid composition to these serpins, the biochemical features of B-43 were different from them. B-43 did not form sodium dodecyl sulfate (SDS)-resistant serpin-proteinase complexes with thrombin, urokinase, pancreatic elastase and plasmin, suggesting that these proteinases were not the targets of B-43. In contrast to GDN/PN-1, B-43 did not have an affinity for heparin. B-43, having different biochemical properties from GDN/PN-1, appears to be an additional serpin expressed in the brain.
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PMID:Purification of a novel serpin-like protein from bovine brain. 884 89

Human myeloblastin (leukocyte proteinase 3) shares many biochemical properties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution or when handled in glass vessels. This poses limits to kinetic experiments requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assays were performed in sodium/potassium phosphate buffer at pH 7.0, ionic strength 0.11, in the presence of 0.05% w/v Triton X-100 and taking care to avoid any contact with glass surfaces. The kinetic parameters of leukocyte elastase and myeloblastin with peptide substrates, irreversible inactivators and glycosaminoglycans were compared under these conditions. MeO-succinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excellent substrate for leukocyte elastase, also proved to be a good substrate for myeloblastin (Km = 16 microM, kcat/Km = 30,600 M(-1)s(-1)). Inactivation of myeloblastin by 3,4-dichloroisocoumarin (ki/Ki = 6,389 M(-1)s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (ki/Ki = 579 M(-1) S(-1)) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectively. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-essential activators in the presence of peptide substrates (activation up to a 6.7-fold factor) and as partial inhibitors (about 50% inhibition at saturation) in the presence of elastin. This property distinguishes myeloblastin from leukocyte elastase, which is always inhibited by glycosaminoglycans, independently of the substrate.
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PMID:Human myeloblastin (leukocyte proteinase 3): reactions with substrates, inactivators and activators in comparison with leukocyte elastase. 906 56

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.
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PMID:Human neutrophil elastase activates human factor V but inactivates thrombin-activated human factor V. 924 37

FK706, sodium 2-[4-[[(S)-1-[[(S)-2-[[(RS)-3, 3, 3-trifluoro-1-isopropyl-2-oxopropyl]aminocarbonyl]pyrrolidin -1-yl]carbonyl]-2-methylpropyl] aminocarbonyl] benzoylamino] acetate, C26H32F3N4NaO7, is a synthetic water-soluble inhibitor of human neutrophil elastase. This compound demonstrated a competitive and slow-binding inhibition of human neutrophil elastase with a Ki of 4.2 nM. In studies using synthetic substrates, FK706 inhibited human neutrophil elastase activity and porcine pancreatic elastase activity with respective IC50 values of 83 and 100 nM. FK706, however, inhibited more weakly, (IC50 values > 340 microM) other serine proteinases such as human pancreatic alpha-chymotrypsin, human pancreatic trypsin and human leukocyte cathepsin G. FK706 also effectively inhibited the hydrolysis of bovine neck ligament elastin (2 mg/ml final concentration) by human neutrophil elastase (4 microg/ml final concentration) with an IC50 value of 230 nM. FK706 protected animals against human neutrophil elastase (50 microg/animal)-induced lung hemorrhage with ED50 values of 2.4 microg/animal by intratracheal administration and 36.5 mg/kg by intravenous administration, respectively. Subcutaneous administration of FK706 significantly suppressed human neutrophil elastase (20 microg/paw)-induced paw edema in mice in a dose-dependent manner (47% inhibition at a dose of 100 mg/kg). These results suggest that FK706 would be a useful tool for investigating the role of human neutrophil elastase in inflammatory disorders associated with an excess of elastase, such as pulmonary emphysema, adult respiratory distress syndrome, septic shock, cystic fibrosis, chronic bronchitis and rheumatoid arthritis.
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PMID:Biochemical and pharmacological characterization of FK706, a novel elastase inhibitor. 938 82

We investigated the effect of human neutrophil elastase (HNE) on tracheal mucociliary transport in anesthetized quails. Topical application of HNE (30-300 microg/kg) to tracheal mucosa dose-dependently decreased mucociliary transport velocity (MCTV). The HNE (300 microg/kg)-induced decrease in MCTV was blocked by ONO-5046 x Na (sodium N-[2-[4-(2,2-dimethylpropionyloxy)phenyl-sulfonylamino]benzo yl]aminoacetate tetrahydrate) (3-30 mg/kg, i.m.), a specific neutrophil elastase inhibitor. Furthermore, we found that HNE increased DNA, fucose and protein contents of tracheal lavages, and the increases were also reverted by ONO-5046 Na. These results indicated that HNE decreased tracheal mucociliary transport, and the decrease may be, at least in part, ascribed to the deterioration of tracheal secretions.
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PMID:Effect of human neutrophil elastase on tracheal mucociliary transport in anesthetized quails. 946 51

In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa alpha-(Arg145) and beta-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC).
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PMID:Neutrophil elastase cleavage of human factor IX generates an activated factor IX-like product devoid of coagulant function. 969 17


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