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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.
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PMID:Interaction of heparin cofactor II with neutrophil elastase and cathepsin G. 231 47

A 20-year-old man on oral substitution of pancreatic enzymes after hemipancreatectomy injected an enzyme preparation of fungal origin intravenously after dissolving it in water. Within a few hours chills, headache, nausea and vomiting, fever of 40.8 degrees C, and shock occurred. The acute illness might have been caused by bacteremia, an anaphylactic reaction, or by direct activation of humoral or cellular mediators by the fungal enzymes. A haemostatic disturbance, particularly a drop in plasminogen, was observed. In vitro, the fungal enzyme preparation stimulated elastase release from isolated neutrophils and eliminated plasmatic inhibitors and plasminogen in normal plasma and whole blood. Human neutrophil elastase complexed to alpha 1-antitrypsin was increased in the patient's plasma, while the levels of the complexes thrombin-antithrombinIII and plasmin-alpha 2-antiplasmin, indicating recent coagulation or fibrinolysis, respectively, were not elevated. Thus, an activation of the neutrophils with release of elastase might have contributed to the observed coagulation disturbances.
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PMID:A unique case of intravenous injection of fungal "pancreatic" enzymes causing shock and proteolysis of haemostatic proteins. 246 40

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.
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PMID:Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin. 248 76

Extravascular, primarily, alveolar fibrin deposition is commonly associated with the alveolitis of many interstitial lung diseases including the interstitial lung disease associated with rheumatoid arthritis (RA). We therefore hypothesized that coagulation pathways, which promote fibrin formation, would be activated in the alveolar lining fluids of patients with rheumatoid interstitial lung disease. To test this hypothesis, we studied the bronchoalveolar lavage (BAL) fluids from patients with rheumatoid interstitial lung disease (n = 7) and patients with RA unassociated with interstitial lung disease (n = 10) to characterize and quantitatively compare the BAL procoagulant material and levels of fibrinopeptide A (FPA), which is cleaved from fibrinogen by thrombin. FPA reactive peptide concentrations were significantly greater in rheumatoid interstitial lung disease than RA when normalized per ml of concentrated BAL fluid (p = 0.02), per mg BAL total protein (p = 0.01) or BAL albumin content (p = 0.03) and correlated with BAL antigenic neutrophil elastase concentrations (r = 0.87). Procoagulant activity was present in similar concentration of BAL of patients with RA and rheumatoid interstitial lung disease and was mainly attributable to tissue factor associated with factor VII (or VIIa). Our results demonstrate that tissue factor and factor VII are endogenous in the alveoli of subjects with RA and interstitial lung disease and could interact with distal coagulation substrates which may enter the alveoli in interstitial lung disease to locally promote fibrin deposition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fibrinopeptide A reactive peptides and procoagulant activity in bronchoalveolar lavage: relationship to rheumatoid interstitial lung disease. 266 53

In 42 patients with septic shock, 29 of whom underwent substitution with antithrombin III concentrate and fresh frozen plasma for coagulation disorders, the proteinase-inhibitor complexes thrombin-antithrombin III and neutrophil elastase-alpha 1 proteinase inhibitor, were elevated on admission. On admission, the elastase complex was significantly higher in the patients receiving substitution (p = 0.0039), but at the endpoint it was higher in the non-survivors (p = 0.0040). The elastase decrease was confined to the substitution group with the thrombin complex decreasing in both groups. Initially the thrombin complex correlated with prothrombin times and factor XIII, while the elastase complex correlated with creatinine, thrombocyte count and prothrombin times in the late stages. Hemostatic disturbance, thrombin generation and neutrophil elastase release were favorably influenced by substitution. Furthermore, in this uncontrolled pilot study, the survival rate was higher in the treated (16 of 29) than in the untreated (1 of 13) patients, although the treated patients initially had pronounced hemostatic disturbances.
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PMID:The disturbance of hemostasis in septic shock: role of neutrophil elastase and thrombin, effects of antithrombin III and plasma substitution. 278 82

Protein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC. In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor Xa was inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor Xa mediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S. These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.
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PMID:The anticoagulant properties of a modified form of protein S. 297 8

The prognosis of septicaemia depends on the occurrence of complications such as shock and coagulation defects. The damage to haemostasis is usually explained by the action of the main coagulation and fibrinolysis enzymes, thrombin and plasmin. This paper presents data concerning the role of a third protease, granulocytic elastase. 82 patients who had been admitted to our hospital with suspected septicaemia were examined. Septicaemia was proven in 22 patients by the growth of microorganisms in blood cultures, and was clinically diagnosed in 9 patients. The plasma levels of neutrophil elastase-like protease complexed to a1antitrypsin (a1AT-ELP) were measured by zone immunoelectrophoresis assay (ZIA). The a1AT-ELP values were significantly increased in the 31 septic as compared to the 51 non-septic patients. In patients with complicated septicaemia, negative correlations of a1AT-ELP with factor XIII and the coagulation inhibitor antithrombin III were demonstrable. Among the patients with septic complications, the 3 who survived exhibited a dramatic decrease of a1AT-ELP, whereas in the other 16 patients who died the levels remained elevated. It might be of therapeutic significance that in 9 patients receiving fresh plasma and AT III-concentrate substitution for DIC the a1AT-ELP levels dropped, whereas they remained high in the other septicaemia patients. There were no correlations between a1AT-ELP and the a2antiplasmin-plasmin complexes (a2AP-P1), but strong correlations with signs of coagulation. The data suggest an interaction of coagulation and elastase release, probably involving the Hageman factor.
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PMID:Participation and interactions of neutrophil elastase in haemostatic disorders of patients with severe infections. 329 74

Neutrophils activated with serum opsonized zymosan, soluble heat-aggregated IgG, and ionophore A23187 in the presence of calcium release a material capable of initially activating factor V. Subsequent inactivation of factor V was only observed with neutrophil releasate derived from IgG and ionophore. In this study we examine the nature of this neutrophil activity and investigate its role in the regulation of factor V/Va. From early in the fractionation it was apparent that the cells contained different enzymes capable of cleaving factor V. The most active of these was isolated and found to be an isomer of human neutrophil elastase. The purified protease caused a dose-dependent activation of isolated factor V to a maximum of threefold. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single-chain factor V was cleaved to form intermediates of 100 and 91 kilodaltons (kD). Coagulant activity correlated with the formation of a 97-kD heavy and 77-kD light chain. On prolonged incubation the formed factor Va(e) was inactivated in association with proteolysis of the 97-kD band to smaller peptides and cleavage of the 77-kD light chain to a molecular weight of 75 kD, which is similar to thrombin-activated factor Va light chain. Neutrophil elastase also caused rapid inactivation of thrombin-activated factor V, factor Va(t). These observations suggest that elastase cleaves factor V at sites distinct from that by thrombin and therefore represents a novel factor V activation pattern. It is proposed that upon neutrophil activation elastase is secreted into the plasma milieu to initiate factor V activation. This serves to generate small amounts of thrombin that, in turn, by positive feedback fully activates factor V and thus amplifies the coagulation reaction.
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PMID:The regulation of human factor V by a neutrophil protease. 330 63

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties of thrombin- and elastase-modified human antithrombin III. 340 16


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