Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.
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PMID:Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation. 133 78

Neuropeptides are considered to play an important role in the modulation of a number of immune functions. Calcitonin gene-related peptide (CGRP), one of the neuropeptides, was found to profoundly inhibit the ability of macrophages to produce H2O2 in response to IFN-gamma or to act as APC. For the inhibition of H2O2 production to occur, preincubation of the macrophages with CGRP was required. Among neuropeptides that are similar in size, calcitonin also prevented macrophage activation but adrenocorticotropic hormone did not. These findings suggest that CGRP and calcitonin play an important role in modulating the ability of macrophages to present Ag and to respond to activating factors.
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PMID:Peptides encoded by the calcitonin gene inhibit macrophage function. 254 3

We describe the simple and rapid enzyme immunoassay of protein C in human plasma with use of a Cobas Fara centrifugal analyzer. The antibody, labeled with horseradish peroxidase, is reacted with antigen (protein C) for 15 min. The peroxidase activity of the resulting antigen-antibody conjugate is measured at 500 nm for 5 min in the presence of excess H2O2, phenol, and 4-aminoantipyrine, as compared with that of free conjugates. Results are calculated from a stored standard curve and expressed as a percentage of the value determined for a pooled specimen of normal adult plasma. The standard curve is linear from 0% to 200%. The CV is generally less than 4% for different concentrations of protein C. In liver cirrhosis, hepatocellular carcinoma, therapy with warfarin, thrombosis, and disseminated intravascular coagulation, protein C concentrations are about 40-70% of normal. Results obtained with the present homogeneous enzyme immunoassay correlated well with those by enzyme-labeled immunosorbent assay (r = 0.97).
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PMID:Protein C in human plasma determined by homogeneous enzyme immunoassay with use of a centrifugal analyzer. 304 78

Leukocytes release lysosomal proteases and reactive oxygen species in response to various stimuli that damage the adjacent endothelial cells. We investigated the effects of granulocyte lysosomal proteases (granulocyte elastase [GE] and cathepsin G [CG] and/or hydrogen peroxide (H2O2) on thrombomodulin (TM) activity of endothelial cells. We wished to determine whether the activated leukocytes damage the nonthrombogenic systems of endothelial cells. When cultured human umbilical vein endothelial cells (HUVECs) were incubated with GE, TM activity of the cells (as judged by the protein C activation capacity) decreased to about 10% of control with the concomitant increase of immunoreactive TM concentration in the conditioned media. CG also decreased TM activity to about 20% of control with the concomitant increase in immunoreactive TM concentration in the conditioned media. The GE- or CG-induced inactivation of TM was not observed in the presence of alpha 1-proteinase inhibitor. Immunoblot analysis showed that CG cleaved purified TM to yield one major fragment with an M(r) of 43,000; TM activity of this fragment was about 10% of the control activity. When purified TM was incubated with GE, TM activity decreased to 10% of control, and no detectable band was found on immunoblotting, suggesting that CG and GE cleave TM into inactive fragments and that GE degrades the epitope structure of TM. Although H2O2 (1.0 mmol/L) enhanced chromium 51 release from prelabeled HUVECs after 30 minutes of incubation, it decreased TM activity only slightly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro. 820 Dec 66

The optimal level of oxygen-dependent microbicidal activity in human neutrophils depends on the generation of highly toxic products, including hypochlorous acid, by hydrogen peroxide in the presence of chloride anion and the neutrophil granule protein myeloperoxidase (MPO). The biosynthesis of MPO is normally restricted to the promyelocytic stage of myeloid development and includes N-linked glycosylation, heme insertion, proteolytic processing, subunit dimerization, and eventual targeting to the azurophilic granule. In the endoplasmic reticulum, MPO precursors interact transiently with calreticulin and calnexin, presumably in their capacity as molecular chaperones. In light of the important role of the MPO-H2O2-chloride system in human host defense, the relatively high prevalence of inherited MPO deficiency was an unanticipated insight provided by the widespread use of automated flow cytometry for the enumeration of leukocytes in clinical specimens. In many cases of inherited MPO deficiency, affected neutrophils have immunochemical evidence of precursor protein but lack the subunits of mature MPO, peroxidase activity, or the ability to chlorinate target proteins. To date, four genotypes have been reported to cause inherited MPO deficiency, each of which results in missense mutations. In the genotype Y173C, the mutant precursor is retained in the endoplasmic reticulum by virtue of its prolonged interaction with calnexin, and it eventually undergoes degradation in the 20S proteasome. In this way, the quality control system operating in the endoplasmic reticulum retrieves malfolded MPO precursors from the biosynthetic pathway and creates the biochemical phenotype of MPO deficiency. Thus MPO deficiency caused by Y173C joins the ranks of cystic fibrosis, protein C deficiency, and other genetic disorders that reflect abnormalities in protein folding.
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PMID:Quality control in the endoplasmic reticulum: lessons from hereditary myeloperoxidase deficiency. 1048 5

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.
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PMID:Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects. 1073 83

Decreased circulating protein C and increased circulating thrombomodulin are markers of the prothrombotic, antifibrinolytic state associated with poor outcomes in sepsis but have not been measured in patients with ALI (acute lung injury)/ARDS (acute respiratory distress syndrome). We measured circulating and intra-alveolar protein C and thrombomodulin in 45 patients with ALI/ARDS from septic and nonseptic causes and correlated the levels with clinical outcomes. Plasma protein C levels were lower in ALI/ARDS compared with normal. Lower levels of protein C were associated with worse clinical outcomes, including death, fewer ventilator-free days, and more nonpulmonary organ failures, even when only patients without sepsis were analyzed. Levels of thrombomodulin in pulmonary edema fluid from ALI/ARDS patients were >10-fold higher than normal plasma and 2-fold higher than ALI/ARDS plasma. Higher edema fluid thrombomodulin levels were associated with worse clinical outcomes. The higher levels in edema fluid compared with plasma suggest local release of soluble thrombomodulin in the lung, possibly from a lung epithelial source. To determine whether lung epithelial cells can release thrombomodulin, A549 cells and primary isolates of human alveolar type II cells were exposed to H2O2 or inflammatory cytokines. Both epithelial cell types released thrombomodulin into the media. In summary, the protein C system is markedly disrupted in patients with ALI/ARDS from both septic and nonseptic causes. The protein C system may be a potential therapeutic target in patients with ALI/ARDS.
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PMID:Protein C and thrombomodulin in human acute lung injury. 1275 94

Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O(6)-benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence. When SNAP-tag is expressed in HEK293T cells and RAW264.7 macrophages as a protein C-terminally fused to the transmembrane domain of platelet-derived growth factor receptor (PDGFR), the tag is presented on the outside of the plasma membrane; conjugation of NBzF-BG with the cell surface SNAP-tag enables detection of H2O2 added exogenously. We also demonstrate molecular imaging of H2O2 that is endogenously produced in phagosomes of macrophages ingesting IgG-coated latex beads. Thus, NBzF-BG, combined with the SNAP-tag technology, should be useful as a tool to measure local production of H2O2 in living cells.
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PMID:Visualization of phagosomal hydrogen peroxide production by a novel fluorescent probe that is localized via SNAP-tag labeling. 2486 9

We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H2O2), largely reduced the active force (Factive), increased the passive force (Fpassive), and decreased the Ca(2+) sensitivity of force production (pCa50) in permeabilized cardiomyocytes. H2O2 alone had significantly smaller effects on Factive and Fpassive and did not alter pCa50. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H2O2 alone and MPO + H2O2 reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca(2+)-regulated active and Ca(2+)-independent passive force production and myofilament Ca(2+) sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation.
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PMID:Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes. 2577 Jun 62