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)

Heparin cofactor II (HCII) is plasma glycoprotein and thrombin inhibitor of the serpin type previously shown to inhibit thrombin in the absence of its N-terminal 74 amino acids, and to be cleaved by neutrophil elastase (NE) at two sites: I66-F67 and V439-G440, the P6-P5 bond of the reactive center loop. We examined the contribution of Val439 to the reaction of HCII with thrombin and NE. Hexahistidine-tagged HCII proteins lacking residues 1-66 (H6delta66HCII) containing either the wild-type Val 439 or one of six substitutions were-expressed in E. coli. The rates of heparin-catalyzed thrombin inhibition of the V439L, C, or R variants were reduced at least 80-fold compared to wild-type H6delta66HCII, while those of the F, S, or W variants were largely unchanged. Following controlled exposure to NE in the presence of heparin, these latter variants retained 3.5- to 4.5-fold more residual anti-thrombin activity than wild-type H6delta66HCII treated in the same manner. This resistance arose due to deflection of NE attack from V439-G440 to secondary sites. The F, S, or W V439 variants exhibited a similar or greater degree of NE resistance when re-expressed as full-length hexahistidine-tagged HCII proteins, suggesting that the I66-F67 NE site is not well recognized in non-glycosylated HCII. Of these full-length variants, the V439F was the most active, exhibiting only a 2-fold reduction in its heparin-catalyzed rate of thrombin inhibition. HCII can therefore be made NE-resistant without severely compromising its capacity to inhibit thrombin.
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PMID:Altering heparin cofactor II at VAL439 (P6) either impairs inhibition of thrombin or confers elastase resistance. 1215 84

The products of the blood clotting reaction, eg, thrombin and fibrinopeptides, have various proinflammatory activities and are suggested to modulate inflammation. The macrophage expression of tissue factor (TF), the clotting initiator, has been shown to cause clotting in the site of the delayed-type hypersensitivity reaction, a cellular immune response. However, the mechanism of the clotting induction in humoral immune response has been insufficiently studied. Therefore, the Arthus reaction, a model of immune-complex diseases, was produced in monkey skin that was examined for TF expression and fibrin deposition. TF antigen was positive on most of polymorphonuclear leukocytes, which were the main leukocytes in the lesions and were identified as neutrophils with an anti-neutrophil-elastase mAb. TF mRNA was detected in neutrophils by in situ hybridization using TF RNA probes, indicating de novo TF synthesis by the leukocytes. Specific binding of activated factor VII onto TF-positive neutrophils suggested the activity of neutrophil TF to trigger the cascade reaction of clotting. The number of TF-positive neutrophils were correlated in time with the intensity and extent of fibrin deposition that was visualized with an mAb specific for fibrin and peaked in 24 hours. Interestingly, the fibrin deposit was partially positive for an mAb specific for neutrophil elastase-digested fibrin. These results show in vivo evidence of a close relationship between neutrophils and both clotting and fibrinolysis in the Arthus reaction and may suggest that these neutrophil functions contribute to the pathogenesis of this hypersensitivity inflammation.
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PMID:New functions of neutrophils in the arthus reaction: expression of tissue factor, the clotting initiator, and fibrinolysis by elastase. 1237 63

Neutrophils and mononuclear cells are implicated in the pathogenesis of several inflammatory conditions including chronic obstructive pulmonary disease (COPD). Neutrophil-derived serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), may interact with mononuclear cells via protease-activated receptors (PARs) which are seven-transmembrane G protein-coupled receptors activated by proteolytic cleavage of the extracellular N-terminus, and which, on activation, induce the release of several mediators and cytokines. We determined whether CG and NE could affect PAR-1 expression and function in mononuclear cells. Human blood mononuclear cells were isolated from 20 healthy donors. Surface and intracellular receptor expression and calcium mobilisation (using the calcium chelator, FLUO3-AM) were studied by fluorescence-assisted cell sorting (FACS analysis). Positive controls, i.e. thrombin (0.1-100 mU/ml) and the PAR-1-activating peptide SFLLRN (100 microM) induced a rapid and transient intemalisation of PAR-1 in monocytes and lymphocytes. CG but not NE had a similar effect. By contrast, in monocytes intracellular calcium mobilisation was induced by thrombin and SFLLRN but not by CG and NE. Thus, CG can induce intracellular PAR-1 sequestration without activation of the receptor, and may act as an antagonist and prevent subsequent activation of PAR-1 in mononuclear cells. These findings may be of relevance to the pathogenesis of COPD.
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PMID:Regulation of protease-activated receptor-1 in mononuclear cells by neutrophil proteases. 1264 29

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis-Menten constant (K(m)) for Chromozym ELA (approximately 245 microM) is much higher than those for the thrombin (approximately 90 microM) and trypsin (approximately 60 microM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1'-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55-60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys(77)-Arg(78), Arg(342)-Met(343), Ala(444)-Ala(445) and Arg(557)-Ile(558). The site Arg(557)-Ile(558) is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala(444)-Ala(445) released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the Aalpha chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val(21)-Glu(22) site.
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PMID:Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida. 1295 13

Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.
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PMID:The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase. 1470 61

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.
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PMID:Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition. 1554 57

The bovine chymotrypsin-bovine pancreatic trypsin inhibitor (BPTI) interaction belongs to extensively studied models of protein-protein recognition. The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. Mutations introduced at the P1 position of BPTI result in a more than five orders of magnitude difference of the association constant values with the protease. To elucidate the structural aspects of the discrimination between different P1 residues, crystal structures of five bovine chymotrypsin-P1 BPTI variant complexes have been determined at pH 7.8 to a resolution below 2 A. The set includes polar (Thr), ionizable (Glu, His), medium-sized aliphatic (Met) and large aromatic (Trp) P1 residues and complements our earlier studies of the interaction of different P1 side-chains with the S1 pocket of chymotrypsin. The structures have been compared to the complexes of proteases with similar and dissimilar P1 preferences, including Streptomyces griseus proteases B and E, human neutrophil elastase, crab collagenase, bovine trypsin and human thrombin. The S1 sites of these enzymes share a common general shape of significant rigidity. Large and branched P1 residues adapt in their complexes similar conformations regardless of the polarity and size differences between their S1 pockets. Conversely, long and flexible residues such as P1 Met are present in the disordered form and display a conformational diversity despite similar inhibitory properties with respect to most enzymes studied. Thus, the S1 specificity profiles of the serine proteases appear to result from the precise complementarity of the P1-S1 interface and minor conformational adjustments occurring upon the inhibitor binding.
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PMID:Crystal structures of five bovine chymotrypsin complexes with P1 BPTI variants. 1554 9

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.
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PMID:Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies. 1600 43

Enzymatic thrombin activity is significantly inhibited only by a few selected natural phenolic compounds (myricetin, rosmarinic acid, caffeic acid phenethyl ester) but more strongly by unsaturated fatty acids like erucic acid and oleic acids. Compared to the inhibitory potential against neutrophil elastase, thrombin activity is rather weakly inhibited by phenolic compounds and fatty acids. Because of the importance of thrombin as a ligand for protease-activated receptor 1 (PAR-1), which is involved in inflammation, the inhibition of thrombin activity by natural compounds might enhance the anti-inflammatory effects of neutrophil elastase inhibition.
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PMID:Inhibition of thrombin activity by selected natural products in comparison to neutrophil elastase. 1614 50

Natural and synthetic caffeic acid esters were assayed for their enzymatic activity versus neutrophil elastase (EC 3.4.21.37) and thrombin (EC 3.4.21.5). Lipophilic caffeic acid esters inhibited neutrophil elastase activity and the inhibition rate was enhanced with increasing length of the aliphatic chain of the alcohol component. The geometry of the chain seems to be more important than the number of carbon atoms. The most inhibitory compound was n-octylcaffeic acid ester with an IC50 value of 1.0 microM. Thrombin activity was only weakly inhibited by the caffeic acid esters thus demonstrating a specificity for neutrophil elastase. Because of its critical role in inflammatory processes, inhibition of neutrophil elastase by caffeic acid esters might be of importance in the treatment of inflammation.
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PMID:Inhibition of neutrophil elastase and thrombin activity by caffeic acid esters. 1632 Sep 52


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