EC:3.4.21.69 - FACTA Search

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)

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.
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PMID:Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin. 1083 87

W215 is a highly conserved residue that shapes the S3 and S4 specificity sites of thrombin and participates in an edge-to-face interaction with residue F8 of the fibrinogen Aalpha chain. Protein C and the platelet receptor PAR-1 carry an acidic residue at P3 and bind to the active site of thrombin without making contact with W215. This suggested that mutation of W215 could dissociate the cleavage of fibrinogen from that of protein C and PAR-1. Replacement of W215 with Phe produces modest effects on thrombin function, whereas the W215Y replacement compromises significantly the catalytic activity toward all chromogenic and natural substrates that are tested. Replacement of W215 with Ala almost obliterates Na(+) binding, reduces the level of fibrinogen cleavage 500-fold, but decreases the levels of protein C activation and PAR-1 cleavage only 3- and 25-fold, respectively. The W215A mutant cleaves PAR-1 with a specificity constant that is more than 13-fold higher than that of fibrinogen and protein C and is the first thrombin derivative to be described that functions as an almost exclusive activator of PAR-1. The environment of W215 influences differentially three physiologically important interactions of thrombin, which should assist in the study of each of these functions separately in vivo.
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PMID:Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C. 1089 Oct 92

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.
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PMID:Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. 1109 6

W215 is a highly conserved residue that shapes the S3 and S4 specificity sites of thrombin. Replacement of W215 with Phe produces modest effects on thrombin function, whereas the W215Y replacement significantly compromises the amidolytic activity toward synthetic and natural substrates. Replacement of W215 with Ala reduces fibrinogen and PAR4 cleavage 500-fold and 280-fold, respectively. On the other hand, the mutant decreases protein C activation and PAR1 cleavage only threefold and 25-fold, respectively. The W215A mutant cleaves PAR1 with a specificity constant more than 13-fold greater than that of fibrinogen and protein C, and 800-fold greater than PAR4. This is the first thrombin derivative to be described that functions as an almost exclusive activator of PAR1. The environment of W215 influences differentially three physiologically important interactions of thrombin, a feature that should assist in the separate study of each of these functions in vivo.
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PMID:Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR1 or protein C. 1146 May 1

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.
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PMID:Temperature-dependent binding of monoclonal antibodies to C hordein. 1182 5

The aim of the present study was to investigate whether the overall oxidation state of plasma proteins is associated with changes of circulating pro- and anticoagulant markers in healthy subjects (n = 99, 49 males, 50 females, aged from 6 to 91 yrs.). The carbonyl content of plasma proteins was measured and validated as an ex vivo index of the overall protein oxidation state due to its correlation with the plasma level of o-tyrosine (r = 0.87, P <0.0001), which is a well known oxidized product of L-phenylalanine. Using a multivariate analysis the carbonyl content of plasma protein was positively associated with procoagulant markers such as prothrombin F1 + 2 (r = 0.28, P = 0.0019) and fibrinopeptide A, (FpA) (r = 0.278, P = 0.003), as well as with the soluble derivative of the endothelial protein thrombomodulin (TM) (r = 0.469, P <0.0001). The procoagulant marker of thrombin activity, FpA, was significantly and positively correlated with the anticoagulant product of thrombin, namely the Protein C activation peptide (PCP), only in the tertile with low protein carbonyl content. At higher tertiles this correlation was no longer observed, thus suggesting a detrimental effect of oxidative stress on the TM/Protein C anticoagulant pathway. In 15 subjects with high carbonyl content of plasma protein, treatment for 18 days with 600 mg/d of vitamin E did not substantially modify the protein carbonyl content, the anticoagulant markers APC/PCP, and all procoagulant markers except F1+2, whose value significantly decreased by 25%. In conclusion, the present study shows that a high plasma protein oxidation ex vivo is associated with an overall hemostatic imbalance, which favors procoagulant markers. Vitamin E treatment in vivo restores only in part the equilibrium between pro- and anticoagulant pathways. This may open the way to further studies aimed at elucidating the mechanisms by which the oxidative stress is linked to activation of the coagulation system in atherothrombotic disorders.
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PMID:Plasma protein oxidation is associated with an increase of procoagulant markers causing an imbalance between pro- and anticoagulant pathways in healthy subjects. 1184 57

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.
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PMID:Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics. 1213 94

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.
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PMID:Expression and function of the endothelial protein C receptor in human neutrophils. 1271 92

The Escherichia coli GABA (gamma-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [(3)H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [(3)H]nipecotic acid and [(14)C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in k (cat)/ K (m)). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [(3)H]nipecotic acid relative to [(14)C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity ( k (cat)/ K (m)) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842-5847] that have a functionally significant CAR-like domain.
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PMID:Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP. 1295 23

We have recently identified in two unrelated patients with bleeding tendency a homozygous mutation causing a deletion of one of the two contiguous Lys(9)/Lys(10) residues in the A-chain of alpha-thrombin (DeltaK9). We used in vitro expression analysis to clarify the role of the deletion of Lys(9) or Lys(10) in the thrombin function. The k(cat)/K(m) value of the hydrolysis by DeltaK9 of the synthetic substrate Phe-Pip-Arg-p-nitroanilide (where Pip represents l-pipecolyl) and fibrinopeptide A was 18- and 60-fold lower, respectively, compared with wild type (WT). Interaction with antithrombin was also reduced in the mutant, the association rate being about 20-fold lower than in the WT thrombin. The sensitivity to sodium ion of DeltaK9 was found significantly attenuated compared with the WT form. DeltaK9 has a very weak platelet-activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ibalpha was unaffected. Likewise, the interaction with protein C was severely impaired, whereas interaction with thrombomodulin had a normal K(d) value. At variance with these findings, both low affinity (basic pancreatic trypsin inhibitor) and high affinity (N-alpha-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) thrombin inhibitors displayed a better binding to DeltaK9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of DeltaK9. A molecular dynamics simulation of the DeltaK9 thrombin in full explicit water solvent provided support to the role of the A-chain in affecting conformation and catalytic properties of the B-chain, especially in some insertion loops of the enzyme, such as the 60-loop, as well as in the geometry of the catalytic triad residues.
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PMID:A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alpha-thrombin. 1472 67

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