Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We compare results of factor V DNA analysis with three different clotting-based assays designed to detect activated protein C (APC) resistance (APCR), using samples from 958 patients undergoing assessment for thrombophilia. The original and most commonly used APTT-based procedure (generating an APTT ratio in presence versus absence of APC), showed the least correlation with DNA findings, with a large overlap between normals and heterozygotes. Using this procedure, over 40% of patients with a normal DNA pattern gave APTT ratio results within the heterozygotes' ratio range, and thus is a poor predictor for factor V DNA Leiden mutation (sensitivity 94.3%, specificity 47.0% [APC ratio cut-off: 3.1]; sensitivity 52.1%, specificity 92.9% [APC ratio cut-off: 2.0]). Two commercially available procedures (protein C impedance [PCI] test and protein C pathway [PCP] test), using modified Russell's viper venom time (RVVT) assays, showed less overlap between normals and heterozygotes than did the APTT-based method. Fewer than 10% of normal individuals gave PCI or PCP test ratio results that fell within the respective heterozygotes' ratio range (PCI: sensitivity 95.3%, specificity 96.0%; PCP: sensitivity 97.3%, specificity 82.4% [APC ratio cut-off: 1.6 and 1.9 respectively]). Use of previously described normalisation procedures (patient's APTT ratio over pooled normal plasma [PNP] APTT ratio) showed little improvement in discriminatory power (sensitivity 96.4%, specificity 44.8% [normalised APC ratio cut-off value: 0.97]; sensitivity 58.8%, specificity 90.1% [normalised APC ratio cut-off: 0.68]). Use of factor V-deficient plasma as sample diluent improved discrimination for all assays, but added considerable time and cost to the testing process. Furthermore, use of factor V-deficient plasma dilutions in the APTT-based test (sensitivity 97.1%, specificity 93.8% [APC ratio cut-off: 2.0]) did not substantially improve discrimination compared with either PCI or PCP performed without factor V-deficient plasma. Overall, a combination of RVVT- and APTT-based tests was found to provide excellent discrimination, particularly negative prediction, with respect to the likely factor V DNA result. Of 567 patients co-tested, all factor V DNA-normal patients (n = 299) gave both PCP-RVVT and APCR-APTT (not prediluted with factor V-deficient plasma) test ratio values > or = 2.2. In conclusion, it is important to recognise the limitation of plasma-based assays, in particular the APTT procedure, to discriminate the factor V mutation.
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PMID:Functional activated protein C resistance assays: correlation with factor V DNA analysis is better with RVVT-than APTT-based assays. 1049 12

These results, obtained in a small series of patients, suggest that both the ProC Global assay and the PCP Test would be suitable, using well-defined cut-off levels, to identify all the carriers of the Factor V Leiden mutation and all the patients with a protein C deficiency or with combined defects of the protein C pathway. For both assays, however, the sensitivity for protein S deficiency was below 60%. These results in selected patients are congruent with those previously reported in the literature about the ProC Global assay, the PCP Test, and other assays evaluating the functionality of the protein C anticoagulant pathway. All demonstrated a weak sensitivity to protein S deficiency, suggesting that protein S plays only a minor role as an APC cofactor in such global assays. A major discrepancy between the two evaluated assays was obtained in the group of patients without abnormality of the protein C pathway. Actually, using the ProC Global assay, more than 40% of the patients had a decreased PCAT-NR while presenting with none of the three tested abnormalities, whereas none of the studied patients had a ratio below 1.80, and only 5 of 143 (3.5%) had a ratio below 2.00 when using the PCP Test. The observation that around 40% of the control patients had a decreased PCAT-NR could suggest the influence of currently unknown defects of the protein C/protein S pathway on the ProC Global assay. It could also be hypothesized that the higher factor VIII levels already reported in patients with a history of thrombosis than in controls had a significant role in the low responsiveness, but this parameter was not tested in the authors' series. In that connection, it is also well established that elevated factor VIII levels both shorten the APTT and reduce the anticoagulant effect of heparin when evaluated using APTT. Actually, some of the samples investigated in this study were obtained during the acute phase of thrombosis. It is not possible to draw out the hypothesis of an association of biologically undetectable minor changes in various factors involved in the protein C anticoagulant pathway; all the individual factors would remain within their normal ranges. Finally, because 40% of the patients without abnormalities of the protein C pathway had a decreased PCAT-NR, the question arises whether the ProC Global assay might in itself be a biologic marker of thrombophilia, independent of its sensitivity for abnormalities of the protein C anticoagulant pathway. In that connection, the correlation between the result of the ProC Global assay and the risk for thromboembolism was recently evaluated by two different groups. In both cases, the preliminary results suggested that a decreased response to the ProC Global assay might be an independent risk factor for venous thrombosis. The two global assays could therefore have distinctly different applications. If the global assays are used in the hemostasis laboratory to screen for abnormalities of the protein C pathway, and thus to rationalize the use of specific assays, the PCP Test should be chosen, because of its high specificity. Because only 3.5% of the control patients had a ratio below 2.00 (and none had a ratio below 1.80), the PCP Test could be accurately used as a first-step assay in the laboratory screening for these abnormalities of the protein C anticoagulant pathway. Using such a flow chart, the specific assays for APC resistance or the identification of the factor V Leiden mutation and protein C would be performed only in case of a ratio below a cut-off defined using receiver operating characteristic (ROC)-analysis in unselected patients. Because of the weak sensitivity of this assay to both constitutional and acquired protein S deficiencies (below 15% using 1.80 as the cut-off level, or 60% using 2.00), the measurement of this parameter had to be performed in all cases. If, on the other hand, the assay is used to screen for risk factors for thrombosis, the ProC Global assay could b
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PMID:Screening for risk factors for thrombosis using a new generation of assays developed to evaluate the functionality of the protein C anticoagulant pathway. 1080 61

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