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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Increased risk of bleeding during oral anticoagulant (OA) treatment may be related to mutations in the precursor of coagulation factor IX. Missense mutations at Ala-10 (Ala-10Thre and Ala-10Val) in the factor IX propeptide lead to impaired carboxylation of factor IX. When patients carrying these mutations are treated with coumarins, functional factor IX levels decrease significantly, leading to an excessively prolonged activated partial thromboplastin time (aPTT) and an increased bleeding risk. Mutations at Ala-10 have been described in northern-European patients, but it is not known whether geographical differences play a role in the prevalence of these mutations. We aimed to analyze the prevalence of mutations at Ala-10 in factor IX in southern-European patients on OA treatment. Patients attending the Oral Anticoagulant Unit of the Hospital Clinic were prospectively included. The aPTT was determined at their normal International Normalized Ratio (INR) control. When the aPTT was excessively prolonged for the INR value, determination of factor IX and genotyping for Ala-10 mutations was performed. A total of 2360 patients were included (1289 men, 1071 women; mean age, 70.5 +/- 12.1 years). Twenty-four patients (16 men, eight women; mean age, 61.0 +/- 16.2 years) had an aPTT over that expected for the INR. The mean aPTT was 69.6 +/- 16.2 s. Only one patient presented with a factor IX level lower than 10%. None of the 24 patients carried mutations at Ala-10. Mutations at Ala-10 in factor IX were non-existent in the southern-European population analyzed, and thus, do not represent a relevant cause of bleeding during OA treatment.
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PMID:No impact of factor IX Ala-10 mutations in acenocoumarol-treated southern Europeans. 1626 29

A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.
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PMID:Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray. 1657 20

The aim of this study was to determine the predictors of mortality among patients infected with Crimean-Congo haemorrhagic fever (CCHF) virus. Among patients with acute febrile syndrome, characterised by malaise, bleeding, leukopenia and thrombocytopenia, who were admitted to hospital during the spring and summer of 2002-2004, 54 had positive IgM and/or PCR results for CCHF virus in blood or tissue. The overall case fatality rate was 7.4%. Among the fatalities, haematemesis (p 0.009), melaena (p 0.001) and somnolence (p 0.022) were more common, the median platelet count was significantly lower (10,600/mL vs. 20,000/mL; p 0.038), the mean prothrombin time (27 s vs. 16 s; p 0.002) and mean activated partial thromboplastin time (73 s vs. 44 s; p < 0.001) were longer, and the mean alanine transferase (ALT) level (1,125 vs. 331; p < 0.001), the mean aspartate transferase (AST) level (3,118 vs. 913; p 0.004) and the mean fibrinogen level (119 vs. 340; p 0.012) were higher. Serum IgM and IgG against CCHF virus was detected in 25% and 0%, respectively, of fatal cases, compared with 94% and 62%, respectively, of cases with favourable outcomes. Oral ribavirin was prescribed to 22 (41%) patients. Of the four fatal cases, it was the intention to prescribe ribavirin to three patients, but this was not possible because of haematemesis and melaena. Higher levels of AST (>or= 700 U/L) and ALT (>or= 900 U/L) are suggested for use as severity criteria. Oral ribavirin was not effective for patients with haematemesis, and intravenous ribavirin is necessary for treatment of CCHF.
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PMID:Analysis of risk-factors among patients with Crimean-Congo haemorrhagic fever virus infection: severity criteria revisited. 1670 Jul 4

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.
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PMID:Purification and characterization of anticoagulant protein from the tabanus, Tabanus bivittatus. 1675 88

Recent studies have indicated that the loop harboring the S1 specificity site (residues 185-189 in chymotrypsin numbering) of coagulation proteases has several charged residues with important structural and functional roles for the catalytic activity of these proteases. This loop is allosterically linked to the Na(+)-binding site in both factor Xa and thrombin. There are three candidate residues (His-185, Glu-186, and Arg-188) on this loop of factor IXa (fIXa) whose side chains can influence the Na(+) binding and the catalytic function of the protease in the intrinsic Xase complex. In this study, we developed a novel expression/purification vector system, substituted all three residues of factor IX individually with Ala, and expressed the mutant zymogens in mammalian cells. Following activation, all three fIXa mutants exhibited normal activity towards a fIXa-specific chromogenic substrate in the presence of Ca(2+) with no obvious requirement for Na(+) in the reaction. Furthermore, all three mutants interacted with factor VIIIa with near normal affinity and catalyzed the activation of factor X in the intrinsic Xase complex with a normal catalytic efficiency. These results suggest that, unlike thrombin and factor Xa, the charged residues of this loop do not play a functional role in modulating the catalytic function of fIXa in the intrinsic Xase complex.
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PMID:Expression, purification and characterization of factor IX derivatives using a novel vector system. 1682 35

Here we report a familial cluster of 3 cases of coxsackievirus B3 infection: a recent history of illness in a woman's 3-year-old son with a coxsackievirus B3-positive stool culture indicated that he probably infected his mother at home during her last week of pregnancy. Consequently, she delivered an infected neonate who developed severe hepatitis, disseminated intravascular coagulation, and bilateral intracranial hemorrhage. The neonate remained well for the first 2 days of life. On the third day, he developed fever (39 degrees C) and poor peripheral circulation. On the fourth day, he developed petechiae and bruises over his chest wall and extremities, and prolonged bleeding was observed over venipuncture sites. Investigations revealed severe thrombocytopenia (platelets: 41 x 10(9)/L) and a markedly deranged coagulation profile (prothrombin time: 19 seconds [reference: < 10 seconds]; activated partial thromboplastin time: > 120 seconds [reference: 24.2-37.0 seconds], serum D-dimers: 6722 ng/mL [reference: < 500 ng/mL]), suggestive of disseminated intravascular coagulopathy. Clinical examination revealed yellow sclera, hepatomegaly (5 cm), and splenomegaly (2 cm), consistent with hepatitis. Serial chest radiographs showed bilateral pleural effusions, and an ultrasound of the abdomen demonstrated ascites. An echocardiogram showed normal cardiac structure and good contractility of both ventricles. However, a cranial ultrasound revealed bilateral grade 2 intraventricular hemorrhages. Serum C-reactive protein increased to 33.9 mg/L. Liver-function tests were also markedly deranged at this time, with maximum values for serum alanine transferase, bilirubin, alkaline phosphatase, and ammonia concentration of 1354 IU/L, 258 micromol/L, 189 IU/L, and 147 micromol/L, respectively. Serum glucose levels were normal. Over the next 3 days, his fever subsided, and his liver function and clotting profile normalized by day 13 after onset of illness. A stool sample from the older brother, collected 14 days after his onset of illness at home, was positive for coxsackievirus B3 by both virus culture and enterovirus reverse-transcription polymerase chain reaction. He had neutralizing coxsackievirus B3 antibody titers of 1:2560 and 1:1280 on days 14 and 28 after his onset of illness, respectively. No virus was cultured from the mother's stool sample, collected 5 days after her onset of illness, but the enterovirus polymerase chain reaction was positive and maternal sera neutralized the coxsackievirus B3 isolated from the neonate. The maternal sera also showed a more than fourfold rise in antibody titer from 1:80 to 1:640 on days 5 and 16 after her onset of illness, respectively. Neonatal antibody titers also showed a more than fourfold rise from < 1:80 to 1:2560 on days 1 and 21 after his onset of illness, respectively. This demonstrates that both the mother and the neonate had had recent coxsackievirus B3 infections. Serially collected neonatal throat swab and stool samples were culture negative for enterovirus by 4 and 8 days after his onset of illness, respectively. However, enterovirus RNA remained detectable by reverse-transcription polymerase chain reaction in these samples for considerably longer, only becoming undetectable by 16, 23, and 41 days after his onset of illness. We show that even mild household infections may have potentially serious consequences for pregnant women and their infants.
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PMID:Probable intrafamilial transmission of coxsackievirus b3 with vertical transmission, severe early-onset neonatal hepatitis, and prolonged viral RNA shedding. 1690 22

Low-molecular-weight heparins (LMWHs) are very important drugs; unfortunately, the routine global hemostasis assays activated partial thromboplastin time and prothrombin time are not sensitive to LMWHs. Here the 50% inhibitory concentration (IC(50)) values of heparin and LMWHs on extrinsic thrombin generation are determined. Pooled normal plasma was supplemented with 0-2 IU/ml unfractionated heparin, 0-2 IU/ml LMWH dalteparin, or 0-20 microg/ml pentosanpolysulfate in 5-ml polystyrole tubes (23 degrees C) and tested in the tissue-factor-triggered extrinsic coagulation activity assay (EXCA): 50 microl plasma + 5 microl tissue factor/CaCl(2), 1 and 2 min incubation time at 37 degrees C (coagulation reaction time for EXCA-1 and EXCA-2); + 100 microl of 2.5 mol/l arginine (pH 8.6), 20 min at room temperature; + 50 microl of 1 mmol/l CHG-Ala-Arg-pNA, 1.25 mol/l arginine; increase in absorbance/time at 23 degrees C; calibrator = 1 IU/ml bovine thrombin in 6.7% human albumin replacing the plasma sample; in EXCA-1, about 1 IU/ml thrombin is generated in pooled unfrozen normal citrated plasma. The IC(50) values in EXCA-1 are 0.1 IU/ml heparin, 0.02 IU/ml LMWH, and 4.7 microg/ml pentosanpolysulfate. In ECXA-2 the IC(50) values are 0.07 IU/ml, 0.01 IU/ml, and 4.6 microg/ml, respectively. The EXCA reflects the efficiency of anticoagulants on plasmatic coagulation. It is suggested to adjust the dosage of LMWH according to the EXCA value; about 30% of normal extrinsic thrombin generation might be the correct dose for prophylactic anticoagulation.
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PMID:Inhibition of extrinsic hemostasis activation by low-molecular-weight heparin. 1710 49

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).
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PMID:A loop of coagulation factor VIIa influencing macromolecular substrate specificity. 1718 39

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988-7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2-3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17-300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.
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PMID:A novel allosteric pathway of thrombin inhibition: Exosite II mediated potent inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids. 1780 13

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8A were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55 degrees C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.
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PMID:Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa. 1829 31


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