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)

Factor VIIIa is a non-covalent heterotrimer of A1, A2, and A3-C1-C2 subunits. Previously, we speculated that the central portion of the A2 subunit, in and around the activated protein C-sensitive bond at Arg562-Gly (Fay, P. J., Smudzin, T.M., and Walker, F.J. (1991) J. Biol. Chem. 266, 20139-20145), is important for macromolecular interactions within the factor Xase enzyme complex. A peptide corresponding to factor VIII residues 558-565, SVDQRGNQ and designated FVIII558-565, was chemically synthesized and inhibited factor Xa generation in a purified system with an apparent KI of 105 microM. Tryptic cleavage of FVIII558-565 eliminated its inhibitory activity, whereas a scrambled sequence version of the peptide possessed < 30% the inhibitory activity of the native version. Overlapping peptides FVIII556-564 and FVIII561-569 were also inhibitory and confirmed the importance of residues in and around the scissile bond for functional factor Xase. Kinetic analysis revealed that peptide-mediated inhibition was non-competitive with respect to factor X. However, increasing factor IXa concentration overcame the observed inhibition. Furthermore, the peptide inhibited the factor IXa-dependent enhancement of factor VIIIa reconstituted from isolated A1/A3-C1-C2 dimer plus A2 subunit. Isolated factor VIII heavy chain (contiguous A1-A2 domains) was cleaved at Arg336 by an equimolar concentration of factor IXa in a reaction that was phospholipid-independent. No proteolysis of the isolated A1 subunit was observed in a similar reaction. These results indicate that the A2 subunit sequence delineated by residues 558-565 contributes to the interaction of cofactor with protease and that this interaction is essential for intrinsic factor Xase activity. Furthermore, that this peptide blocks both factor Xase activity and the capacity of factor IXa to stabilize the labile factor VIIIa heterotrimer suggest that this latter property is of physiologic significance.
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PMID:Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site. 805 Nov 50

Resistance to activated protein C (APCR), in the majority of cases due to the point mutation Arg 506 Gln of the factor V gene, has emerged as the most important hereditary cause of venous thromboembolism. Using an activated thromboplastin time (aPTT) based method in the presence of APC together with a DNA technique based on the polymerase chain reaction, we investigated 37 children with venous (V: n=19) or arterial (A: n=18) thromboembolism and 196 age-matched healthy controls for the presence of this mutation. In the control group 10 children were detected to be heterozygous for the factor V Leiden mutation, indicating a prevalence of 5.1%. 10/19 children (52%) with venous thrombosis and 7/18 (38%) patients with arterial thromboembolism showed the common factor V gene mutation. Additional inherited coagulation disorders were found in 1/10 (V:10%) and 2/7 (A:28%) APC-resistant patients. Inherited coagulation disorders without APCR were diagnosed in 3/9 (V: 33%) and 2/11 (A:18%) children. Furthermore, we diagnosed exogenous risk factors in 6/10 (V: 60%) and 2/7 (A: 28%) children with thrombosis and APCR. These data are evidence that APCR combined with exogenous reasons may play an important role in the early manifestation of thromboembolism during infancy and childhood.
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PMID:Resistance to activated protein C (APCR) in children with venous or arterial thromboembolism. 1036 46

Resistance to activated protein C (APC) is the most common cause of familial thrombophilia. The partial thromboplastin time (PTT)-based test for resistance to APC has been widely employed as a screening test for this disorder. However, the utility of this test for screening is not well characterized. More than 90% of patients with resistance to APC have the G1691A mutation in factor V (factor V Leiden). The authors studied the ability of a commercial APC resistance assay to correctly identify the factor V Leiden genotype in 130 individuals. At the recommended assay cut-off value of 2, the sensitivity of the APC resistance assay was 50%, with a specificity of 98%. Increasing the cut-off value increased the sensitivity but decreased the specificity of the test. Receiver operating characteristic (ROC) curve analysis indicated that the test was of intermediate utility. There was considerable overlap in APC ratios in the range of 2 to 3 between subjects with a normal factor V genotype and heterozygotes for factor V Leiden. The authors conclude that the APC resistance assay in its present form is not a useful screening test for factor V Leiden heterozygotes. Until the performance of this assay is improved, patients should have molecular diagnostic testing performed to determine their factor V Leiden status.
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PMID:Sensitivity and specificity of the APC resistance assay in detection of individuals with factor V Leiden. 916 71

We studied both inherited and acquired activated protein C (APC) resistance in a group of 22 patients with primary antiphospholipid syndrome (APS). The APC resistance genotype was assessed using a PCR-based analysis for the factor V R506Q (Leiden) mutation. One patient with primary APS was found to be heterozygous for the factor V Leiden mutation. He and other family members were affected by severe thrombophilia and had a familial form of primary APS. The APC resistance phenotype was assessed by measuring the prolongation of the activated partial thromboplastin clotting time in response to APC. It was found in five out of six patients with APS, in one of them transiently. We have found that the APC resistance phenotype is more frequent than the genotype in primary APS. It would seem that patients with thrombophilia should be investigated for APC resistance even if found to have antiphospholipid antibodies and/or lupus anticoagulants.
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PMID:Activated protein C resistance phenotype and genotype in patients with primary antiphospholipid syndrome. 873 42

Protein C is a major regulatory protein critical to physiologic anticoagulation. When activated, it selectively degrades the activated forms of factors V and VIII, thereby, down-regulating blood coagulation. Using an activated partial thromboplastin time (APTT) assay, Dahlback et al. recently reported that some individuals with thrombophilia show a poor in vitro anticoagulant response to activated protein C (APC-Resistance). Subsequent studies identified a point mutation in the gene for factor V as the underlying cause of APC-Resistance. The incidence of APC-Resistance in patients with recurrent thromboembolic events approaches 50%. The APC-Resistance phenotype is also present in approximately 5% of normal Caucasian subjects. In an attempt to develop a more sensitive and specific test system, we evaluated an assay based on Textarin(Pentapharm, Basel, Switzerland). Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern Brown Snake) activates prothrombin in the presence of phospholipid (PL), factor V and calcium ions. Based on Textarin's requirement for factor V, we developed a Textarin time assay to test for APC-Resistance. We evaluated this test system in normal subjects and the following patient populations: stable orally anticoagulated, previously diagnosed factor V Leiden, and therapeutically heparinized samples. We found the Textarin assay to be a sensitive and specific test system to identify APC-Resistance. The phenotypic Textarin APC-Resistance test correlated more closely with the genotypic abnormality of factor VR506Q than the APTT-APC-Resistance test.
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PMID:APC-resistance as measured by a Textarin time assay: comparison to the APTT-based method. 887 45

Resistance to the anticoagulant effects of activated protein C (APC) is now considered the most prevalent cause of inherited thrombophilia. The great majority of patients with activated protein C resistance (APCR) have a missense mutation in the factor V molecule (factor V Leiden, FVR506Q) resulting in defective inactivation of factor Va due to a loss of an APC cleavage site. The diagnosis of APCR has been based upon the inability of APC to prolong the activated partial thromboplastin (aPTT) clotting time in subjects with APCR. However, this assay has a number of deficiencies which limit its general use. We have evaluated a newly described one-stage tissue factor dependent factor V coagulation assay for APCR in 117 patients and controls and compared the results of this assay in a blinded manner to a polymerase chain reaction (PCR) based assay for the molecular defect of factor V Leiden. 43% (50/117) of the patients studied were receiving coumadin or heparin, or had a lupus anticoagulant. The tissue factor dependent factor V assay had 100% specificity and sensitivity for factor V Leiden and successfully predicted a homozygous state in the three documented homozygotes. The PCR-based assay for factor V Leiden resulted in a single false positive assay due to a silent A to C transition at nucleotide 1692 resulting in the loss of the Mnl restriction endonuclease cleavage site. The single-stage tissue factor dependent factor V assay is a highly sensitive and generally applicable assay for APCR.
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PMID:Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance. 894

The aim of the study is to determine if a novel thrombophilia mechanism (factor V Leiden) that is associated with resistance to activated protein C (APC) is in itself a risk factor for the development of ischemic stroke (IS). Sixty-six controls and 66 patients with IS were included in an unmatched case-control study. In the group of patients selected for this study, other causes of IS were ruled out. APC resistance was considered if activated partial thromboplastin time (aPTT) measured in the presence of APC was less than 2.2 times prolonged when compared to aPTT in the absence of APC (APC ratio < 2.2). Digestion with a restriction enzyme of a previously amplified exon 10 of the gene that encodes for factor V was used to detect the presence of the factor V mutation. We identified 5 patients (prevalence: 7.5%) with APC resistance (mean age: 31 years, range: 6-52 years). Mutation in factor V gene was confirmed in three of them. In the control group we detected 3 (4.5%) low APC ratios, all of them carrying specific factor V mutation. We cannot conclude a significant association between APC resistance and IS [odds ratio: 1.72; chi 2 Mantel and Haenszel was 0.53 (p = 0.4673) and exact Fisher's test p = 0.3589] but these 5 young adults suffered an episode of IS having APC resistance as the only prothrombotic condition. In conclusion, these results cannot prove a statistical association between APC resistance and IS. Further studies must be done in order to confirm that there is no relationship between APC resistance and IS in young adults when major risk factors are excluded.
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PMID:Low prevalence of the factor V Leiden among patients with ischemic stroke. 909 81

Blood samples from 104 patients with clinically suspected thrombophilia were analyzed for coagulation factor V Leiden mutation (1691, G-->A) by allele-specific polymerase chain reaction. In 86 individuals (82.7%), the mutation was not detectable, whereas 15 patients (14.4%) were heterozygous and three patients (2.9%) were homozygous for factor V Leiden mutation. Plasma samples from these individuals were also tested for functional resistance of coagulation factor V to activated protein C (activated protein C resistance). This test was performed on a Schnitger-Gross coagulometer using an activated partial thromboplastin time-based activated protein C resistance test modified by applying a 1 : 5 dilution with factor V-deficiency plasma. All the individuals negative for factor V Leiden mutation were also negative in the functional activated protein C resistance test. On the other hand, all patients carrying the mutation revealed pathologic results in the activated protein C resistance test. The cutoff value for the activated protein C resistance index (> or = 1.7 = negative) was determined by testing 31 male and female blood donors. One of them was heterozygous for factor V Leiden mutation and had an activated protein C resistance index of 1.4, whereas those without factor V Leiden mutation had an activated protein C resistance index of 1.9 +/- 0.1 (mean +/- SD). Patients with clinically suspected thrombophilia without factor V Leiden mutation had an activated protein C resistance index of 2.1 +/- 0.2 (mean +/- SD), whereas patients heterozygous for the mutation had an index of 1.5 +/- 0.1 (mean +/- SD). Within the group of patients carrying the mutation, the activated protein C resistance test even distinguished between heterozygous and three homozygous (activated protein C resistance 1.0 to 1.2) carriers. The data demonstrate that the activated protein C resistance test in the presence of factor V-deficiency plasma provides a clear-cut discrimination between normal wildtype and carriers of factor V Leiden mutation with a sensitivity and specificity of 100%. Verification of positive activated protein C resistance tests can be performed easily with a simple and reliable polymerase chain reaction protocol for the 1691, G-->A mutation.
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PMID:Discrimination between normal wildtype and carriers of coagulation factor V Leiden mutation by the activated protein C resistance test in the presence of factor V deficient plasma. 915 66

The most common commercially available test measuring activated protein C (APC) resistance relies on the the anticoagulant response to added APC in an activated partial thromboplastin time (APTT) based method. Another method is a Russell Viper venom time (RVVT) based system. To improve the specificity for factor V Leiden of the APTT based method, pre-dilution of test plasma in FV-deficient plasma has recently been recommended. In this study we tested the relative suitabilities of the APTT-based system, the RVVT-based system and their corresponding assays modified by pre-dilution in FV-deficient plasma, for screening asymptomatic subjects, a group of thrombophilic patients (in particular those with low APC ratios), patients on oral anticoagulants, and patients with lupus anticoagulant (LAC). We found the RVVT-based assay to be superior to the APTT-based method in the separation of normals from those with FV Leiden mutation both in asymptomatic subjects and in the thrombophilic patient group. Both modified assays demonstrated a sensitivity and specificity of 100% for FV Leiden, as verified by genotyping in asymptomatic subjects, thrombophilic patients and patients on oral anticoagulants, with the modified RVVT-based assay giving better separation between normals and FV Leiden. Inhibition of phospholipid-dependent coagulation by LAC antibodies rendered the APTT-based system less suitable than the phospholipid-rich RVVT-based one, and as nine of the 20 LAC-positive patients were on warfarin, we showed only the modified RVVT assay to be a reliable predictor of factor V Leiden in this patient group.
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PMID:A comparison between two activated protein C resistance methods as routine diagnostic tests for factor V Leiden mutation. 921 78

Resistance to activated protein C is a recently detected phenomenon that has gained a rapid acceptance as a major risk factor for venous thromboembolism. The phenotypic expression of resistance to activated protein C is characterized by a poor response to the anticoagulant activity of activated protein C, a key enzyme in the down-regulation of blood coagulation, which causes a disposition for a hypercoagulable state. At least 90% of the cases with resistance to activated protein C are explained by a point mutation in the gene for coagulation factor V, resulting in replacement of an Arg to Gln at position 506 (factor V:Q506, often denoted factor V Leiden), one of the three activated protein C cleavage sites in activated factor V. The mutation is inherited as an autosomally dominant trait and has a prevalence of 2% to more than 10% in the general Caucasian population. A number of clinical studies, using different inclusion criteria, show a prevalence of activated protein C resistance of 20-60% among patients with venous thromboembolism. The actual thrombotic risk is moderate with an odds ratio of 5-7 but its high prevalence makes it by far the most important inherited risk factor known today, even higher than the sum of contributions from inherited deficiencies of antithrombin, protein C and protein S. Recent data suggest that activated protein C resistance, which is not due to factor V:Q506 and which appears to be acquired, is also a risk factor for venous thrombosis and for cerebral ischaemic disease. A decreased response to activated protein C is common during pregnancy and during use of oral contraceptives, but the clinical relevance of these findings have yet to be determined. The activated protein C resistance phenotype is typically diagnosed with an activated partial thromboplastin time-based assay, which detects factor V:Q506-dependent as well as acquired activated protein C resistance. However, the sensitivity and specificity for the factor V mutation are usually below 90%. Coagulation instruments with a turbidimetric or photometric clot detection principle generally provide a better performance as compared to electromechanical instruments. The activated partial thromboplastin time test requires careful control of preanalytical variables and platelet contamination should be below 1% since otherwise a falsely low activated protein C response will be obtained. A sensitivity and specificity of close to 100% for factor V:Q506 is obtained in a modified activated partial thromboplastin time test using predilution of sample plasma with factor V deficient plasma. The influence of preanalytical variables in this assay is minor. A number of polymerase chain reaction-based methods, some of them allele-specific, have been published, which provide convenient and objective confirmation of the factor V mutation. Thrombotic events are often triggered through the presence of a combination of inherited and circumstantial risk factors. The high prevalence of activated protein C resistance raises the issue whether it would be cost-beneficial to screen for this trait in connection with surgery, pregnancy and oral contraceptives. Some data already support this, but prospective studies will be necessary to delineate under which circumstances this might be implicated.
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PMID:Activated protein C resistance--a major risk factor for thrombosis. 926 26


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