Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0684275 (haemophilia)
 10,958 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.
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PMID:Expression of blood clotting factor VIII:C gene in capillary endothelial cells. 162 40

In five patients with hemophilia B and detectable Factor IX antigen, altered reactivity to a specific polyclonal antibody fraction or monoclonal anti-Factor IX antibodies was noted. Amplification of selected portions of their Factor IX genes by polymerase chain reaction allowed rapid identification of a single base transition in each of the five families tested. In a patient with severe hemophilia and an altered calcium binding domain, a G to A transition in exon II changed the codon for Glu-27 to Lys (Factor IXSeattle 3). Patients from two families with mild hemophilia with decreased reactivity to a MAb that binds to a site within the sequence coded for by exon IV had a G to A transition changing the codon for Gly-60 to Ser (Factor IXDurham). Two unrelated patients with moderately severe hemophilia lacked reactivity to another murine monoclonal anti-Factor IX which binds to an epitope in the amino-terminal half of the heavy chain of Factor IXa. In these patients, exon VIII contained a G to A transition changing Arg-248 to Gln (Factor IXSeattle 4).
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PMID:Three point mutations in the factor IX genes of five hemophilia B patients. Identification strategy using localization by altered epitopes in their hemophilic proteins. 247 24

Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.
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PMID:Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo. 271 93

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immuno-depletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain. The immunodepleted plasma contained less than 0.002 u/ml factor VIII coagulation activity (VIII:C) less than 0.0001 u/ml von Willebrand factor antigen and 1-2 g/l fibrinogen, while the levels of other clotting factors were unchanged. This immunodepleted plasma was compared with commercial factor VIII deficient plasma obtained from a severe haemophilia A patient as substrate in the one-stage factor VIII assay. Plasmas obtained from 20 normal subjects and 28 patients with von Willebrand's disease or haemophilia A were assayed for VIII:C using the two substrates. The results were very highly correlated (r = 0.96). The columns have high capacity and can be regenerated at least 10 times. Large-scale production of a substrate for factor VIII assays free of virus contamination is now feasible.
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PMID:Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies. 311 89

An immunoassay has been developed for the measurement of factor VIII heavy chain (FVIII-HC). IgG from a human inhibitor plasma with specificity for FVIII-HC and FVIII-light chain (FVIII-LC) was coated to microplates with loose wells. After washing, the plates were incubated with sample and after another wash 125I-FVIII-HC was added so that the amount of bound tracer was dependent on the amount of FVIII-HC in sample. When EDTA was included in the assay buffer the assay response was increased 3-fold for normal human plasma. This indicated that the antibody was reactive with a determinant hidden in the FVIII-HC/FVIII-LC complex as EDTA splits the complex. The sensitivity of the assay was 0.004 units/ml (1 unit/ml in normal human plasma pool). Together with a previously published assay for FVIII-LC it has now become possible to measure the relative amounts of FVIII-HC and FVIII-LC in haemophilia A plasma and to study the separate expression of FVIII-HC and FVIII-LC by recombinant DNA technology. Patients with severe haemophilia A had FVIII-HC levels below 0.01 units/ml. However, inhibitor patients in high dose FVIII-treatment showed up to 50 times higher levels of FVIII-HC than FVIII-LC and FVIII:C, indicating the presence of FVIII/anti-FVIII-LC immune complexes. Thus, dependent on assay specificity plasma samples can show very variable content of FVIII:antigen.
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PMID:Radioimmunoassay for quantitative measurement of factor VIII-heavy chain. 312 21

DNA sequence analysis of the gene coding for the variant protein, factor IXLong Beach (FIXLB), has identified a transition mutation in an otherwise normal factor IX (FIX) gene. Genomic DNA clones spanning 35 kilobase (kb) pairs of the FIXLB gene were isolated. A gene analysis strategy that specifically characterized exons and their flanking intron sequences predicted the entire amino acid sequence of FIXLB. A thymine to cytosine transition causes the substitution of a threonine codon (ACA) for an isoleucine codon (ATA) in exon VIII of the FIXLB gene. This mutation results in an amino acid substitution at residue 397 of the FIX zymogen and the phenotypic display of hemophilia-B. Previous studies revealed that activated purified FIXLB (FIXaLB) had normal Ca2+, phospholipid, and factor VIIIa binding characteristics. However, FIXaLB activated factor X or factor VII (with their cofactors Ca2+ and phospholipid) at significantly reduced rates, suggesting that the defect in FIXaLB lies near or within the catalytic triad of the FIX heavy chain. Identification of an amino acid substitution near the carboxy-terminus of the FIXaLB heavy chain supports the earlier characterization of this variant protein. Moreover, our data identify a residue in the catalytic domain of FIXa essential for normal function.
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PMID:Genetic defect responsible for the dysfunctional protein: factor IXLong Beach. 340 2

Two-site immunoradiometric assays (IRMAs) for factor IX antigen (IX:Ag) were developed using a monoclonal antibody (RFF-IX/1) on the solid-phase and either another monoclonal antibody (RFF-IX/4) or a human polyclonal inhibitor antiserum as tracer (M-M and M-I IRMA respectively). The lower sensitivity limits of these two assays for IX:Ag in normal reference plasma were 4 X 10(-4) (M-M IRMA) and 2 X 10(-4) (M-I IRMA) units/ml. In 20 samples of normal plasma, levels of factor IX coagulation activity (IX:C) and of factor IX antigen measured by both IRMAs were highly correlated. Mean values of approximately 1.0 units/ml were obtained in all three assays. In normal serum, IX:Ag levels were lower with means of 0.84 (M-M IRMA) and 0.83 (M-I IRMA) units/ml. 4/25 patients with haemophilia B were CRM neg., two were CRM + and the remaining 19 patients were CRMr variants. In two of these, IX:Ag was detectable by M-I IRMA whilst IX:C and IX:Ag measured by M-M IRMA were undetectable. In plasma from a fetus subsequently terminated on eugenic grounds, IX:C and IX:Ag by both M-M and M-I IRMA were undetectable. In warfarin-treated plasma (n = 12), the level of IX:C was low (mean 0.39 units/ml). The levels of IX:Ag measured by M-M IRMA (mean of 0.80 units/ml) and by M-I IRMA (0.70 units/ml) showed a discrepancy. M-M IRMA reflects the real amount of IX:Ag in warfarinized plasma because both monoclonal antibodies bind to epitopes distant from the light chain carboxylated region. Western blotting of denatured factor IX demonstrated that RFF-IX/1 binds an epitope that is lost after XIa activation. RFF-IX/4 binds the heavy chain. Antigen measured after activation but without denaturing showed loss of 60% reactivity after XIa activation but no change after RVV activation. These data indicate a binding site for RFF-IX/1 within the activation peptide (residues 146-180).
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PMID:Studies on immunological assay of vitamin-K dependent factors. III. A double monoclonal immunoradiometric assay for factor IX antigen. 348 43

The genetics of haemophilia B and the structure-function relationships of factor IX interactions with cofactors and substrates have been reviewed. Emphasis has been placed on contributions to our understanding made by analysis of variants. Amino acid substitutions at or near the site of activation lead to inactive factor IX or to factor IX species with decreased clotting activity. Release of the activation peptide is necessary for optimal interaction of factor IX with its cofactors and substrates. Abnormalities in the calcium binding region, whether Gla independent or dependent, also decrease clotting activity. The defects in haemophilia Bm variants somehow affect factor VII-tissue factor interactions with factor X. Other mutations may affect the factor IX heavy chain, probably at or near the active site. Amino acid substitutions may cause conformational changes in factor IX that interfere with other interactions such as with antithrombin III and factor VIII. Recombinant DNA techniques have been employed to analyse normal and abnormal factor IX genes. DNA sequence analysis of factor IX cDNA clones revealed the primary structure of the mature protein and a predicted leader peptide. Knowledge of the primary sequence of factor IX allowed identification of the specific defect in the factor IX Chapel Hill variant. Analysis of normal factor IX genomic clones has determined that the 35 kb gene is composed of eight coding exons and seven intervening sequences. Sequence analysis of the CRM+ variants will identify mutations disrupting the normal interactions of factor IX. Southern analysis of CRM- variants has revealed gross factor IX gene deletions in some cases. Such deletions have been employed for carrier deletion in some families. Restriction fragment length polymorphisms in the factor IX gene have also proven useful for carrier identification. Manipulations of the cloned factor IX gene to make specific mutations in vitro and improvements in the technology for expression of deliberately modified genes will further elucidate the relationships between factor IX structure and function.
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PMID:Structure and function of factor IX: defects in haemophilia B. 389 39

A murine hybridoma cell line that produces a monoclonal IgG1 antibody to human factor IX was established to provide a conformational probe for the clotting factor and its genetic variants. The antibody inhibited factor IX procoagulant activity, but did not appreciably interfere with the cleavage of factor IX by factor XIa nor with the binding of antithrombin-III-heparin complex to factor IXa. The antigen-solid-phase-antibody complex could be readily dissociated by relatively low concentrations of guanidine or sodium dodecyl sulfate, but only partially by high concentrations of urea. After gel electrophoresis and blotting of reduced samples of factor IXa, the antibody bound exclusively to the heavy chain. Sensitive immunoradiometric assays were developed using insolubilized monoclonal or polyclonal antibodies. Bovine factor IX had little cross-reactivity with the monoclonal antibody. Of 55 patient samples representing different pedigrees with hemophilia-B, antigen levels by the two assays were in excellent agreement in 49. There were 2 severely affected patients whose levels were too low to quantitate in the monoclonal antibody assay. A third, who had the lowest level of all by polyclonal antibody testing, and 3 less severely affected patients had no detectable antigen in the monoclonal antibody assay system (less than 0.03 U/dl). The latter 3 had at least 100-500 times as much antigen by polyclonal antibody testing. It is proposed that these 3 individuals have structural defects involving the epitope recognized by the monoclonal antibody and that they are due to amino acid substitutions between residues 188 through 359. Furthermore, it is suggested the substitutions lead to abnormal kinetic properties.
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PMID:Monoclonal antibody to an epitope on the heavy chain of factor IX missing in three hemophilia-B patients. 619 35

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.
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PMID:Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa. 749 34


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