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PubMed2001

A factor VIII minigene comprising the truncated intron I of factor IX highly improves the in vitro production of factor VIII.

The biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.

Animals↗
PubMed2000

Inactivation and clearance of viruses during the manufacture of high purity factor IX.

Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained with other viruses of a similar or larger size, such as HIV, HBV and HCV.Overall, these studies indicate that MonoFIX-VF is a product with an extremely high level of viral safety.

Animals↗
PubMed1986

Partial factor IX protein in a pedigree with hemophilia B due to a partial gene deletion.

A partial gene product was identified in a pedigree with hemophilia B due to a partial deletion of the Factor IX gene (Chen, S.-H.,S. Yoshitake, P.F. Chance, G.L. Bray, A.R. Thompson, C.R. Scott, and K. Kurachi, 1985, J. Clin. Invest., 76:2161-2164). Levels of this mutant protein in plasma of affected family members studied ranged from 24 to 36 ng/ml (0.6-0.9 U/dl or percent of normal) by a solid-phase immunoassay which is sensitive and specific for the calcium-dependent conformation of human Factor IX. No Factor IX antigen could be detected in patients' plasmas by a non-calcium-requiring monoclonal anti-Factor IX antibody (less than 2 ng/ml). The unconcentrated urine from the five affected family members and four obligate heterozygotes the five affected family members and four obligate heterozygotes tested contained calcium-dependent Factor IX antigen levels ranging from 64 to 160 ng/ml (1.6-4.0 U/dl) and from 10 to 68 ng/ml (0.25-1.7 U/dl), respectively. Of nine normal volunteers screened, three had detectable calcium-dependent antigen in unconcentrated first morning-voided urines with 9.6-16.8 ng/ml (0.24-0.42 U/dl), while the remaining six had detectable urinary antigen only after a 10-fold concentration. Abnormal and normal urinary Factor IX antigen species were concentrated, immunoaffinity purified, electrophoresed, immunoblotted, and distinguished by autoradiography after incubation with 125I-polyclonal calcium-requiring anti-Factor IX. After reducing purified or concentrated samples, a single abnormal 36,000-mol-wt band was identified in the urines from the four affected family members and four obligate heterozygotes tested. Electrophoresis of the reduced urinary Factor IX antigen from the one normal subject tested showed a broad 15,000-20,000-mol-wt band. This normal band was smaller than the species in patients' urines, and was seen as a minor component in the samples from the heterozygotes. No abnormal antigen could be detected in urine from the two other female family members tested. Thus, abnormal urinary Factor IX antigen represents a marker for the presence of the hemophilic Factor IX gene in this family.

Antibodies↗
PubMed1982

Factor IX Deventer-evidence for the heterogeneity of hemophilia BM.

Factor IX Deventer was isolated from the plasma of a patient with severe hemophilia B. The patient was classified as BM because of an abnormal prolongation (2.1 times) of the ox-brain prothrombin time, that could be corrected by addition of antifactor IX serum. Experiments with the isolated factor IX Deventer showed that one of the two peptide bonds involved in the proteolytic activation of factor IX cannot be cleaved by physiological or non-physiological activators (XIa and RVV-X, respectively). Such a defect can explain why the molecule has no procoagulant activity. At present it is not clear why this defect makes factor IX Deventer such an effective inhibitor of the ox-brain prothrombin time. It is proposed that hemophilia BM is a heterogeneous disorder.

Factor IX↗
PubMed1985

An intragenic deletion of the factor IX gene in a family with hemophilia B.

A family of seven patients severely afflicted with hemophilia B has been studied for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (less than 10% of normal) factor IX antigen in urine and no detectable inhibitors in sera to factor IX protein. Based on the DNA hybridization analysis, these patients showed a partial intragenic deletion in their factor IX gene. The deletion included two exons (exons V and VI) coding for the amino acid sequence from number 85 to 195 of the factor IX protein. The deleted portion of the gene contained the entire factor IX activation peptide. The length of the deletion was estimated to be 10 +/- 0.3 kilobase pairs. This specific gene has been named FIXSeattle. In this family both the deletion and a Taq 1 restriction fragment length polymorphism can be used as a useful marker for accurate detection of female carriers of the deficient factor IX gene.

Chromosome Deletion↗
PubMed1984

Assay of factor IX antigen using an enzyme immuno assay.

An Enzyme Linked immuno Sorbent Assay (ELISA) was developed for the measurement of factor IX:Ag. The results obtained in 90 control subjects, 56 hemophilia B patients and 40 patients under oral anticoagulant treatment were compared with factor IX:C and factor IX:Ag levels according to Electro Immuno-Assay (EIA). In healthy volunteers the mean factor IX:C level was 106.7 U/dl, the mean factor IX:Ag level was 96.8 U/dl according to EIA procedure and 101.5 U/dl according to ELISA method. Using ELISA, 10 hemophiliacs had no detectable antigen, and 13 had minute amounts of IX:Ag. All these patients are classified as B-. Among the others 21 had reduced antigen and are considered as BR and 12 had normal level of IX:Ag and are B+. Patients taking oral anticoagulants had not only a decreased factor IX:C level but also a reduced factor IX:Ag level which is always lower when using EIA procedure in the presence of EDTA than according to ELISA method.

Antigens↗
PubMed1994

In vitro mutagenesis study of two critical glutamic acids in the calcium binding loop of the factor IX heavy chain.

We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium's absence, factor IXGlu245Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245Lys's conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens↗
PubMed1993

Tyrosine 69 of the first epidermal growth factor-like domain of human factor IX is essential for clotting activity.

Epidermal growth factor (EGF)-like domains are present in many extracellular proteins including clotting factor IX. Those EGF-like domains with the consensus amino acid residues Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where * denotes a beta-hydroxylated residue) bind calcium. Previous NMR studies of the isolated first EGF-like domain of human factor IX suggested that the first 3 consensus residues Asp-47, Asp-49, and Asp-64 were direct ligands for calcium. We now show from mutagenesis studies that Tyr-69 is not a direct ligand for calcium but is essential for the clotting activity of intact factor IX, specifically for the factor VIIIa-dependent activation of factor X. Surprisingly, Tyr-69 is not required for beta-hydroxylation of Asp-64.

Amino Acid Sequence↗
PubMed1989

Effects of plasma collection systems and processing parameters on the quality of factor IX concentrate.

Using pilot-scale production of our present factor IX (II and X) concentrate, we have studied the effects of starting plasma source and processing parameter on two in-vitro indicators of product quality - yield and thrombogenic potential. Plasma source did not affect factor IX yield but had a marked effect on thrombogenic potential. Factor IX concentrates produced from plasma derived through centrifugation-based technology showed significantly higher thrombogenic potential than products derived from plasma derived through a filtration-based system. Removal of Cohn fraction I prior to ion-exchange chromatography resulted in a drop in factor IX yield and thrombogenic potential, as did heat treatment to 80 degrees C for 72 h. We conclude that a membrane-filtration-based plasmapheresis system may be the preferred method of plasma procurement for factor IX concentrate production.

Chemical Fractionation↗
PubMed1976

Study of a proposed international standard for blood coagulation factor IX.

An International Collaborative Study was organized to establish a standard for factor IX. Two freeze-dried concentrate preparations, C1 and C2, and one freeze-dried plasma P were compared with each other, with fresh normal plasmas and with local standards in 13 laboratories. One of the concentrate preparations (C1) contained heparin and this gave rise to non-parallel assays in laboratories testing concentrate C1 in dilutions containing more than 0.05 i.u. of heparin per ml. Assays of factor IX showed good precision for both plasma and concentrate in all laboratories; no systematic effect of method, operator or day of assay was detected. The plasma preparation P and the concentrate preparation C2 were compared with 59 individual fresh normal plasma samples, and a mean potency ratio of 0.78 (95% confidence limits 0.73-0.84) for plasma and 5.62(95% confidence limits 5.13-6.16) for the concentrate C2 obtained. Only 21 estimates of concentrate C1 in terms of fresh plasma were obtained giving a mean potency ratio of 3.85 (95% confidence limits 1.87-7.92). The estimated loss of potency for freeze-dried plasma stored at -20 degrees C is approximately 0.4% per year. The concentrate C2 is apparently more stable and only very small losses occurred even at higher storage temperatures. All participants agreed that the preparation C2 would be suitable to serve as an International Standard for factor IX; they also agreed that the figure assigned for the unitage should be based on the number of ml of 'average fresh normal plasma' estimated to contain the factor IX activity of one ampoule of the preparation. It is proposed to recommend to the World Health Organization that the preparation of factor IX concentrate C2, in ampoules coded 72/32, be considered for establishment as the International Standard for factor IX, and that the international unit for factor IX be assigned on the basis of 5.62 units per ampoule of this preparation.

Blood Preservation↗
PubMed2000

Pharmacokinetics and pharmacodynamics of a humanized monoclonal antibody to factor IX in cynomolgus monkeys.

The pharmacokinetics and pharmacodynamics (PK/PD) of a humanized anti-Factor IX IgG1 monoclonal antibody (SB 249417, FIX mAb) were studied in Cynomolgus monkeys. Single i.v. bolus doses of 1, 3, or 10 mg/kg of FIX mAb were administered. The total FIX mAb concentration, activated partial thromboplastin time (aPTT), and Factor IX activity were monitored for up to 4 weeks after dosing. In the monkey, FIX mAb had a plasma clearance of 0.6 ml/h/kg and a steady-state volume of distribution of approximately 70 ml/kg. The elimination phase half-life (3.8 days) was considerably less than other humanized IgG1 mAbs in the monkey, for which there is no binding to endogenous antigen. The suppression of Factor IX activity and the prolongation of aPTT were rapid and dose dependent. The time for aPTT values to return to basal levels (25-170 h) increased with increasing dose. A mechanism-based PK/PD model consistent with the stoichiometry of binding (2:1) was developed to describe the Factor IX activity and aPTT response time course. The model incorporated Factor IX synthesis and degradation rates that were interrupted by the sequestration of Factor IX by the antibody. aPTT values were related to free Factor IX activity. This model was able to describe the PD profiles from the three dose levels simultaneously. The estimated Factor IX half-life was 11 h and the third-order association rate constant was 3.96 x 10(3) microM(-2) h(-1). The PK/PD modeling was useful in summarizing the major determinants (endogenous and antibody-ligand binding) controlling FIX mAb-related effects.

Animals↗
PubMed1984

Isolation and characterization of human factor IX cDNA: identification of Taq I polymorphism and regional assignment.

Hemophilia B or Christmas disease is an X-linked condition caused by absent or reduced levels of functional coagulation factor IX. Based upon the peptide sequence of bovine factor IX, we synthesized a 17-base pair oligonucleotide probe to screen a human liver cDNA library. A recombinant clone was identified with a 917-nucleotide insert whose sequence corresponds to 70% of the coding region of human factor IX. This factor IX cDNA was used to probe restriction endonuclease digested human DNA to identify a Taq I polymorphism associated with the genomic factor IX gene as well as to verify that there is a single copy of this gene per haploid genome. The factor IX cDNA was also used to map the locus for factor IX to a region from Xq26 to Xqter. The cloning of human factor IX cDNA and identification of a Taq I polymorphism and its regional localization will provide a means to study the molecular genetics of hemophilia B and permit linkage analysis with nearby loci.

Amino Acid Sequence↗
PubMed2003

Application of Fmoc-amino acid carrying an unmasked carbohydrate to the synthesis of the epidermal growth factor-like domain of bovine blood coagulation factor IX.

The epidermal growth factor (EGF)-like domain of the bovine blood coagulation factor IX (45-87) carrying glucose at Ser(53) was synthesized by a solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids. The introduction of Ser(53) was carried out using the benzotriazolyl ester of Fmoc-serine carrying an unmasked glucose. The remaining sequence was also introduced using the benzotriazolyl ester. HPLC analysis of the crude peptide shows that acylation of the free hydroxyl group of the glucose was not significant, demonstrating that the amino acid carrying an unmasked carbohydrate is a useful building block for solid-phase synthesis.

Amino Acid Sequence↗
PubMed1995

High-purity factor IX concentrates for treatment of hemophilia B: relative purity and thrombogenic potential.

Constituents other than factor IX have been implicated as etiologic agents for thrombotic complications in patients receiving prothrombin complex concentrates (PCCs). In vitro studies, in vivo animal models, and clinical evaluations in patients with hemophilia B indicate that high-purity factor IX concentrates contain significantly fewer potentially thrombogenic contaminants than PCCs. A recent in vitro study from our laboratory used highly sensitive assays to analyze the relative purity of these newer products. The following products were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting: Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC (now known as Replinine). The mean specific activity of the high-purity factor IX products ranged from 68 IU factor IX/mg (Aimafix) to 246 IU factor IX/mg (Mononine). SDS-PAGE analysis under reducing and nonreducing conditions showed that Mononine had the fewest contaminating bands. The immunoblot to detect factor IX showed a dominant factor IX band for all products, visible light chain of factor IX for all products except Aimafix, and another contaminating band visible at 49,500 daltons for all products except 9MC. High molecular weight contaminants were apparent for some products. Factor VIIa was detected in some lots of Immunine, Nanotiv and 9MC. Factor X and prothrombin contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv and 9MC. Thus, Mononine, Nanotiv and 9MC demonstrated the highest purity but no product was totally free of contaminants.

Blood Coagulation Factors↗
PubMed1998

Blood coagulation factor IX residues Glu78 and Arg94 provide a link between both epidermal growth factor-like domains that is crucial in the interaction with factor VIII light chain.

Recently, we established that mutations at calcium-binding sites within the first epidermal growth factor (EGF)-like domain of activated factor IX affect its interaction with factor VIIIa (Lenting, P. J., Christophe, O. D., ter Maat, H., Rees, D. J. G., and Mertens, K. (1996) J. Biol. Chem. 271, 25332-25337). In the present study, we have investigated the functional role of residue Glu78, which is not involved in calcium binding. Glu78 is also located in the first EGF-like domain and, when mutated to Lys, is associated with severe hemophilia B. Because Glu78 is conserved in related vitamin K-dependent proteins, it is difficult to understand how a mutation at this position is associated with factor IX-specific function. In this study, we addressed the hypothesis that Glu78 exerts its biological activity by interacting with another residue. One candidate was found to be the second EGF-like domain residue, Arg94, which is also associated with severe hemophilia B when mutated. We constructed a series of mutants that included mutations at position 78 alone (Glu78 to Lys/Glu78 to Asp) or at both positions 78 and 94 (Glu78 to Lys and Arg94 to Asp). The functional parameters of immunopurified and activated mutants were compared with normal activated factor IX. Mutants were indistinguishable from normal factor IXa in cleaving the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide or activating factor X in the absence of factor VIIIa. In contrast, in the presence of factor VIIIa, factor IXa Glu78 to Asp and factor IXa Glu78 to Lys/Arg94 to Asp were stimulated to the same extent as normal factor IXa, whereas factor IXa Glu78 to Lys was markedly less stimulated (140-fold versus 2,000-fold). This suggests that residues 78 and 94 should carry an opposite charge for a normal interaction of factor IXa to factor VIIIa. This hypothesis was confirmed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy (Ser558-Gln565) or light chain (Glu1811-Lys1818) and in direct binding studies. We propose that residues 78 and 94 link both EGF-like domains and thereby maintain the integrity of the factor VIII light chain binding site.

Animals↗
PubMed1994

Haemophilia B Leyden: the effect of mutations at position +13 on the liver-specific transcription of the factor IX gene.

Haemophilia B Leyden is characterized by low plasma levels (< or = 1-13% of normal) of blood coagulation factor IX during childhood. After the onset of puberty, plasma factor IX levels gradually rise, probably under the influence of androgens. Single point mutations have been detected in the factor IX promoter at -21, -20, -6, -5, +6, +8 and +13. This paper examines how two of these mutations (a deletion of an A, and an A-->G substitution at +13) interfere with normal factor IX gene transcription. It is shown that both mutations do impair factor IX promoter activity in transiently transfected HepG2 cells. The mutations at +13 lie in a region (+1 to +18) that is considered to contain a binding site for the CCAAT/enhancer binding protein. Transactivation by the CCAAT/enhancer binding protein alpha of the wild-type and mutated factor IX promoter (-192 to +38) resulted in an approximately four-fold and approximately two-fold, respectively, increase of CAT activity. Gel mobility shift assays revealed that the binding of the CCAAT/enhancer binding protein alpha is disrupted by both the deletion of an A, and the A-->G substitution at +13. The role of two additional bZIP factors, D-site binding protein and liver-enriched transcriptional activator protein, in the binding and activation of the +13 factor IX promoter region was examined. The D-site binding protein binds to the factor IX promoter region (+1 to +18) in gel mobility shift assays. The deletion of an A at +13 does not interfere with the binding of the D-site binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1999

The three in-frame ATG, clustered in the translation initiation sequence of human factor IX gene, are required for an optimal protein production.

Three in-frame potential methionine codons have been identified in human factor IX gene and are clustered at amino acids -46, -41 and -39. In view of initiating a gene therapy approach, human factor IX production has been evaluated after modifications of these first three in-frame translation start sites. To characterize the most efficient translation initiation context, five factor IX cDNA expression vectors directed by CMV promoter-enhancer were generated. These vectors contained different starting site combinations including one, two or three ATG. A quantitative analysis of factor IX production in stably transfected CHO cells and in a rabbit reticulocyte lysate cell free system revealed the ability of all single site to generate fully active factor IX. However, the factor IX production level increased with the ATG number and the wild type (WT) cDNA bearing the 3 ATG induced the highest protein production. A truncated intron I of factor IX, previously suggested of having an expression-augmenting activity, was also placed in the WT factor IX cDNA. In stably transfected CHO cells, a 8-fold increase in protein production was measured. These results show that at least in vitro, the presence of the three ATG seems to be crucial for a maximal factor IX production. The data also suggest that both the three ATG and the truncated intron I are required for an optimal factor IX production in a perspective of a human gene therapy of haemophilia.

Animals↗