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PubMedDate not supplied

Chromatographic removal of hepatitis B virus from a factor IX concentrate. Experimental studies in chimpanzees.

Non-A, non-B hepatitis virus can be removed from a factor IX concentrate by a hydrophobic chromatographic step added to the ordinary fractionation process. The efficacy of this procedure for removal of hepatitis B virus (HBV) was evaluated in chimpanzees. A well-defined hepatitis B virus (HBV) inoculum was added to a factor IX preparation and this preparation was subjected to chromatography with octanohydrazide-Sepharose 4B at a high salt concentration and then injected intravenously into two chimpanzees. A control chimpanzee was inoculated with the part of the factor IX/HBV preparation that had not been chromatographed. The two chimpanzees that received the treated material remained free of any serologic or biochemical evidence of hepatitis B infection during a 12-month follow-up, whereas the control chimpanzee had hepatitis B. After a later HBV challenge, the two healthy animals also had hepatitis B. The hydrophobic binding procedure seems to be useful for the adsorption of viral agents in blood components.

Animals↗
PubMed2005

Crystallization of an anti-factor IX antibody and its complex.

Initiation, propagation and regulatory processes of blood coagulation occur at the cell surface. During blood coagulation, many coagulation factors are anchored onto cell-surface membranes through their N-terminal carboxyglutamic acid-rich (Gla) domains found on such vitamin K-dependent blood coagulation factors as factors VII, IX, X, prothrombin, factors C and S. 10C12 is a conformation-specific calcium-dependent anti-factor IX antibody, which is directed at the calcium-stabilized Gla domain and interferes with factor IX-membrane interaction. In a variety of animal models, 10C12 has been demonstrated to be effective anticoagulant in attenuating thrombosis without severe bleeding. The crystallization of the Fab fragment of 10C12 and its complex with the Gla domain of human factor IX was not trivial. Here, the crystallization conditions and unusual aspects of this crystallization process are reported.

Animals↗
PubMed1990

Laser nephelometer evaluation of factor IX.

Laser Nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetic of the reaction between antigen and antibody. In a few instances the method was also applied in the characterization of abnormal molecules. We developed assays for the measurement of Factor IX antigen and the results were compared with those obtained by conventional immunological methods such as rocket immunoelectrophoresis. Plasmas from patients with haemophilia B, on coumarin treatment, with liver cirrhosis were studied. A standard reference curve was obtained using pooled normal plasma. The factor IX levels obtained by laser nephelometer correlated fairly well with those obtained by electroimmunoassay.

Antigen-Antibody Complex↗
PubMed1989

A semi-continuous method for purification of factor IX complex from human plasma.

We report the development of a semi-continuous method for preparation of factor IX complex from human plasma using ion exchange resins. Traditionally, stirred batch adsorption has been used due to the high pressure drops and low flow rates associated with soft gels in packed columns. Batch methods, however, typically involve higher labor costs and are more cumbersome in process environments. In the semi-continuous process, cryo-supernatant plasma is pumped through a 'stirred column' containing the resin. At both lab and pilot scale, higher recoveries of factor IX (FIX) were obtained at decreased total process times, compared to batch adsorption. A residence time of 15 min was found to be sufficient for capture of 95% of the FIX in the starting plasma. In the pilot plant, 550 liters of plasma was passed through a 50-liter column containing 8.5 liters of resin, yielding a 68% recovery of FIX. The results suggest that the recovery of FIX depends on the mode of contact (batch, continuous or packed column) between plasma and resin.

Blood Coagulation Factors↗
PubMed1989

Controlled prospective study of factor IX concentrate therapy and immunodeficiency.

In 1983, six patients who were exclusively treated with factor IX (FIX) concentrate (greater than 40,000 units/yr) were prospectively matched for age and dosage to six patients treated exclusively with factor VIII (FVIII) concentrate and to six normal male controls. At baseline evaluation between October 1983 and May 1984, both groups had significantly decreased absolute T helper cell counts (mean of 452/microliters and 505/microliters for FIX- and FVIII-treated groups, respectively) compared to normal (1,157/microliters). By August 1988, three of the six FIX-treated group have developed AIDS and two are seropositive for antibody to the human immunodeficiency virus (HIV). Four of the six FVIII-treated group have HIV seropositivity or disease. The other three patients (1/6 for FIX and 2/6 for FVIII) declined HIV antibody testing. Our results support other studies showing a dose-related risk of HIV exposure for FIX concentrate-treated patients and do not support the view that FIX concentrate was intrinsically safer than FVIII concentrate.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Comparison of factor IX methylation on human active and inactive X chromosomes: implications for X inactivation and transcription of tissue-specific genes.

Maintenance of dosage compensation for housekeeping genes on the human X chromosome is mediated through differential methylation of clustered CpG nucleotides associated with these genes. To determine if methylation has a role in maintaining inactivity of X-linked genes which show tissue-specific expression, we examined the locus for blood clotting Factor IX. The analysis encompassed 91% of the HpaII and HhaI sites in the 41-kb region that includes the presumed promoter region, 5 kb of 5'- and 4 kb of 3'-flanking sequences. Although there are sex differences in methylation of the locus in leukocytes, the methylation pattern in liver, where the gene is expressed, is essentially the same for loci on the active and inactive X chromosome. The lack of differences in methylation of active and inactive genes makes it unlikely that methylation within the locus has a role in expression of the Factor IX gene. These findings, along with the absence of clustered CpG dinucleotides within the Factor IX locus, suggest that functional differences in DNA methylation related to X chromosome dosage compensation may be limited to CpG clusters. In any event, dosage compensation seems to be maintained regionally, rather than locus by locus.

Factor IX↗
PubMed1994

A comparison of the allelic frequencies of ten DNA polymorphisms associated with factor VIII and factor IX genes in Thai and Western European populations.

The frequency of five factor VIII gene intragenic and linked DNA polymorphisms and five factor IX gene intragenic polymorphisms was studied in Thai females. The polymorphisms in the FVIII gene were detected by restriction enzymes BclI, XbaI, BglI and at linked loci DX13 (DXS15) and St14 (DXS52) by BglII and TaqI, respectively, and in the FIX gene by MseI, DdeI, XmnI, TaqI and HhaI. With the exception of the BglI restriction fragment length polymorphism (RFLP), which is absent in Thais, factor VIII polymorphism frequencies were similar in Thais and Caucasians. Combined use of XbaI and TaqI/St14 resulted in a heterozygosity rate of greater than 90% in Thai females. For FIX, the recently described MseI RFLP in the 5' flanking region was the most informative polymorphism in Thais, 43% of females being heterozygous. The other four polymorphisms added little to the overall heterozygosity rate. The appropriate polymorphisms were used to track defective factor VIII and IX genes through 22 Thai pedigrees with haemophilia to enable carrier status to be assigned to female family members. The information obtained during this study will form the basis for carrier detection and prenatal diagnosis of haemophilia A and B by DNA polymorphism analysis in Thailand.

Alleles↗
PubMed1989

Mapping of monoclonal antibodies to human factor IX.

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF-like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.

Amino Acid Sequence↗
PubMed1991

Direct carrier detection in hemophilia B kindreds: use of modified primers (mutagenic primers) for enzymatic amplification of the factor IX gene.

Rapid direct detection of point mutations in hemophilia B kindreds was performed by analyzing the restriction fragments of the factor IX gene amplified by polymerase chain reaction (PCR). The family members of the two patients, HB 5 and HB 6, whose mutant factor IX gene had previously been defined by sequence analysis, were examined in this study. Since there are no restriction endonucleases available for detecting each mutation directly, we designed modified primers which were substituted one nucleotide near the mutated positions. Following PCR with these primers, new Mbo I or Alu I cleavage sites for normal alleles were created. In each family, the restriction fragment revealed the carriership of each patient's mother. This simple methodology to detect mutations of interest is useful for genetic counseling in sporadic cases of hemophilia B.

Alleles↗
PubMed1995

No detectable alterations in immunogenicity following terminal severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX (HP9) concentrates.

We used monoclonal antibody ELISAs, antigen molecular size distribution, competition ELISA and neonatal mouse immune tolerance methods to detect potential neoantigen formation and increased immunogenicity following severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX concentrates. To provide positive controls, concentrates were heated in solution (70 degrees C for 2 h) to produce denaturation on purpose. The competition ELISA applied to factor IX proved particularly useful for quantifying differences between the positive control and the dry-heated/unheated concentrates. None of the test systems employed by us indicated any detectable neoantigen formation or any alteration in immunogenicity following terminal severe dry-heat treatment of the high-purity concentrates, and this finding is supported by clinical experience so far.

Antigens↗
PubMed1998

In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides.

A chimeric RNA/DNA oligonucleotide was constructed to induce a sequence mutation in the rat factor IX gene, resulting in prolonged coagulation. Oligonucleotides were targeted to hepatocytes in cell culture or in vivo by intravenous injection. Nucleotide conversion was both site-specific and dose-dependent. The mutated gene was associated in vivo with significantly reduced factor IX coagulant activity and a marked prolongation of the activated partial thromboplastin time. The results demonstrate that single base-pair alterations can be introduced in hepatocytes in situ by RNA/DNA oligonucleotides, suggesting a potentially powerful strategy for hepatic gene repair without the use of viral vectors.

Animals↗
PubMed2001

Pharmacokinetics of recombinant factor IX in relation to age of the patient: implications for dosing in prophylaxis.

The aims of this study were to investigate possible age-related changes in the disposition of factor IX procoagulant activity (FIX:C) after administration of recombinant factor IX (rFIX) and to translate the pharmacokinetic findings into suggestions for dosing of rFIX during prophylactic treatment of haemophilia B. Pharmacokinetic data were available from a previous study on 56 patients, aged 4-56 years (one of whom was excluded from analysis). FIX:C curves during prophylactic dosing were computer-simulated from the single-dose data. Clearance and volume of distribution at steady state of FIX:C increased linearly with body weight of the patients, consequently increasing during childhood and adolescence but remaining fairly constant during adulthood. The terminal half-life of FIX:C showed no correlation with age, while in vivo recovery (in U dL(-1) per U kg(-1) given) tended to increase. Computer-predicted trough levels of exogenous FIX:C during repeated doses of rFIX (50 U kg(-1)) and, conversely, doses (in U kg(-1)) needed to maintain a 1-U dL(-1) trough level showed little or no dependence on age. There was considerable interindividual variation in disposition and required doses of rFIX, emphasizing the need for individual dose titration. Dosing of rFIX according to lean body mass instead of body weight did not reduce this variability. During prophylaxis a 1-U dL(-1) trough level can normally be maintained by dosing every 2-3 days, the former schedule resulting in, on average, a 45% lower consumption of rFIX.

Adolescent↗
PubMed2001

Posttranslational modifications of recombinant myotube-synthesized human factor IX.

Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

1-Carboxyglutamic Acid↗
PubMed1987

Single-dose pharmacokinetics of factor IX evaluated by model-independent methods.

We studied the pharmacokinetic data of 13 subjects with hemophilia B treated with a single-dose of a Factor IX concentrate (Bebulin TIM2, N = 9; Preconativ, N = 4). The decay curves of Factor IX were evaluated by model-independent methods and the following pharmacokinetic parameters (mean +/- SD) were estimated: clearance (ml/h/kg) = 4.99 +/- 2.01; mean residence time (h) = 22.9 +/- 10.6; volume of distribution (ml/kg) = 99.9 +/- 35.5. The in vivo recovery (59.8% +/- 16.9%) was found to be inversely correlated with the volume of distribution. No significant difference in the pharmacokinetic parameters was found between patients treated with Preconativ and those treated with Bebulin. A model-dependent compartmental evaluation of the 13 decay curves showed that the two-compartment model was better than the one-compartment model in 7 cases (53.8%), but the improvement of fit resulting from the two-compartment model was statistically significant in only 2 cases (15.4%).

Factor IX↗
PubMed1994

[A new polymorphism in the human factor IX gene, useful for determining carriers of hemophilia B].

A new Taq I polymorphism in Alu repeat 4 of the human factor IX gene is reported. This polymorphism is associated with a C-T transition at the 72-bp position of the Alu repeat consensus sequence. A simple PCR system for testing of this structural anomaly, with internal control of restriction hydrolysis, was developed. The frequency of the new polymorphic site and its linkage with other polymorphisms of the factor IX gene were also evaluated. The new polymorphism was used for establishing hemophilia B carriers.

Base Sequence↗
PubMed1985

[Recombination between the fragile site Xq27 and the gene for coagulation factor IX].

Using the Taq I restriction polymorphism of a factor IX probe, we analysed the segregation of this gene and that of the fragile site Xq27. The ancestor of this family was a healthy carrier male. Of twelve informative meioses, at least four recombinations were detected. The hypothesis of a particular instability of the distal part the long arm of the X chromosome is reconsidered.

Adolescent↗
PubMed1995

[Concentrate of coagulation factor IX: viral validation of the thermoinactivation process].

The coagulation factor IX obtained at the Institute of Haematology and Transfusion in Warsaw was subjected to virological validation. Two lipide enveloped viruses were used: HSV-1 and Sindbis which may be a model for the viruses of B and C types in hepatitis. It was shown that heating of the freeze-drying preparations at 80 degrees C during 72 hrs leads to full inactivation of the added model viruses.

Biological Products↗