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PubMedDate not supplied

Thrombogenicity of factor IX concentrates: in vitro and in vivo (rabbit) studies.

The reproducibility and correlation between the NAPTT, TGt50 in (vitro) tests and the Wessler (rabbit) stasis thrombus (in vivo) model have been studied using 10 different factor IX concentrates. The TGt50 test was more reproducible than the NAPTT and the overall reproducibility of the rabbit model was poor. The low reproducibility of the rabbit model appeared to be largely confined to those factor IX concentrates which showed a poor correlation between the NAPTT and TGt50 results. The TGt50 test emerged as the in vitro test which correlated most closely with the in vivo (rabbit) test. It is concluded that the NAPTT and TGt50 test are measuring different thrombogenic moieties in factor IX concentrates and that further studies are required to elucidate this phenomenon.

Animals↗
PubMed1985

A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z.

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).

Amino Acids↗
PubMed2001

Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA.

Long-term expression of coagulation factor IX (FIX) has been observed in murine and canine models following administration of recombinant adeno-associated viral (rAAV) vectors into either the portal vein or muscle. These studies were designed to evaluate factors that influence rAAV-mediated FIX expression. Stable and persistent human FIX (hFIX) expression (> 22 weeks) was observed from 4 vectors after injection into the portal circulation of immunodeficient mice. The level of expression was dependent on promoter with the highest expression, 10% of physiologic levels, observed with a vector containing the cytomegalovirus (CMV) enhancer/beta-actin promoter complex (CAGG). The kinetics of expression after injection of vector particles into muscle, tail vein, or portal vein were similar with hFIX detectable at 2 weeks and reaching a plateau by 8 weeks. For a given dose, intraportal administration of rAAV CAGG-FIX resulted in a 1.5-fold or 4-fold higher level of hFIX compared to tail vein or intramuscular injections, respectively. Polymerase chain reaction analysis demonstrated predominant localization of the rAAV FIX genome in liver and spleen after tail vein injection with a higher proportion in liver after portal vein injection. Therapeutic levels of hFIX were detected in the majority of immunocompetent mice (21 of 22) following intravenous administration of rAAV vector without the development of anti-hFIX antibodies, but hFIX was not detected in 14 immunocompetent mice following intramuscular administration, irrespective of strain. Instead, neutralizing anti-hFIX antibodies were detected in all the mice. These observations may have important implications for hemophilia B gene therapy with rAAV vectors.

Animals↗
PubMed1982

Factor IX antigen by a rapid staphylococcal protein A-membrane binding radioimmunoassay: results in haemophilia B patients and carriers and in fetal samples.

Staphylococcal protein A-membranes have been used with isolated, radiolabelled factor IX and specified rabbit antisera for modification of a radioimmunoassay. The current method is a rapid 4 h procedure and dilution curves of plasma parallel those of isolated, unlabelled protein. Non-specific binding is 5%; the assay readily detects concentrations as low as 0.6 u/dl. Carrier detection of haemophilia B was improved and/or confirmed by the demonstration of factor IX antigen in excess of clotting activity in nine of 15 women tested from pedigrees in which the affected members had excess circulating antigen. Of 15 new haemophilia B pedigrees examined, 13 had antigen levels which were in two-fold or greater excess over their clotting activities; all but three were considerably below normal, however. To diagnose haemophilia B in newborns at risk, levels in three cord blood samples were tested; two were positive and the third was normal. Six fetal blood samples were assayed and contained from 4 to 20 u/dl factor IX antigen; levels correlated with fetal age.

Antigens↗
PubMed1999

Production of highly purified clotting factor IX by a combination of different chromatographic methods.

A highly enriched preparation of human clotting factor IX was produced by a combination of adsorption chromatography, hydrophobic interaction chromatography and heparin affinity chromatography. The introduction of adsorption chromatography with a hydroxyaminopropyl support allows the capture step to be carried out directly from the cryoprecipitate-depleted plasma with a chromatographic column in flow-through mode. This replaces the batch procedure used until now. The other two chromatographic steps are designed in such a way that the eluate from the preceding step can be directly applied, without any intermediate treatment of the sample. This cuts the period of time required for the process by almost 50%, and increases the yield considerably. The isolated factor IX contains practically no contaminants and has a specific activity over 200 IU/mg of protein.

Adsorption↗
PubMed1992

Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo.

We have explored the use of primary myoblasts as a somatic tissue for gene therapy of acquired and inherited diseases where systemic delivery of a gene product may have therapeutic effects. Mouse primary myoblasts were infected with replication-defective retroviruses expressing canine factor IX cDNA under the control of a mouse muscle creatine kinase enhancer and human cytomegalovirus promoter. The infected myoblasts were injected into the hindlegs of recipient mice and levels of secreted factor IX protein were monitored in the plasma. We report sustained expression of factor IX protein for over 6 months without any apparent adverse effect on the recipient mice.

Animals↗
PubMed2002

Magnesium and manganese ions accelerate tissue factor-induced coagulation independently of factor IX.

The purpose of the present study was to assess the effect of magnesium and manganese ions on tissue factor (TF)-induced coagulation and the possible role of factor IX therein. When magnesium chloride or manganese chloride were added in low concentrations to normal human plasma, the human (recombinant) TF-induced coagulation time was shortened. At higher concentrations, magnesium and manganese prolonged the TF-induced coagulation time. Maximum shortening of the coagulation time was obtained at a concentration of 0.5 mmol/l Mn or 2 mmol/l Mg in plasma. Shortening of the TF-induced coagulation time by magnesium and manganese was also observed in factor IX-deficient plasma. A comparison was made between TF preparations from human, rabbit, and bovine brain. The accelerating effect of magnesium was greater with human than with rabbit brain TF. Using bovine brain TF, the clotting time was not shortened by magnesium. Activated factor X-induced coagulation of normal plasma was not accelerated by magnesium. From these experiments, it is inferred that activation of factor X by factor VII-TF can be accelerated by magnesium and manganese ions independently of factor IX.

Animals↗
PubMed1968

An investigation of three patients with Christmas disease due to an abnormal type of factor IX.

Three patients with Christmas disease whose plasma was shown to have a prolonged one-stage prothrombin time with ox brain thromboplastin have been investigated. These patients have an inhibitor for the reaction between factor X, factor VII, and ox brain extract. The abnormal constituent responsible for this inhibitor appears to be factor IX whuch is functionally inactive but antigenically indistinguishable from normal factor IX. It is proposed that patients might be classified into haemophilia B(+) for patients with this defect (Christmas disease(+)) and haemophilia B(-) (Christmas disease(-)) for patients who have classical Christmas disease.

Animals↗
PubMed1992

Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

Adolescent↗
PubMed1975

Purification and some characteristics of the coagulation factor IX from human plasma.

Non-activated coagulation factor IX was purified approx. 10,000-fold from human plasma. The final product was electrophoretically homogeneous and comprised a tingle polypeptide chain with a molecular weight of about 70,000 and a pI of 4.3-4.45. The N-terminal amino acid was glycine. The amino acid and the carbohydrate contents were analysed and a monospecific antiserum to the factor was raised in rabbits.

Amino Acids↗
PubMed1995

[Characterization of human coagulation factor IX cDNA expressed in cultured cells and in transgenic mice].

Two recombinant genes containing human coagulation factor IX cDNA driven by SV40 early promoter or by mouse MT promoter were introduced into the cultured mouse fabroblasts, respectively. It was found that both of them could be expressed in the cultured cells. Then, the two recombinant genes were microinjected into the male pronuclei of fertillized mouse eggs for generating transgenic mice harbouring the introduced genes, respectively. However, we did not observed that the two genes could be expressed in transgenic mice. These results suggest that the in vivo expression of human coagulation factor IX cDNA be controlled by some cis regulatory element (s).

Animals↗
PubMed1998

Consequences of factor IX mutations in 26 families with haemophilia B.

Haemophilia B is due to a variety of mutations within the factor IX gene. In the Seattle series, 26 additional unrelated families have had a mutation identified within the past 2 years. Of these, 11 were common recurrent point mutations identifiable by rapid restriction digest screening; eight of these probably represent founder mutations. 15 others were identified by sequencing amplified coding region fragments; eight are novel. Two each had frameshift and donor splice mutations and 11 had missense mutations. Five of these mutations associated with normal levels of circulating dysfunctional factor IX were computer modelled into coordinates for factor IXa.

Factor IX↗
PubMed1982

The stability of factor IX concentrate during storage.

A storage study of factor IX concentrate was performed on 35 randomly selected batches that had been prepared between 1976 and 1980. Vacuum stability, dissolution time, F IX procoagulant activity, NAPTT, and thrombin generation were measured immediately after the manufacturing process and compared in this study with the results obtained from the spare samples analysed in 1981. No significant changes during storage at cold room temperature for up to 5 years were established. The product can thus be considered to be stable for 5 years at + 4 degrees C.

Drug Stability↗
PubMed1990

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

It has been suggested that epidermal growth factor-like (EGF-like) domains, containing conserved carboxylate residues, are responsible for the high-affinity calcium binding exhibited by a number of vitamin K-dependent plasma proteins involved in the control of the blood coagulation cascade. These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S. To test this hypothesis we have expressed the first EGF-like domain from human factor IX (residues 46-84) using a yeast secretion system, and examined calcium binding to the domain. Using 1H-NMR to measure a calcium-dependent shift assigned to Tyr69 we have detected a high-affinity calcium binding site (Kd = 200-300 microM). We suggest that other EGF-like domains of this type may have similar calcium binding properties. In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.

Amino Acid Sequence↗
PubMed1991

Identification of haemophilia B patients with mutations in the two calcium binding domains of factor IX: importance of a beta-OH Asp 64----Asn change.

The polymerase chain reaction procedure (PCR) coupled with direct sequencing has been used to screen a panel of haemophilia B patients. This analysis has identified, amongst others, several mutations in the functionally important gla and type B EGF domains of factor IX, both of which are known to bind calcium. Type B EGF domains are widely distributed in proteins; located within these domains are highly conserved amino acid residues important for the formation of a high-affinity calcium binding site. One prominent feature of these domains is a highly conserved beta-hydroxylated Asp or Asn residue. Of particular interest is the identification of one patient, with a substitution of the beta-hydroxy Asp-64 residue normally present in factor IX for Asn. This change results in a functionally defective factor IX molecule with altered calcium binding properties. To explain the functional abnormality caused by this substitution of one amino acid residue for another which is commonly found at the equivalent position in other proteins with type B EGF domains, we propose the existence of additional conserved residues within this domain, which are important for calcium binding, and which correlate with whether the beta-hydroxylated residue is Asp or Asn.

Base Sequence↗
PubMedDate not supplied

Therapy with factor IX concentrate resulting in DIC and thromboembolic phenomena.

The development of subclinical DIC in a patient with factor IX hemophilia receiving concentrate replacement therapy during surgery is discussed with respect to pertinent laboratory features. Subsequent thromboembolic phenomena are presented in the context of current literature. Of significance is the failure of heparin given with the factor IX concentrate to prevent DIC. The value of adequate laboratory monitoring during therapy is stressed.

Adult↗
PubMed1996

Three novel and one C31133T (Arg-338-->Stop) mutations of antihemophilic factor IX gene detected in Taiwan.

BACKGROUND: Hemophilia-B is caused by mutation of the coagulation factor IX (F.IX) gene. The gene is about 34 kilobases in length and contains 8 exons ranging from 25 to 548 base pairs. There is no common mutation pattern of this gene in any population as there is in thalassemia. The hope is to localize the mutations in patients for use in making rapid familial surveys and for prenatal diagnoses. METHODS: Diagnoses for a male patient with a newly-discovered mutation in Taiwan and three others with different novel mutations of F.IX gene were made from the results of prothrombin time (PT), activated partial thromboplastin time (APTT), assay of coagulation factors activity and factor IX antigen (F.IX:Ag) and also by assay of its inhibitor. The polymerase-chain-reaction (PCR)-based direct sequencing method was applied to evaluate all of the 8 exons and their flanking regions of F.IX gene for these patients. RESULTS: The four male patients are all severe hemophilia-B. In the three patients with novel mutation of F.IX gene, two have a very low F.IX:Ag of less than 1% and one has 73%, but no one has any inhibitor. One of the four mutations is A to C transversion at nucleotide (nt) 10458 resulting in tyrosine (69) to serine change in epidermal growth factor (EGF) type B domain. Although the F.IX:Ag is present, the F.IX activity (F.IX:C) is markedly deficit owing to the change of beta-hydroxylase recognition site. Another two have mutations at the splicing junctions. They are G to C transversion at nt 118, i.e. the first base of IVS-1 splicing donor, and G to T transversion at nt 17668, the last base of IVS-4 splicing recipient. These mutations may cause splicing failure and subsequently result in deficiency of both F.IX activity and antigen. The other mutation that has never previously presented in this country is a C to T transition (arginine-338 to stop) at nt 31133 that results in early termination of translation. CONCLUSIONS: The G to C mutation at nt 118 creates an Alu I restriction site and the C to T mutation at nt 31133 abolishes a Taq I site. Therefore, familial survey and rapid prenatal diagnosis for these patients by PCR-based direct sequencing method or by the restriction of enzyme cutting method pointing at the known mutation sites are certainly possible to achieve in our patients.

Base Sequence↗
PubMed1998

The formulation of recombinant factor IX: stability, robustness, and convenience.

A lyophilized recombinant factor IX (rFIX) formulation has been developed that is stable and contains no preservatives. No blood or plasma products are used in the production or formulation of rFIX. The formulation contains 10 mmol/L histidine, 0.26 mol/L glycine, 1% sucrose, and 0.005% polysorbate-80 (pH 6.8). Polysorbate-80 acts as a protectant for the protein from freezing-induced damage (eg, aggregation). Sucrose provides protection to the protein in the freeze-dried state. Glycine provides for a high-quality cake morphology. Histidine provides optimal buffering stability at the desired pH and minimizes aggregate formation upon storage in the lyophilized state. This optimized combination of excipients provides a high degree of long-term stability, as demonstrated by a variety of analytical methods, including clotting assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), size-exclusion chromatography (SEC), peptide mapping, oligosaccharide fingerprinting, and reverse-phase high-performance liquid chromatography (HPLC). The rFIX product is easy to reconstitute and demonstrates excellent stability in solution after reconstitution.

Chemistry, Pharmaceutical↗