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PubMed1988

Large-scale production and properties of a solvent-detergent-treated factor IX concentrate from human plasma.

A human solvent-detergent (SD)-treated factor IX concentrate has been produced from cryoprecipitate-poor plasma using DEAE-Sepharose CL-6B and heparin-Sepharose CL-6B chromatography. The DEAE eluate was incubated with an SD mixture [0.3% tri(n-butyl) phosphate-1% Tween 80, 6-h at 24 degrees C] which was found to inactivate, in less than 1 h, more than 3.8 log10 of vesicular stomatitis virus and more than 4.8 log10 of Sindbis virus; the SD was removed by a subsequent heparin adsorption step. The specific activity of the concentrate was 10.9 +/- 1.3 IU factor IX: c/mg protein (n = 15). The factor IX coagulant to antigen ratio was 0.7 +/- 0.1. The concentrate was essentially free of factors II, VII and X, and protein C. The usual major contaminants of prothrombin complex concentrate (PCC) were absent: the concentrate contained about 94% alpha-1 proteins, and only 4 major proteins were resolved by SDS-PAGE (respective apparent molecular weight: 130, 86, 76 and 69 kilodaltons), and by crossed immunoelectrophoresis against an anti-PCC serum. The nonactivated partial thromboplastin time was equivalent to that of PCC; the product was devoid of factor IXa, of other activated procoagulant factors and of coagulant-active phospholipids (removed with SD in the heparin breakthrough fraction). Animal studies using the Wessler test and acute-toxicity test in rabbits revealed no adverse side effects. SD treatment could thus be used to inactivate viruses in factor IX concentrate and improve the safety of replacement therapy in hemophilia B.

Adsorption↗
PubMed1994

Primary myoblast-mediated gene transfer: persistent expression of human factor IX in mice.

Primary myoblast-mediated gene transfer was tested for its ability to mediate a persistent expression of recombinant human factor IX in SCID mice. Mouse primary myoblasts were transduced with factor IX retroviral vectors, LIXSN, which uses the retroviral long-terminal repeats as the promoter, or dLMMBAIX, which uses muscle creatine kinase enhancer and beta-actin promoter to drive factor IX transcription. In vitro, myoblasts transduced with either LIXSN or dLMMBAIX expressed recombinant human factor IX with full biological activity. Upon implantation of transduced myoblasts into skeletal muscles of SCID mice, a sustained systemic expression of factor IX at a level of 10-30 ng/ml plasma was achieved. This was further supported by the presence of recombinant factor IX protein and mRNA in muscle tissues after 5 months of myoblast implantation. Intramuscular implantation of the transduced myoblasts resulted in a gene transfer which was confined locally to the region of injection, with no dissemination to other organs and tissues including testis. Additionally, basic fibroblast growth factor co-injected with primary myoblasts significantly improved the expression level of recombinant factor IX in vivo. These results demonstrate that primary myoblast-mediated gene therapy for hemophilia B is feasible and safe, and can be optimized by using cytokines or other conditions which augment myoblast survival and fusion with myofibers.

Animals↗
PubMed2005

No impact of factor IX Ala-10 mutations in acenocoumarol-treated southern Europeans.

Increased risk of bleeding during oral anticoagulant (OA) treatment may be related to mutations in the precursor of coagulation factor IX. Missense mutations at Ala-10 (Ala-10Thre and Ala-10Val) in the factor IX propeptide lead to impaired carboxylation of factor IX. When patients carrying these mutations are treated with coumarins, functional factor IX levels decrease significantly, leading to an excessively prolonged activated partial thromboplastin time (aPTT) and an increased bleeding risk. Mutations at Ala-10 have been described in northern-European patients, but it is not known whether geographical differences play a role in the prevalence of these mutations. We aimed to analyze the prevalence of mutations at Ala-10 in factor IX in southern-European patients on OA treatment. Patients attending the Oral Anticoagulant Unit of the Hospital Clinic were prospectively included. The aPTT was determined at their normal International Normalized Ratio (INR) control. When the aPTT was excessively prolonged for the INR value, determination of factor IX and genotyping for Ala-10 mutations was performed. A total of 2360 patients were included (1289 men, 1071 women; mean age, 70.5 +/- 12.1 years). Twenty-four patients (16 men, eight women; mean age, 61.0 +/- 16.2 years) had an aPTT over that expected for the INR. The mean aPTT was 69.6 +/- 16.2 s. Only one patient presented with a factor IX level lower than 10%. None of the 24 patients carried mutations at Ala-10. Mutations at Ala-10 in factor IX were non-existent in the southern-European population analyzed, and thus, do not represent a relevant cause of bleeding during OA treatment.

Acenocoumarol↗
PubMed1992

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.

Antibodies, Monoclonal↗
PubMed1990

A mutation adjacent to the beta cleavage site of factor IX (valine 182 to leucine) results in mild haemophilia Bm.

The genetic basis of a mild form of haemophilia Bm has been investigated. The patient under investigation has a mild bleeding disorder and has never experienced spontaneous bleeds. Factor IX coagulant activity (FIX:C) was 0.15 units/ml and factor IX antigen (FIX:Ag) 1.32 units/ml. The prothrombin time performed with an ox brain thromboplastin was 65 s (normal plasma 31 s). Studies of the abnormal factor IX protein in this patient showed a normal molecular weight and normal calcium binding properties. Activation of the mutant factor IX with factor XIa showed normal proteolytic cleavage. DNA sequence from the eight factor IX exons and flanking introns was amplified from this patient using the polymerase chain reaction. The amplified material was subjected to direct chain termination nucleotide sequencing. The only nucleotide sequence alteration found was a G----C transversion at nucleotide 20,524, changing the amino acid encoded at residue 182 from valine to leucine. This residue is one amino acid removed from the beta cleavage site of factor IX. This residue is highly conserved in other vitamin K dependent serine proteases and we propose that its alteration in this patient is responsible for his mild haemophilic phenotype, and for the abnormal interaction of this factor IX protein with the extrinsic system of coagulation.

Aged↗
PubMed1988

Inhibition of prothrombin activation by factor X and factor IX Gla-peptides.

In the present study, human factor X and factor IX were each digested with chymotrypsin, and the Gla-peptide from each protein was purified by QAE-Sephadex chromatography. The effect of each Gla-peptide on the activation of human prothrombin by a complex of factor Xa, phospholipid, and calcium was studied using an amidolytic assay for generated thrombin. Prothrombin activation was half-maximally inhibited by factor X Gla-peptide at a concentration of 0.7 microM. Factor IX Gla-peptide was markedly less inhibitory and inhibited this reaction half-maximally at a concentration of 3.7 microM. Kinetic analyses revealed that the factor X Gla-peptide inhibited this reaction in an apparent competitive manner, whereas the factor IX Gla-peptide yielded an exponential Dixon plot. Heat decarboxylation experiments revealed that 3-4 gamma-carboxyglutamic acid residues are critical for the expression of inhibitory activity in each peptide. These studies indicate that, in spite of their structural homology, the ability of each of these Gla-peptides to act as a prothrombinase inhibitor is markedly different.

Factor IX↗
PubMed1996

Localization of a calcium-dependent epitope to the amino terminal region of the Gla domain of human factor IX.

We have used site-directed mutagenesis to define the epitope of calcium-dependent monoclonal antibodies to human factor IX. We demonstrate that the calcium-specific epitope includes residues 1-11 of factor IX, with apparent contributions from other regions of the protein. Antibodies JK.IX-1, -3, and -4 had critical portions of their epitopes in the first eleven amino acids of the Gla domain. These three antibodies could bind to a chimera containing the factor VII Gla domain, in which surface residues 3, 4, 5, 9, 10, and 11 were modified to those of factor IX. In contrast, the epitope of JK.IX-2 was unaffected by mutations in residues 3-11 of factor IX, but was dependent on the amino terminal tyrosine residue. Furthermore, the calcium-dependent monoclonal antibodies, JK.IX-1, -3, and -4, whose epitope include residues 3 through 11. inhibit factor IX's binding to endothelial cells, for which the binding site on factor IX has been localized to this region. Our results, together with previous studies, confirm the existence of discrete calcium and metal-dependent epitopes within the Gla domain of factor IX and show that the calcium-specific epitope lies near the amino terminus region of this domain.

1-Carboxyglutamic Acid↗
PubMed1979

Electroimmunoassay of factor IX in patients with liver damage and vitamin K unresponsive coagulation disorder.

Factor IX antigen and activity were assayed in a group of patients with liver disease and in a group of patients in coumarin therapy. In patients with liver disease there was a similar decrease in activity and antigen. On the other hand factor IX activity is decreased in coumarin treatment with factor IX antigen remaining normal. Factor IX electrophoretic mobility in liver disease is normal.

Blood Coagulation Disorders↗
PubMed1994

Factor IX Bm Kiryu: a Val-313-to-Asp substitution in the catalytic domain results in loss of function due to a conformational change of the surface loop: evidence obtained by chimaeric modelling.

Factor IX Kiryu is a naturally occurring mutant of factor IX that has 2.5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor IX Kiryu was purified from a patient's plasma by immunoaffinity chromatography with a calcium-dependent anti-factor IX monoclonal antibody column. It was cleaved normally by factor XIa in the presence of Ca2+, yielding a two-chain factor IXa. However, the resulting factor IXa showed only 1.5% of the normal factor IXa in terms of factor X activation in the presence of factor VIII, phospholipids, and Ca2+, and had 20% of the normal esterase activity for Z-Arg-p-nitrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single-strand conformational polymorphism analysis and DNA sequencing of the amplified DNA revealed a missense point mutation, a T-to-A substitution at nucleotide number 31,059 of the factor IX Kiryu gene. This mutation resulted in the amino acid substitution of Val-313 by Asp in the catalytic domain. Restriction enzyme analysis of the amplified DNA showed that the mutation was inherited from the patient's mother. The chimaeric method was employed to construct a model of the serine protease domain of factor IXa, and the resultant model suggested that the Val-313 to Asp substitution altered the conformation of the substrate-binding site. These data combined with our previous findings on a Gly-311-to-Glu mutant of factor IX suggest that the loop conformation from Gly-311 to ARg-318 is important for the expression of coagulant activity.

Amino Acid Sequence↗
PubMed1979

A new treatment strategy for hemophilia B: incorporation of factor IX into red cell ghosts.

Although patients with severe hemophilia B have lifelong spontaneous hemorrhage and crippling hemarthroses, patients with 0.01--0.03 U/ml Factor IX activity have a milder disease state. The clinical condition of severely affected individuals could potentially be improved by prolonging the half-life of transfused Factor IX. The feasibility of incorporating Factor IX into red cell ghosts was suggested by resealing experiments with similar sized molecules such as albumin. We have prepared resealed red cell ghosts containing human Factor IX and X. Human red cells were subjected to hypotonic lysis at 0 degrees C, pH 6.0. Commercial prothrombin complex concentrate was dissolved in the lysing medium immediately prior to the addition of the red cells. After being returned to isotonicity, the red cell ghosts were annealed at 37 degrees C for 30 minutes and then washed extensively. When intact red cell ghosts were tested, no Factor IX or X activity could be demonstrated. After disruption of the red cell ghost membranes with 3M urea or 2% Triton X-100, the procoagulants could be quantitatively recovered. Similar recovery of the clotting factors could be demonstrated from the lysate and the early wash samples. Red cells from Factor IX and X deficient patients served equally well as those from normal subjects. Red cell ghosts prepared in similar fashion but not exposed to the procoagulants had negligible clotting activity. We have demonstrated that human clotting factors can be incorporated into red cell ghosts. The ability of this system to prolong the biological half-life of Factor IX is under investigation.

Erythrocyte Membrane↗
PubMed1986

Binding of iron by factor IX. Possible role for beta-hydroxyaspartic acid.

In concentrations exceeding 1 mg/ml, bovine factor IX exhibits a pink color that arises from a broad absorption band with a lambda max = 500 nm. Analysis by x-ray fluorescence reveals the presence of iron but no other transition metals in the factor IX preparation. Quantitative analysis by atomic absorption spectroscopy indicates that 1 g atom of iron is bound tightly to 1 mol of factor IX. The iron is removed slowly (t1/2 = 3 h) by EDTA. In contrast, prothrombin binds no detectable iron, and factor X binds less than 0.2 g atom/mol. alpha-Hydroxybutyrate chelates Fe3+ with sufficient stability to preclude formation of [Fe(OH)3]n. It is proposed that factor IX binds iron with physiologically significant affinity and that the beta-hydroxyaspartate residue in factor IX is a chelator for the bound metal.

Animals↗
PubMed1993

Disseminated intravascular coagulation in a patient with haemophilia B during factor IX replacement therapy.

We report a case of haemophilia B, who developed disseminated intravascular coagulation (DIC) with massive bleeding following an administration of factor IX complex concentrates. A 22-year-old male with severe haemophilia B had a bone fracture of the fibula and haemorrhage in the ankle joint because of a traffic accident. Factor IX complex concentrates were given to keep the plasma factor IX level more than 80% for surgery. 60 h after the infusion he showed epistaxis, haematuria and swelling of the injured ankle with evidence of DIC. A small dose of monoclonal antibody (mAb)-purified factor IX concentrates with heparin and gabexate relieved the haemorrhage. A high dose of the mAb-purified product attained more than 90% levels of plasma factor IX without coagulopathy. This observation emphasizes that currently available factor IX concentrates have thrombogenicity to induce DIC. Highly purified factor IX concentrates are needed for the high-dose replacement in haemophilia B.

Adult↗
PubMed2001

Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

Antibody Specificity↗
PubMed1991

Expression of human factor IX in rat capillary endothelial cells: toward somatic gene therapy for hemophilia B.

In aiming to develop a gene therapy approach for hemophilia B, we expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression (0.84 and 3.6 micrograms per 10(6) cells per day for CECs and NIH 3T3 cells, respectively) as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX of 68 kilodaltons identical to purified plasma factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including gamma-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

Animals↗
PubMed1998

Membranous glomerulonephritis and nephrosis post factor IX infusions in hemophilia B.

Children with hemophilia B who receive exogenous factor IX infusions may rarely develop inhibitors to the exogenous factor IX and require desensitization. Nephrotic syndrome has recently been described in some of these children. We report the renal clinicopathological findings in a child with severe factor IX deficiency, requiring induction of an immune tolerance protocol, who developed nephrotic syndrome. Renal biopsy revealed peripheral capillary wall thickening and a spike appearance consistent with membranous glomerulonephritis. Electron microscopy showed prominent deposits throughout the thickness of the basement membrane. Factor IX dose reduction was accompanied by reversal of the child's nephrotic syndrome without relapses.

Child, Preschool↗
PubMed2000

Activation of factor IX zymogen results in exposure of a binding site for low-density lipoprotein receptor-related protein.

The interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and either coagulation factor IX or its active derivative factor IXa was studied. Purified factor IX was unable to associate with LRP when analyzed by surface plasmon resonance. By contrast, factor XIa-mediated conversion of factor IX into factor IXa resulted in reversible dose- and calcium-dependent binding to LRP. Active-site blocking of factor IXa did not affect binding to LRP, whereas LRP binding was efficiently inhibited in the presence of heparin or antibodies against factor IX or LRP. The factor IXa-LRP interaction could be described by a 2-site binding model with equilibrium dissociation constants of 27 nmol/L and 69 nmol/L. Consistent with this model, it was observed that factor IXa binds to 2 different recombinant receptor fragments of LRP (denoted cluster II and cluster IV) with equilibrium dissociation constants of 227 nmol/L and 53 nmol/L, respectively. The amount of factor IXa degraded by LRP-deficient cells was 35% lower than by LRP-expressing cells, demonstrating that LRP contributes to the transport of factor IXa to the intracellular degradation pathway. Because ligand binding to LRP is often preceded by binding to proteoglycans, the contribution of proteoglycans to the catabolism of factor IXa was addressed by employing proteoglycan-deficient cells. Degradation of factor IXa by proteoglycan-deficient cells proceeded at a 83% lower rate than wild-type cells. In conclusion, the data presented here indicate that both LRP and proteoglycans have the potential to contribute to the catabolism of factor IXa.

Animals↗
PubMed1980

Post-natal development of factor IX.

Post-natal development of clotting activity and of antigen level of Factor IX was evaluated in 111 healthy, breastfed, newborn infants, aged 1-30 days. Of these, 80 had received at birth 2 mg of vitamin K1 orally. Factor IX clotting activity was determined by one-stage assay and antigen level by electroimmunoassay. On the 1st day both antigen level and clotting activity were low and the ratio was 1.01. There was a significant postnatal increase of the two activities of Factor IX during the first three days of life; thereafter both remained constant. No statistical difference inFactor IX activity was found with oral administration of vitamin K1 after the birth. During the first month of life both clotting activity and antigen level of Factor IX were low as compared to adult values. There was no correlation with age. The Factor IX protein of newborns did not show molecular heterogeneity by crossed-immunoelectrophoresis.

Antigens↗
PubMed1995

Structure of the calcium ion-bound gamma-carboxyglutamic acid-rich domain of factor IX.

We have determined the Ca(II)-bound structure of factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-carboxyglutamic acid-rich and aromatic amino acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in factor IX. Protons in the 1-47 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo-factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in factor IX.

1-Carboxyglutamic Acid↗