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PubMed1997

Complete deletion of factor IX gene and inhibition of factor IX activity in a labrador retriever with hemophilia B.

Hemophilia B is a heritable bleeding disorder caused by mutations in the gene coding for coagulation factor IX. The defect has been identified in purebred and mixed-breed dogs. Management of affected dogs requires transfusion of canine blood products supplying active factor IX. Production of inhibitors to factor IX is a complication of transfusion therapy that has been documented as affecting human patients. Risk for producing coagulation inhibitors is greatest for patients having large factor IX gene deletions. To our knowledge, this is the first report of canine factor IX inhibitor production in dogs. The affected dog had clinically severe hemophilia B caused by complete deletion of the factor IX gene and developed resistance to transfusion. Comprehensive evaluation of hemophilic dogs, including assays of specific factor activity, concentration, and factor inhibition, enhances diagnosis and management of this bleeding disorder. Characterization of the molecular defect causing hemophilia is useful for genetic counseling and identifying individuals at highest risk for producing coagulation inhibitors.

Animals↗
PubMed1995

Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression.

Recombinant adenoviruses containing the canine factor IX (FIX) cDNA were directly introduced in the hind leg muscle of mice. We show that (i) in nude mice, high expression (1-5 micrograms/ml in plasma) of FIX protein can be detected for > 300 days; (ii) in contrast, expression of FIX protein was transient (7-10 days) in normal mice; (iii) CD8+ lymphocytes could be detected within 3 days in the infected muscle tissue; (iv) use of beta 2-microglobulin and immunoglobulin M heavy chain "knockout" mice showed that lack of sustained expression of FIX protein is due to cell-mediated and humoral immune responses; (v) normal mice, once infected with recombinant adenovirus, could not be reinfected efficiently for at least 30 days due to neutralizing viral antibodies; and, finally, (vi) using immunosuppressive drugs, some normal mice can be tolerized to produce and secrete FIX protein for > 5 months. We conclude that currently available adenoviral vectors have serious limitations for use for long-term gene therapy.

Adenoviridae↗
PubMed1999

Kinetics of factor IX activity differ from that of factor IX antigen in patients with haemophilia B receiving high-purity factor IX replacement.

Pharmacokinetic studies in haemophilia B have found in vivo recovery of FIX (FIX) to be uniformly lower than the factor VIII recovery in haemophilia A. We hypothesized that this lower recovery could result from rapid binding to high-affinity receptors on platelets and endothelium. To test this hypothesis, we evaluated the kinetics of FIX activity and protein in haemophilia B patients. Twelve patients were enrolled in a double dosing, crossover study with two high-purity FIX concentrates, AlphaNine SD and MonoNine. Subjects were given 40 U kg-1 of FIX concentrate and blood samples were taken at 15, 30, and 60 min. A second infusion of 40 U kg-1 was given after the 60 min blood sample and further blood samples removed at 15, 60, 120, and 360 min after the second dose. Patients were infused with the alternate concentrate at least 7 days later. Plasma samples were assayed for FIX activity by coagulation assay and antigen by RIA. FIX antigen in the infused concentrates was measured and quantified as microg U-1. There was no difference between the two FIX concentrates (AlphaNine vs. MonoNine) in the initial (15 min) activity (57% +/-1 19% vs. 53% +/-1 12%) and antigen (62% +/-1 16% vs. 55% +/-1 19%) recoveries. Recoveries after the second FIX dose were not statistically different than those observed after the first FIX dose. In one patient, a doubling of the initial infusion dose did not increase FIX recovery after the second FIX dose. However, the recovery of FIX antigen was significantly greater than the recovery of FIX activity and the differences became more significant in the post-15 min samples. We calculated a ratio of plasma FIX antigen to FIX activity in microg U-1. Average antigen to activity ratio increased from 5.8 +/-1 1.9 microg U-1 at 15 min to 7.1 +/-1 2.2 microg U-1 at 60 min. At 420 min the ratio increased to 9.3 +/-1 2.4 microg U-1. Although these studies failed to demonstrate a significant FIX receptor pool, they did demonstrate a phenomenon of progressive loss of biologic activity of the FIX protein after infusion of FIX concentrates.

Blood Platelets↗
PubMed1989

Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo.

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.

Amino Acid Sequence↗
PubMed1977

Isolation and characterization of factor IX Chapel Hill: comparison to normal human factor IX.

A classification scheme for patients with hemophilia B has been devised. Assessment of factor IX antigen and factor IX coagulant activity has confirmed the genetic heterogeneity of hemophilia B and has shown that some affected patients have normal amounts of factor IX-like protein. An abnormal factor IX, factor IX Chapel Hill, has been isolated from one of these variants and compared structurally and functionally to the purified normal human factor IX molecule. The major difference between factor IX Chapel Hill and normal human factor IX appears to be defective activation of the abnormal molecule by factor XIa and calcium.

Factor IX↗
PubMed1980

Activation of 125I-factor IX and 125I-factor X: effect of tissue factor and factor VII, factor Xa and thrombin.

Activation of Factor IX and Factor X was studied by adding 125I-Factor IX or 125I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor Xa produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added 125I-Factor IX or 125I-Factor X, tissue factor and Ca2+ ions, the percentage of total radioactivity in the heavy chain peak of 125I-IXa and the heavy chain peak of 125I-Xa increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of 125I-Factor IX and percent cleavage of 125I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Factor IX by the tissue factor-Factor VII reaction produce represents a physiologically significant step in normal haemostasis.

Blood Coagulation↗
PubMed1995

Variability of in vivo recovery of factor IX after infusion of monoclonal antibody purified factor IX concentrates in patients with hemophilia B. The Mononine Study Group.

Monoclonal antibody purified factor IX concentrate, Mononine (Armour Pharmaceutical Company, Kankakee, Illinois, USA), is a recently developed replacement factor concentrate for the treatment of patients with hemophilia B. The pharmacokinetic properties of monoclonal antibody purified factor IX concentrate (MAb Factor IX concentrate) have been evaluated in only small samples of patients, and little is known about those factors that might influenced in vivo recovery of factor IX after infusion is a larger patient population. In vivo recovery of factor IX was therefore evaluated for 80 different indications in 72 patients who received MAb Factor IX concentrate for the management of spontaneous or trauma-induced bleeding, or as prophylaxis with surgery. The average recovery after infusions for presurgical pharmacokinetic analysis (mean +/- standard deviation) was 1.28 +/- 0.56 U/dl rise per U/kg infused (range 0.41-2.80), and the average recovery after all infusions for treatment was 1.23 +/- 0.49 U/dl rise per U/kg infused (range - 0.35-2.92). Recovery values for multiple MAb Factor IX doses in a given patient were also variable; the average recovery was 1.22 +/- 0.53 U/dl rise per U/kg given, and standard deviations ranged from 0.03 to 1.26. Patient age, weight, and MAb Factor IX concentrate dose minimally but significantly influenced factor IX recovery. There was no significant effect of either race, history of previous thrombotic complications during treatment with other replacement factor concentrates, or bleeding state on recovery. All of the patients treated with this preparation experienced excellent hemostasis, and no thrombotic complications were observed.

Adolescent↗
PubMed1991

Characterization of a mouse factor IX cDNA and developmental regulation of the factor IX gene expression in liver.

A mouse factor IX cDNA was isolated and characterized. The cDNA was 1,837 bp in length and contained the coding region as well as short 5' and 3' untranslated sequences. Northern blot analysis of liver RNA showed two mRNA species of 3.2 kb (major) and 2.2 kb (minor) for the mouse factor IX. An antisense RNA probe prepared from the mouse cDNA was employed to determine the steady state level of factor IX mRNA in mouse liver at various developmental stages. The factor IX mRNA level was very low (2-5% of the adult level) during the gestational period until day -3 (gestational day 17) followed by a rapid increase at day -2 through birth. This phase of rapid increase was followed by a gradual increase before it reached the adult level at around 20 to 24 days. At birth, the factor IX mRNA level was found to be at about 43% of that of the adult. The mRNA levels in mouse liver agreed well with the plasma factor IX activity levels. These results indicate that reduced factor IX activity in newborns is due to the low levels of factor IX mRNA available for translation.

Amino Acid Sequence↗
PubMed1992

Coagulation factor IX: successful surgical experience with a purified factor IX concentrate.

The use of plasma-derived coagulation factor concentrates has been marked by the transmission of viral agents. Infusions of factor IX complex concentrates have been additionally complicated by inappropriate thrombosis. Use of these concentrates in the neonate, in those with liver disease, and in surgical patients results in increased risk for this complication. Twenty patients have been infused with a purified coagulation factor IX concentrate for fall-off and recovery studies. A two-compartment model indicated an initial phase half-life of 4.06 +/- 2.86 hr and a beta phase half-life of 20.0 +/- 3.8 hr following the administration of AlphaNine, Coagulation Factor IX (Human). In vivo recovery was 62.7% +/- 13.8%, with an average factor IX coagulant level of 73% +/- 16% at 15 min after the infusion of a mean dose of 45 U/kg. Thirteen previously transfused patients with hemophilia B underwent major orthopedic or general or dental surgery using this purified factor IX. Operative outcomes were excellent in all patients. No excessive bleeding was noted. There was no laboratory or clinical evidence for a disseminated intravascular coagulopathy. The excellent surgical outcomes observed in this multitransfused group with biochemical evidence for active liver disease demonstrates the utility and safety of a purified coagulation factor IX concentrate.

Adult↗
PubMed1982

Factor IX Zutphen. A genetic variant of blood coagulation factor IX with an abnormally high molecular weight.

A genetic variant of blood coagulation factor IX has been isolated from the plasma of a patient with severe hemophilia B (congenital factor IX deficiency). The isolated variant--factor IX Zutphen--has a strongly reduced affinity for binding of Ca2+, it cannot be cleaved proteolytically by factor XIa, and it has a molecular weight of about 90,000, which is much higher than the 65,000 found for normal factor IX and acarboxy factor IX. Available data indicate that these unique properties of factor IX Zutphen are connected with the presence of an extra polypeptide (molecular weight 33,000) that is linked to the factor IX molecule by a disulfide bond and thus prevents effective binding of Ca2+ or factor XIa. Such a model might explain the extremely low specific coagulant activity of factor IX Zutphen.

Blood Protein Electrophoresis↗
PubMed1996

Hepatitis A transmission by factor IX concentrates.

Factor IX concentrates unlike factor VIII concentrates have not to date been associated with the transmission of hepatitis A virus (HAV). A retrospective study by reverse transcriptase polymerase chain reaction on a batch of factor IX concentrate used to treat two haemophilia B patients who developed jaundice and IgM anti-HAV antibodies within 50 days of factor IX administration in 1985 revealed the presence of HAV RNA. These findings indicate that factor IX concentrates can transmit HAV and that appropriate viral inactivation steps to inactivate nonenveloped viruses as well as enveloped viruses are necessary to ensure the safety of factor IX concentrates.

Adult↗
PubMed1983

The activation of Factor IX by tissue factor-Factor VII in a bovine plasma system lacking Factor X.

The activation of Factor IX by tissue factor-Factor VII has been studied in a bovine plasma system under conditions that minimize the activation of Factor VII. The plasma was defibrinated, then passed twice through a column of anti-Factor X coupled to Sepharose in order to lower the Factor X level below its limit of assay (ca. 5 ng/ml), and once through an anti-Factor IX column to remove Factor IX. Varying levels of tritium-labelled Factor IX were then added back to the plasma, permitting measurement of its activation upon the addition of tissue factor and Ca2+. Despite the absence of significant levels of Factor X in the system, the course of Factor IX activation was initially characterized by some upward curvature, which suggested activation of the plasma Factor VII during the incubation. In order to obtain linear activation of Factor IX three proteolytic inhibitors were added to the system: 1) a Factor Xa inhibitor, 1,2-bis-(5-amidinobenzimidazole)-ethane, 2) aprotinin, and 3) heparin. Under these conditions the apparent Km of non-activated Factor VII (+ tissue factor) on Factor IX was 17.3 +/- 2.5 nM (SE), and the maximum velocity was 0.12 nM/min. In parallel experiments the plasma Factor VII was activated by first treating the plasma with Factor Xa for 30 seconds before the addition of inhibitors and the final addition of substrate. Under these conditions the maximum velocity rose to 4.2 nM/min, and the Km increased to 53.3 +/- 6.0 nM (SE). This change in the Km is highly significant (P less than 0.002), and indicates that the activation of Factor IX by nonactivated plasma Factor VII cannot be due only to traces of Factor VIIa in the plasma. At least in part, activation of Factor IX in the presence of tissue factor is suggested to be a result of the action of Factor VII itself.

Animals↗
PubMed1991

Purification and characterization of rabbit factor IX and its existence as a two-chain factor IX alpha in circulating plasma.

The long term objective of this study is to immunodeplete rabbits of factor IX as a means of developing a rabbit model for hemophilia B for use in studies of tissue factor-dependent blood coagulation. As a first step, we have purified rabbit factor IX by basically two different methods: (1) conventional chromatography utilizing DEAE-Sephadex and heparin-agarose column chromatography (2) immunoaffinity chromatography on monoclonal anti-rabbit factor IX IgG column. Purified rabbit factor IX migrated as a single band with an apparent molecular mass of 76 kD on nonreduced SDS-PAGE. On reduced SDS-PAGE the majority of factor IX migrated as a two-chain molecule (molecular masses 51 and 28 kD) and a faint band corresponding to 78 kD. We have shown that the purification of rabbit factor IX as a two-chain molecule is not due to the partial proteolysis of factor IX during its purification from commercially obtained rabbit plasma. Analysis of 3H-labelled rabbit factor IXa on SDS-PAGE revealed that, in contrast to human factor IXa, carbohydrate was found associated with the heavy chain of activated factor IX (H beta) after release of the activation peptide. Further understanding of the molecular properties of rabbit factor IX and the generation of neutralizing monoclonal and polyclonal antibodies will facilitate the development of a rabbit model for hemophilia B.

Amino Acid Sequence↗
PubMed2003

Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5.

Anti-human factor IX monoclonal antibody, A-5 (Mab A-5), has been widely used in structure-function studies for factor IX. Mab A-5 recognizes the catalytic domain of human factor IX (FIX). Regions important for Mab A-5 binding have previously been localized to the amino terminus of the heavy chain of factor IX, encompassing amino acid residues 181-310 [Blood (74) 971]. We have selected 20 positions in this region for alanine-scanning mutagenesis. We found that Mab A-5 failed to react with the recombinant factor IX mutants with substitutions at positions 228 and 252. Mab A-5 also reacted to a lesser extent to FIXD276A (factor IX with alanine substitution for aspartic acid at residue 276) and FIXK201A/D203A (double alanine substitutions at residues 201 and 203). The apparent dissociation rate constants (K(D)) in binding Mab A-5 were 6.0 x 10(-9), 1.4 x 10(-8) and 2.0 x 10(-8) M, for wild-type FIX, FIXK201A/D203A and FIXD276A, respectively. The increased K(D) values of the two FIX mutants are mainly owing to the increased dissociation rates. These affected residues constitute a surface opposite from the factor VIIIa binding surface. We conclude that the epitope for Mab A-5 is on a surface composed of residues 228, 252, 276, and 201 or 203. This surface, which may not be important for factor VIII binding, contains a strong antigenic region on factor IX.

Amino Acid Substitution↗