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PubMed1983

Haemorrhage and factor IX deficiency in pituitary insufficiency.

Acquired isolated factor IX deficiency is rare. A case presenting with haemorrhage after dental extraction is described. The factor IX deficiency was associated with pituitary insufficiency and was completely corrected following the administration of thyroxine. This contrasts with a previously reported case in which both thyroxine and cortisone were required for complete correction of the factor IX deficiency. The underlying mechanism and the consequences are briefly discussed.

Hemophilia B↗
PubMed1998

The manufacturing process for recombinant factor IX.

Advances in recombinant DNA manufacturing technology have now made possible the production of a highly purified and active recombinant factor IX (rFIX) product. Recombinant factor IX was developed by (1) stable insertion of the genes for both factor IX and PACE-SOL (a truncated, soluble serine protease needed to enhance the capacity of cells to remove the amino-terminal propeptide from rFIX) into Chinese hamster ovary cells; (2) selection of a cell line that was capable of expressing high amounts of active rFIX while growing in bioreactors containing a completely defined culture medium that does not contain blood or plasma products; and (3) inclusion of four independent chromatography steps, none of which require monoclonal antibodies. Furthermore, rFIX has been extensively tested to demonstrate similarity to plasma-derived factor IX and has been shown to be a consistent, high-purity product. For example, a high-specific-activity product (276+/-23 IU/mg) has been consistently produced throughout 65 consecutive batches from five consecutive manufacturing campaigns. Thus, rFIX offers a consistent and high-purity source of factor IX treatment for patients with hemophilia B.

Animals↗
PubMed1988

Immunoaffinity purification of factor IX from commercial concentrates and infusion studies in animals.

Thrombosis and transmission of viral diseases are the principal adverse effects of current replacement therapy for factor IX deficiency when using heat-treated concentrates of vitamin K-dependent coagulation factors. More highly purified factor IX preparations could decrease the risk of disease transmission, reduce patient exposure to allogeneic proteins, and reduce the risk of thrombosis. In this study, two immunoaffinity-purified factor IX preparations from commercial vitamin K-dependent coagulation factor concentrates had specific activities of 134 and 155 U/mg. Crude concentrates and purified factor IX preparations were tested for thrombogenicity in rabbits. One of two crude concentrates tested in the stasis-thrombosis assay caused large thrombi at doses of 50 U/kg. Purified factor IX from this concentrate was not thrombogenic at 106 to 234 U/kg. A heparin-treated concentrate that was not active in the stasis model at 100 U/kg caused significant (P less than .05) delayed consumption of rabbit fibrinogen, platelets, antithrombin III antigen, and factor VIII activity at the same dose. Factor IX prepared from this concentrate caused no consumption of coagulation factors at 214 to 243 U/kg despite the presence of trace amounts of activated factor IX. These results indicate that more highly purified preparations could reduce the risk of thrombosis in replacement therapy for hemophilia B. Also, at least for the preparations tested, factor IX and factor IXa were not the thrombogenic components of the crude concentrates.

Animals↗
PubMed1988

A second BamHI DNA polymorphism and haplotype association in the factor IX gene.

A second BamHI DNA polymorphism has been identified in the factor IX gene in an American black population at an allelic frequency of 0.13. This site has been localized within 500 basepairs (bp) 5' to the XmnI intron 3 polymorphic site and increases the heterozygosity of black females at the factor IX gene locus. In addition, haplotype analysis of factor IX genes at five polymorphic loci indicates that although there is conservation of sequences between the races, factor IX genes show more heterogeneity in an American black population and thus more heterozygosity is observed in black females compared with whites. This increased heterogeneity is due to DNA polymorphisms unique to black populations and to linkage equilibrium between the most 5' and 3' polymorphic sites in factor IX genes in blacks.

Alleles↗
PubMed1989

Blood clotting factor IX Kashihara: amino acid substitution of valine-182 by phenylalanine.

Hemophilia B Kashihara is a severe hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. In addition, purified factor IX Kashihara is not activated by purified factor XIa in the presence of calcium ions. Amino acid sequence analysis of one of the tryptic peptides isolated from factor IX Kashihara indicated that Val-182 (equivalent to Val-17 in the chymotrypsin numbering system) had been replaced by Phe. No substitution was found in the members of the catalytic triad His-221, Asp-269, and Ser-365 of factor IX Kashihara. The Val-to-Phe replacement found in factor IX Kashihara appears to sterically hinder the cleavage of Arg 180-Val 181 by factor XIa required for the activation of this zymogen.

Amino Acids↗
PubMed1997

Human factor IX binds to specific sites on the collagenous domain of collagen IV.

The primary region of factor IX that mediates binding to bovine aortic endothelial cells resides in residues 3-11 of the N-terminal region known as the Gla domain. Recently, it was proposed that the observed binding to endothelial cells is actually a measure of the interaction between factor IX and collagen IV (Cheung, W. F., van den Born, J., Kuhn, K., Kjellen, L., Hudson, B. G., and Stafford, D. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11068-11073). To confirm that factor IX binds to collagen IV and to examine the specificity of this interaction, we used scanning force microscopy to examine factor IX binding to collagen IV. We imaged collagen IV in the presence and the absence of factor IX and observed specific interactions between factor IX and collagen IV. Our results demonstrate that factor IX binds to collagen IV at specific sites in the collagenous domain approximately 98 and approximately 50 nm from the C-terminal pepsin-cleaved end.

Animals↗
PubMed1986

Induction of split tolerance and clinical cure in high-responding hemophiliacs with factor IX antibodies.

An approach to the problem of inducing tolerance in patients with hemophilia complicated by high-responding antibodies is described. Thus, in four patients with severe hemophilia B and high-responding antibodies against factor IX, it has been possible to modify the immune response by giving high doses of intravenous IgG in combination with cyclophosphamide and factor IX, followed by regular factor IX treatment. In three of the patients, the in vivo recovery and half-life of infused factor IX coagulant activity (IX:C) are now normal, while the fourth patient has been converted to a low responder. Hip replacement surgery has been performed successfully in one patient. The tolerant state in these four patients is characterized, and they have all been found to have complexes between factor IX antigen and a "new" antibody without IX:C inhibitory activity. The disappearance rate of the complexed factor IX antigen (i.e., lacking IX:C activity) is considerably prolonged, and the persistence in the circulation of this (probably modified) factor IX molecule may be crucial, since tolerance to factor IX treatment was only induced when immunocomplexes were produced. Since earlier treatment of the patients with cyclophosphamide and factor IX, but without IgG, failed to induce tolerance, it appears to be the IgG that is the prerequisite.

Adolescent↗
PubMed1983

Human factor IX inhibitors: immunochemical characteristics and treatment with activated concentrate.

Plasma was obtained from two patients with severe factor IX deficiency who had developed specific inhibitors of factor IX. Immunochemical characterization of the inhibitors by coagulation inhibitor neutralization assays and by immunoelectrophoretic methods demonstrated that both were IgG antibodies. One of the antibodies appeared to be monoclonal in origin with IgG subclass 4 heavy chains and lambda light chains. The other appeared to be oligoclonal and contained IgG subclass 1 and subclass 4 heavy chains and kappa and lambda light chains. One of the patients was treated with conventional, non-activated factor IX concentrate and with activated factor IX concentrate (Feiba) for repeated bleeding episodes. Administration of Feiba resulted in a progressive shortening of the kaolin cephalin clotting time and was followed by a good clinical response. Infusion of non-activated factor IX concentrate failed to induce clinical resolution of haemarthroses and had minimal effect on laboratory tests. The presence of circulating immune complexes could not be demonstrated in this patient.

Adult↗
PubMed1998

Anaphylactoid reactions and nephrotic syndrome--a considerable risk during factor IX treatment in patients with haemophilia B and inhibitors: a report on the outcome in two brothers.

Anaphylaxis/anaphylactoid reactions have recently been reported after few treatments with factor IX concentrates in patients with haemophilia B at the same time as inhibitors to factor IX were demonstrated. In some of these cases nephrotic syndrome has appeared during immune tolerance induction (ITI) with high doses of factor IX concentrates. Gene deletions seem to be associated with a high risk of developing antibodies to factor IX. This report presents two brothers with deletion of 1 bp in exon f of the factor IX gene. Both showed anaphylactoid reactions and they were desensitized using slow i.v. injections of factor IX. At the time of anaphylaxis, inhibitors of factor IX in a low titre could be demonstrated. The elder brother responded well after a short time on ITI and has no spontaneous bleedings on regular prophylaxis although in a somewhat higher dose than expected. On the other hand, in spite of comparable regimens, the younger brother has so far been resistant to ITI. Moreover, during treatment with extremely high doses of factor IX concentrate he developed nephrotic syndrome which only slowly subsided after treatment with corticosteroids and withdrawal of factor IX.

Anaphylaxis↗
PubMed1979

A genetic variant of factor IX with decreased capacity for Ca2+ binding.

A genetic variant of factor IX is described that behaves identically to PIVKA/IX (the precursor factor IX molecule induced by the absence of vitamin K or presence of vitamin K antagonists, acarboxy factor IX). It shows an increased electrophoretic mobility in the presence of Ca2+, a low affinity for adsorption to A1(OH)3 and a very low specific coagulant activity. This variant of factor IX has been demonstrated in the plasma of a patient with severe haemophilia B and in the plasmas of a number of possible carriers from the probands' pedigree.

Binding Sites↗
PubMed1988

A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred.

We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3' of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5' of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.

Blood Coagulation Tests↗
PubMed1989

Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior.

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.

Base Sequence↗
PubMed1995

Localization of a metal-dependent epitope to the amino terminal residues 33-40 of human factor IX.

Metal binding sites within the Gla domain of vitamin K-dependent coagulation factors have been divided into nonspecific metal sites and calcium-specific sites. We demonstrate here that five residues within the Gla domain of factor IX are responsible for the reactivity with the metal-dependent factor IX monoclonal antibody, A-7. First we demonstrate that modifying any one of three residues within this site in factor IX abolishes the binding of A-7. To confirm the specificity of the antibody, the Gla domain of factor VII was changed at residues 32, 33, 34, 38 and 39 to the homologous residues of human factor IX. These changes were sufficient to generate a factor VII Gla domain with an A-7 binding site of the same affinity as that in factor IX. The site identified is one of the two major surfaces of the Gla domain and may represent the metal-dependent binding site.

1-Carboxyglutamic Acid↗
PubMed1991

Factor IX Amagasaki: a new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities.

Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. We identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate factor X in the presence of factor VIII, phospholipids, and Ca2+, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311----Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

Amino Acid Sequence↗
PubMed1993

Increased thrombin generation in a child with a combined factor IX and protein C deficiency.

We report a quantitative protein C deficiency combined with a factor IX deficiency in a one-year-old boy. The inheritance of the two deficiency states was independent, the factor IX defect coming from the mother and the protein C defect from the father. Both factor IX activity and antigen were below 1%, and protein C activity as well as antigen were close to 27% of normal values. This association raises a real therapeutic and prognostic question. Protein C deficiency is indeed associated with a significant thrombotic risk and some factor IX concentrates seem to carry a potential thrombogenicity, particularly following infusion of repeated doses. We evaluated in this patient the potential activation of the coagulation system by measuring the levels of prothrombin fragment F1 + 2 at the basal state and after a single administration of 20 U/kg of a high purity factor IX concentrate. We found an unexpected basal activation of the hemostatic system before infusion (F1 + 2 = 1.6 nmol/L), which further increased during 8 hours. Despite the clinical predominant expression of the hemophilic trait, our results seem to assess the biologic prevalence of the protein C deficiency. This emphasizes the need for a careful follow-up after infusions of repeated doses of factor IX, as used during a surgical procedure. Furthermore, this raises the question of the prognosis because the risk of thrombotic manifestations associated with a protein C deficiency increases with age. Finally, these results highlight a part of the in vivo activation process of prothrombin in case of failure of the intrinsic pathway of coagulation. The protein C defect seems to be responsible for an upregulation of the prothrombin activation through the extrinsic pathway.

Factor IX↗
PubMed2001

The distinct roles that Gln-192 and Glu-217 of factor IX play in selectivity for macromolecular substrates and inhibitors.

In this paper, we report functional characterization of positions 192 and 217 (chymotrypsinogen numbering system) in human factor IX and discuss the distinction and similarity of these two sites among the blood coagulation factors. Recombinant factor IXQ192E (residue glutamine at position 192 replaced by glutamic acid), IXQ192K, IXE217D, and IXE217R proteins exhibited 11%, 46%, 39%, and 2% of the wild-type factor IX's clotting activity, respectively. Binding of these variants to factor VIIIa (FVIIIa) was inefficient compared to that of wild-type factor IX, and the dissociation constants doubled for IXQ192E, 3-fold higher for IXQ192K and 4-fold higher for both IXE217D and IXE217R. In the presence of FVIIIa, all variant factor IX hydrolyzed factor X at the catalytic efficiencies correlating with respective clotting activities. However, FVIIIa greatly enhanced the catalytic efficiency of both IXE217 variants to a greater extent (approximately 7 x 10(4)-fold) as compared to its effect on the wild-type factor IXa and the other two IXQ192 variants [by a factor of (1-2) x 10(4)]. Moreover, while both IXQ192 variants demonstrated small substrate selectivity similar to that of wild-type factor IXa, the selectivity of both IXE217 variants was greatly altered. Mutations at position 192 disturbed the interaction of factor IXa with physiological inhibitors. Although all variants formed an SDS-stable complex with antithrombin III (ATIII) equally well in the presence of heparin and were readily inhibited by ATIII in the absence of heparin, activated IXQ192K exhibited a slower stable complex formation with ATIII without heparin. On the other hand, only IXQ192E showed decreased interaction with TFPI. Our results demonstrate that positions 192 and 217 play different roles unique to factor IX in specifying the interaction of factor IX with substrates and inhibitors.

Antithrombin III↗
PubMed1983

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products.

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca2+. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (TM) of 54.5 +/- 0.5 degrees C and a calorimetric enthalpy (delta Hc) of 105 +/- 15 kcal/mol, in the absence of Ca2+. In the presence of Ca2+ concentrations sufficient to saturate its sites on Factor IX, the Tm value is increased to 57.0 +/- 0.5 degrees C and the delta Hc is virtually unchanged. When the activation intermediate, Factor IX alpha, is similarly analyzed in the absence of Ca2+, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca2+, Factor IX alpha displayed thermograms characterized by a TM of 51.0 +/- 0.5 degrees C and a delta Hc of 109 +/- 10 kcal/mol. The activated product, Factor IXa alpha, in the absence of Ca2+ (the values in the presence of saturating Ca2+ are given in parentheses), undergoes thermal denaturation with a TM of 54.5 +/- 0.5 degrees C (57.0 +/- 0.5 degrees C) and a delta Hc of 158 +/- 10 kcal/mol (156 +/- 10 kcal/mol). Similarly, the terminal-activation product, Factor IXa beta, displays a TM of 51.5 +/- 0.5 degrees C (54.0 +/- 0.5 degrees C) and a delta Hc of 85 +/- 5 kcal/mol (126 +/- 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a TM of 54.0 +/- 0.5 degrees C (55.0 +/- 0.5 degrees C) and a delta Hc of 102 +/- 10 kcal/mol (118 +/- 10 kcal/mol), whereas its activated form, Factor Xa beta, possesses a TM of 55.0 +/- 0.5 degrees C (55.0 +/- 0.5 degrees C) and a delta Hc of 92.0 +/- 5 kcal/mol (136 +/- 10 kcal/mol). These studies indicate that, for many of these proteins, Ca2+ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.

Animals↗
PubMed1992

Purified factor IX using monoclonal immunoaffinity technique: clinical trials in hemophilia B and comparison to prothrombin complex concentrates.

Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.14 U/dL (mean +/- SD) increase per 1U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 +/- 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

Antibodies, Monoclonal↗