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PubMed1996

High purity factor IX and prothrombin complex concentrate (PCC): pharmacokinetics and evidence that factor IXa is the thrombogenic trigger in PCC.

Recent studies using assays for surrogate markers of thrombogenicity in man have demonstrated that activation of the coagulation system occurs following infusion of clinical doses of prothrombin complex concentrates (PCC) but not after the same doses of high-purity factor IX concentrates (HP-FIX) in patients with haemophilia B. Here we have investigated the mechanism of such thrombogenesis by applying assays that detect early-through to late-events in coagulation system activation in a pharmacokinetic cross-over study of 50 IU/kg PCC and a new HP-FIX product in haemophilia B patients. Satisfactory recoveries and half-lives were observed for both concentrates. HP-FIX caused no increases in thrombin-antithrombin III complex (TAT), prothrombin activation peptide fragment F1+2 (F1+2), factor X activation peptide (FXAP) or factor VIIa (FVIIa). In contrast the same dose of factor IX in the form of PCC was followed by significant increases over pre-infusion levels of TAT, F1+2 and FXAP, but not FVIIa. Elevations of FIXAP occurred after both HP-FIX and PCC but did not reach normal levels and were attributed to normalisation of the FIX concentration in those patients whose levels of FIXAP were initially low. We conclude that the thrombogenic trigger associated with PCC infusion occurs at the level of factor X activation. In the absence of any increase in FVIIa, we would attribute this to the likely presence of FIXa in the PCC.

Adult↗
PubMed1996

Safety and efficacy of purified factor IX concentrate and antifibrinolytic agents for dental extractions in hemophilia B.

This study evaluated the safety and efficacy of combined treatment with epsilon-aminocaproic acid or tranexamic acid and monoclonal antibody purified factor IX (MAb factor IX) for prophylaxis against bleeding in eight hemophilia B patients undergoing nine dental extraction procedures. All patients achieved excellent hemostasis without clinical evidence of thrombosis. There were no significant changes in hemoglobin or hematocrit or in markers of hemostatic system activation (prothrombin fragment F1+2, fibrinopeptide A, and fragment B beta 15-42) after surgery. Thus, a highly purified factor IX concentrate and antifibrinolytic therapy can be effectively and safely combined in hemophilia B patients undergoing dental extractions.

Adult↗
PubMed1985

Characterisation of the tolerant state in a patient with haemophilia B after removal of high-titre factor IX antibodies.

In a patient with severe haemophilia B and antibodies against factor IX in high titre, and known for many years to be a really high responder, it was possible to suppress the secondary antibody response after treatment with high doses of intravenous IgG (Gammonativ, KabiVitrum AB) combined with factor IX and cyclophosphamide. After 2 further treatments, including IgG, a total disappearance of the IX:C inhibitor was noted. The patient now treats himself at home with weekly infusions of only factor IX concentrate. Survival of IX:C is normal, though IX antigen (IX:Ag), as measured with the original antibody in an immunoradiometric assay, persists for at least a week after concentrate infusion. We have not been able to demonstrate an antiidiotypic antibody. Instead the antigenic factor IX material circulates complexed to a 'new' antibody without anticoagulant activity. Theoretically at least, the persistence of such immune complexes may be important for sustaining the patient's tolerance to the anticoagulant antibody's epitope. The initial IgG treatment appears as a crucial factor for induction of tolerance in this case.

Adult↗
PubMed2001

Characterization of clotting factor IX in plasma-derived preparations by electrophoretic techniques.

Clotting factor IX preparations from human plasma (pdFIX) have been characterized using electrophoretic methods like sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Factor IX prior to and after activation with factor XIa was separated by one- and two-dimensional polyacrylamide gel electrophoresis and on isoelectric focusing gels. The main differences between the band patterns of the two pdFIX preparations are due to their purity. Vitronectin was identified by immunological techniques as major accompanying plasma protein, separated from factor IX and characterized by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed1999

Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity.

We used a first-generation adenovirus vector (AVC3FIX5) to assess whether human factor IX could be expressed and detected in the rhesus macaque, which we have shown does not make high-titer antibodies to human factor IX protein. Three animals received 1 x 10(10) to 1 x 10(11) plaque-forming units per kilogram by intravenous injection. Human factor IX was present within 24 hours of vector administration and peaked 4 days later at 4,000 ng/mL in the high-dose recipient, and lower levels were seen in the intermediate-dose recipient. No human factor IX was detected in the low-dose recipient's plasma. Serum cytokine analysis and early hypoferremia suggested a dose-dependent acute-phase response to the vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared concomitant with high-titer antihuman factor IX antibody development. There was substantial, dose-dependent, dose-limiting liver toxicity that was manifest as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. Of particular interest was prolongation of the thrombin clotting time, an indicator of decreased fibrinogen or fibrinogen dysfunction. All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. Our data suggest a narrow therapeutic window for first-generation adenovirus-mediated gene transfer. The development of antihuman factor IX antibodies and abnormalities of fibrinogen in the rhesus macaque is of concern for application of adenovirus (or other viral) vectors to hemophilia gene therapy.

Adenoviridae↗
PubMed1996

The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX.

In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.

Amino Acid Sequence↗
PubMed1983

Monoclonal antibody to an epitope on the heavy chain of factor IX missing in three hemophilia-B patients.

A murine hybridoma cell line that produces a monoclonal IgG1 antibody to human factor IX was established to provide a conformational probe for the clotting factor and its genetic variants. The antibody inhibited factor IX procoagulant activity, but did not appreciably interfere with the cleavage of factor IX by factor XIa nor with the binding of antithrombin-III-heparin complex to factor IXa. The antigen-solid-phase-antibody complex could be readily dissociated by relatively low concentrations of guanidine or sodium dodecyl sulfate, but only partially by high concentrations of urea. After gel electrophoresis and blotting of reduced samples of factor IXa, the antibody bound exclusively to the heavy chain. Sensitive immunoradiometric assays were developed using insolubilized monoclonal or polyclonal antibodies. Bovine factor IX had little cross-reactivity with the monoclonal antibody. Of 55 patient samples representing different pedigrees with hemophilia-B, antigen levels by the two assays were in excellent agreement in 49. There were 2 severely affected patients whose levels were too low to quantitate in the monoclonal antibody assay. A third, who had the lowest level of all by polyclonal antibody testing, and 3 less severely affected patients had no detectable antigen in the monoclonal antibody assay system (less than 0.03 U/dl). The latter 3 had at least 100-500 times as much antigen by polyclonal antibody testing. It is proposed that these 3 individuals have structural defects involving the epitope recognized by the monoclonal antibody and that they are due to amino acid substitutions between residues 188 through 359. Furthermore, it is suggested the substitutions lead to abnormal kinetic properties.

Antibodies, Monoclonal↗
PubMed1986

A de novo intragenic deletion of the potential EGF domain of the factor IX gene in a family with severe hemophilia B.

We have studied a family of three patients who were severely afflicted with hemophilia B without inhibitor for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (30% of normal) factor IX antigen. DNA hybridization analysis demonstrated that these patients had a partial intragenic deletion in their factor IX gene. This 2.8-kb deletion included exon d and the surrounding sequences. This exon codes for the amino acid sequence from No. 47 through 84 of the factor IX protein and contains its first potential EGF domain; the de novo occurrence of the mutation in the grandfather's germ cells was established by linkage analysis. This specific gene has been named F IXStrasbourg.

Chromosome Deletion↗
PubMedDate not supplied

[Methylation of the factor IX gene--a basic reason for the mutation causing hemophilia B].

The analysis of 750 mutations in the gene of blood coagulation factor IX has been made for 806 patients with haemophilia B. It has been found that 40% of all point mutations take place in 11 "hot spots", i.e., methylated CG sites, and are CG-->TG or CA transitions. Mechanism proposed explains the high rate of these transitions by m5C deamination during replicative DNA methylation and by mistakes of G/T-repair. This is why mutations constantly occur de novo in the factor IX gene, and haemophilia B maintains on a high level. Asymmetry of C-->T and G-->A transitions is found in some CG sites at different DNA chains, and is related to occurring "missense" mutations, which usually escape from the detection. Summing up these substitutions, CG methylation of the factor IX gene contributes to 50% of all point mutations. Thus, mutations in the CG sites take place in overall 48 times more frequently than in other sites of the gene. It has been found that at least 35 new CG sites are a result of sporadic mutations, and methylation and mutations of these sites may cause 14% of substitutions in the factor IX gene. The "hot spots" for T-->C transitions in codon of Ile397 can be a result both of "founder effect" and of recessive mutations in such CG site in mother's parents. It is shown that methylated CTCG sites, as well as G/T-repair mistakes, can be potential sources of 5.4% of mutations in the gene. Thus, from 50 to 70% of all mutations may be the result of the gene methylation in human genome. The analysis of duplex frequencies in the factor IX gene has shown that the loss of 60 CG sites and the accumulation of 8% of mutations may be a result of "fossil" CG methylation. The remained 20 CG sites are located in the codons of those amino acids which positions are more critical for the factor IX activity. It has been proposed that the most radical way in haemophilia B therapy can be a protection of the factor IX gene from methylation in the human genome.

Factor IX↗
PubMed1999

Surface loop 199-204 in blood coagulation factor IX is a cofactor-dependent site involved in macromolecular substrate interaction.

In factor IX residues 199-204 encompass one of six surface loops bordering its substrate-binding groove. To investigate the contribution of this loop to human factor IX function, a series of chimeric factor IX variants was constructed, in which residues 199-204 were replaced by the corresponding sequence of factor VII, factor X, or prothrombin. The immunopurified and activated chimeras were indistinguishable from normal factor IXa in hydrolyzing a small synthetic substrate, indicating that this region is not involved in the interaction with substrate residues on the N-terminal side of the scissile bond. In contrast, replacement of loop 199-204 resulted in a 5-25-fold reduction in reactivity toward the macromolecular substrate factor X. This reduction was due to a combination of increased K(m) and reduced k(cat). In the presence of factor VIIIa the impaired reactivity toward factor X was largely restored for all factor IXa variants, resulting in a more pronounced stimulation by factor VIIIa compared with normal factor IXa (3 to 5 x 10(4)-fold versus 5 x 10(3)-fold). Inhibition by antithrombin was only slightly affected for the factor IXa variant with the prothrombin loop sequence, whereas factor IXa variants containing the analogous residues of factor VII or factor X were virtually insensitive to antithrombin inhibition. In the presence of heparin, however, all chimeric factor IXa variants formed complexes with antithrombin. Thus the cofactors heparin and factor VIIIa have in common that they both alleviate the deleterious effects of mutations in the factor IX loop 199-204. Collectively, our data demonstrate that loop 199-204 plays an important role in the interaction of factor IXa with macromolecular substrates.

Antithrombins↗
PubMed2002

Does inflammatory proteolytic activity contribute to the increased factor IX activation peptide in men at high risk of coronary heart disease? A preliminary study.

In the Second Northwick Park Heart Study, the activation peptides of factor IX (FIXpep) and factor X (FXpep) were measured in 1261 middle-aged men by double-antibody radioimmunoassay. During follow-up 147 men who had a first coronary heart disease (CHD) event were found to have had an increased FIXpep (p = 0.003) and a reduced FXpep (p = 0.05) at baseline compared with those remaining CHD-free (controls). Plasma FIXpep and FXpep were positively associated, but the rate of rise in FIXpep with increasing FXpep was higher in cases than controls (p for interaction = 0.01). In a sample of 87 controls, FIXpep was positively and independently related to the concentrations of a polymorphonuclear-specific fibrinogen degradation product (p = 0.036) and FXpep (p = 0.004), but in larger samples no statistically significant associations were found either with C-reactive protein or with fibrinogen concentration. The findings suggested that the increased FIXpep in men at high CHD-risk may have been partly due to the generation of factor IX inactivation peptides by inflammatory proteolysis and their recognition together with true FIXpep in the radioimmunoassay. Direct evidence for this hypothesis requires development of assays for human elastase-specific factor IX inactivation peptides.

Case-Control Studies↗
PubMed2004

Lysine 5 and phenylalanine 9 of the factor IX omega-loop interact with phosphatidylserine in a membrane-mimetic environment.

The binding of factor IX to cell membranes requires a structured N-terminal omega-loop conformation that exposes hydrophobic residues for a highly regulated interaction with a phospholipid. We hypothesized that a peptide comprised of amino acids Gly4-Gln11 of factor IX (fIX(G4)(-)(Q11)) and constrained by an engineered disulfide bond would assume the native factor IX omega-loop conformation in the absence of Ca(2+). The small size and freedom from aggregation-inducing calcium interactions would make fIX(G4)(-)(Q11) suitable for structural studies for eliciting details about phospholipid interactions. fIX(G4)(-)(Q11) competes with factor IXa for binding sites on phosphatidylserine-containing membranes with a K(i) of 11 microM and inhibits the activation of factor X by the factor VIIIa-IXa complex with a K(i) of 285 microM. The NMR structure of fIX(G4)(-)(Q11) reveals an omega-loop backbone fold and side chain orientation similar to those found in the calcium-bound factor IX Gla domain, FIX(1-47)-Ca(2+). Dicaproylphosphatidylserine (C(6)PS) induces HN, Halpha backbone, and Hbeta chemical shift perturbations at residues Lys5, Leu6, Phe9, and Val10 of fIX(G4)(-)(Q11), while selectively protecting the NHzeta side chain resonance of Lys5 from solvent exchange. NOEs between the aromatic ring protons of Phe9 and specific acyl chain protons of C(6)PS indicate that these phosphatidylserine protons reside 3-6 A from Phe9. Stabilization of the phosphoserine headgroup and glycerol backbone of C(6)PS identifies that phosphatidylserine is in a protected environment that is spatially juxtaposed with fIX(G4)(-)(Q11). Together, these data demonstrate that Lys5, Leu6, Phe9, and Val10 preferentially interact with C(6)PS and allow us to correlate known hemophilia B mutations of factor IX at Lys5 or Phe9 with impaired phosphatidylserine interaction.

Calcium↗
PubMed2004

An antibody specific for coagulation factor IX enhances the activity of the intrinsic factor X-activating complex.

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (kcat) of the enzyme complex increased 2- to 3-fold whereas the Km for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.

Animals↗
PubMed1985

The role of factor VIII in the activation of human blood coagulation factor X by activated factor IX.

The role of factor VIII in the activation of human factor X by factor IXa, Ca2+ and phospholipid has been investigated. Factor VIII stimulated the factor Xa formation after activation by factor Xa or thrombin; the activity of thrombin-activated factor VIII was about 4-fold that of factor Xa-activated factor VIII. The isolated procoagulant moiety of the factor VIII complex behaved identically to the complete complex, whereas the von Willebrand factor moiety did not participate in the factor Xa formation. Thrombin-activated factor VIII complex (factor VIIIa) was used to study the effect of factor VIIIa in kinetic experiments. The results revealed a complex kinetic behaviour, including substrate inhibition and non-linearity of the reaction rate with the enzyme concentration. Using previously obtained insight into the kinetics of factor X activation in the absence of factor VIII, the results were found to support the hypothesis that factor VIIIa participates in the factor Xa formation in a complex with phospholipid-bound factor IXa; the formation of the factor VIIIa-factor IXa complex then increases the catalytic efficiency of the factor IXa by 500-fold.

Blood Coagulation↗
PubMed1994

Large-scale preparation and biochemical characterization of a new high purity factor IX concentrate prepared by metal chelate affinity chromatography.

Metal chelate affinity chromatography on copper-charged Chelating Sepharose has been used to purify a factor IX concentrate from 4,000- to 5,000-kg pools of human plasma, with an overall yield of 194 IU/kg. Unwanted proteins and solvent-detergent reagents added to inactivate lipid-enveloped viruses were removed during the chromatographic step. The freeze-dried product was > 80% pure factor IX with a mean specific activity of > 160 IU/mg protein. The concentrate showed no evidence of clotting factor activation by in vitro tests for potential thrombogenicity or by direct assay for activated factor IX. The concentrate did not exhibit proteolytic activity against a range of synthetic peptide chromogenic substrates. Full functional factor IX activity was retained and there was no evidence of protein degradation. Metal chelate affinity chromatography therefore appears to present less physicochemical challenge to the protein than other factor IX purification methods, while allowing the preparation of a clinical factor IX concentrate at a large scale.

Chelating Agents↗
PubMed1989

Defective propeptide processing and abnormal activation underlie the molecular pathology of factor IX Troed-y-Rhiw.

Affected members of a South Wales haemophilia B family (from Troed-y-Rhiw) were shown by Western blotting and immunoperoxidase detection to have a factor IX molecule of higher than normal molecular weight which also shows impaired calcium binding. Gene cloning and DNA sequencing revealed the same arginine to glutamine mutation at position -4 of the propeptide that has been found in two previously described factor IX variants which circulate with the propeptide still attached. The mutation also abolishes a HaeIII restriction enzyme recognition site. A potential carrier was shown to be normal both by Western blotting and DNA studies. The way in which the attached propeptide interferes with normal factor IX function was investigated by activation studies with crude normal and patient factor IX Troed-y-Rhiw preparations using Western blotting and detection with iodinated immunopurified polyclonal antifactor IX serum. We demonstrate that the -4 mutation appears to block cleavage between the Arg145-Ala146 peptide bond in the activation peptide, thus preventing the normal activation of factor IX Troed-y-Rhiw. A small amount of normally processed factor IX is produced, implying that the -4 mutation does not completely prevent propeptide cleavage, thus accounting for the low levels of factor IX activity measured in the plasma of affected family members.

Base Sequence↗