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PubMed1987

Molecular interactions of the intrinsic activation complex of coagulation: binding of native and activated human factors IX and X to defined phospholipid vesicles.

The assembly of proteins of the intrinsic activation complex has been partially elucidated. In the present study we examine the association of gamma-carboxylated serine proteinase zymogens factors IX and X, and their proteolytically activated counterparts factors IXa and Xa to unilamellar lipid vesicles of defined composition using three types of physical measurement. Utilizing relative light scatter to estimate the dissociation constants for binding in the presence of calcium ions, it appears that factor IXa (0.93 +/- 0.37 microM) may preferentially associate with phospholipids relative to factor IX (0.35 +/- 0.08 microM). In contrast, factor X (0.34 +/- 0.14 microM), the substrate for factor IXa, appears to bind to phospholipid with a higher affinity than factor Xa (0.58 +/- 0.13 microM). These observations are compatible with the hypothesized dynamics where the forward 'traffic' is facilitated by favoring the association of factor IXa with factor X. The dissociation constants were estimated by molecular exclusion chromatography (1.1 - 2.5 microM) and do not reflect these relative and ordered differences in association with lipid vesicles. Quasi-elastic light scatter analyses indicate that each protein appears to saturate the same vesicle surface, consistent with competition for similar surface lipids, although the molecular shell formed by factor Xa (36 A) is smaller, suggesting that it has a different packing on the phospholipid surface than the other proteins (64-79 A). The pattern of preferential affinities for phospholipid is consistent with a kinetically functional forward traffic through the reaction precursors to products, and suggests that these preferential affinities may assist in the ordering of the four proteins in the intrinsic activation complex.

Calcium↗
PubMed1994

The rates and patterns of deletions in the human factor IX gene.

Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of > or = 21 bp) are uncommon. We have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions > 20 bp. Eleven of these are > 2 kb (range > 3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For our sample of 203 independent mutations, we estimate the "baseline" rates of deletional mutation per base pair per generation as a function of size. The rate for large (> 2 kb) deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (< 20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversions, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA.

Base Sequence↗
PubMed2000

Decline of factor VIII and factor IX inhibitors during long-term treatment with NovoSeven.

Recombinant factor VIIa (rFVIIa) (NovoSeveng) is used to treat bleeding episodes in hemophilia A and B patients with inhibitor antibodies against factor VIII (FVIII) and factor IX. rFVIIIa has been studied in home treatment of mild-to-moderate joint, muscle, and mucocutaneous bleeds to assess safety and efficacy. Treatment with other factor concentrates was allowed according to treating physician's judgment. Blood samples were drawn before study start and after 6 and 12 months. It has thus been possible to follow the inhibitor titres during this period. Analyses of 53 patients (49 hemophilia A, four hemophilia B) showed inhibitor levels up to 1,208 BU/ml before study start. Based on the first analysis, hemophilia A patients were divided into high responders (> 5 BU/ml; 28 patients), low responders (> 1 and < 5 BU/ml; 15 patients) and very low responders (< or = 1 BU/ml; six patients). In high responders receiving rFVIIa as only treatment, FVIII inhibitor titre decreased to one-third of the initial level. For high responders receiving other factor treatments such as FVIII or prothrombin complex concentrates, inhibitor titre remained unchanged. Titres for low responders and very low responders remained unchanged independent of treatment. Thus, when rFVIIa is used as the only coagulation factor to treat hemophilia A/B high-responder inhibitor patients, inhibitor level declines significantly.

Factor IX↗
PubMed1999

Gene therapy expression vectors based on the clotting Factor IX promoter.

The liver is one of the prime targets for gene therapy, and the correction of defects in a variety of clotting factor genes is one of the main goals of liver-directed therapies. The use of transcriptional regulatory elements derived from these genes may provide for the optimal expression of transduced genes. We have applied our knowledge of the promoter structure of the clotting Factor IX gene to design optimized expression vectors for use in gene therapy. The activity of the proximal promoter has been augmented by the introduction of a multimerized upstream site which we have previously shown to be a prime regulator of the pro- moter. Introduction of this element increases promoter activity at least 20-fold over the proximal promoter alone when assayed in the human liver cell line Hep G2. This optimized promoter is significantly more active than the SV40 enhancer/early promoter. The expression of the optimized Factor IX promoter is also more persistent in the short term. The inclusion of a liver-specific locus control region, derived from the apolipoprotein E/C locus, did not further augment expression levels. These Factor IX vectors also exhibit a high degree of tissue specificity, as measured by transfection into breast and muscle cell lines.

Cell Line↗
PubMed1996

Characterization of gamma-carboxyglutamic acid residue 21 of human factor IX.

We investigated the functional role of gamma-carboxyglutamic acid (Gla) residue 21 of human factor IX, using site-directed mutagenesis to change the glutamic acid residue to aspartic acid (FIX21D). FIX21D had reduced activity in an activated partial thromboplastin time (aPTT) assay and was activated by factor XIa more slowly than wild-type factor IX (FIXwt). FIX21D underwent normal, two-stage calcium-dependent intrinsic fluorescence quenching, indicating that a folding event similar to that seen in FIXwt occurred upon the addition of calcium ions. Antibody A-7, which recognizes factor IX-specific residues at positions 33-40, bound FIX21D as well as FIXwt; however, the calcium-specific monoclonal antibody, JK-IX-2, whose epitope includes residues 1 and 22, did not recognize FIX21D. FIX21D bound phosphatidylserine/phosphatidylcholine (PS/PC) vesicles with Kd approximately 10-fold greater than FIXwt, as measured by a fluorescence light scattering assay. Finally, although FIXwt binds endothelial cells with a Kd of 2.8 nM, FIX21D did not bind endothelial cells. Molecular modeling simulations of FIXwt and FIX21D indicate that mutating Gla 21 to Asp causes structural changes in residues 3-5 and 8-10, as well as in two exposed calcium ions, consistent with the reduced function of FIX21D. Immunological and intrinsic fluorescence quenching assays and the molecular dynamics simulations suggest normal folding in the C-terminal region of the Gla domain. Thus we hypothesize the FIX21D has reduced JK-IX-2 and phospholipid and endothelial cell binding due to localized structural changes in residues 3-10 and the exposed calcium ions. Our study suggests that the Gla 21 to Asp mutation disrupts function in the N-terminal region of the Gla domain without affecting structure in the C-terminal Gla domain region.

1-Carboxyglutamic Acid↗
PubMed1995

Structure of the metal-free gamma-carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy.

The gamma-carboxyglutamic acid-rich domain of blood coagulation Factor IX is required for the binding of the protein to phospholipid membranes. To investigate the three-dimensional structure of this domain, a synthetic peptide corresponding to residues 1-47 of Factor IX was studied by 1H NMR spectroscopy. In the absence of metal ions, the proton chemical shift dispersion in the one-dimensional NMR spectrum indicated that the peptide contains regular structural elements. Upon the addition of Ca(II) or Mg(II), large chemical shift changes were observed in the amide proton and methyl proton regions of the spectrum, consistent with the conformational transitions that metal ions are known to induce in native Factor IX. The apopeptide was studied by two-dimensional NMR spectroscopy at 500 MHz to determine its solution structure. Protons were assigned using total correlation spectroscopy, nuclear Overhauser effect spectroscopy, and double quantum-filtered correlation spectroscopy experiments. Intensities of cross-peaks in the nuclear Overhauser effect spectrum were used to generate a set of interproton distance restraints. The structure of the apopeptide was then calculated using distance geometry methods. There are three structural elements in the apopeptide that are linked by a flexible polypeptide backbone. These elements include a short amino-terminal tetrapeptide loop (amino acids 6-9), the disulfide-containing hexapeptide loop (amino acids 18-23), and a carboxyl-terminal alpha helix (amino acids 37-46). Amide hydrogen exchange kinetics indicate that the majority of the peptide is solvent accessible, except in the carboxyl-terminal element. The structured regions in the apopeptide are insufficient to support phospholipid binding, indicating the importance of additional structural features in the Ca(II)-stabilized conformer.

1-Carboxyglutamic Acid↗
PubMed1996

Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX.

The blood coagulation and regulatory proteins that contain gamma-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to gamma-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca2+, Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

Amino Acid Sequence↗
PubMed1994

Markers of hypercoagulability in patients with hemophilia B given repeated, large doses of factor IX concentrates during and after surgery.

Purer factor IX (FIX) concentrates have been produced for the treatment of hemophilia B in the attempt to reduce the risk of thrombotic complications associated with the use of prothrombin complex concentrates. To evaluate ex vivo whether or not FIX concentrates activate the coagulation system in conditions associated with a high risk for thrombosis, we measured markers of hypercoagulability in 10 patients with hemophilia B who underwent surgery, mainly orthopedic procedures, covered by multiple concentrate infusions (40-80 U/kg/day). Postinfusion plasma levels of prothrombin fragment 1 + 2 and factor X activation peptide did not differ significantly from the presurgical levels, neither before nor after each concentrate dose. Therefore, it appears that prolonged treatment of patients with hemophilia B undergoing high risk surgical procedures with high doses of FIX concentrate does not cause systemic activation of coagulation. This suggests that purified FIX concentrates are preferable to prothrombin complex concentrates for conditions associated with an increased risk of thrombosis.

Adolescent↗
PubMedDate not supplied

Test method for the absence of active clotting factors in dried human blood coagulation factor IX complex.

Adverse effects possibly due to the active clotting factors are sometimes reported upon administration of some preparations of Factor IX complex having conformed to the specifications in the Japanese Minimum Requirements for Biological Products. The author, therefore, proposed the revision of the test method for the absence of active clotting factors in the Minimum Requirements by introducing calcium ion into the test medium.

Calcium↗
PubMed1992

Circulating human or canine factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle.

We have used retroviral vectors to introduce human or canine factor IX cDNAs into cultured primary murine and canine myoblasts and into the established murine myoblast cell line C2C12. In all cases, the stably infected cells produced biologically active factor IX in culture and secreted detectable amounts into the culture medium both before and after differentiation of the cells into myotubes. Myoblasts and differentiated myotubes are therefore capable of performing all the posttranslational modifications of the coagulation factor required for biological activity. We have grafted the genetically modified myoblasts into skeletal muscles of nude mice and have detected stable levels of circulating human factor IX for up to two months after grafting. We propose that grafting genetically modified primary myoblasts or established myoblast cell lines into skeletal muscle may represent a useful approach to gene therapy for a variety of genetic diseases, including intrinsic muscle disease and defects in circulating proteins as in the hemophilias.

Animals↗
PubMed2000

Sensitivity of the reaction time of the resonance thrombogram for factor VIII:C and factor IX deficiencies in the blood of dogs with haemophilia A or B.

The sensitivity of the reaction time of the resonance thrombogram (RTG-r) was examined for two instruments based on 105 canine samples with a reduced factor VIII:C activity and 26 samples with a reduced factor IX activity. These samples were taken from dogs suffering from haemophilia A (n=18) or B (n=3) before and at different times after the substitution therapy with fresh frozen plasma. The RTG-r sensitivity was low; with reference to the total of all examined samples, it was between 0.39 and 0.65 for the factor VIII:C activity and between 0.46 and 0.62 for the factor IX activity, depending on the instrument and the testing procedure used. Low sensitivity was shown especially by the fact that even some of the samples with a factor VIII:C activity < 5% showed false negative results. Moreover, the correlation found between RTG-r and factor VIII:C activity (Spearman's rank correlation coefficient, r(S)=-0.556 to -0.700) as well as between factor IX activity and RTG-r (r(S)=-0.380 to -0.612) was only moderate. The low sensitivity of the RTG towards a reduced activity of the haemophilia factors VIII:C and IX contradicts the exclusive use of the RTG for monitoring haemophilic dogs as well as a global screening test for the haemostasis of dogs.

Animals↗
PubMed1990

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.

Autoantibodies↗
PubMed1999

Antithrombotic efficacy of a novel murine antihuman factor IX antibody in rats.

A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg x kg(-1) x h(-1)), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl(3)-patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U x kg(-1) x min(-1)), whereas the latter rendered the blood incoagulable (aPTT>1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18+/-0.49 to 1.80 +/-0.3 mg, P<0.001), increasing vessel patency time (from 14.9+/-0.9 minutes to 58.3+/-1.7 minutes, P<0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle- versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (P<0.01), extended vessel patency time (P<0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8+/-0.7 to 3.3 +/-0.6 mL (P<0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.

Anesthesia↗
PubMed1999

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Animals↗
PubMed1976

Incidence of viral hepatitis after administration of factor IX concentrates.

A high frequency of viral hepatitis has been reported after treatment with the human factor IX concentrate 'Konyne'. Clinical trials with 'Konyne' and a similar factor IX concentrate, called 'Preconativ', was started in Sweden 1969. During the first 2 years, 26 patients were treated with either one or both preparations. Nine patients developed viral hepatitis within 6 months after treatment. 'Preconativ' alone was introduced on the Swedish market in 1971. During the period 1971-1974, another 26 hemophiliacs were treated but only two cases of hepatitis have occurred. Selection of donors and screening for hepatitis B surface antigen in donor blood used for the manufacturing of 'Preconativ', might be contributing factors to this low hepatitis incidence.

Factor IX↗
PubMed1984

Regional localization on the human X chromosome and polymorphism of the coagulation factor IX gene (hemophilia B locus).

Hemophilia B is an X-linked disease caused by a functional deficiency in coagulation factor IX. A cDNA clone corresponding to factor IX has been used to detect homologous sequences in the human genome. All DNA fragments hybridizing to the probe, under medium- or high-stringency conditions, are X-linked, and the patterns obtained suggest that a single large (greater than or equal to 20 kilobases) gene is detected. The gene has been mapped to the q26-q27 region of the long arm of the X chromosome by hybridization to DNA from a panel of human-mouse hybrid cell lines. A search for restriction fragment length polymorphisms using seven restriction enzymes has led to the detection of a Taq I polymorphism, with allelic frequencies of about 0.71 and 0.29. This genetic marker should be useful for the detection of carriers of the hemophilia B trait and for prenatal diagnosis in informative families and, more generally, for the establishment of a linkage map of the human X chromosome.

Chromosome Mapping↗
PubMed1996

Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B.

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

Alginates↗
PubMed2000

Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro.

We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.

3' Untranslated Regions↗