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PubMed2002

Site-specific genomic integration produces therapeutic Factor IX levels in mice.

We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.

Animals↗
PubMed1993

Structural analysis of O-linked sugar chains in human blood clotting factor IX.

Type and structural analysis of O-linked sugar chains in human blood clotting factor IX was performed by the pyridylamination method developed for O-linked sugar chains [Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126]. O- and N-linked sugar chains were released with hydrazine, and then N-acetylated, followed by pyridylamination. The type of sugar chain was determined by reducing-end analysis of the pyridylaminated (PA-) sugar chains. Sugar chains with PA-GalNAc at the reducing terminal and that with PA-Fuc [Nishimura, H. et al. (1992) J. Biol. Chem. 267, 17520-17525] were obtained besides known sugar chains with PA-Glc from the Xyl-Glc-Ser type and those with PA-GlcNAc from asparagine-linked sugar chains. The sugar chains with PA-GalNAc were identified as mono- and disialyl Gal beta 1-3GalNAc by two-dimensional HPLC mapping. The structure of the sugar chain with PA-Fuc was Neu5Ac alpha 2-6Gal beta 1-4GlcNAc beta 1-3Fuc, as determined by exoglycosidase digestion, methylation analysis, and Smith degradation.

Carbohydrate Conformation↗
PubMed1992

Physical and biochemical characterization of five commercial resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation factor IX (FIX) to homogeneity using monoclonal antibodies (MAb) that bind FIX in the presence of divalent cations. The MAb is immobilized on Sepharose CL2B, a soft gel with a low pressure tolerance as well as poor large-scale performance characteristics, including low reusability, and resin crumbling and deterioration. In this study, we examined several commercially available resin supports. Aside from Sepharose CL2B, we studied two other cross-linked agaroses, as well as two synthetic polymer supports. Immobilization chemistries included cyanogen bromide activation of agarose, 2-fluoro-2 methylpyridinium toluene-4-sulfonate activation of one of the synthetic polymer as well as aldehyde group reduction by NaCNBH3 to form secondary amine linkages on one of the cross-linked agaroses. To determine the feasibility of using the resins in large-scale immunoaffinity chromatographic purification of FIX, we studied physical and biochemical properties of the resins. The physical characteristics studied included the crushability of the resins under pressure as well as ability to support increasing flow-rates at increasing pressures. The biochemical examination of the various resins focused on efficiency of antigen capture by the immobilized antibody ligand and the effect of flow-rate on MAb efficiency, where we found that very low flow-rates slightly increased the capacity of the MAb. The results demonstrate a straightforward method of assessing the feasibility of using particular resins in large-scale affinity purification.

Antibodies, Monoclonal↗
PubMed1994

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.

Amino Acid Sequence↗
PubMed2002

The limitation of factor IX coagulant activity determination in the diagnosis of hemophilia B carriers.

A preliminary study of factor IX coagulant activity (FIX:C) for determining hemophilia B carriers was conducted at the Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital in Bangkok. Twenty-eight females (8 obligate, 20 potential carriers) from 17 hemophilia B families were enrolled in the study. Additionally, 25 normal females were included. They were not pregnant and not using oral contraceptives. Then, three cut-off levels of FIX:C including 50 per cent which was the commonly used level; 57 per cent which was the mean-2 SD of normal females and 75 per cent which was the level reported by Knobe and Ljung in 1999 were used for the diagnosis of hemophilia B carriers. The sensitivities of these three cut-off levels were 12.5 per cent (1/8) for 50 per cent, 37.5 per cent (3/8) for 57 per cent and 50 per cent (4/8) for 75 per cent. Also, the specificities were 100 per cent (25/25) for both 50 and 57 per cent, and 96 per cent (24/25) for 75 per cent. Although the low cut-off levels of 50 per cent and 57 per cent had low sensitivities, they yielded a high specificity (100%) compared to the higher level of 75 per cent. In the present study, the sensitivity of the cut-off level at 75 per cent was much lower than that of the study by Knobe and Ljung (93%) since the presented sample size of obligate carriers was rather small. So, enrollment of more subjects should be further carried out. In conclusion, FIX:C determination alone showed a limitation in the diagnosis of hemophilia B carriers. The addition of genetic analysis of linkage analysis or mutation detection is required for a definite diagnosis.

Adolescent↗
PubMed1988

Orthotopic liver transplantation totally corrects factor IX deficiency in hemophilia B.

Orthotopic liver transplantation has been performed for a growing range of liver-based inborn errors of metabolism. Previous authors have documented that liver transplantation can reverse the coagulation defect of hemophilia A. In this article we report total correction of factor IX deficiency in a patient with hemophilia B and blood product-related liver disease. Intraoperative bleeding was not excessive, and the donor liver produced normal amounts of factor IX immediately.

Adolescent↗
PubMed1992

Biology of factor IX.

Hemophilia B, one of two common hereditary bleeding disorders, is caused by a deficiency of factor IX in the circulation. Molecular mechanisms of hemophilia B are highly heterogeneous including gene deletions, insertions, complex rearrangements, and a large number of point mutations. Currently, hemophilia B is treated by plasma protein replacement therapy. This therapy is effective but exposes patients to possible side effects and complications such as infection of blood-borne pathogens including hepatitis viruses and HIV-1. Intensive efforts to develop alternative, safer therapies for hemophilia B, including somatic gene therapy, are now under way.

Amino Acid Sequence↗
PubMed2004

Cryoprecipitate-removed plasma 'cryo-removed plasma' as a source of factor IX in the treatment of haemophilia B.

Fresh frozen plasma (FFP) is the main source of factor IX (FIX) in the treatment of bleeding episodes of haemophilia B in the Philippines. Cryoprecipitate-removed plasma otherwise known in the Philippines as cryosupernate, is a by-product of cryoprecipitate preparation. These blood products expire in storage or are just thrown- away because of less demand for clinical use. By theory, this product should have almost the same amount of FIX as in FFP, therefore can be used in the treatment of haemophilia B. There is no local data on the actual FIX content of the cryoprecipitate-removed plasma. Hence, the authors established these data to support the use of this product. Eighty-three bags of cryoprecipitate-removed plasma received from three different blood banks in Manila, Philippines were tested for FIX activity using an activated partial thromboplastin time (APTT)-based one-stage FIX assay. The FIX content in each bag of cryoprecipitate-removed plasma was calculated by multiplying its volume in mL with that of FIX activity per mL of plasma measured in vitro. The total mean FIX content per bag was 212.20 U (+/-88.98) exceeding the contents set by the American Association of Blood Banks (AABB, 70-90 U). The mean FIX activity per bag was 127.62% (+/-38.23) with the mean volume of 164.28 mL (+/-52.23). Statistically significant difference on volume (P = 0.000) was found across the three sources resulting to a significant variation of the actual FIX content (P = 0.000).

Factor IX↗
PubMed2001

Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo.

Naked DNA transfer of a high-expressing human factor IX (hFIX) plasmid yielded long-term (over 1 1/2 years) and therapeutic-level (0.5-2 microg/ml) gene expression of hFIX from mouse livers. The expression cassette contained a hepatic locus control region from the ApoE gene locus, an alpha1-anti-trypsin promoter, hFIX cDNA, a portion of the hFIX first intron, and a bovine growth hormone polyadenylation signal. In contrast, a hFIX plasmid containing the expression cassette without effective regulatory elements produced initially low-level gene expression that rapidly declined to undetectable levels. Southern analyses of the cellular DNA indicated that the majority of the input genome from either vector persisted as episomal forms of the original plasmids. Together with RT-PCR analyses of the transcripts, these data indicated that at least two processes are critical for sustained gene expression: persistence of vector DNA and transcriptional/posttranscriptional activation. Liver regeneration after partial hepatectomy resulted in a significant decline in transgene expression, further suggestive of decreased episomal plasmid maintenance rather than transgene integration. Transaminase levels and liver histology showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage (< 5% of hepatocytes), which was rapidly repaired within 3 to 10 days and resulted thereafter in histologically normal tissue. No significant differences were observed between rapid injection of plasmid and saline control solutions. Transient, very low level antibodies directed against hFIX did not prevent the circulation of therapeutic levels of the protein. Gene transfer of hFIX plasmid DNA into liver elicited neither transgene-specific cytotoxic effect nor long-term toxicity. These results demonstrate that long-term expression of hFIX can be achieved by nonviral plasmid transfer and suggest that this occurs independent of integration.

Animals↗
PubMed1995

Role of calcium(II) ions in the recognition of coagulation factors IX and X by IX/X-bp, an anticoagulant from snake venom.

IX/X-bp, an anticoagulant protein isolated from the venom of the habu snake Trimeresurus flavoviridis, has a structure homologous to the carbohydrate-recognition domains of C-type (Ca(2+)-dependent) animal lectins, and it binds to the gamma-carboxyglutamic acid (Gla) domains of coagulation factors IX and X in a Ca(2+)-dependent fashion. In the present study, we elucidated the role of Ca2+ ions in this binding. The binding of 125I-labeled IX/X-bp to both coagulation factors required about 1 mM Ca2+ ions in this at pH 7.5. A decrease in the pH to 6.5 had a striking negative effect on the binding, and the Ca(2+)-requirement curve was shifted rightward. We investigated the binding of Ca2+ ions to IX/X-bp directly by equilibrium dialysis and identified two independent binding sites with different affinities. At pH 7.5, the apparent Kd values for these sites were 25 and 200 microM, respectively. When the pH was decreased to 6.5, the affinity of the high-affinity binding site was reduced only slightly but that of the low-affinity site was reduced considerably. Moreover, it was evident from observations of Ca(2+)-induced changes in the intrinsic fluorescence that IX/X-bp underwent a conformational change upon binding of Ca2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

The propeptides of human protein C, factor VII, and factor IX are exchangeable with regard to directing gamma-carboxylation of these proteins.

The specificity of the propeptide sequence in directing vitamin K-dependent post-translational gamma-carboxylation has been assessed by examination of the extent of processing of chimeric constructs of blood coagulation factor VII (fVII), factor IX (fIX) and protein C (PC). One chimera consisted of a protein in which the gamma-carboxyglutamic acid (Gla)/helical stack domain of PC (amino acid residues 1 to 46) was replaced by that of fIX (residues 1 to 47) in an otherwise intact PC. Another consisted of the same construction of a fVII/PC Gla domain-based mutant protein. The final chimera contained the leader/propeptide sequence of PC (residues -42 to -1) replaced by that of fIX (residues -46 to -1). In each case, all Glu-precursor Gla residues in the Gla domains of the proteins were fully processed to Gla. These results demonstrate that the propeptides of fIX and PC are capable of directing gamma-carboxylation of the Gla regions of either protein, that the propeptide of PC can fully function in gamma-carboxylation of the Gla region of fVII, and further suggest that, with regard to gamma-carboxylation, communications between the propeptides and Gla domains in intact proteins are general in nature.

Carboxylic Acids↗
PubMed1995

Improved virus safety and purity of a chromatographically produced factor IX concentrate by nanofiltration.

A virus removal system based on tangential flow filtration was introduced into a Factor IX production process. Beside the intended virus reduction potency of filter membranes, an additional purification effect could be achieved. This purification effect was evaluated in detail by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion HPLC. High-molecular-mass impurities were retained by the membrane, thus increasing the specific activity of the product.

Blood-Borne Pathogens↗
PubMed1991

Restriction fragment length polymorphisms associated with the factor VIII and factor IX genes in Polynesians.

New Zealand Maoris (72 X chromosomes) have been compared with Pacific Island Polynesians (121 X chromosomes) and Caucasian New Zealanders (51 X chromosomes) as a control group to determine the allelic frequency of six RFLPs associated with the genes for two X linked diseases (haemophilia A and haemophilia B). RFLPs examined were BclI, XbaI, and BglI within the factor VIII gene, the factor VIII extragenic TaqI system, and the factor IX intragenic TaqI and XmnI sites. The information obtained facilitates the design of strategies for both carrier detection and prenatal diagnosis of haemophilia A within these groups. Strong linkage disequilibrium was observed between the factor VIII BclI and XbaI sites in Polynesians. Genetic counselling for Polynesians with haemophilia B continues, however, to rely on phenotypic diagnosis. The RFLP data from the two separate loci on the X chromosome in Polynesians show similarities with Chinese and Japanese populations, reinforcing theories of an early Polynesian ancestry originating in east Asia.

Alleles↗
PubMed1992

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.

Amino Acid Sequence↗
PubMedDate not supplied

Treatment with a pure factor IX concentrate in a patient with moderate hemophilia B undergoing bilateral total hip replacement.

Recently, a pure factor IX concentrate, licensed in Sweden as Immunine, was prepared through ion exchange and hydrophobic chromotography. Virus inactivation included two steps of steam treatment. Reconstituted Immunine could be kept at room temperature for at least 7 days without any substantial loss of activity. A patient with moderate hemophilia B undergoing bilateral total hip replacement was continuously infused with Immunine by use of a pump. There were no bleeding complications or signs of thrombosis, and the operation markedly improved the patient's range of motion and quality of life.

Antithrombin III↗
PubMed1987

The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.

Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing.

Amino Acid Sequence↗
PubMed1990

Deduced amino acid sequence of mouse blood-coagulation factor IX.

A mouse fetal liver cDNA library was screened with a cDNA clone encoding human blood coagulation factor IX protein (hBCFIX). A bacteriophage lambda clone was isolated and the nucleotide sequence of a 2710-bp insert was determined. An open reading frame of 459 amino acids (aa) was identified within the sequence that has an 80% sequence similarity with hBCFIX. The cDNA contains a long 3'-untranslated sequence similar to that of BcfIX gene from human and canine sources. However, instead of a sequence that might form two hair-pins such as those found in hBcfIX, a (GA)16 repeat that has been reported to form H-DNA [Htun and Dahlberg, Science 241 (1988) 1791-1796; Johnston, Science 241 (1988) 1800-1804] was found in the 3'-untranslated region. The predicted aa sequence of mouse BCFIX serves as a comparative sequence for identifying key residues within hBCFIX where epitopes recognized by monoclonal antibodies produced from an immunized mouse are compared with respect to the human and mouse primary BCFIX sequence.

Amino Acid Sequence↗
PubMed2004

Efficient delivery of human clotting factor IX after injection of lentiviral vectors in utero.

AIM: To explore gene transfer feasibility for human clotting factor IX (hFIX) mediated by recombinant lentivirus in utero. METHODS: ICR mice fetus at 17-19 d gestation were received lentiviral vectors carrying hFIX cDNA under the control of liver specific promoter by intrahepatic injection. The expression and distribution of hFIX cDNA and possible immune responses against the hFIX were assessed by ELISA, PCR, RT-PCR, and immunohistochemistry, respectively. RESULTS: The serum hFIX protein were detected at different time points in all newborn mice, the highest level of hFIX was 50 microg/L and lasted for more than 30 d. Anti-hFIX antibody was not detected. hFIX cDNA was detected in liver, spleen, and heart. The expression of hFIX cDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effect associated with gene transfer was detected. CONCLUSION: The efficient delivery of hFIX can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy for hemophilia in utero.

Animals↗