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PubMed1998

A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refolded in vitro adopt the native fold. In contrast, the Gly --> Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.

Amino Acid Substitution↗
PubMed1980

In vitro thrombogenicity tests of factor IX concentrates. II: effects of phospholipids and heparin.

Measurement of the total phospholipid (and that portion active in coagulation) in factor IX concentrates revealed no correlation with in vitro tests of potential thrombogenicity, except in the case of the recalcification time and the thrombin generation test which may detect coagulant phospholipid as well as the presence of thrombogenic enzymes. This is probably due to separation of the prothrombin complex proteins from most phospholipid during ion-exchange chromatography. Although low levels of phospholipid remain in the final product these are apparently insufficient to effect appreciable activation of factor IX concentrates despite low levels of antithrombin III. Two tests which measure the formation of thrombin and factor Xa after recalcification of concentrates were affected by the addition of exogenous phospholipid. However this is a relative effect such that differences are quantitative rather than qualitative. Heparin addition during production of factor IX concentrate was found to have only minor effects on the results of in vitro thrombogenicity tests of the final product. This was confirmed in the laboratory by incubation of unheparinised products with heparin for periods of up to 6 hr.

Blood Coagulation Tests↗
PubMed1980

In vitro thrombogenicity tests of factor IX concentrates. I. A survey of available assays.

Various factor IX concentrates have been examined in a number of in vitro tests of thrombogenicity. The results suggest that some tests are superfluous as in concentrates with activity in any of these tests activation is revealed by a combination of the non-activated partial thromboplastin time, the thrombin (or Xa) generation time and factor VIII inhibitor bypassing activity tests. Assay of individual coagulant enzymes revealed that most concentrates contained more factor IXa than Xa. However only a small number of concentrates, chiefly those that had been purposefully activated, contained appreciable amounts of either enzyme.

Blood Coagulation Tests↗
PubMed2001

Factor IX gene polymorphisms in Indian population.

Hemophilia B is an X-linked, recessive disorder of hemostasis, caused by a defect in coagulation factor IX. To date, several restriction fragment length polymorphisms (RFLPs) have been identified within the gene for human factor IX. The incidence of these RFLPs differs significantly in different populations. In the present study, we analyzed the heterozygosity frequency and the allele frequency of three common intra- and extragenic polymorphic sites of the factor IX gene in Indian population. The main objective was to test the informativeness of two intragenic markers Dde I and Xmn I and one extragenic marker Hha I for carrier detection and prenatal diagnosis. The method used was polymerase chain reaction (PCR) and RFLP, which is economical yet simple to perform. In Indian population Dde I marker showed an informativeness of 69.0% followed by 38.0% for Hha I and 23.0% for Xmn I. The cumulative informativeness of these three markers was found to be 80 to 82%. A comparison of the heterozygosity rates of these three markers with the other ethnic groups showed that Indian population had almost similar pattern with the Caucasians and American blacks but differed significantly from the Orientals including Japanese, Chinese and Malays.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1999

Reported in vivo splice-site mutations in the factor IX gene: severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations.

Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.

Computer Simulation↗
PubMed1993

Synergy between transcription factors DBP and C/EBP compensates for a haemophilia B Leyden factor IX mutation.

Haemophilia B Leyden is characterized by low childhood levels of factor IX which gradually increase after puberty, eventually resulting in a return to health. The disease is the result of single nucleotide substitutions within a 40 bp region encompassing the major transcriptional start site. We have characterized transcription factor binding sites within the factor IX promoter. Five sites were identified and a Leyden mutation at nucleotide -5 was shown to interfere with the binding of proteins to one of three newly identified sites. The correlation between the post-pubertal recovery of these mutants and the induction of the transcription factor DBP led us to the discovery of a synergistic interaction between DBP and C/EBP responsible for the recovery of normal transcriptional activity of the -5 mutant promoter and may play a role in the resolution of other Leyden mutants.

Adolescent↗
PubMed1994

Determinants of the factor IX mutational spectrum in haemophilia B: an analysis of missense mutations using a multi-domain molecular model of the activated protein.

A multi-domain molecular model of factor IXa was constructed by comparative methods. The quaternary structure of the protein was assembled by docking individual domains through consideration of their shape complementarity, polaric properties and the location of cross-reacting material positive/negative (CRM+/-) variants on domain surfaces. Some 217 different missense mutations in the factor IX (F9) gene were then selected for study. Using maximum likelihood analysis, missense mutations affecting highly conserved amino acid residues of factor IX were shown to be 15-20 times more likely to result in haemophilia B than those affecting non-conserved residues. However, about one quarter of this increase in likelihood of clinical observation could be attributed to the magnitude of the amino acid exchange. Missense mutations in structurally conserved residues were found to be 2.1-fold more likely to come to clinical attention than those in structurally variable residues. Missense mutations in residues whose side chains were inwardly pointing were 3.6-fold more likely to be observed than those in surface residues. These observations imply a complex hierarchy of sequence/structure conservation in the protein. The severity of the clinical phenotype correlated with both the extent of the evolutionary sequence conservation of the residue at the site of mutation and the magnitude of the amino acid exchange. Further, the substitution of residues exhibiting minimal side chain solvent accessibility was associated disproportionately with severe haemophilia compared with that of surface residues. Clusters of CRM+ mutations were observed at factor IX-specific residues on the surface of the molecule. These clusters may reflect factor IX-specific docking interactions. The likelihood that a given factor IX mutation will come to clinical attention is therefore a complex function of the sequence characteristics of the F9 gene, the nature of the amino acid substitution, its precise location and immediate environment within the protein molecule, and its resulting effects on the structure and function of the protein.

Factor IX↗
PubMed2003

Pharmacokinetics of factor VIII and factor IX.

A survey of principal pharmacokinetic (PK) studies on factor VIII (FVIII) and factor IX (FIX) plasma- and rDNA-derived concentrates, analysed by means of the PKRD program, has been performed. Notwithstanding the accurate definition of the study design, released in 1991 by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC-ISTH), a large variability of PK parameters has been pointed out. In the majority of the PK studies, the size of the population is small. In this situation, a careful individualization of haemophilia therapy is strongly recommended. The tailored prediction of loading and maintenance dosages and the need for strict control of trough FVIII/IX levels are mandatory not only to decrease the risk of bleeds but also to spare financial resources. Recently, the old problem of FVIII assay standardization has again become a concern among physicians, especially after the introduction of B-domain deleted rFVIII concentrate. The discrepancies between the widely used one-stage clotting assay and the chromogenic substrate assay seem to be solved by the introduction of a product-specific laboratory standard.

Cross-Over Studies↗
PubMed1990

Two mutations of the factor IX gene including a donor splice consensus deletion and a point mutation in a Dutch patient with severe hemophilia B.

The abnormal factor IX gene of a patient with severe hemophilia B (hemophilia B Ursem) was selected for study. All of the coding and their flanking regions and parts of the 5'- and 3'-untranslated regions of the factor IX gene were amplified from the patient's genomic DNA by using the polymerase chain reaction (PCR). By analyzing the nucleotide sequence of the PCR products we have identified two mutations in the patient's factor IX gene, viz. a tetranucleotide deletion (GAGT, nt 6492 to 6495) or (TGAG, nt 6491 to 6494) in the 5'-donor splice site consensus at the exon 2-intron B boundary, and a point mutation at nucleotide 31103 in the catalytic domain (exon 8) of factor IXa, which changes the codon for valine 328 (GTT) to one for isoleucine (ATT). PCR-amplified exon 8 from 45 normal males and 55 normal females had the codon for valine-328. We propose that the deletion within the donor splice-site consensus is the cause of the disease in this individual, whereas the substitution of valine-328 by isoleucine may be a neutral variant which is, at least, very rare in the normal population. In a family study the DNA sequence of the patient's mother shows both the G to A transition in exon 8 and the 5'-donor splice consensus deletion in intron B in one allele.

Amino Acid Sequence↗
PubMed1992

Subcutaneous injection of factor IX for the treatment of haemophilia B.

Haemophilia B patients are normally treated, either prophylactically or in response to bleeding episodes, by frequent intravenous injections of factor IX purified from blood donors. Here we show in model animal experiments that purified human factor IX, when injected subcutaneously, is rapidly (in 3-11 h) and reasonably efficiently (30-40% of an equivalent intravenous dose) transported at least partly by the lymphatic drainage of the skin into the bloodstream, mostly in a biologically active form. This suggests that patients could be treated prophylactically by subcutaneous rather than intravenous injection, where the short delay in raising plasma factor IX to haemostatic levels would be clinically acceptable. More generally, our studies emphasize that the subcutaneous route of injection should be useful for other therapeutic proteins, including other clotting factors, which have to be delivered to the bloodstream, as long as their half-life is at least a few hours allowing time for transport into the general circulation.

Animals↗
PubMed1985

Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level.

In order to establish the relative importance of genetic factors on the variation in plasma concentration of coagulation factors VIII and IX, these parameters were determined in 74 monozygotic and 84 like-sexed dizygotic twin pairs. The twins belonged to two age groups: 33-39 years and 57-62 years. Factor VIII was determined as factor VIII coagulant antigen (VIIICAg) and as factor VIII-related antigen (VIIIRAg). Factor IX was determined as factor IX antigen (IXAg). A higher value for each coagulation factor was found in the older-age group compared to the younger group, whereas no difference was found between the sexes. A significant correlation was found between values for VIIIRAg and VIIICAg (r = .56). For VIIICAg, it could be demonstrated that the age effect was secondary to the age effect on VIIIRAg. The concentration of VIIICAg and VIIIRAg varied among ABO blood types, being lowest in type O individuals, higher in A2 individuals, and highest in A1 and B individuals. The effect of the ABO locus on VIIICAg was secondary to an effect on VIIIRAg. Analysis of variance revealed a significant genetic influence on the variance of VIIICAg and VIIIRAg with a heritability estimate of .57 for VIIICAg and .66 for VIIIRAg. This is in agreement with a previous hypothesis of an effect of several autosomal genes on factor VIII concentration. Thirty percent of the genetic variance of VIIIRAg was due to the effect of ABO blood type. The ABO locus is therefore a major locus for the determination of factor VIII concentration. No significant genetic effect on the variation in plasma concentration of IXAg could be detected.

ABO Blood-Group System↗
PubMed2002

In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor.

The fundamental hypotheses behind fetal gene therapy are that it may be possible (1) to achieve immune tolerance of transgene product and, perhaps, vector; (2) to target cells and tissues that are inaccessible in adult life; (3) to transduce a high percentage of rapidly proliferating cells, and in particular stem cells, with relatively low absolute virus doses leading to clonal transgene amplification by integrating vectors; and (4) to prevent early disease manifestation of genetic diseases. This study provides evidence vindicating the first hypothesis; namely, that intravascular prenatal administration of an adenoviral vector carrying the human factor IX (hFIX) transgene can induce immune tolerance of the transgenic protein. Following repeated hFIX protein injection into adult mice, after prenatal vector injection, we found persistence of blood hFIX and absence of hFIX antibodies in 5 of 9 mice. Furthermore, there was substantial hFIX expression after each of 2 reinjections of vector without detection of hFIX antibodies. In contrast, all adult mice that had not been treated prenatally showed a rapid loss of the injected hFIX and the development of high hFIX antibody levels, both clear manifestations of a strong immune reaction.

Adenoviridae↗
PubMed1991

[Isolation of factor IX concentrate from cryosupernatant of donor's plasma].

Soviet anion-exchangers were comparatively studied to specify an optimal carrier useful for isolation of factor ix of the blood coagulation system from donor's blood cryosupernatant. The main criteria of gel selection were the yield and purification degree of factor IX, as well as its specific activity and concentration in eluates. It has been shown that DEAE-agarose 6B-CL, possessing pronounced activity with respect to specific sorption of factor IX, meets these requirements to the greatest degree.

Blood Donors↗
PubMed1987

Possible absence of common polymorphisms in coagulation factor IX gene in Japanese subjects.

Four distinct intragenic polymorphisms in the coagulation factor IX gene which have been reported to be important for family diagnosis of Caucasian hemophilia B were studied in 51 normal Japanese subjects (21 males and 30 females). High-molecular-weight DNA prepared from peripheral blood lymphocytes were digested with endonuclease, Ddel, Mspl, Taql or Xmnl, and were studied by Southern blot analysis with factor IX complementary DNA as a probe. None of the minor fragments produced by these enzymes was found in the normal Japanese DNA samples tested, although the probe detects minor allelic forms in control Caucasian DNA samples. Our data suggest that the frequent polymorphic sites found in Caucasians are possibly absent in the Japanese population.

Asian People↗
PubMed2003

Application of semi-industrial monolithic columns for downstream processing of clotting factor IX.

It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branović et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.

Chromatography, Liquid↗
PubMed1985

Linkage and recombination between fragile X-linked mental retardation and the factor IX gene.

Linkage analysis on a family with fragile X-linked mental retardation was performed using a Taq 1 restriction fragment length polymorphism detected by a cloned human coagulation factor IX cDNA. Two affected brothers in this sibship were found to have different factor IX RFLP alleles, indicating a recombinational event occurred between the two genes. Our data therefore indicate that the gene responsible for fragile X-linked mental retardation is not as tightly linked to the factor IX gene as the previously published data may suggest.

Alleles↗
PubMed1987

Antithrombin antigen of high molecular weight associated with neoantigen in hemophilic plasma after factor IX concentrate therapy.

These studies were performed to investigate the cause(s) of the cathodal shift of mobility seen in crossed immunoelectrophoresis of antithrombin antigen in plasma of hemophilic patients after factor IX concentrate therapy. These plasmas were shown to contain antithrombin neoantigen with apparent identity to the neoantigen present in normal serum but not present in normal plasma. Sephacryl S-200 gel chromatography of serum demonstrated that the neoantigen eluted with the first two, early eluting protein peaks; thus the neoantigen had a higher molecular weight than native antithrombin. When the chromatographic fractions containing the neoantigen were studied by crossed immunoelectrophoresis, they were found to contain antithrombin antigen of more cathodal mobility than normal. Sephacryl S-200 chromatography of factor IX concentrate-treated hemophilic plasma also showed an early eluting peak of antithrombin antigen of more cathodal mobility than normal in crossed immunoelectrophoresis. The mobility of this peak was identical to the cathodal peak found in normal serum and in early eluting fractions from chromatography of normal serum. These results support the conclusion that factor IX concentrate-treated hemophilic plasma contained a non-functional, high molecular weight form of antithrombin, associated with the presence of neoantigen, which may represent complexed and/or modified antithrombin produced by the action of the concentrates in vivo.

Antigens↗
PubMed1993

Expression of human factor IX cDNA in mice by implants of genetically modified skin fibroblasts from a hemophilia B patient.

Double-copy retroviral vector containing human factor IX cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor IX at a rate of 3420 ng/10(6) cells/24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. It was demonstrated that human factor IX was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng/ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B.

Animals↗