Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Factor IX”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1991

Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397.

Factor IXLong Beach has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IXwild type and the two mutants (factor IXVal and factor IXThr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IXVal and factor IXwild type had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IXThr had only 5% clotting activity compared with normal factor IX, a slightly lower Km and significantly reduced kcat, using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXawild type-factor X model and the factor IXaThr-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond.

Base Sequence↗
PubMed1992

Effects of gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX.

Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.

1-Carboxyglutamic Acid↗
PubMed1978

Purification and characterization of an abnormal factor IX (Christmas factor) molecule. Factor IX Chapel Hill.

Human Factor IX (Christmas factor) was isolated from the plasma of a patient with mild hemophilia B. The patient's plasma contained 5% Factor IX clotting activity but 100% Factor IX antigenic activity as determined by immunological assays, which included inhibitor neutralization and a radioimmunoassay for Factor IX. This abnormal Factor IX is called Factor IX Chapel Hill (Factor IXCH). Both normal Factor IX and Factor IXCH have tyrosine as the NH2-terminal amino acid. The two proteins have a similar molecular weight, a similar amino acid analysis, the same number of gamma-carboxyglutamic acid residues (10 gamma-carboxyglutamic acid residues), and a similar carbohydrate content. Both exist as a single-chain glycoprotein in plasma. The major difference between normal Factor IX and Factor IXCH is that the latter exhibits delayed activation to Factor IXa in the presence of Factor XIa and Ca2+. Thus, Factor IXCH differs from other previously described abnormal Factor IX molecules.

Adult↗
PubMed1978

Immunologic studies of factor IX (Christmas factor). II. Immunoradiometric assay of factor IX antigen.

A solid-phase two-site immunoradiometric assay has been developed which measures factor IX antigen levels as low as 0.0004 u per ml of plasma. In normal individuals, the factor IX antigen level correlated with the factor IX procoagulant level. In haemophilia B, 14 patients had markedly reduced antigen levels (less than 0.06 u/ml) and five had normal levels (greater than 0.60 u/ml).

Antigens↗
PubMed1992

Hemophilia B caused by five different nondeletion mutations in the protease domain of factor IX.

Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G----T, Ser365Ile; IXVarel: nucleotide 31,214A----G, Ser365Gly; IXMechtal: nucleotide 31,211G----C, Asp364His; IXDreihacken: nucleotide 30,864G----A, Arg248Gln; and IXMonschau: nucleotide 30,855A----T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT----GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal. The Ser365Gly and Ser365Ile mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the alpha-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had approximately 41% and Glu245Val had approximately 17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca(2+)-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1977

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

Amino Acid Sequence↗
PubMed1979

Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.

The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.

Amino Acid Sequence↗
PubMed1978

Activation of human factor IX (Christmas factor).

Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.

Amino Acid Sequence↗
PubMed1991

Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX.

Factor IX is a blood clotting protein that contains three regions, including a gamma-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF-like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel beta-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp28, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the beta-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.

1-Carboxyglutamic Acid↗
PubMed1992

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.

Amino Acid Sequence↗
PubMed1977

Inhibitor-neutralisation assay and electro-immuno assay of human factor IX (Christmas factor).

A rabbit antibody specifically precipitating human factor IX has been used in the assay of factor IX antigen. The results obtained with two different methods (inhibitor-neutralisation assay and electro-immunoassay) have been compared in a group of healthy individuals and in a group of hemophilia B patients and carriers. In general, identical results are obtained with both methods, except in some hemophilia B+ carriers and patients, where the electroimmuno assay gives 1.5-2.0 times higher levels. Results obtained by electroimmuno assay are more accurate and reproducible than those obtained by inhibitor-neutralisation assay, which is of importance for its potential use in carrier detection.

Adult↗
PubMed1991

Standardization of factor IX: standards for "purified" factor IX concentrates.

An international collaborative study was carried out to determine the suitability of the current WHO II-IX-X concentrate standard, 84/681, for assigning potency to the more highly purified factor IX concentrates. Three Coagulation Factor IX (Human) preparations and one Factor IX Complex preparation were assayed by the one stage method against WHO 84/681 following predilution to 1.0 u/ml in buffer, 1% albumin, or factor IX deficient plasma. There were no cases of non-parallelism between any of the preparations and the current WHO standard. Predilution of the Coagulation Factor IX (Human) preparations in 1% albumin or factor IX deficient plasma gave similar potency values. Predilution in buffer gave significantly lower (p less than 0.01) potency values. For the Factor IX Complex preparation, potency estimates were significantly different (p less than 0.01) with each prediluent. The overall precision was similar within each predilution for all preparations with predilution in buffer being less precise than predilution in albumin or in deficient plasma. WHO standard 84/681 appears to be a suitable standard for the potency determination of the more highly purified factor IX preparations. Predilution in 1% albumin or factor IX deficient plasma is recommended as they give equivalent results with the least variability.

Factor IX↗
PubMed1991

Three distinct point mutations in the factor IX gene of three Japanese CRM+ hemophilia B patients (factor IX BMNagoya 2, factor IX Nagoya 3 and 4).

Enzymatic DNA amplification and complete sequence analysis were used to investigate human factor IX coding sequences in three CRM+ hemophilia B patients. In a patient with severe hemophilia B and a markedly prolonged ox-brain prothrombin time, a C to T transition in exon VI changed the codon for Arg180 to Trp (factor IX BMNagoya 2). This mutation would impair the cleavage by factor XIa required for activation of the zymogen. In a patient with mild hemophilia B, a G to A transition in exon VI changed the codon for Arg145 to His(factor IX Nagoya 3). This substitution also would be predicted to preclude the cleavage of factor IX by factor XIa at this peptide bond (Arg145-Ala146). Furthermore, this point mutation creates a new NlaIII restriction site which provides a quick and reliable method for carrier detection in the affected family members. A patient with severe hemophilia B (factor IX Nagoya 4) had a G to A transition in exon II changing the codon for Glu21 to Lys. This novel point mutation is assumed to impair the function of factor IX by disrupting the calcium binding of factor IX.

Amino Acid Sequence↗