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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↗
PubMed1989

Factor VIII influences binding of factor IX and factor X to intact human platelets.

To investigate the influence of factor VIII on the binding of factors IX and X to the surface of intact human platelets, washed collagen-stimulated platelets were incubated with factor IX and factor X in the presence or absence of factor VIII. Platelets were then lysed and IX:Ag and X:Ag were determined in the platelet lysate. Platelet bound IX:Ag was higher after incubation of platelets with factor IX in the presence of native or activated factor VIII than after incubation of platelets with factor IX alone. Thrombin degraded factor VIII did not support binding of factor IX to stimulated platelets. Preactivation of factor IX enhanced binding to platelets; influence by factor VIII on binding to platelets was greater with native factor IX than with preactivated factor IX. Presence of native or activated factor VIII also increased binding of factor X to platelets. In contrast to factor IX, preactivation of factor X did not influence binding to platelets, but in the presence of factor VIII preactivation of factor X increased binding of factor X to platelets. Our data suggest that mediation of binding of factors IX and X to the surface of platelets is one of the mechanisms by that factor VIII exerts its cofactor function in the activation of factor X.

Blood Platelets↗
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↗
PubMed2000

Zymogen factor IX potentiates factor IXa-catalyzed factor X activation.

Intrinsic factor X activation is accelerated >10(7)-fold by assembly of the entire complex on the activated platelet surface. We have now observed that increasing the concentration of zymogen factor IX to physiologic levels ( approximately 100 nM) potentiates factor IXa-catalyzed activation of factor X on both activated platelets and on negatively charged phospholipid vesicles. In the presence and absence of factor VIIIa, factor IX (100 nM) lowered the K(d,appFIXa) approximately 4-fold on platelets and 2-10-fold on lipid vesicles. Treatment of two factor IX preparations with active-site inhibitors did not affect these observations. Autoradiographs of PAGE-separated reactions containing either (125)I-labeled factor IX or (125)I-labeled factor X showed that the increased factor X activation was not due to factor Xa-mediated feedback activation of factor IX and that there was increased cleavage of factor X heavy chain in the presence of factor IX in comparison with control reactions but only in the presence of both the enzyme and the surface. Since plasma concentrations of prothrombin, factor VII, protein C, or protein S did not by themselves potentiate factor Xa generation and did not interfere with the potentiation of the reaction of factor IX, the effect is specific for factor IX and is not attributable to the Gla domain of all vitamin K-dependent proteins. These observations indicate that under physiologic conditions, plasma levels of the zymogen factor IX specifically increase the affinity of factor IXa for the intrinsic factor X activation complex.

Binding Sites↗
PubMed1984

erythro-beta-Hydroxyaspartic acid in bovine factor IX and factor X.

To localize beta-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing beta-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of beta-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.

Amino Acid Sequence↗
PubMed2000

Activation of factor IX by factor XIa.

Blood coagulation factor IX is activated during hemostasis by two distinct mechanisms. Activation through factor VIIa/tissue factor occurs early in the course of fibrin clot formation. Activation by factor XIa appears to be important for maintaining the integrity of the clot over time. In general, coagulation proteases are activated on a phospholipid surface in the presence of a protein cofactor. Until recently, activation of factor IX by factor XIa was thought to be the exception to this rule, as phospholipid has no effect on the reaction and no cofactor had been identified. These curious observations suggest that factor IX is activated by factor XIa in the fluid phase. A large amount of new evidence now indicates that factor IX activation by factor XIa occurs on the surface of activated platelets. The data suggest, however, that this reaction differs significantly from other protease-substrate interactions on the platelet surface. This is likely to be due, in part, to the unusual structure of the factor XI molecule.

Amino Acid Sequence↗
PubMed1998

Some human inhibitor antibodies interfere with factor VIII binding to factor IX.

Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.

Antibodies, Blocking↗
PubMed1974

A comparison of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor).

A comparison has been made of the electrophoretic behavior, chemical composition, amino-terminal sequence, and immunological properties of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor). Some immunological crossreactivity was found between the antibody to prothrombin and factor X although prothrombin and factor X differ substantially in amino-acid and carbohydrate composition. Considerable amino-acid sequence homology was found in the amino-terminal portion of prothrombin, factor IX, and the light chain of factor X. These data provide further evidence to support the hypothesis that at least three of the vitamin K-dependent clotting factors have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed2005

Factor XIa dimer in the activation of factor IX.

Factor XI, unlike other coagulation proteins, is a homodimer of two identical subunits linked by a single disulfide bond formed by Cys321. The present study was undertaken to understand the physiological significance of the dimeric nature of factor XI. We have expressed a mutant FXI/G326C in which the Gly326 residue of factor XI has been mutated to Cys326, reasoning that Cys321 would form an intrachain disulfide bond with Cys326 as in prekallikrein, a plasma protein that exists as a monomer even with 58% amino acid sequence identity and a domain structure very similar to factor XI. No free thiol could be detected in the expressed protein, and it migrated as a monomer on nonreduced SDS-PAGE. In physiological buffer, however, the protein was found to exist in a state of monomer-dimer equilibrium as assessed by gel-filtration chromatography and ultracentrifugation studies (K(d) approximately 36 nM). Functional studies revealed that FXI/G326C was indistinguishable from plasma factor XI in a plasma-clotting assay and in a factor IX activation assay both in the presence and absence of activated platelets even at concentrations at which less than 5% of the mutant exists as dimers. We conclude that, for optimal function in the presence of activated platelets, a preformed dimer of factor XI is not required.

Amino Acid Substitution↗
PubMed1983

The structures of the carbohydrate moieties of bovine blood coagulation factor IX (Christmas factor).

Bovine blood coagulation factor IX (Christmas factor) contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis. Bovine factor IX contained two unique penta- and tetrasialyl triantennary sugar chains with the structures shown below in addition to tetra-, tri-, and disialyl biantennary sugar chains of Sia alpha 2 leads to 3 Gal beta 1 leads 3(Sia alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Sia alpha 2 leads to 3Gal beta 1 leads to 3(Sia alpha 2 leads to 6)GlcNac beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Sia alpha 2 leads to 3Gal beta 1 leads to 3(Sia alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, and Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and their partially desialized forms.

Acetylation↗
PubMed1986

The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions.

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.

Animals↗
PubMed2006

An RNA aptamer that discriminates bovine factor IX from human factor IX.

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.

Animals↗
PubMed1983

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X.

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.

Amino Acid Sequence↗
PubMed2000

The tissue factor region that interacts with substrates factor IX and Factor X.

The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.

Amides↗
PubMed1994

Nanofiltration, a new specific virus elimination method applied to high-purity factor IX and factor XI concentrates.

We have validated the use of two new regenerated multilayered structured cellulose membranes (BMM), Planova 15 N and Planova 35 N, with respective mean pore sizes of 15 and 35 nm, as a new filtration system to eliminate viruses in highly purified factor IX and factor XI concentrates. Virus spiking experiments indicated that single dead-end filtration on the membranes could remove more than 5.7-7.8 log10 of human immunodeficiency virus, bovine viral diarrhoea virus, porcine pseudorabies virus, reovirus type 3, and simian virus 40, as well as the small non-enveloped viruses, poliovirus Sabin type 1 and bovine parvovirus. In vitro control tests and animal studies (Wessler stasis model, rat hypotension model) of the two concentrates did not reveal any significant differences with the non-nanofiltered material. Viral filtration of plasma derivatives on porous polymeric membranes might be an essential step in the improvement of their viral safety.

Factor IX↗
PubMed1989

Molecular cloning of a cDNA encoding canine factor IX.

Factor IX (F.IX) is a vitamin K-dependent plasma protein, a deficiency of which results in hemophilia B. A canine model of hemophilia B exists; attempts to use this model for gene transfer experiments or characterization of the hemophilic defect require elucidation of normal canine F.IX structure. We report the isolation and characterization of the coding region for canine F.IX cDNA. Canine F.IX possesses 86% identity at the amino-acid level with human F.IX. The leader peptide, Gla domain, EGF domains, and the carboxy-terminal portion of the heavy chains show extensive sequence conservation between the canine and human. All Glu residues undergoing gamma-carboxylation in humans are conserved in canines. The complete coding sequence for canine F.IX has been determined, and the derived translation product has been analyzed. A similar approach should allow identification of the causative mutation in canine hemophilia B. Furthermore, this clone may prove a valuable resource in gene transfer experiments for this disease.

Amino Acid Sequence↗
PubMed1995

Comparison of the recovery and half-life of a high-purity factor IX concentrate with those of a factor IX complex concentrate. Factor IX Study Group.

BACKGROUND: Recovery and half-life estimations were carried out to compare a high-purity factor IX concentrate with an established factor IX complex concentrate. STUDY DESIGN AND METHODS: Two high-purity factor IX concentrates, which are identical except for the presence or absence of heparin (Immuninehep-plus and Immuninehep-minus), were evaluated in two independent crossover studies using an intermediate-purity factor IX complex concentrate (Bebulin) as reference drug. RESULTS: In the Immuninehep-plus crossover study (n = 27), Immuninehep-plus and Bebulin had, respectively, a recovery of 0.90 +/- 0.26 and 0.84 +/- 0.23 IU per dL per IU per kg, a compartmental half-life of 17.11 +/- 6.18 and 15.94 +/- 4.69 hours, and an effective half-life of 16.51 +/- 3.48 and 16.48 +/- 4.26 hours. In the Immuninehep-minus crossover study (n = 26), Immuninehep-minus and Bebulin had, respectively, a recovery of 0.92 +/- 0.31 and 1.02 +/- 0.36 IU per dL per IU per kg, a compartmental half-life of 17.42 +/- 5.60 and 18.77 +/- 6.27 hours, and an effective half-life of 16.39 +/- 4.44 and 16.48 +/- 4.28 hours. Equivalence tests indicated that the recovery and half-life of Immunine, with or without heparin, are equivalent to those of Bebulin. CONCLUSION: The equivalence in pharmacokinetics and bioavailability indicates that the dosage schedule for Immunine should be the same as or very similar to that of Bebulin. The high specific activity of the former, however, allows administration at lower volumes.

Adolescent↗
PubMed1985

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B).

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

Amino Acid Sequence↗