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PubMed1990

Mutations in hemophilia Bm occur at the Arg180-Val activation site or in the catalytic domain of factor IX.

Hemophilia Bm is characterized by a strikingly prolonged plasma ox brain prothrombin time. In an attempt to find an explanation for this phenomenon we have analyzed various aspects of the Bm variants factor IX Deventer, factor IX Milano, factor IX Novara, and factor IX Bergamo. Proteolytic cleavage by factor XIa was normal in two Bm variants, but absent at the Arg180-Val bond in the other two. In the latter variants Arg180 was replaced by either Trp or Gln, whereas Val181----Phe and Pro368----Thr replacements have occurred in the variants that were normally cleaved by factor XIa. In all four variants the Bm effect could be neutralized with a single monoclonal antibody against factor IX. Also, after treatment with factor XIa, none of the Bm variants reacted with antithrombin III (in contrast to normal factor IXa). Purified factor IX Deventer (one of the variants with a replacement of Arg181), either with or without pretreatment with factor XIa, was found to be a more effective competitive inhibitor of the factor VIIa-tissue factor-induced factor X activation than similarly treated normal factor IX. In addition, this inhibitory effect was much more pronounced when bovine tissue factor was used instead of human tissue factor. We propose that the normal activation of factor IX not only produces a conformational change around the active site serine that allows efficient substrate binding and catalysis, but that the same conformational change is instrumental in effectively dissociating factor IXa from the activating factor VIIa-tissue factor complex. Amino acid replacements that disrupt this conformational transition directly (e.g. Pro368----Thr near the catalytic center) or indirectly (mutations at the Arg180-Val activation site) therefore lead to a combination of 1) the loss of coagulant activity and 2) an inhibitory effect in the ox brain prothrombin time assay.

Amino Acid Sequence↗

PubMed1999

An Arg/Ser substitution in the second epidermal growth factor-like module of factor IX introduces an O-linked carbohydrate and markedly impairs activation by factor XIa and factor VIIa/Tissue factor and catalytic efficiency of factor IXa.

Factor IXR94S is a naturally occurring hemophilia B defect, which results from an Arg 94 to Ser mutation in the second epidermal growth factor (EGF)-like module of factor IX. Recombinant factor IXR94S was activated by factor XIa/calcium with an approximately 50-fold reduced rate and by factor VIIa/tissue factor/phospholipid/calcium with an approximately 20-fold reduced rate compared with wild-type factor IX. The apparent molecular mass of the light chain of factor IXaR94S was approximately 6 kD higher than that of plasma or wild-type factor IX, which was not corrected by N-glycosidase F digestion. This result indicated the presence of additional O-linked carbohydrate in the mutant light chain, probably at new Ser 94. The initial rate of activation of factor X by factor IXaR94S in the presence of polylysine was 7% +/- 1% of the initial rate of activation of factor X by plasma factor IXa, and the kc/Km for activation of factor X by factor IXaR94S/factor VIIIa/phospholipid/calcium was 4% +/- 1% of the kc/Km for activation of factor X by plasma factor IXa/factor VIIIa/phospholipid/calcium. The reduced efficiency of activation of factor X by factor IXaR94S in the tenase enzyme complex was due to a 58-fold +/- 12-fold decrease in kcat with little effect on Km. In conclusion, the R94S mutation had introduced an O-linked carbohydrate, which markedly impaired both activation by factor XIa and turnover of factor X in the tenase enzyme complex.

Amino Acid Substitution↗

PubMed1986

Two allotypes of factor IX present in haemophilia B.

Factor IX antigen (IX:Ag) was measured with three different immunoradiometric assays (IRMAs) in 30 healthy people and 43 patients with haemophilia B of varying severity. Two of the IRMAs were based on monoclonal antibodies capable of differentiating between two genetically determined molecular variants of normal factor IX. Most patients with severe hemophilia B lacked demonstrable IX:Ag. The factor IX variant that is undetectable with one of the monoclonal antibodies used was present in 2 out of 6 families with moderate haemophilia B and in 1 out of 6 families with mild haemophilia B. The existence of allotypes of factor IX in hemophilia B may have practical implications for carrier detection and prenatal diagnosis.

Alleles↗

PubMed1993

PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway.

Factor IX is synthesized as a precursor polypeptide which requires proteolytic cleavage of the propeptide for functional activity. Expression of factor IX at high levels in Chinese hamster ovary (CHO) cells results in the secretion of a mixture of profactor IX and mature factor IX. We have studied whether the processing of profactor IX may be mediated by the recently discovered subtilisin-like serine proteases PACE/furin and/or PACE4. Co-transfection of a PACE expression vector with a profactor IX expression vector resulted in the secretion of fully processed factor IX. In contrast, co-transfection of a PACE4 expression vector with a profactor IX expression vector did not increase processing of profactor IX to the mature form. A factor IX Arg-to-Thr mutation at the P1 position (residue 39) destroyed the ability for PACE to process profactor IX. Amino-terminal sequence analysis demonstrated that processing mediated by PACE occurred at the authentic site within profactor IX. The specificity of profactor IX processing by PACE was also evaluated by transfection of a vector encoding the serine protease inhibitor alpha 1-antitrypsin. Expression of wild-type alpha 1-antitrypsin, which does not inhibit PACE, did not influence processing of profactor IX mediated by co-expression of PACE. In contrast, the alpha 1-antitrypsin Pittsburgh mutant, which inhibits PACE, inhibited profactor IX processing activity mediated by transfected PACE as well as the endogenous CHO cell propeptide processing enzyme. Pulse-chase labeling indicated that PACE processed profactor IX late within the secretory pathway, although a secreted soluble mutant PACE was also capable of processing profactor IX in the conditioned medium. The results implicate PACE as a candidate for the enzyme that processes profactor IX in vivo.

Animals↗

PubMed2001

Model for a factor IX activation complex on blood platelets: dimeric conformation of factor XIa is essential.

Human coagulation factor XI (FXI) is a plasma serine protease composed of 2 identical 80-kd polypeptides connected by a disulfide bond. This dimeric structure is unique among blood coagulation enzymes. The hypothesis was tested that dimeric conformation is required for normal FXI function by generating a monomeric version of FXI (FXI/PKA4) and comparing it to wild-type FXI in assays requiring factor IX activation by activated FXI (FXIa). FXI/PKA4 was made by replacing the FXI A4 domain with the A4 domain from prekallikrein (PK). A dimeric version of FXI/PKA4 (FXI/PKA4-Gly326) was prepared as a control. Activated FXI/PKA4 and FXI/PKA4-Gly326 activate factor IX with kinetic parameters similar to those of FXIa. In kaolin-triggered plasma clotting assays containing purified phospholipid, FXI/PKA4 and FXI/PKA4-Gly326 have coagulant activity similar to FXI. The surface of activated platelets is likely to be a physiologic site for reactions involving FXI/FXIa. In competition binding assays FXI/PKA4, FXI/PKA4-Gly326, and FXI have similar affinities for activated platelets (K(i) = 12-16 nM). In clotting assays in which phospholipid is replaced by activated platelets, the dimeric proteins FXI and FXI/PKA4-Gly326 promote coagulation similarly; however, monomeric FXI/PKA4 has greatly reduced activity. Western immunoblot analysis confirmed that activated monomeric FXI/PKA4 activates factor IX poorly in the presence of activated platelets. These findings demonstrate the importance of the dimeric state to FXI activity and suggest a novel model for factor IX activation in which FXIa binds to activated platelets by one chain of the dimer, while binding to factor IX through the other.

Blood Coagulation↗

PubMed1986

Gene for human factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C.

Factor X is one of six vitamin K dependent proteins known to be involved in blood coagulation, the others being factor VII, factor IX, prothrombin, protein S, and protein C. In the present studies, recombinant bacteriophage containing overlapping DNA inserts coding for the gene for human factor X have been isolated and characterized. These DNA inserts code for almost the entire gene for factor X, extending from the prepro leader peptide through the 3' noncoding region of the transcription product. The organization of the gene for factor X was established by DNA sequencing to identify the location of the introns and exons in the gene. Seven introns and eight exons were identified and their intron/exon boundaries established. The seven introns interrupt the coding sequence at essentially identical locations in the amino acid sequence as the introns in the genes for human factor IX and protein C. In addition, the introns in the gene for factor X divide the coding sequence into discrete exons that code for potential structural and functional domains of the protein. This information provides strong evidence to support the suggestion that the vitamin K dependent proteins present in plasma have evolved from a single, common gene and that this ancestral gene arose through a process that involved the assembly of small protein coding units of DNA into a single gene.

Amino Acid Sequence↗

PubMed1989

In vitro carboxylation of a blood coagulation factor IX precursor produced by recombinant-DNA technology.

Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.

Animals↗

PubMed1990

Characterization of the functional defect in factor IX Alabama. Evidence for a conformational change due to high affinity calcium binding in the first epidermal growth factor domain.

Factor IX Alabama is a factor IX variant in which a glycine has been substituted for Asp47 in the first epidermal growth factor (EGF) domain. The structural defect in factor IX Alabama results in a molecule with 10% of normal coagulant activity. The interactions of immunoaffinity-purified factor IX Alabama with its activator, cofactors, and substrate have been investigated to determine the functional defect in the variant. Factor IX Alabama is activated by factor XIa/calcium at near normal rates. Calcium fluorescence-quenching experiments indicate that high affinity calcium binding in the first EGF domain is not altered in factor IX Alabama. The active site of factor IXa Alabama is fully competent to activate factor X in the absence of calcium when using polylysine as a surface to catalyze the reaction. Factor IXa Alabama has only 64% of normal factor IXa activity in the presence of 300 microM CaCl2 in the polylysine-catalyzed system although apparent high affinity calcium binding constants are similar. Factor IXa Alabama has 52-60% of normal activity in a calcium/phospholipid vesicle system. The addition of factor VIIIa to the phospholipid vesicle system decreases the relative rate of factor IXa Alabama to 18-19% of normal. Three-dimensional computer-aided models of the first EGF domain of normal factor IX and factor IX Alabama indicate no major structural alterations resulting from the glycine substitution for Asp47. The model of the first EGF domain of normal factor IX predicts a calcium-binding site involving Asp47, Asp49, Asp64, and Asp65. Our binding data, however, indicate that Asp47 is not necessary to form the high affinity binding site. We conclude that Asp47 in normal factor IX coordinates to the bound calcium, inducing a conformational change in the molecule essential for proper interaction with factor X and factor VIIIa.

Binding Sites↗

PubMed2006

Characterization of a monoclonal antibody B1 that recognizes phosphorylated Ser-158 in the activation peptide region of human coagulation factor IX.

Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as gamma-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K(d) value of 5 nm in the presence of Ca(2+) (EC(50) = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca(2+) dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca(2+). These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca(2+)-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.

Amino Acid Sequence↗

PubMed1990

Recombination between two 14-bp homologous sequences as the mechanism for gene deletion in factor IX Seattle 1.

Factor IXSeattle 1 is a 10-kb intragenic deletion identified in a family that has hemophilia B. By sequencing across the site of the deletion, we discovered at the deletion junction a 13-bp sequence (5' . . . TAGAA-GTTCACTT . . . 3') that was homologous to two 14-bp sequences 10 kb apart in introns D and F of the normal factor IX gene. The presence of these homologous sequences in two different regions of the normal gene allows us to propose that genetic recombination has occurred between the sequences, resulting in the gene deletion. The precise recombination site was able to be localized to one of 5 bp (5' . . . AGTTC . . . 3') in the middle of the homologous sequences. The exact length of the deletion is 10,000 bp.

Base Sequence↗

PubMed1998

Structural determination of lipid-bound human blood coagulation factor IX.

Human coagulation factor IX (FIX) is a serine protease which binds to a negatively charged phospholipid surface in the presence of Ca ions (Ca2+). FIX two-dimensional (2-D) crystals were obtained by the lipid layer crystallisation technique under near physiological conditions. The 2-D projection map of the protein was calculated to a resolution of 3 nm using electron crystallographic analysis. The structural organisation of membrane-bound FIX is discussed and compared with the known X-ray crystallographic data.

Binding Sites↗

PubMed1996

Identification of the endothelial cell binding site for factor IX.

We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined.

Animals↗

PubMed2003

Purification, crystallization and preliminary crystallographic analysis of AHP IX-bp, a zinc ion and pH-dependent coagulation factor IX binding protein from Agkistrodon halys Pallas venom.

A new coagulation factor IX binding protein, AHP IX-bp, has been purified from Agkistrodon halys Pallas venom and estimated to be an AB heterodimer of about 25 kDa consisting of two chains (an A chain of 15.5 kDa and a B chain of 15 kDa) linked by one or more disulfide bonds. The N-terminal sequence of AHP IX-bp has been determined and aligned with C-type lectin-like proteins. The protein has a high sequence similarity to some snake-venom C-type lectin-like proteins. AHP IX-bp binds to coagulation factor IX but not to coagulation factor X. Moreover, AHP IX-bp shows binding to coagulation factor IX in both zinc ion-dependent and pH-dependent manners. The affinity between AHP IX-bp and coagulation factor IX is higher under neutral or weakly alkaline conditions than under weakly acidic conditions. Single crystals of AHP IX-bp grown at pH 6.5 and 7.5 diffract X-rays to 2.0 and 1.8 A resolution, respectively. Both crystals are isomorphous and belong to the space group P1; only one AB heterodimer is present in the unit cell.

Agkistrodon↗

PubMed1997

Multicenter retrospective study on the utilization of FEIBA in France in patients with factor VIII and factor IX inhibitors. French FEIBA Study Group. Factor Eight Bypassing Activity.

Factor VIII or factor IX replacement is frequently impossible in inhibitor-developing hemophiliacs, because of the level of the inhibitor titer. Activated prothrombin complex concentrates are one of the available options to treat the bleeding episodes in such patients. However, the efficacy of these products and the associated thrombogenic risk, particularly in prolonged administration such as employed during surgeries, are important concerns for hemophilia care providers. We performed a multicenter retrospective study to evaluate the use of FEIBA (Factor Eight Bypassing Activity) in France, and data is presented on 433 bleeding episodes, including surgical procedures, concerning 60 patients from 15 hemophilia centers. The efficacy was judged as good or excellent in 352 episodes (81.3%), poor in 73 episodes (16.9%) and non-existent in 8 episodes (1.8%). Minor and major surgical procedures were successfully performed using FEIBA as a second-line therapy after human or porcine factor VIII, and in some occasions FEIBA was utilized as the only substitution product. The tolerance was assessed as good in 428 episodes (98.8%), but in 5 cases adverse effects were reported. Only 3 patients out of 52 regularly evaluated (5.8%) were HIV-seropositive, and for two of them the seroconversion occurred prior to the first use of FEIBA. In contrast, 80.4% of the patients were HCV-seropositive. An anamnestic response after the administration of FEIBA was noted in 31.5% of cases. This study points out the main features of the use of FEIBA in France, and particularly the low HIV seroprevalence in the patients treated. The good efficacy and the excellent tolerance still confer to this product a place to consider in the therapeutic options for the treatment of inhibitor-developing hemophiliacs or in acquired hemophilia.

Adolescent↗

PubMed1998

IgE-mediated allergy and desensitization to factor IX in hemophilia B.

BACKGROUND: We describe two patients with factor IX deficiency and high levels of inhibitors to factor IX who developed anaphylaxis to factor IX. OBJECTIVE: The aim of this study was to develop a skin test, RAST, and desensitization protocol for factor IX allergy. METHODS: The patients were evaluated by skin test and RAST to factor IX. They also underwent desensitization to factor IX. RESULTS: Both patients had positive skin test and RAST reactions to factor IX. Control subjects had negative reactions. Both patients were successfully desensitized to factor IX by using two different desensitization protocols. The patients' skin test and RAST reactions to factor IX converted to negative after desensitization. CONCLUSIONS: IgE-mediated reactions to factor IX do occur and may be diagnosed with the use of skin test and RAST. Patients with this type of reaction may be successfully desensitized to factor IX.

Child↗

PubMed2000

Surface-loop residue Lys316 in blood coagulation Factor IX is a major determinant for Factor X but not antithrombin recognition.

The active site of activated Factor IX (FIXa) and related blood-coagulation enzymes is surrounded by a number of highly variable surface loops, which contribute to the characteristic substrate specificity of each individual enzyme. FIX residue Lys(316) is located in one of these loops and mutation of this residue to Glu is associated with haemophilia B. In the present study we investigated the functional role of Lys(316) in human FIXa by analysing the purified and activated FIX mutants FIXa-K316E and FIXa-K316A. FIXa-K316E was indistinguishable from normal FIXa in binding the competitive active-site inhibitor p-aminobenzamidine. In addition, substitution of Glu for Lys(316) had no significant effect on the reactivity towards various synthetic tripeptide substrates. Inhibition by the macromolecular inhibitor antithrombin was only slightly reduced for both FIXa mutants (less than 2-fold). In contrast, proteolytic activity of FIXa-K316E towards the natural substrate Factor X (FX) was virtually lacking, while the Lys(316) to Ala mutation resulted in a more than 10-fold reduction in FX activation. Thus residue Lys(316) plays a key role in FIXa activity towards FX. The requirement for Lys at position 316 for FX activation was also evident in the presence of the cofactor activated Factor VIII, although to a lesser extent than in its absence. These data demonstrate that Lys(316) specifically determines the reactivity of FIXa towards its natural substrate FX, but not to synthetic peptide substrates or antithrombin.

Antithrombins↗

PubMed1997

Recombinant factor IX.

Despite the introduction of recombinant preparations of factor VIII and recombinant factor VII and VIIa, patients with other forms of hemophilia, especially hemophilia B, have remained at increased risk for blood borne viruses because of a lack of clinically utilizable preparations of recombinant factor IX. This report describes the state of current tests with a recently licensed preparation of recombinant factor IX, BeneFix, from Genetics Institute. Structurally, functionally, and therapeutically, recombinant factor IX is comparable to monoclonal plasma-derived factor IX. The only observed difference between recombinant and plasma factor IX is the recovery in pharmacokinetic studies where recombinant factor IX recovery was approximately 72% that of a plasma factor IX product. This difference is attributed to be due to minor differences in the post-translational modification of recombinant factor IX compared to plasma. These studies demonstrate that recombinant factor IX is effective in the treatment of hemophilia B and has the safety profile expected from a product prepared by recombinant technology.

Animals↗

PubMed1987

Thrombogenicity of factor IX complex: in vivo investigation.

Factor IX Complex therapy has been associated with thrombosis when used in patients with liver disease, hemophilia B and hemophilia A with inhibitors to Factor VIII when administered repetitively and in high doses. Three mechanisms for inducing thrombogenicity have been proposed: activated coagulants, coagulant-active phospholipid content and/or the high zymogen level attained in recipients. A new animal model using Russel Viper Venom (RVV) as an in vivo stimulus of coagulation has been used to investigate the role of high levels of zymogens in the induction of thrombosis. The quantity of RVV tolerated by rabbits infused with Factor IX Complex is reduced 100-fold. Infusion of Factor X or prothrombin also reduces the lethal dose of RVV. In contrast, Coagulation Factor IX devoid of other coagulants has little effect. However other animal data indicate that the amount of activated coagulants can play a role in the in vivo thrombogenicity of Factor IX Complex.

Animals↗