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PubMed1992

The endothelial cell binding determinant of human factor IX resides in the gamma-carboxyglutamic acid domain.

The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a Kd of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the gamma-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, we performed competitive binding experiments between 125I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. Our data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.

1-Carboxyglutamic Acid↗
PubMed1997

gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.

1-Carboxyglutamic Acid↗
PubMed1988

A factor IX mutation, verified by direct genomic sequencing, causes haemophilia B by a novel mechanism.

A novel factor IX gene mutation (factor IX London 2) has been characterized. This causes severe crm+ haemophilia B as the patient's plasma shows normal factor IX antigen level and less than 1% clotting activity. Sequence analysis of the entire cloned coding and promoter regions revealed a single point mutation: a G----A transition at position 31,119. This region of the patient's DNA was amplified in vitro by the polymerase chain reaction and the nucleotide change was confirmed by direct sequencing of the amplified products. The mutation results in the substitution of the arginine at position 333 by glutamine. This arginine residue is absolutely conserved in the catalytic domain of normal human and bovine factor IX, X and prothrombin. The substitution by glutamine causes the loss of a positive charge from the surface of the factor IX London 2 protein. This mutation pinpoints a previously unknown, functionally critical feature of factor IX which may be involved in substrate or co-factor binding.

Base Sequence↗
PubMed1986

Calcium-specific immunoassays for factor IX: reduced levels of antigen in patients with vitamin K disorders.

Polyclonal rabbit anti-factor IX antisera were fractionated to establish solid-phase immunoassays recognizing calcium-dependent and non-calcium-dependent epitopes. The assays were greater than 99.9% specific for factor IX and sensitive to 0.05 U/dl plasma or 2 ng/ml purified factor IX. For the calcium-dependent fraction, an absolute requirement of divalent metal ions was found, and Sr(II), Mn(II), and Mg(II) could substitute for Ca(II). On immunoblots of reduced, electrophoresed factor IXa, the 125I-calcium-dependent antibody fraction bound to the amino-terminal light chain. Plasma sampled from 13 patients receiving warfarin and one with cephalosporin-related vitamin K deficiency had a mean level of calcium-dependent factor IX antigen of 22 U/dl, comparable to the 24 jU/dl average of factor IX procoagulant activity; these two results were highly correlated. Antigen levels determined by either the polyclonal or a monoclonal, non-calcium-dependent anti-factor IX assays ranged from 1.7-fold to 6.0-fold greater than the corresponding levels of factor IX procoagulant activity or calcium-dependent antigen level for each subject's plasma. The difference reflects inactive, circulating factor IX. In contrast, factor IX antigen levels determined by an assay using a monoclonal, calcium-dependent anti-factor IX were from one half to one thirteenth as much as those measured by the polyclonal, calcium-dependent immunoassay. The disparity between results of calcium-dependent assays suggests that some Gla residues near the amino terminus of factor IX are relatively less important for normal procoagulant function of factor IX than others, are more sensitive to the effects of vitamin K antagonism or deficiency, and are important for the epitope recognized by this particular calcium-dependent, monoclonal antibody.

Adult↗
PubMed1995

Low risk of viral infection after administration of vapor-heated factor VII concentrate or factor IX complex in first-time recipients of blood components. International Factor Safety Study Group.

BACKGROUND: Vapor-heated human factor VII concentrate and human factor IX complex are both obtained from prothrombin complex, undergo similar methods of manufacture, and are subjected to an identical two-step vapor-heating process for virus inactivation. STUDY DESIGN AND METHODS: Intermediate-purity vapor-heated human factor VII concentrate and vapor-heated human factor IX complex were monitored for safety with regard to viral infection in the context of an International Factor Safety Study, a prospective study that follows the revised recommendations from the International Congress of Thrombosis and Hemostasis (ICTH). Because the rarity of the respective hereditary deficiencies would have made separate analyses unrealizable, the results were combined for the final analysis. Entry required that patients have no history of transfusion with any blood derivative. After the first infusion of the study drug, patients were monitored for 6 months for the development of non-A, non-B hepatitis (NANBH) and infection with hepatitis B virus (HBV) and for 15 months for infection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV). An event was defined as a positive result on any test for any infection. An alanine aminotransferase level more than 2.5 times the upper limit of normal on two consecutive occasions was defined as an event for NANBH. HBV infection was monitored with tests for three different HBV markers: the HBV surface antigen, antibody against the HBV surface antigen, and antibody against HBV core antigen. HCV and HIV infection were monitored with tests for HCV and HIV antibodies. RESULTS: The 25 patients who completed the study (1 has not completed the study and 1 dropped out) received a total of 434 infusions comprising 17 different production lots of the concentrates. Twenty patients were analyzable for NANBH and 25 for HCV and HIV infection. Since most patients had been given HBV vaccination, only 4 patients were analyzable for this end point. None of the patients showed evidence of having developed an event. These data satisfy ICTH criteria when the products are considered together, but vapor-heated factor VII concentrate does not qualify alone because there were only five patients in this group. CONCLUSION: Vapor-heated factor VII concentrate and vapor-heated factor IX complex are associated with a low risk of viral infection. Preliminary results are also presented, indicating that the concentrates are safe with regard to inhibitor development.

Adult↗
PubMed1990

Development of a sensitive and rapid chromogenic factor IX assay for clinical use.

A chromogenic factor IX assay is developed which requires only two time-dependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XIa, thrombin, CaCl2, and phospholipids. Then factor XIa activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXa, factor VIIIa, Ca ions, and phospholipids) rapidly activates factor X. Finally, ethylenediaminetetraacetic acid plus a chromogenic substrate are added to stop the reaction and to measure formed factor Xa. Factor Xa formation is proportional to the plasma factor IX concentration (from 0 to 140%). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 degrees C. A new isolation procedure for factor VIII is described. Factor VIII is purified from bovine plasma in a few steps with a yield of 20% and a 8,000-fold purification.

Animals↗
PubMed2002

Pharmacokinetic and pharmacodynamic modeling of humanized anti-factor IX antibody (SB 249417) in humans.

BACKGROUND: SB 249417 is a humanized anti-factor IX/IXa antibody that, on administration to rats and monkeys, produces an immediate suppression of factor IX activity and prolongation of activated partial thromboplastin times (aPTT). OBJECTIVE: Our objective was to establish the pharmacokinetics of SB 249417 and to explore its effects on factor IX activity levels and aPTT in humans. METHODS: In this phase I, single-blind, randomized, placebo-controlled, parallel-group, single intravenous infusion study, individual and mean data from a total of 26 healthy volunteers at 5 dosing levels were analyzed. A 2-compartment pharmacokinetic model was used in the analysis of total SB 249417 concentration-time profiles. A modified indirect-response model was used, with the total concentration indirectly serving as the driving force for the suppression of free factor IX concentration (as assessed by factor IX activity). The aPTT was related to factor IX activity with a biexponential equation, and a population approach was used to generate posterior parameter estimates for the individual fittings. RESULTS: Mean parameter estimates from individual fittings are 0.092 L/kg for volume of distribution, 0.15 L/kg for steady-state volume of distribution, and 0.0021 L/kg per hour for systemic clearance. The model described well the factor IX activity and aPTT time course in response to SB 249417 at 5 dose levels. The estimated half-life of factor IX in blood was 21 hours. CONCLUSIONS: This model was stable and robust in fitting both mean and individual data. Endogenous factor IX baseline levels and dose were the major determinants of the decline in factor IX activity during the infusion period. Thereafter the recovery of factor IX activity was governed solely by the endogenous factor IX turnover rate.

Adult↗
PubMed1979

The binding of calcium to the activation products of bovine factor IX.

Binding isotherms of Ca2+ to the bovine Factor IX activation intermediates and products, i.e. Factor IXalpha, Factor IXa alpha, and Factor IXa beta have been examined. At pH 7.4, Factor IX alpha possesses at least two strong Ca2+ sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 3.7 mM. Bovine Factor IXa alpha also contains at least two Ca2+ binding sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 1.3 mM. Factor IXa beta, the ultimate activation product of Factor IX, in the intrinsic system, likewise contains at least two strong Ca2+ sites, of average KD 0.1 mM, as well as seven additional weaker sites, possessing an average KD of 1.0 mM. The Ca2+-binding properties of the above proteins are similar to those of their precursor molecule, Factor IX, which we have earlier shown to possess at least two strong Ca2+ sites, with an average KD of 0.1 mM, and 11 weaker sites, of average KD 1.3 mM (Amphlett, G.W., Byrne, R., and Castellino, F.J. (1978) J. Biol. Chem. 253, 6774-6779). Circular dichroism analysis of all of the above proteins was consistent with the molecules possessing a low alpha-helical content, and a high quantity of beta structure and random coil conformations.

Animals↗
PubMed1994

Membrane binding properties of the factor IX gamma-carboxyglutamic acid-rich domain prepared by chemical synthesis.

The fully gamma-carboxylated peptides based upon the complete and truncated Gla/aromatic amino acid stack domains of human Factor IX were prepared by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. A 47-residue peptide Factor IX-(1-47) and a 42-residue peptide Factor IX-(1-42), both containing 12 residues of L-gamma-carboxyglutamic acid, were purified by high performance liquid chromatography and oxidized to form the disulfide bond. Quantitative gamma-carboxyglutamic acid analysis of Factor IX-(1-47) and Factor IX-(1-42) indicated the presence of 12.1 and 11.2 gamma-carboxyglutamic acid residues/mol of peptide, respectively; no glutamic acid was detected. As monitored by fluorescence quenching, calcium ions induced the prototypical conformational transition in Factor IX-(1-47), but not in Factor IX-(1-42), that is observed with Factor IX. Half-maximal quenching of the intrinsic fluorescence of Factor IX-(1-47) was observed at Ca(II) concentrations of about 50 microM. Factor IX-(1-47) bound to the conformation-specific antibodies, anti-Factor IX:Mg(II) and anti-Factor IX:Ca(II)-specific in the presence of metal ions. Factor IX-(1-47) bound to phospholipid membranes, as monitored by energy transfer from intrinsic fluorophores to dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-phosphatidylethanolamine incorporated into a lipid bilayer composed of phosphatidylserine:phosphatidylcholine. In contrast, Factor IX-(1-42) bound poorly to these same membranes. Factor IX-(1-47) did not inhibit Factor XIa activation of Factor IX but did inhibit the activation of Factor X by Factor IXa bound to Factor VIII in the presence of calcium ions and phospholipid. These results show that phospholipid membrane binding is a property of the Gla/aromatic amino acid stack domain and that the Factor IX-(1-47) peptide, prepared by chemical synthesis, preserves the membrane binding properties and the metal-induced conformational transitions observed in native Factor IX. These results indicate that Factor IX-(1-47) but not Factor IX-(1-42) is a suitable model for structural studies of Factor IX-membrane interaction.

1-Carboxyglutamic Acid↗
PubMed1987

Factor IX metal ion-dependent antigen assays for measurement of warfarin effect.

Factor IX metal ion-dependent antigen was assayed using monoclonal antibodies in 521 samples obtained after prothrombin time testing in patients treated with warfarin. Factor IX metal ion-dependent antigen was less than measured Factor IX clotting activity and less than total Factor IX antigen adsorbable to aluminium hydroxide, suggesting that the metal ion-dependent antigen assay measures a subpopulation of circulating Factor IX in patients treated with oral anticoagulants. There was a graded decrease of Factor IX metal ion-dependent antigen as prothrombin times increased in patient samples. In two hospitals, the median prothrombin times and Factor IX antigen levels were 19 and 20 seconds and 0.10 and 0.11 U/mL (kU/L), respectively. This study shows that immunoassays measure the biologic effect of warfarin and provide information that may supplement the prothrombin time test for patient monitoring. Factor IX metal ion-dependent antigen assays may be useful in efforts to standardize laboratory tests for warfarin effect.

Anticoagulants↗
PubMed1994

Characterization of a factor IX variant with a glycine207 to glutamic acid mutation.

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

Benzamidines↗
PubMed1986

Rabbit polyclonal antibodies against the calcium-dependent conformation of factor IX and their application in solid phase immunoradiometric assays.

Two subpopulations of antibodies were isolated from rabbit polyclonal antiserum directed against human factor IX: one against the Ca(II)-dependent conformation of factor IX and one against the Ca(II)-independent conformation of factor IX. The two subpopulations were used for the development of immunoradiometric assays (IRMA's) for factor IX:Ca(II)Ag and factor IX:NonCa(II)Ag respectively. Ranges for the concentration of factor IX:Ca(II)Ag and factor IX:NonCa(II)Ag were established in plasmas of healthy volunteers, patients treated with oral anticoagulants and hemophilia B patients. In the group of patients using oral anticoagulant therapy a progressively reduced ratio of factor IX:Ca(II)Ag to factor IX:NonCa(II)Ag was observed with increasing intensity of oral anticoagulant treatment. Variant factor IX molecules from hemophilia B patients (CRM-, CRM(Red) and CRM+) with a defective Ca(II) binding or defective conformational transition induced by Ca(II) binding, were identified. These defects are absent in variant factor IX molecules from one hemophilia Bm patient and from patients with hemophilia B Leyden.

Administration, Oral↗
PubMed1989

Factor IX Kawachinagano: impaired function of the Gla-domain caused by attached propeptide region due to substitution of arginine by glutamine at position -4.

Factor IX Kawachinagano (KWC) is a mutant factor IX protein initially recognized in a patient with severe haemophilia B, who had 46% of normal factor IX antigen and no detectable clotting activity. Previous studies indicated that factor IX KWC was not activated by factor XIa in the presence of Ca ions. In the present study, we purified and analysed factor IX KWC at a structural level in an attempt to clarify the nature of the impaired reaction with factor XIa. Kinetic studies showed that activation of factor IX KWC by factor X activator from Russell's viper venom (RVV-X) was normal, whereas activation by factor XIa was defective. Amino acid sequence analysis of tryptic peptides and direct analysis of the NH2-terminal sequence of factor IX KWC demonstrated that this mutant factor IX retained the propeptide region of 18 amino acids due to a substitution of arginine-(-4) by glutamine. These data suggested that the Gla-domain of factor IX KWC was dysfunctional, although the total gamma-Gla content, measured by alkaline-hydrolysis, was normal. We assumed that this attached propeptide region of the molecule directly interferes with the adjacent NH2-terminus and prevents the metal-induced conformational changes which are essential for biological activity of normal factor IX.

1-Carboxyglutamic Acid↗
PubMed1981

Labeled factor IX kinetics in patients with hemophilia-B.

Labeled factor IX was infused five time into four patients with hemophilia-B. Ten-minute plasma recovery average 35% (SD +/- 2) and the mean T 1/2 beta-phase elimination was 23 hr (+/- 5). No alteration in the postinfusion 125I-factor-IX could be detected by radioautography of plasma samples run on polyacrylamide gels or on crossed-immunoelectrophoresis. Label was excreted into the urine as free 125I-iodide. Kinetics were similar when the labeled preparation was infused alone or with a commercial concentrate containing unlabeled factor IX. Infusion of factor IX in man is best described by a two-compartment open pharmacokinetic model where factor IX is distributed in a space larger than the plasma volume.

Animals↗
PubMedDate not supplied

Evaluation of two viral inactivation methods for the preparation of safer factor VIII and factor IX concentrates.

We report here the results of our evaluation of two procedures to eliminate viruses in factor VIII and factor IX coagulation factor concentrates. Both procedures were equally effective in the in vitro destruction of marker viruses. However, in a controlled infectivity test in chimpanzees, treatment at 60 degrees C for 20 hours inactivated greater than 500 and less than 10,000 chimpanzee infectious doses (CID) of hepatitis B virus, while treatment at 98 degrees C for 30 minutes inactivated less than 500 CID. Both methods were successful in preventing infection with an undetermined amount of an indeterminate non-A, non-B hepatitis agent. The 60 degrees C, 20-hour treatment method rendered 5.25 logs of the putative acquired immune deficiency syndrome virus, human T-cell lymphotrophic virus III/lymphadenopathy virus, added to factor VIII or factor IX concentrates, undetectable. Heat-treated factor VIII and factor IX complex concentrates prepared by these methods were tested against corresponding untreated control lots. There was no significant difference in the plasma recovery or plasma half-life of the factor (p greater than 0.05). The treated concentrates were equivalent to the control concentrates with respect to vital signs, clinical laboratory studies, and adverse reactions. The heat-treated concentrates appeared bioequivalent to the untreated concentrates with the additional benefit of inactivation of potentially present infectious viruses.

Animals↗
PubMed1998

Identification of residues in the Gla-domain of human factor IX involved in the binding to conformation specific antibodies.

The binding of Ca2+ induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(II) antibodies recognize a conformational epitope (FIX') that can be induced by several metal ions such as Ca2+, Mg2+, Mn2+ and Ba2+, while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX*) that can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) pp. 7605-7612). The latter conformation is essential for the function of factor IX. In this study we tried to identify residues in the Gla-domain of factor IX which are involved in binding to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For this we substituted residues in recombinant human factor IX for those of factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodies. Re-introduction of factor IX specific residues increased the binding to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicating that the structural integrity of the Gla-domain was not seriously affected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg(II) antibodies, while residues 1-11 are important for binding to anti-factor IX:Ca(II) antibodies.

Amino Acid Sequence↗
PubMed1980

Removal of hepatitis-B-virus infectivity from factor-IX complex by hepatitis-B immune-globulin. Experiments in chimpanzees.

Hepatitis-B-virus infectivity can be removed from a heat-labile clotting factor concentrate by the addition of hepatitis-B immune-globulin. Three chimpanzees were each inoculated with samples of factor-IX complex (factor IX) which had been deliberately contaminated with 10(3.5) chimpanzee infectious doses of hepatitis-B virus from a known infectious inoculum. Two of these factor IX samples had been incubated with hepatitis-B immune-globulin after the addition of hepatitis-B virus. 10 weeks after inoculation hepatitis-B infection developed in the chimpanzee inoculated with untreated factor IX. Hepatitis B did not develop in the two which received treated factor IX; no serloigical evidence of hepatitis B could be detected during 52 weeks of evaluation.

Animals↗
PubMed1995

A method to estimate effects of amino acid substitutions in blood coagulation factor IX from hemophilia B patients.

Hemophilia B is a hereditary disease caused by defects in coagulation factor IX. Accumulation of sequence data in the hemophilia B database makes it possible to study this disease at the molecular level. The most common mutations reported in the database are amino acid substitutions. Activity of factor IX in a patient's blood depends on a position of the substitution and combination of original and substituting amino acids. We have developed a method to estimate the effects of amino acid substitutions in factor IX of hemophilia B patients. We adopted two kinds of scores: (1) amino acid homology matrices, and (2) distances obtained from physical-chemical properties of amino acids. We found that: (1) the distance obtained from molecular volume shows the highest correlation with factor IX activity among four physical-chemical properties of amino acids, and (2) Dayhoff's 250PAM matrix gives the highest correlation among three homology matrices. We performed a multiple regression analysis for the estimation of factor IX activity by using four amino acid parameters and obtained a distance matrix, which gives the multiple correlation coefficient 0.6. Hemophilia is a hereditary, X-linked, recessive hemorrhagic disorder. About three-fourths of patients with hemophilia have the A type which is due to deficient factor VIII activity. The B type is less frequent than the A type and is due to deficient factor IX activity. Factor IX is a vitamin K dependent plasma protein that participates in the middle phase of blood coagulation. The entire nucleotide sequence of the factor IX gene was reported by Yoshitake et. al. There have been reported a variety of defects in the factor IX gene from hemophilia B patients, and these are summarized in the hemophilia B database. The effects of amino acid substitutions on the activities of factor IX depends on two factors. One is the combination of native and hemophilic amino acids, the differences of physical-chemical properties of the amino acids. Another is the position of mutation in factor IX protein. In this study, we analyzed missense mutations in the database described with factor IX activity values. Among them, the cases with double mutations were excluded from the analysis, leaving 255 cases. The number of unique mutations are 127. We have introduced distances between 20 amino acids by using the following four physical-chemical properties: (1) Molecular volume, (2)Hydropathy, (3) Polar requirement, and (4) Isoelectric point. We also adopted three homology matrices. These matrices are symmetric and composed of 20 x 20 elements, corresponding to amino acid pairs: (1)Dayhoff's 250 PAM matrix, (2) SCM (Structure-derived Correlation Matrix), and (3) SSM (Structure Superposition Matrix). This analysis shows that the highest correlation with factor á¿ activity is 0.47 given by molecular volume. The second one is 0.4 of Dayhoff's 250 PAM matrix. The multiple regression analysis, using four physicalchemical properties of amino acids, gives a correlation coefficient of 0.60. The amino acid distance obtained by the multiple regression analysis provides a useful measure to estimate the severity of the disease caused by a missense mutation. For example, if the distance between native and hemophilic amino acids is less than 1.5, the ratios of severe cases (activity is less than or equal to 1% of the normal), moderate cases (less than or equal to 4%), and mild cases are 67%, 27% and 6%, respectively.

Amino Acid Sequence↗