Factor IX Denver, ASN 346→ASP Mutation Resulting in a Dysfunctional Protein with Defective Factor VIIIa Interaction

SummaryHemophilia B is a sex-linked recessive bleeding disorder characterized by the presence of either a decreased amount of normal factor IX (FIX) or the presence of a dysfunctional FIX. We have identified a unique mutation in a family with mild hemophilia B. DNA analysis of family members revealed a single base transition in the 8th exon of the FIX gene predicting an amino acid change of Asn 346→Asp in the catalytic domain. The FIX variant, named FIX Denver, was purified from proband plasma. Kinetic studies of factor X (FX) interactions with normal FIXa or FIXa Denver and phospholipid (PL) showed little difference in kcat, but a significant difference when factor VIIIa (FVIIIa) was included in the reaction. Using kinetic assays to infer the Kd of FIXa for FVIIIa, normal FIXa had a Kd of 0.095 nM while that of FIXa Denver was 9.85 nM. The major defect caused by this point mutation is a marked decrease in the affinity of FIXa Denver for factor VIIIa.

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Regions 301–303 and 333–339 in the catalytic domain of blood coagulation Factor IX are Factor VIII-interactive sites involved in stimulation of enzyme activity

Biochemical Journal ◽

10.1042/bj3390217 ◽

1999 ◽

Vol 339 (2) ◽

pp. 217-221 ◽

Cited By ~ 5

Author(s):

Joost A. KOLKMAN ◽

Peter J. LENTING ◽

Koen MERTENS

Keyword(s):

Catalytic Domain ◽

Coagulation Factor ◽

Factor Ix ◽

Factor X ◽

Factor Ixa ◽

Factor Viiia ◽

Α Helix ◽

Factor X Activation ◽

Affinity Interaction ◽

Stimulation Of

The contribution of the Factor IX catalytic domain to Factor VIIIa binding has been evaluated by functional analysis of Factor IX variants with substitutions in α-helix region 333–339 and region 301–303. These regions were found to play a prominent role in Factor VIIIa-dependent stimulation of Factor X activation, but do not contribute to the high-affinity interaction with Factor VIIIa light chain. We propose that complex assembly between Factor IXa and Factor VIIIa involves multiple interactive sites that are located on different domains of these proteins.

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Hemophilia B with Mutations at Glycine-48 of Factor IX Exhibited Delayed Activation by the Factor VIIa-tissue Factor Complex

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614078 ◽

2000 ◽

Vol 84 (10) ◽

pp. 626-634 ◽

Cited By ~ 8

Author(s):

Pai-Chih Wu ◽

N. Hamaguchi ◽

Yi-Shing Yu ◽

Ming-Ching Shen ◽

Shu-Wha Lin

Keyword(s):

Calcium Ions ◽

Factor Viia ◽

Factor Ix ◽

Hemophilia B ◽

Factor X ◽

Extrinsic Pathway ◽

Catalytic Function ◽

Factor Ixa ◽

Factor Viiia ◽

Human Factor Ix

SummaryGly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IX>Malmö27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%–27% normal factor IX’s clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X; in the presence of only phospholipids and calcium ions, both mutants showed a 4∼7-fold elevation in K m, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2∼3-fold decrease in the catalytic function was observed with the mutant factor IXa’s, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent K d for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine thromboplastinbased PT assays. Taken together, our results suggest that mutations at Gly-48 altered the interaction of factor IX with its extrinsic pathway activator (VIIa-TF complex), its macromolecular substrate (factor X), and its cofactor (factor VIIIa).

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Markers of Hypercoagulability in Patients with Hemophilia B Given Repeated, Large Doses of Factor IX Concentrates during and after Surgery

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642515 ◽

1994 ◽

Vol 71 (06) ◽

pp. 737-740 ◽

Cited By ~ 18

Author(s):

E Santagostino ◽

P M Mannucci ◽

A Gringeri ◽

G Tagariello ◽

F Baudo ◽

...

Keyword(s):

High Risk ◽

Ex Vivo ◽

Factor Ix ◽

Hemophilia B ◽

Coagulation System ◽

Prolonged Treatment ◽

Orthopedic Procedures ◽

Factor X ◽

Prothrombin Complex Concentrates ◽

Increased Risk

SummaryPurer factor IX (FIX) concentrates have been produced for the treatment of hemophilia B in the attempt to reduce the risk of thrombotic complications associated with the use of prothrombin complex concentrates. To evaluate ex vivo whether or not FIX concentrates activate the coagulation system in conditions associated with a high risk for thrombosis, we measured markers of hypercoagulability in 10 patients with hemophilia B who underwent surgery, mainly orthopedic procedures, covered by multiple concentrate infusions (40-80 U/kg/day). Postinfusion plasma levels of prothrombin fragment 1+2 and factor X activation peptide did not differ significantly from the presurgical levels, neither before nor after each concentrate dose. Therefore, it appears that prolonged treatment of patients with hemophilia B undergoing high risk surgical procedures with high doses of FIX concentrate does not cause systemic activation of coagulation. This suggests that purified FIX concentrates are preferable to prothrombin complex concentrates for conditions associated with an increased risk of thrombosis.

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Development of Monospecific Antibodies to Human Factor IX

10.1055/s-0039-1680660 ◽

1977 ◽

Author(s):

Cheryl Y. Tiarks ◽

Chin-Hai Chang ◽

Liberto Pechet

Keyword(s):

Neutralizing Antibodies ◽

Human Factor ◽

Human Albumin ◽

Factor Ix ◽

Hemophilia B ◽

Red Cross ◽

Factor X ◽

Normal Plasma ◽

Human Factor Ix ◽

Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

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The role of the second growth-factor domain of human factor IXa in binding to platelets and in factor-X activation

Biochemical Journal ◽

10.1042/bj3100427 ◽

1995 ◽

Vol 310 (2) ◽

pp. 427-431 ◽

Cited By ~ 14

Author(s):

S S Ahmad ◽

R Rawala ◽

W F Cheung ◽

D W Stafford ◽

P N Walsh

Keyword(s):

Growth Factor ◽

Human Factor ◽

Factor Ix ◽

Factor X ◽

Binding Studies ◽

Factor Ixa ◽

Equilibrium Binding ◽

Activated Platelets ◽

Factor Viiia ◽

Factor X Activation

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd, app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.

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Releasing the brakes in coagulation Factor IXa by co-operative maturation of the substrate-binding site

Biochemical Journal ◽

10.1042/bcj20160336 ◽

2016 ◽

Vol 473 (15) ◽

pp. 2395-2411 ◽

Cited By ~ 5

Author(s):

Line Hyltoft Kristensen ◽

Ole H. Olsen ◽

Grant E. Blouse ◽

Hans Brandstetter

Keyword(s):

Coagulation Factor ◽

Factor Ix ◽

Fine Tuning ◽

Structural Basis ◽

Factor X ◽

Multifaceted Approach ◽

Factor Ixa ◽

Activated Platelets ◽

Factor Viiia ◽

Multiple Regions

Coagulation Factor IX is positioned at the merging point of the intrinsic and extrinsic blood coagulation cascades. Factor IXa (activated Factor IX) serves as the trigger for amplification of coagulation through formation of the so-called Xase complex, which is a ternary complex of Factor IXa, its substrate Factor X and the cofactor Factor VIIIa on the surface of activated platelets. Within the Xase complex the substrate turnover by Factor IXa is enhanced 200000-fold; however, the mechanistic and structural basis for this dramatic enhancement remains only partly understood. A multifaceted approach using enzymatic, biophysical and crystallographic methods to evaluate a key set of activity-enhanced Factor IXa variants has demonstrated a delicately balanced bidirectional network. Essential molecular interactions across multiple regions of the Factor IXa molecule co-operate in the maturation of the active site. This maturation is specifically facilitated by long-range communication through the Ile212–Ile213 motif unique to Factor IXa and a flexibility of the 170-loop that is further dependent on the conformation in the Cys168–Cys182 disulfide bond. Ultimately, the network consists of compensatory brakes (Val16 and Ile213) and accelerators (Tyr99 and Phe174) that together allow for a subtle fine-tuning of enzymatic activity.

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A candidate activation pathway for coagulation factor VII

Biochemical Journal ◽

10.1042/bcj20190595 ◽

2019 ◽

Vol 476 (19) ◽

pp. 2909-2926

Author(s):

Tina M. Misenheimer ◽

Kraig T. Kumfer ◽

Barbara E. Bates ◽

Emily R. Nettesheim ◽

Bradford S. Schwartz

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Factor Viia ◽

Coagulation Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Contact Activation ◽

Coagulation Factor Vii ◽

Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

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Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B

Blood ◽

10.1182/blood.v72.5.1633.bloodjournal7251633 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1633-1638

Author(s):

AR Thompson ◽

SH Chen ◽

KJ Smith

Keyword(s):

White Women ◽

Dna Analysis ◽

Solid Phase ◽

Factor Ix ◽

Hemophilia B ◽

Carrier Status ◽

Strong Linkage Disequilibrium ◽

White Families ◽

African Black ◽

Diagnostic Role

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.

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Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽

10.1182/blood.v79.2.398.bloodjournal792398 ◽

1992 ◽

Vol 79 (2) ◽

pp. 398-405 ◽

Cited By ~ 26

Author(s):

R Rawala-Sheikh ◽

SS Ahmad ◽

DM Monroe ◽

HR Roberts ◽

PN Walsh

Keyword(s):

Binding Sites ◽

Factor Xa ◽

Catalytic Efficiency ◽

Factor Ix ◽

Factor X ◽

Apparent Dissociation Constant ◽

Factor Ixa ◽

Factor Viiia ◽

Carboxyglutamic Acid ◽

Factor X Activation

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

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Two Distinct Mutations Cause Severe Hemophilia B in Two Unrelated Canine Pedigrees

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614374 ◽

1999 ◽

Vol 82 (10) ◽

pp. 1270-1275 ◽

Cited By ~ 11

Author(s):

Weikuan Gu ◽

James Catalfamo ◽

Jharna Ray ◽

Kunal Ray ◽

Marjory Brooks

Keyword(s):

Catalytic Domain ◽

Stop Codon ◽

Spontaneous Mutation ◽

Mammalian Species ◽

Donor Site ◽

Splice Junction ◽

Factor Ix ◽

Hemophilia B ◽

Acceptor Site ◽

Plasma Factor

SummaryThe molecular defects causing severe factor IX deficiency were identified in two distinct canine breed-variants. Both defects were associated with an absence of plasma factor IX coagulant activity and antigen. A large deletion mutation was found in 1 breed variant, spanning the entire 5’ region of the factor IX gene extending to exon 6. An approximately 5 kb insertion disrupted exon 8 of the second breed-variant. This insertion was associated with alternative splicing between a donor site 5’ and acceptor site 3’ to the normal exon 8 splice junction, with introduction of a new stop codon. The resultant transcript lacked most of the factor IX catalytic domain and 3’ untranslated region. Molecular analyses of canine hemophilia B define an experimental model for study of inhibitor formation and gene therapy strategies, and provide insight into spontaneous mutation mechanisms in the factor IX gene and on the X chromosome of mammalian species.

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