Antibody to C1 Domain of Factor VIII Alters Interaction of Factor Xase Complex with Factor X.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1738-1738
Author(s):  
Gary E. Gilbert ◽  
Anu Bhimavarapu ◽  
Patricia Price ◽  
Marc Jacquemin
Keyword(s):  
Factor Viii ◽  
Factor Ix ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Apparent Affinity ◽  
Factor Ixa ◽  
Factor Viiia ◽  
C1 Domain ◽  
Gla Domain

Abstract The role of the C1 domain in function of factor VIII has not been clearly defined. In contrast, functional interactions have been identified for the three A domains and the C2 domain. We hypothesized that the C1 domain of factor VIII participates in both phospholipid binding and interaction with factor X and/or factor IXa. We evaluated inhibition of the factor Xase complex by LE2E9, a human inhibitor IgG4k mAb against C1. We utilized altered catalytic activity of the factor Xase complex in a defined assay to report the inhibition by LE2E9. Inhibition by LE2E9 was also evaluated when soluble phosphatidylserine replaced vesicles to support the factor Xase complex and when Gla-domainless factor X was the substrate. The deglycosylated form of LE2E9 was also evaluated to better define the mechanism through which LE2E9 exerts its effect. We found that LE2E9 bound to factor VIIIa with an apparent KD of 0.5 nM. The apparent affinity of factor VIIIa for sonicated phospholipid vesicles of phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine 4:20:76 increased 3-fold in the presence of LE2E9. The apparent affinity of factor VIIIa for factor IXa was not significantly changed. The KM of the factor VIIIa-factor IXa complex was 20 ± 2 nM with LE2E9 vs. 40 ± 2 nM without. LE2E9 decreased the Vmax by 77 ± 6% indicating that the affinity of factor X for the factor Xase complex is increased while the rate of cleavage is decreased. When Gla-domainless factor X was used as the substrate for the factor Xase complex, LE2E9 did not inhibit activity indicating that inhibition occurs via an interaction that involves the factor X Gla domain. When the factor VIIIa-factor IX complex was supported by dihexanoyl phosphatidylserine rather than phospholipid vesicles the inhibition of Vmax was 47% indicating that the inhibitory effect does not require a phospholipid bilayer. Deglycosylated LE2E9 did not significantly change the KM but decreased the Vmax by 22% while both antibodies bound to factor VIII with the same affinity. These results suggest that LE2E9 inhibition relates largely to interaction of a carbohydrate moiety with factor VIII or factor X rather than binding the core C1 epitope. We conclude that LE2E9 decreases the KM, and the Vmax for the factor VIIIa-factor IXa complex, but only when the factor X Gla domain is present. These results suggest that in the factor Xase complex the C1 domain of factor VIII is intimately associated with the Gla domain of factor X and that interaction between these domains enhances the kcat for the factor VIIIa-factor IXa complex.

Membrane-Interactive Amino Acids in the Factor VIII C1 Domain Are Cooperative with C2 Domain Epitopes for Membrane Binding and Cofactor Function.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 848-848
Author(s):  
Junhong Lu ◽  
Steven W. Pipe ◽  
Hongzhi Miao ◽  
Marc Jacquemin ◽  
Gary E. Gilbert
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Membrane Binding ◽  
Binding Motif ◽  
C2 Domain ◽  
Factor X ◽  
Apparent Affinity ◽  
Factor Ixa ◽  
C1 Domain ◽  
Membrane Interactive

Abstract Abstract 848 Background: Factor VIII functions as a cofactor in blood coagulation. When released from a non-covalent complex with von Willebrand factor (vWf), activated factor VIII assembles with factor IXa on phosphatidylserine (PS)-containing membranes to form the factor Xase complex. Binding to PS-containing membranes amplifies the activation of factor X by several orders of magnitude. Factor VIII is composed of three A domains, one B domain and two C domains (C1 and C2). The role of C2 domain, including the orientation with respect to membrane surface, vWf-binding motif, and protein-protein contact sites among Xase complex, are relatively well-documented. Recently, the position of the C domains in the factor VIII crystal structure suggested a possible role for the C1 domain in membrane binding. We recently confirmed the participation of K2092 and F2093 of the factor VIII C1 domain in membrane binding (Meems et al. Blood 2009 First edition Aug 18). This work explores the participation of additional C1 domain amino acids and the way the corresponding motif(s) cooperate with motifs of the C2 domain for membrane binding. Methods: Four factor VIII C1 domain mutants encompassing the lower surface of the C1 domain (Arg2090/GLy2091, Lys 2092/Phe2093, Gln2042/Tyr2043, and Arg2159) had individual or paired amino acids mutated to alanine. Mutants were produced in COS-1 cells and purified by immunoaffinity chromatography. The specific activities of these mutants were assessed in a commercial PTT assay as well as phospholipid-limiting and phospholipid-saturating factor Xase assay. Their affinities to factor IXa and factor X were measured by titration experiments using different concentrations of factor IXa and factor X, respectively. Binding to plasma vWf was evaluated in a competition, solution phase enzyme-linked immunosorbent assay (ELISA). The cooperative role of C1 and C2 domains in membrane-binding for cofactor activity was carried out using C1 mutants and antibodies against established membrane-interactive C2 domain motifs, ESH4 and BO2C11. Results: In a competition ELISA for vWf, the affinity of Arg2159 was reduced more than 50-fold, while the other mutants were normal. All mutants had reduced specific activity (range 24-61% of wild type) in a commercial PTT assay containing excess phospholipid. All mutants had decreased apparent affinity for vesicles with limiting (4%) PS by 33, 5, 20, and 18-fold for Arg2090/GLy2091, Gln2042/Tyr2043, Arg2159, and Lys 2092/Phe20933, respectively. However, addition of excess vesicles led to near normal activity for Arg2159. Mutants Arg2090/GLy2091 and Gln2042/Tyr2043 both had 4-fold decreased apparent affinity for factor X and 77% and 84% reduction in Vmax even when phospholipid and factor X were in excess. Mutant Lys 2092/Phe2093 had normal apparent affinity for factor IXa and factor X but > 91% reduction in Vmax. These results indicate that the C1 domain affects interaction with factor X and the Vmax of the factor Xase complex aside from the effect on membrane affinity. To further explore the role of membrane-binding motif in the Xase complex, the activities of mutants were tested with the C2 domain membrane-interactive epitopes blocked by mAb's BO2C11 or ESH4. For WT factor VIII, ESH4 and B02C11 decreased apparent affinity for vesicles of 15% PS by 6-fold and 5-fold, and decreased the Vmax by 0 and 89%, respectively. BO2C11 completely inhibited the activity of Arg2090/GLy2091, Lys 2092/Phe2093, and Arg2159 while ESH4 decreased apparent affinity 2-7-fold for the three mutants. ESH4 decreased the Vmax by 2-5-fold for the mutants. Thus, the intact membrane-binding motif in C1 can independently support Xase activity although the C1 motifs and both C2 membrane-interactive epitopes are required for full activity. Conclusion: Amino acids Arg2090/GLy2091, Lys2092/Phe2093 , Gln2042/Tyr2043, and Arg2159 of the factor VIII C1 domain participate in membrane binding. Our data suggest that engagement of the C1 domain through these residues, together with the ESH4 and the BO2C11 epitopes of the C2 domain, cooperatively influence alignment or an allosteric effect that alters activity for the assembled factor Xase complex. Disclosures: Pipe: Baxter: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Wyeth: Speakers Bureau; Inspiration Biopharmaceuticals: Research Funding; CSL Behring: Honoraria.

Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly

1999 ◽  
Vol 82 (08) ◽  
pp. 209-217 ◽  
Author(s):  
Patrick Celie ◽  
Joost Kolkman ◽  
Peter Lenting ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Three Dimensional ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor X Activation

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.

Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Dependent Manner ◽  
C2 Domain ◽  
Factor X ◽  
Calcium Dependent ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Gla Domain

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

scholarly journals Dissimilar interaction of factor VIII with endothelial cells and lipid vesicles during factor X activation

1997 ◽  
Vol 323 (3) ◽  
pp. 735-740 ◽  
Author(s):  
Herm-Jan M. BRINKMAN ◽  
Pauline KOSTER ◽  
Koen MERTENS ◽  
Jan A. van MOURIK
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Binding Sites ◽  
Blood Platelets ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Factor Viiia ◽  
Factor X Activation ◽  
Sustained Activation

A localized and regulated cascade of proteolytic events is a prerequisite for normal haemostasis. The activation of factor X by activated factor IX (factor IXa) in the presence of activated factor VIII (factor VIIIa) is essential for the formation of a fibrin clot at sites of vascular injury. We observed sustained activation of factor X on the surface of vascular endothelial cells, whereas, in agreement with others, on synthetic negatively charged phospholipid vesicles and activated blood platelets factor X activation is transient and starts to decline a few minutes after the onset of the reaction. We examined the mechanism responsible for these differences in factor X activation. Procoagulant membrane and solution were analysed separately for the occurrence of factor VIII and its activation fragments. On negatively charged phospholipid vesicles, on dissociation of factor VIIIa, the 67 kDa light-chain fragment remains associated with the lipid membrane. As a result, factor VIII-binding sites remain occupied, and dampening of factor X activation occurs. In contrast, on monolayers of endothelial cells, no residual factor VIIIa fragments associated with the cell membrane were observed. During endothelial-cell-mediated activation of factor X, accumulation of factor VIIIa fragments was observed in the solution phase only. This finding suggests that, on endothelial cells, factor VIII-binding sites remain accessible for further factor VIII binding, guaranteeing sustained activation of factor X. These data demonstrate that the nature of the procoagulant membrane contributes to the regulation of the cofactor activity of factor VIII and thereby affects the progress of factor X activation.

scholarly journals Membrane-binding properties of the Factor VIII C2 domain

2011 ◽  
Vol 435 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Valerie A. Novakovic ◽  
David B. Cullinan ◽  
Hironao Wakabayashi ◽  
Philip J. Fay ◽  
James D. Baleja ◽  
...  
Keyword(s):  
Factor Viii ◽  
Structural Integrity ◽  
Membrane Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Motif ◽  
C2 Domain ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa–Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.

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

1995 ◽  
Vol 310 (2) ◽  
pp. 427-431 ◽  
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.

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

2016 ◽  
Vol 473 (15) ◽  
pp. 2395-2411 ◽  
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.

scholarly journals 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

1999 ◽  
Vol 339 (2) ◽  
pp. 217-221 ◽  
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.

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

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
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.

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

2000 ◽  
Vol 84 (10) ◽  
pp. 626-634 ◽  
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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