Antibody ESH4, Against the Factor VIII C2 Domain, Causes a Remote Change near the Factor IXa Active Site and Inhibition That Is Related to Membrane Composition

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3355-3355
Author(s):  
Valerie A Novakovic ◽  
Junhong Lu ◽  
Gary E. Gilbert
Keyword(s):  
Factor Viii ◽  
Active Site ◽  
Hemophilia A ◽  
Membrane Binding ◽  
Bleeding Risk ◽  
C2 Domain ◽  
Membrane Composition ◽  
Factor Viii Activity ◽  
Factor Ixa ◽  
Factor Viiia

Abstract Abstract 3355 Introduction: Development of antibodies against factor VIII is a common complication of therapy for hemophilia A and can cause acquired hemophilia A in previously normal subjects. Dominant epitopes for inhibitory antibodies reside on the C2 domain of factor VIII, which has been shown to be important for membrane binding. Although acquired hemophilia A is associated with a prolonged activated partial thromboplastin time (aPTT), the relative bleeding risk does not correlate well with factor VIII activity levels. Thus there is a need for basic insights that explain the discrepancies between factor VIII activity values and bleeding risk in these patients. mAb ESH4, directed against the factor VIII C2 domain, interferes with membrane binding and is a prototypic factor VIII inhibitor. ESH4 is a type II inhibitor, with residual factor VIII activity in the presence of saturating antibody concentrations. Thus, exploration of the inhibitory mechanism of ESH4 may offer insights into bleeding risk assessment of antibodies that inhibit phospholipid binding. Methods: Binding of fluorescein-labeled factor VIII to phospholipid membranes supported on glass microspheres was measured in the presence and absence of ESH4 using flow cytometry. The effect of ESH4 on factor VIII activity was measured using a two-stage amidolytic factor Xase assay and sonicated lipid vesicles with either low (4%) or high (15%) levels of PS. The factor Xase assay was also performed using platelets as the phospholipid source. Factor IXa, with its active site labeled using fluorescein-EGR chloromethyl ketone (Fl-EGRck), was mixed with sonicated phospholipid vesicles, factor VIIIa, and factor × and the anisotropy of the fluorescein molecule was measured to test the effect of ESH4 on the factor IXa active site. Results: Saturating concentrations of ESH4 inhibited 40% of factor VIII activity in a commercial aPTT assay. In a defined assay, inhibition of factor VIII activity was directly related to the phospholipid composition and concentration. In the presence of saturating phospholipid and factor X, ESH4 caused over 60% decrease in the Vmax for vesicles with either 4% or 15% PS. To determine the mechanism through which ESH4 inhibits membrane-bound factor VIII activity, we measured the fluorescence anisotropy of factor IXa-Fl-EGRck. ESH4 decreased anisotropy of the factor VIIIa-factor IXa-factor × complex from 0.280 ± 0.002 to 0.272 ± 0.002 on 15% PS vesicles and from 0.275 ± 0.002 to 0.262 ± 0.001 on 4% PS vesicles, indicating that ESH4 alters the Vmax through a change near the factor IXa active site, remote from the C2 domain-membrane interface. ESH4 decreased the apparent affinity 4-fold for membranes of 4% PS (KD = 4.8 ± 0.4 mM without and 21 ± 4 mM with ESH4) but only 2-fold on 15% PS vesicles (KD = 1.3 ± 0.2 mM without and 2.5 ± 0.5 mM with ESH4). Direct membrane binding studies of fluorescein-labeled factor VIII indicated a reduction in affinity and number of binding sites consistent with the results from the factor Xase assay. The apparent affinity for factor × in the presence of saturating phospholipid and ESH4 was higher on 15% PS vesicles (KM = 129 ± 18 nM) than on 4% PS vesicles (KM= 284 ± 30 nM). Together, these results indicate that ESH4 can decrease factor VIII activity through three mechanisms: (1) decreased membrane affinity (2) decreased activity of membrane-bound factor VIII and (3) differential affinity of the factor Xase complex for factor X. Because two of these mechanisms are influenced by membrane composition we asked whether the degree of inhibition by ESH4 might differ on platelets stimulated to different degrees. Platelets stimulated by thrombin express limited PS in a reversible manner while platelets stimulated by > 1 μM A23187 have complete PS exposure. ESH4 showed 80% inhibition of Xase activity on platelets stimulated with thrombin vs. 40% inhibition on platelets stimulated with A23187, similar to the aPTT assay. Conclusions: Our results indicate that ESH4 disruption of factor VIII C2 domain engagement with the membrane has a remote effect at the factor IXa active site. Inhibition of factor VIII activity by ESH4 is sensitive to membrane composition and concentration through two mechanisms. These results highlight the need to better understand how membrane binding activates the factor VIIIa-factor IXa complex and to develop clinical assays that measure factor VIII activity on clinically relevant membrane types and concentrations. Disclosures: No relevant conflicts of interest to declare.

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.

Partial Reconstitution of Factor VIII Activity from a Mild Crm+ Hemophilia A Patient by Replacement of the Defective A2 Domain

1998 ◽  
Vol 79 (05) ◽  
pp. 943-948 ◽  
Author(s):  
W. C. Pieneman ◽  
P. Fay ◽  
E. Briët ◽  
P. H. Reitsma ◽  
R. M. Bertina
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Wild Type ◽  
Coding Region ◽  
Factor Viii Activity ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor Viii Antigen ◽  
A2 Domain ◽  
Reduced Activity

SummaryWe further characterised the abnormal factor VIII molecule (factor VIII Leiden) of a Crm+, mild hemophilia A patient with a factor VIII activity of 0.18 IU/ml and a factor VIII antigen of 0.95 IU/ml. Mutation analysis of the coding region, promoter and 3’ untranslated region of the factor VIII gene revealed the presence of a C to T substitution at codon 527. This nucleotide change predicts the replacement of an arginine to tryptophan in the A2 domain close to a suggested binding site for factor IXa. Since a previous study of this mutant factor VIII protein suggested that this protein had a reduced affinity for factor IXa, position 527 in the protein might be involved in the interaction with factor IXa.In this study we gathered evidence for our hypothesis that the Arg to Trp mutation at position 527 is the cause of the reduced activity of factor VIII Leiden. Replacement of the mutated A2 domain by wild type A2 domain partially corrected the defect.Factor VIII from normal and factor VIII Leiden plasma was concentrated by cryoprecipitation, activated with thrombin and incubated with excess wild type A2 domain. Competition with excess isolated human A2 domain resulted in a partial reconstitution of the factor VIIIa activity of thrombin treated factor VIII Leiden. This supports the hypothesis that the mutation in the A2 domain is the cause of the reduced factor VIII activity.

Effect of Membrane Binding Upon Intramolecular Dynamics of the Factor VIII C2 Domain.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3168-3168 ◽  
Author(s):  
Dennis Pantazatos ◽  
Christopher Gessner ◽  
Virgil Woods ◽  
Gary E. Gilbert
Keyword(s):  
Exchange Rate ◽  
Factor Viii ◽  
Membrane Binding ◽  
Phospholipid Vesicles ◽  
C2 Domain ◽  
Amide Hydrogen ◽  
Binding Peptide ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Membrane Interactive

Abstract Abstract 3168 Poster Board III-109 Factor VIII functions as a membrane-bound cofactor in the Factor Xase complex. Binding to a phospholipid membrane enhances activity of the factor VIIIa-factor IXa complex approx. 100,000-fold. While membrane binding increases the net affinities of factor VIIIa for factor IXa and factor X, the major effect of membrane binding is upon the catalytic activity of the assembled complex. The mechanism through which activity is enhanced remains largely unknown. The C2 domain of factor VIII contains the major membrane-binding function. The x-ray crystal structures of the C2 domain and, subsequently, of intact factor VIII have enabled hypotheses about the mechanism of membrane binding and the enhancement of activity. Identified functional motifs of the C2 domain include two pairs of hydrophobic, membrane-interactive amino acids at the tips of “spikes,” on the lower end of the C2 domain and a putative factor IXa-binding peptide on an upper surface. Hydrogen/Deuterium Exchange Mass Spectrometry (DXMS) has proven to be an effective method for characterizing protein conformational change induced by ligand interaction. Amide backbone hydrogens of a protein readily exchange with those in the solvent, contingent upon physical contact of the solvent hydrogen with the amide hydrogen. Therefore the exchange rate in a particular region of the protein is dependent on amide hydrogen solvent accessibility. DXMS is performed by isotopic labeling of a protein at various time points with deuterium oxide (D2O) followed by quenching the exchange and proteolysis to produce overlapping peptides spanning the length of the sequence. The degree of labeling is assessed using liquid chromatography coupled with mass spectrometry analysis. Using this analysis a stability map of the protein can be determined that represents changes in global and local structural dynamics in bound and unbound conditions. We have utilized DXMS to characterize the dynamics of the factor VIII C2 domain (fVIII-C2) in solution and when bound to phospholipid vesicles. fVIII-C2 was produced in E. coli and purified by immobilized metal affinity chromatography and ion exchange chromatography. Preliminary studies indicated that digestion on serial pepsin and V8 protease columns produced peptides spanning the entire fVIII-C2 structure, with overlapping peptides spanning the membrane-interactive “spikes” and the putative factor IXa-interactive peptide. Exchange was rapid on the membrane-interactive spikes in the absence of phospholipid vesicles, confirming flexibility of these structures. Upon binding of fVIII-C2 to phospholipid vesicles, the exchange rate decreased particularly in spike 3 (from 73% to 49% at 10s labeling, upon addition of phospholipid vesicles). The extent of the region with slowed exchange implied increased structural rigidity in addition to any solvent protection that occurs upon immersion of the hydrophobic side chains into the membrane. A marked decrease in exchange rate was also observed in the putative FIXa binding peptide (from 85% to 21% at 10s labeling, upon addition of phospholipid vesicles), while the exchange rates of most of the peptides from the core beta-barrel of fVIII-C2 were not significantly affected. These observations indicate that membrane engagement alters the mobility of membrane interactive spikes and suggest that membrane binding has an allosteric effect on the putative factor IXa binding site. Membrane-induced allosteric changes in the factor VIII C2 domain may contribute to the major enhancement of factor VIIIa-factor IXa activity that occurs upon membrane binding. Disclosures No relevant conflicts of interest to declare.

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.

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.

Factor VIII Ise (R2159C) in a Patient with Mild Hemophilia A, an Abnormal Factor VIII with Retention of Function but Modification of C2 Epitopes

1997 ◽  
Vol 77 (05) ◽  
pp. 0862-0867 ◽  
Author(s):  
Hiroshi Suzuki ◽  
Midori Shima ◽  
Morio Arai ◽  
Kazuhiko Kagawa ◽  
Katuyuki Fukutake ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Missense Mutation ◽  
Hemophilia A ◽  
Sscp Analysis ◽  
C2 Domain ◽  
Factor Viii Activity ◽  
Normal Plasma ◽  
Factor Viii Antigen

SummaryWe found a patient with mild hemophilia A who had no detectable factor VIII antigen (FVIII:Ag), as shown by two-site ELISA using inhibitor alloantibodies (TK). We then analyzed A2, A2/B, and C2 antigen of the patient's DDAVP-induced FVIII using several anti-FVIII monoclonal antibodies. Factor VIII activity (FVIII : C) was increased from 12 to 42 Uldl by the administration of DDAVP. The DDAVPinduced increases in the A2 and A2/B antigens were 40 and 36 Uldl, respectively. However, the increase in the C2 antigen was only 7.5 Uldl. SSCP analysis and subsequent sequencing demonstrated an Arg to Cys transition at codon 2159. The anti-FVII1:C titer of monoclonal antibody, NMC-VIII15 which recognized the C2 domain, against normal plasma was 450 Bethesda Ulmg of IgG. However, the titer against DDAVP-treated patient's plasma was only 15 Bethesda Ulmg. We also tested DDAVP-induced increase in the FVIII : Ag in another mild hemophilia A patient with the same mutation at Arg2159. Increase in his C2 antigen levels was only 19% of those in the A2 and A2/B antigen. We designate this abnormal FVIII as FVIII Ise. Our results show that a missense mutation at Arg2159 to Cys modifies the antigenicity of the C2 domain.

scholarly journals Factor VIII activity and bleeding risk during prophylaxis for severe hemophilia A: a population pharmacokinetic model

Haematologica ◽  
2020 ◽  
pp. haematol.2019.241554 ◽  
Author(s):  
Andreas Tiede ◽  
Faraizah Abdul Karim ◽  
Victor Jiménez-Yuste ◽  
Robert Klamroth ◽  
Sandra Lejniece ◽  
...  
Keyword(s):  
Factor Viii ◽  
Population Pharmacokinetic Model ◽  
Pharmacokinetic Model ◽  
Hemophilia A ◽  
Bleeding Risk ◽  
Population Pharmacokinetic ◽  
Severe Hemophilia ◽  
Factor Viii Activity

The Factor VIIII C2 Domain Shows Stereospecificity for Phosphatidyl-L-Serine and Increases Factor IXa Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3090-3090 ◽  
Author(s):  
Valerie A Novakovic ◽  
James D Baleja ◽  
Gary E. Gilbert
Keyword(s):  
Factor Viii ◽  
Conformational Change ◽  
Membrane Binding ◽  
Coagulation Cascade ◽  
Phospholipid Membranes ◽  
C2 Domain ◽  
Factor X ◽  
Lower Affinity ◽  
Factor Ixa ◽  
C1 Domain

Abstract Background: Factor VIII is an essential cofactor in the blood coagulation cascade and shows greatly increased activity when bound to phospholipid membranes. While high proportions of various negatively-charged phospholipids support binding of factor VIII, only phosphatidyl-L-serine (Ptd-L-Ser) supports stereospecific affinity when present at physiologically relevant proportions. Factor VIII binds to phospholipid membranes primarily through its C2 domain, but binding is also mediated by the C1 domain (Meems et al., abstract ASH 2008). However, the membrane-binding properties of the isolated C2 domain and the relationship of the C2 domain to factor IXa and factor X have not been fully studied. Methods: The factor VIII C2 domain (fVIII-C2) and a mutant in which residue Val2223 was replaced by Cys (fVIII-C2C) were produced in E. coli and purified from cytosol by metal ion affinity chromatography followed by cation exchange chromatography. fVIII-C2C was labeled with fluorescein maleimide (fVIII-C2C-fluor). Binding studies were performed by flow cytometry using membranes supported by glass microspheres (lipospheres) and by fluorescence resonance energy transfer between fVIII-C2 and dansyl-labeled vesicles. The factor Xase assay was utilized to infer the capacity of fVIII-C2 to influence membranebinding and protein-protein interactions. Results: fVIII-C2C-fluor bound to liposphere membranes containing at least 10% Ptd-LSer. The KD for binding to lipospheres was 150 ± 40 nM. The KD for fVIII-C2 binding to sonicated vesicles of composition Ptd-L-Ser:PE-dansyl:PC 20:5:75 was 230 ± 30 nM indicating that fVIII-C2 and fVIII-C2C-fluor bind with similar affinities. Binding was measurable at pH 6.0 but was at least 10-fold lower affinity at pH 7.8. Change of pH from 6.0 to 7.8 was associated with a change in intrinsic fluorescence and was not associated with increased light scatter, suggesting conformational change rather than formation of dimers or aggregates. Sonicated vesicles with 15% Ptd-L-Ser competed with lipospheres for binding of fVIII-C2C-fluor with 3-fold higher affinity than vesicles with 15% Ptd-D-Ser. Phosphatidylinositol or phosphatidic acid-containing vesicles bound fVIII-C2C-fluor with at least 4-fold lower affinity than Ptd-L-Ser. fVIII-C2 did not compete with factor VIII-fluor for binding to lipospheres at pH 6 or 7.8. At pH 6.0 the factor Xase assay was inhibited by addition of fVIII-C2 with a plateau of 30% inhibition at 1.5 μM. In the absence of intact factor VIII, fVIII-C2 enhanced the activity of factor IXa by 2-fold. The enhanced activity correlated to a reduced KM but not an altered apparent affinity for phospholipid vesicles. Conclusions: These results indicate that fVIII-C2 binds membranes containing Ptd-L-Ser in a stereospecific manner with approximately 30-fold lower affinity than intact factor VIII. Membrane binding is pH-dependent, likely requiring a conformational change in fVIII-C2. Lack of competition with intact factor VIII implies that fVIII-C2 does not recognize the initial membrane contact site of the full protein. Partial inhibition of the factor Xase complex and enhancement of factor IXa activity in the absence of intact factor VIIIa implies that fVIII-C2 binds either factor IXa or factor X. We speculate that the conformational change enabling membrane binding is caused by an acidic microenvironment in intact factor VIII produced by proximity to the C1 domain and/or the phospholipid membrane.

scholarly journals A Human Alloantibody Interferes With Binding of Factor IXa to the Factor VIII Light Chain

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2347-2352 ◽  
Author(s):  
Karin Fijnvandraat ◽  
Patrick H.N. Celie ◽  
Ellen A.M. Turenhout ◽  
Jan W. ten Cate ◽  
Jan A. van Mourik ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Functional Characterization ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Number Of Patients

Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa–factor VIIIa complex assembly.

scholarly journals A Human Alloantibody Interferes With Binding of Factor IXa to the Factor VIII Light Chain

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2347-2352 ◽  
Author(s):  
Karin Fijnvandraat ◽  
Patrick H.N. Celie ◽  
Ellen A.M. Turenhout ◽  
Jan W. ten Cate ◽  
Jan A. van Mourik ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Functional Characterization ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Number Of Patients

Abstract Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa–factor VIIIa complex assembly.

Sign in / Sign up

Export Citation Format

Share Document