A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3181-3189 ◽  
Author(s):  
Junhong Lü ◽  
Steven W. Pipe ◽  
Hongzhi Miao ◽  
Marc Jacquemin ◽  
Gary E. Gilbert
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Membrane Binding ◽  
Factor X ◽  
Wild Type ◽  
C2 Domains ◽  
C1 Domain ◽  
Interactive Surface ◽  
Type Factor ◽  
Membrane Interactive

Abstract Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the Vmax for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the Vmax of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase 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.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

Factor VIII Arg2304 → His Binds to Phosphatidylserine and Is a Functional Cofactor in the Factor X-ase Complex

2001 ◽  
Vol 85 (02) ◽  
pp. 260-264 ◽  
Author(s):  
Deborah Lewis ◽  
Karen Moore ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Mammalian Cells ◽  
Exchange Chromatography ◽  
Light Chains ◽  
Factor X ◽  
Wild Type ◽  
Functional Protein ◽  
Type Factor ◽  
Circulating Levels

SummaryFour factor VIII light chain constructs containing hemophilia A mutations at R2304 and R2307 were prepared and expressed in mammalian cells. These mutations are located in a putative phosphatidylserine binding site identified by peptide studies (spanning amino acids 2303-2332). The levels of all four mutants in conditioned medium were significantly less than wild type by immunoprecipitation and ELISA. R2304H and wild type factor VIII light chains were concentrated by cation exchange chromatography from medium. R2304H and wild type factor VIII light chains bound immobilized phosphatidylserine similarly. The reconstituted cofactor activity of R2304H factor VIII light chain was slightly greater than wild type factor VIII light chain. These results are consistent with the recently reported crystal structure of factor VIII C2 domain that suggests R2304H is not directly involved in phospholipid binding. The observed clinical phenotype is probably due to decreased circulating levels of a functional protein.

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.

Molecular simulation studies of human coagulation factor VIII C domain-mediated membrane binding

2015 ◽  
Vol 113 (02) ◽  
pp. 373-384 ◽  
Author(s):  
Jiangfeng Du ◽  
Kanin Wichapong ◽  
Tilman M. Hackeng ◽  
Gerry A. F Nicolaes
Keyword(s):  
Factor Viii ◽  
Membrane Binding ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
Haemophilia A ◽  
C2 Domain ◽  
Missense Mutations ◽  
C2 Domains ◽  
C1 Domain ◽  
Dynamics Simulations

SummaryThe C-terminal C domains of activated coagulation factor VIII (FVIIIa) are essential to membrane binding of this crucial coagulation cofactor protein. To provide an overall membrane binding mechanism for FVIII, we performed simulations of membrane binding through coarsegrained molecular dynamics simulations of the C1 and C2 domain, and the combined C-domains (C1+C2). We found that the C1 and C2 domain have different membrane binding properties. The C1 domain uses hydrophobic spikes 3 and 4, of its total of four spikes, as major loops to bind the membrane, whereas all four of its hydrophobic loops of the C2 domain appear essential for membrane binding. Interestingly, in the C1+C2 system, we observed cooperative binding of the C1 and C2 domains such that all four C2 domain spikes bound first, after which all four loops of the C1 domain inserted into the membrane, while the net binding energy was higher than that of the sum of the isolated C domains. Several residues, mutations of which are known to cause haemophilia A, were identified as key residues for membrane binding. In addition to these known residues, we identified residues from the C1 and C2 domains, which are involved in the membrane binding process, that have not been reported before as a cause for haemophilia A, but which contribute to overall membrane binding and which are likely candidates for novel causative missense mutations in haemophilia A.

scholarly journals Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity

Blood ◽  
2012 ◽  
Vol 120 (9) ◽  
pp. 1923-1932 ◽  
Author(s):  
Gary E. Gilbert ◽  
Valerie A. Novakovic ◽  
Randal J. Kaufman ◽  
Hongzhi Miao ◽  
Steven W. Pipe
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Membrane Binding ◽  
Fold Increase ◽  
Factor V ◽  
Factor X ◽  
C2 Domains ◽  
Hydrophobic Amino Acids ◽  
Increased Activity ◽  
Distinct Membrane

Abstract Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FVMTTS/Y). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FVMTTS/Y, had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FVMTTS/Y showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.

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.

A FVIII C1 Domain Loop Containing Phe2093 Contributes Affinity to the Binding of Recombinant FVIII C1C2 Proteins to Activated Platelets

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3069-3069
Author(s):  
Ting-Chang Hsu ◽  
Kathleen P. Pratt ◽  
Arthur R. Thompson
Keyword(s):  
Membrane Binding ◽  
Cysteine Residue ◽  
Wild Type ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Mutant Proteins ◽  
Activated Platelets ◽  
C1 Domain ◽  
Beta Hairpin ◽  
The Difference

Abstract Factor VIII (FVIII) circulates bound to von Willebrand factor, and upon proteolytic activation it dissociates and attaches to activated membranes, e.g. at the platelet surface, that expose negatively-charged phosphatidylserine. This membrane association is mediated entirely by the FVIIIa light chain (A3-C1-C2), and the C2 domain is known to be a primary contributor to the membrane affinity. We previously demonstrated that the C1 domain, which is homologous to C2, also contributes to the affinity for activated platelets, (Hsu et al., Blood111, 200–208, 2008). Our earlier work showed that the platelet affinity of recombinant C1C2, as well as the total number of binding sites per platelet, were significantly higher than those measured for recombinant C2. Thus C1 either interacts with the platelet surface directly, or it stabilizes a conformation of C2 that promotes membrane binding. In this study, the affinities for activated platelets of a series of mutant C1C2 proteins were evaluated to determine residues involved in FVIII binding to platelets. C1C2 and C2 proteins were generated with a free cysteine residue substituted for the wild-type serine at position 2296 in C2 (S2296C does not disrupt the protein structure or affect membrane binding), to which a sulfyhydryl-linked fluorescein probe was attached covalently (C1C2* and C2*). Single or double alanine substitutions were introduced at the two beta-hairpin turns in C2 that mediate membrane binding (M2199A, F2200A, L2251A, L2252A), and at C1 residue F2093, which aligns with C2 residue L2252. Washed platelets were activated with SFLLRN-amide, incubated for 1 hr with the wild-type or mutant proteins, and analyzed by flow cytometry on a FACSCaliber. The relative binding affinities were estimated by using multiple protein concentrations and comparing the fluorescent signals to the values for saturation binding of C1C2* and C2*. Alanine substitutions at all of these positions resulted in decreased binding of C1C2*, comparable to the difference in affinity of C2* versus C1C2* (figure 1A). C1C2-F2093C was then generated, and the introduced sulfhydryl was blocked by adding free cysteine, thus introducing the bulky, charged cystine residue at this putative membrane-binding site (C1C2-F2093C-C). Binding assays utilizing detection by monoclonal antibody ESH8, which does not interfere with membrane attachment, followed by a PE-labeled secondary antibody showed that C1C2-F2093C-C had markedly reduced binding to activated platelets compared to C1C2 (figure 1B). These results are consistent with the hypothesis that the C1 domain contacts the membrane directly when FVIIIa becomes attached to the activated platelet surface and indicate that F2093 contributes significantly to this interaction. Figure Figure

Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 domain

2017 ◽  
Vol 89 ◽  
pp. 34-41
Author(s):  
Lydia Castro-Núñez ◽  
Johanna M. Koornneef ◽  
Mariska G. Rondaij ◽  
Esther Bloem ◽  
Carmen van der Zwaan ◽  
...  
Keyword(s):  
Factor Viii ◽  
Cellular Uptake ◽  
Membrane Binding ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
C1 Domain

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.

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