Antigenicity of putative phospholipid membrane-binding residues in factor VIII

Barrow, Rachel T.; Healey, John F.; Jacquemin, Marc; Saint-Remy, Jean-Marie; Lollar, Pete

doi:10.1182/blood.v97.1.169

Cited by 64 publications

(66 citation statements)

References 31 publications

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“…Crystal structure analysis has demonstrated that binding of the C2 domain to phospholipid membranes involves three hydrophobic "feet" containing residues (35,36). The factor IXa-interactive site that we have identified within residues 2228 -2240 appears, therefore, to be in close proximity to, but not likely to be overlapping, the phospholipid-binding region.…”

Section: Effects Of Synthetic C2 Peptides On Rc2 and Egr-factor Ixa Imentioning

confidence: 86%

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

Soeda

Nogami

Nishiya

et al. 2009

Journal of Biological Chemistry

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Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dosedependently (K d ‫؍‬ 108 nM). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ϳ95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 proteasecleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228 -2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228 -2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.

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Section: Effects Of Synthetic C2 Peptides On Rc2 and Egr-factor Ixa Imentioning

confidence: 86%

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

Soeda

Nogami

Nishiya

et al. 2009

Journal of Biological Chemistry

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“…This observation is consistent with the identification of a major epitope for factor VIII inhibitors in this part of factor VIII. 6,29 Based on their reactivity with factor VIII/V hybrid 8A, it is likely that residues contained within the carboxyterminus of the C2 domain also contribute to binding of DP-5-encoded scFv (Figure 3). Recently, the 3-dimensional structure of DP-5-encoded human monoclonal antibody BO2C11 in complex with the C2 domain has been determined.…”

Section: Discussionmentioning

confidence: 99%

Two classes of germline genes both derived from the VH1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII

Brink¹,

Bril²,

Turenhout³

et al. 2002

Blood

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Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V H ) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V H 1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V H gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino-and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V H gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII. IntroductionHemophilia A is an X-linked bleeding disorder that is characterized by the absence or dysfunction of blood coagulation factor VIII. Current treatment of hemophilia A consists of infusion of therapeutic amounts of factor VIII that can evoke an immune response in some patients. The presence of neutralizing antibodies to factor VIII, commonly termed factor VIII inhibitors, presents a serious complication of hemophilia care. 1 The biochemical properties of factor VIII inhibitors have been extensively studied with emphasis on the epitope specificity and mode of action of these antibodies. Binding sites for inhibitors have been identified within the A2, A3, and C2 domains of factor VIII. [2][3][4][5][6] In general, heterogeneous mixtures of anti-factor VIII antibodies are present in plasma from patients with factor VIII inhibitors, and in more than 80% of inhibitor plasmas antibodies directed against the C2 domain are present. 7 Epitope mapping of anti-C2 domain antibodies revealed the presence of an inhibitor binding site comprising amino acid residues Val2248-Ser2312 of the C2 domain. 3 A second inhibitor epitope in the C2 domain has been attributed to region Glu2181-Val2243. 6 Antibodies reactive with the C2 domain prevent factor VIII from binding to phospholipid surfaces and von Willebrand factor. 8,9 Both findings are in agreement with the presence of binding sites for phospholipids and von Willebrand factor in the C2 domain. 10,11 An add...

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“…These results are consistent with the BO2C11 epitope identified here. 53 Only 2 point mutations associated with hemophilia A, A2201P and V2223M, have been identified within the antibody interface identified here. The patients carrying these mutations suffered from relatively mild bleeding disorders.…”

Section: Org Frommentioning

confidence: 99%

Structure of a factor VIII C2 domain–immunoglobulin G4κ Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII

Spiegel

Jacquemin

Saint-Remy

et al. 2001

Blood

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Antigenicity of putative phospholipid membrane-binding residues in factor VIII

Cited by 64 publications

References 31 publications

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

Two classes of germline genes both derived from the VH1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII

Structure of a factor VIII C2 domain–immunoglobulin G4κ Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII

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