Deletion of alanine 2201 in the FVIII C2 domain results in mild hemophilia A by impairing FVIII binding to VWF and phospholipids and destroys a major FVIII antigenic determinant involved in inhibitor development.

The C2 domain of factor VIII (FVIII) mediates FVIII binding to von Willebrand factor (VWF) and phospholipids (PLs), thereby determining the stability and the activity of FVIII. A deletion of Ala2201 (Del2201) was identified in the FVIII C2 domain of 2 unrelated patients with mild hemophilia A (FVIII:C 11%-33%). This mutation prevents FVIII binding to a human monoclonal antibody recognizing the C2 domain and inhibiting FVIII binding to VWF and phospholipids. By comparison to healthy FVIII, Del2201 FVIII had a significantly reduced binding to VWF, which likely contributes to reduced FVIII levels in plasma. Del2201 FVIII interaction with phospholipids was evaluated in an FXa generation assay, using various concentrations of synthetic phospholipid vesicles mimicking an activated platelet surface. At the lowest phospholipid concentration allowing FXa generation, Del2201 FVIII activity was reduced 3-fold. This is the first report of a mutation altering FVIII binding to phospholipids and occurring in patients with hemophilia A.

Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor.

Hemophilia A is a life-threatening, hemorrhagic, X-linked recessive disorder resulting in deficient factor VIII (FVIII) activity. After the infusion of therapeutic FVIII, 25% of patients develop anti-FVIII antibodies that inhibit FVIII procoagulant activity, thus precluding further administration of FVIII. Here we report a novel approach aimed at neutralizing the activity of FVIII inhibitors by peptide epitope surrogates. To illustrate our concept, we chose the human anti-FVIII monoclonal antibody, Bo2C11, as a representative of anti-FVIII antibodies and a phage-displayed peptide library approach to obtain surrogate peptides. We selected a series of constrained dodecapeptides with the core sequence W-NR, which specifically interacts with the combining site of Bo2C11. The peptides mimic the epitope recognized by Bo2C11 and are able to inhibit specifically and in a dose-dependent manner the binding of Bo2C11 to FVIII. Peptide 107, in particular, neutralized the activity of Bo2C11 in vitro and restored normal hemostasis in hemophilic mice. Thus, the use of peptide decoys may be a promising new approach for the neutralization of pathologic antibodies.

Epitope specificity of anti-FVIII antibodies during immune tolerance therapy with factor VIII preparation containing von Willebrand factor.

The study aimed at characterizing the putative changes in the epitope specificity of anti-FVIII antibodies during a successful immune tolerance treatment of the haemophilia A patient with the factor VIII (FVIII) preparation containing the von Willebrand factor (VWF). At the beginning of treatment, anti-FVIII inhibitory antibodies recognizing predominantly the light chain of FVIII were prevalent and persisted throughout the treatment. More detailed characterization of the FVIII antibody epitope specificity by using GST-fusion proteins corresponding to different FVIII domains revealed the prevalence of C1-domain-specific antibodies, while a remarkably lower amount of antibodies were targeted at the C2 and the a3 domains of the FVIII light chain and towards the A2 and the A1 domain of the FVIII heavy chain. The epitope specificity of antibodies remained rather unchanged throughout treatment except the elevated level of C2-domain-specific FVIII antibodies after a temporary interruption of treatment. The patient's antibodies were unable to interfere with the FVIII binding to VWF or to phospholipids, but inhibited FXa generation and the binding of FX to FVIII on the phospholipid monolayer. Thus, a unique pattern of the epitope specificity of FVIII antibodies and the mechanism to inhibit FVIII:C activity by FVIII-light-chain-specific antibodies were characterized.