Figure 1.
Figure 1. Interaction of healthy and mutated plasma FVIII with BO2C11. (A) Location of residues involved in FVIII binding to BO2C11 in the 3-dimensional structure of the C2 domain. The C2 domain is represented as ribbon diagram. The hydrophobic and basic residues mediating binding to PLs are highlighted as Corey-Pauling-Kultun (CPK) spheres. Arrowheads indicate residues mediating FVIII binding to BO2C11 as determined by crystallization of an Fab fragment of BO2C11 bound to a recombinant C2 domain.20 The human monoclonal antibody BO2C11 was obtained by immortalization of B lymphocytes of a patients with hemophilia A with a high-titer inhibitor. BO2C11 is representative of C2 inhibitor insofar as it belongs to the immunoglobulin G4 (IgG4) isotype and inhibits FVIII binding to both VWF and PLs.14 The figure was drawn using MolMol,21 using the published coordinates of the C2 domain.7 (B) Inhibition of healthy and mutated FVIII by BO2C11. Plasma from Del2201 of patients 1 and 2 and from healthy pool plasma were incubated with BO2C11 at 10 μg/mL for 2 hours at 37°C, and the residual FVIII activity was measured in a chromogenic assay. Results are expressed as residual FVIII activity (mean ± SD). (C) FVIII binding to VWF in plasma. Plasma from patient 1 or 2, diluted 20-fold, was incubated with either Sepharose beads coated with anti-VWF antibody or control Sepharose. Following centrifugation, FVIII was measured in the supernatant of anti-VWF Sepharose (free FVIII) and of control Sepharose (total FVIII) by using a FVIII chromogenic assay. FVIII bound to VWF was calculated by subtracting free FVIII from total FVIII. The fraction of FVIII bound to VWF is expressed as the percentage of total FVIII (mean ± SD). (D) Cofactor activity of plasma FVIII was evaluated in a chromogenic assay by using various concentrations of synthetic phospholipid vesicle containing 9% phosphatidyl-serine, 25% phosphatidyl-choline, 31% phosphatidyl-ethanolamine, 15% sphingomyelin, and 20% cholesterol and representative of PLs expressed at the surface of activated platelets. At each PL concentration, FXa generation was compared with that observed in the presence of a known concentration of healthy plasma FVIII. For each FVIII variant, results are expressed as the percentage (mean ± SD) of the FVIII activity measured at the highest PL concentration tested (4 μM).

Interaction of healthy and mutated plasma FVIII with BO2C11. (A) Location of residues involved in FVIII binding to BO2C11 in the 3-dimensional structure of the C2 domain. The C2 domain is represented as ribbon diagram. The hydrophobic and basic residues mediating binding to PLs are highlighted as Corey-Pauling-Kultun (CPK) spheres. Arrowheads indicate residues mediating FVIII binding to BO2C11 as determined by crystallization of an Fab fragment of BO2C11 bound to a recombinant C2 domain.20  The human monoclonal antibody BO2C11 was obtained by immortalization of B lymphocytes of a patients with hemophilia A with a high-titer inhibitor. BO2C11 is representative of C2 inhibitor insofar as it belongs to the immunoglobulin G4 (IgG4) isotype and inhibits FVIII binding to both VWF and PLs.14  The figure was drawn using MolMol,21  using the published coordinates of the C2 domain.7  (B) Inhibition of healthy and mutated FVIII by BO2C11. Plasma from Del2201 of patients 1 and 2 and from healthy pool plasma were incubated with BO2C11 at 10 μg/mL for 2 hours at 37°C, and the residual FVIII activity was measured in a chromogenic assay. Results are expressed as residual FVIII activity (mean ± SD). (C) FVIII binding to VWF in plasma. Plasma from patient 1 or 2, diluted 20-fold, was incubated with either Sepharose beads coated with anti-VWF antibody or control Sepharose. Following centrifugation, FVIII was measured in the supernatant of anti-VWF Sepharose (free FVIII) and of control Sepharose (total FVIII) by using a FVIII chromogenic assay. FVIII bound to VWF was calculated by subtracting free FVIII from total FVIII. The fraction of FVIII bound to VWF is expressed as the percentage of total FVIII (mean ± SD). (D) Cofactor activity of plasma FVIII was evaluated in a chromogenic assay by using various concentrations of synthetic phospholipid vesicle containing 9% phosphatidyl-serine, 25% phosphatidyl-choline, 31% phosphatidyl-ethanolamine, 15% sphingomyelin, and 20% cholesterol and representative of PLs expressed at the surface of activated platelets. At each PL concentration, FXa generation was compared with that observed in the presence of a known concentration of healthy plasma FVIII. For each FVIII variant, results are expressed as the percentage (mean ± SD) of the FVIII activity measured at the highest PL concentration tested (4 μM).

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