PS membrane and PS-bound fVIII C2 ELISA results for factor VIII C2 domain variants and inhibitory antibodies.

<p>(A) Comparison of human (open circles) and porcine (closed squares) factor VIII C2 domains binding to PS membrane surfaces. (B) Comparison of human factor VIII C2 domain (open circles) with the human K2227A C2 domain mutant (closed squares). (C) Comparison of human factor VIII C2 domain (open circles) with the human K2227E C2 domain mutant (closed squares). (D) Binding of G99 (open circles) and 3E6 (closed circles) to PS-bound fVIII C2 domain. Bound His₆-C2 was detected with Ni-NTA-alkaline phosphatase, and bound IgG was detected with a AP-conjugated goat anti-mouse mAb.</p

X-ray crystal structure of the porcine factor VIII C2 domain.

<p>(A) Ribbon diagram presentation of the 1.7 Å X-ray crystal structure. Displayed residues are solvent-exposed hydrophobic and basic residues proposed to interact with platelet surfaces. (B) Superposition of the human (pdb#: 1D7P, magenta) and porcine (pdb#: 4MO3, green) factor VIII C2 domain X-ray crystal structures. (3) Sequence alignment of human and porcine factor VIII C2 domains. Highlighted residues represent sequence differences (orange: 3E6 mAb binding region, blue: G99 mAb binding region, red: BO2C11 mAb binding region, cyan: G99 and BO2C11 binding region).</p

Factor VIII C2 domain-specific inhibitory antibody epitopes and membrane binding models.

<p>(A) Ribbon diagram representation of the fVIII C2 domain bound to different classes of C2-specific inhibitory antibodies. Each X-ray crystal structure was superimposed with the C2 domain structure and residues involved in the different epitopes are shown by stick representation (red: fVIII C2 domain; green: G99 mAb, a non-classical BC epitope; blue/cyan: 3E6 mAb, a classical A epitope; yellow: BO2C11 mAb, a classical AB epitope). (B) Proposed PS membrane binding models for the fVIII C2 domain (left: old PS binding model, including the non-classical epitope with residues K2227 and V2223; right: new PS binding model, including both 3E6 and BO2C11 classical antibody epitopes, which centers at residue R2320).</p

Structural representation of sequence differences proximal to inhibitory antibody epitopes.

<p>(A) The 3E6 mAb epitope face. (B) The G99 mAb epitope face. Proximal residues are defined as <5 Å C2 domain/mAb intermolecular distances (orange: 3E6 mAb binding region, blue: G99 mAb binding region, red: BO2C11 mAb binding region, cyan: G99 and BO2C11 binding region, green: >5 Å from all mAbs)</p

ELISA of human and porcine C2/mAb interactions.

<p>Binding of human (open circles) and porcine (closed squares) His₆-C2 domain to immobilized (A) 3E6 mAb and (B) G99 mAb. Bound His₆-C2 was detected with Ni-NTA-alkaline phosphatase. (C) Bethesda assay for 3E6 and G99 with human or porcine BDD fVIII.</p

Electrostatic surface potentials for the human and porcine factor VIII C2 domain structures.

<p>(A) The 3E6 mAb epitope face. (B) The G99 mAb epitope face. The surface potential calculations were performed with APBS with surface potential values of ±5 kT/e, and the surface were generated with PyMol (blue: positive charge, red: negative charge).</p