Structure of Blood Coagulation Factor VIII in Complex With an Anti-C2 Domain Non-Classical, Pathogenic Antibody Inhibitor

Abstract

Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.

Keywords: antibody binding; antibody inhibitors; blood coagulation; factor VIII; x-ray crystallography.

Figure 1

Crystal structure of ET3i bound to the G99 F_AB fragment. (A) Cartoon representation of the B domain deleted bioengineered fVIII construct (ET3i) bound to the variable domain of G99 inhibitor antibody. Porcine A1 and A3 (pA1 and pA3) domains are colored dark red and pink, respectively, and human A2, C1, and C2 (hA2, hC1, and hC2) domains are colored cyan. Heavy and light chains of the G99 F_AB fragment (G99_HC and G99_LC) are colored green and purple, respectively. N-acetylglucosamine modifications are depicted as sticks. (B) Ribbon diagram of aligned C2 domain (cyan) and G99 heavy and light chains (green and purple, respectively) from the ET3i:G99 crystal structure and of the isolated C2 domain bound to G99 (grey; PDB ID: 4KI5) in stereo view. (C) Stick representation of the G99 epitope from the ET3i:G99 (cyan) and C2:G99 (grey, faded) crystal structures. (D, E) Electrostatic contacts between ET3i epitope 2222-2229 (cyan) and G99 heavy chain (green) and light chain (purple). Dotted lines depict hydrogen bonds (distance ≤ 5 Å). (F) Hydrophobic residues along the ET3i epitope 2269-2282 (cyan) buried by the G99 light chain (purple).

Figure 2

Structural alignment of free ET3i and ET3i:G99 crystal structures. (A) Alignment of free ET3i (grey) and ET3i:G99 (A1, dark red; A3, pink; A2/C1/C2, cyan). Insets depict intramolecular contacts that are broken in the ET3i:G99 structure. (B) Alignment of the C2 domain from free ET3i structure (grey) and ET3i:G99 structure (cyan). (C) Structure of the C2 domain from the ET3i:G99 complex colored as a function of RMSD from alignment with free ET3i. White, low RMSD shifts; orange, large RMSD shifts.

Figure 3

Anti-C domain inhibitors reduce atomic B-factors on solvent-exposed loops. (A, B) Normalized atomic B-factors averaged for each epitope in the (A) C1 domain and (B) C2 domain from free ET3i (black, PDB ID: 6MF0), ET3i:G99 (blue, PDB ID: 7KBT), and ET3i:2A9 (red, PDB ID: 7K66). (C) Cartoon representation of the C1 and C2 domains from the free ET3i crystal structure (PDB ID: 6MF0, model A). Antibody epitopes are highlighted (2A9, magenta; G99, green; 3E6, cyan; BO2C11, orange).