OC 44.1 - Structural studies of blood coagulation factor VIII bound to patient-derived inhibitory antibodies

Background: Coagulation factor VIII (FVIII) promotes hemostasis by binding to activated factor IX on activated platelet surfaces to form the intrinsic tenase complex. The formation of antibody inhibitors against FVIII occurs in approximately 30% of congenital hemophilia A patients receiving FVIII replacement therapy, significantly complicating treatments. By identifying the FVIII regions that bind to pathogenic inhibitors, FVIII replacement therapies can be engineered with reduced immunogenicity.

Aims: We aimed to identify the FVIII amino acid residues that bind to two patient-derived inhibitory antibodies- NB33 and NB11B2.

Methods: We performed cryogenic electron microscopy to determine the structures of B domain-deleted FVIII bound to NB33 (FVIII:NB33) and NB11B2 (FVIII:NB11B2).

Results: The FVIII:NB33 structure was solved at a resolution of 4.19 Å, revealing the NB33 epitope localizes to FVIII residues R2090-I2096 and I2158-K2159 which constitute several membrane-binding loops in the C1 domain. These results support the inhibitory mechanism of NB33 blocking FVIII binding to von Willebrand factor and activated platelet membranes. The 3.38 Å structure of FVIII:NB11B2 represents the first structure of FVIII bound to an anti-A2 domain inhibitory antibody. Structural analysis revealed the NB11B2 epitope encompasses FVIII residues S470-P472, R490-K493, and P502-E507 which partially overlaps with the binding region for thrombin, suggesting NB11B2 disrupts FVIII function by inhibiting activation at the A1-A2 junction. Interestingly, the NB33 and NB11B2 epitopes overlap with putative binding regions for lipoprotein receptor-related protein 1 (LRP1) which mediates FVIII hepatic clearance and antigen presentation in dendritic cells.

Conclusion(s): These results elucidated the structural basis for inhibition of FVIII by two patient-derived antibodies. While NB33 and NB11B2 disrupt FVIII function through discrete mechanisms, both epitopes overlap with known LRP1 binding regions. In addition to providing a structural basis for engineering FVIII with reduced immunogenicity, these results support a critical role for surface-exposed positively charged residues in driving LRP1-mediated clearance at the A2 and C1 domains.