November 18, 2024
Journal Article
Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hot-spot on the C1 domain
Abstract
Background: Hemophilia A arises from dysfunctional or deficient coagulation factor VIII (FVIII) and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with VWF and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined. Objective: To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition. Methods: FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy (cryo-EM). Results: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprised of FVIII residues S2040-Y2043, K2065-W2070, and R2150-H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting these residues represent a shared antigenic region on the C1 domain between FVIII-/- mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the VWF D’ domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain. Conclusion: These results describe an antigenic “hot-spot” on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.Published: November 18, 2024