Chemistry Department, Western Washington University, Bellingham, Washington, USA.
Medicines and Healthcare products Regulatory Agency, South Mimms Laboratories, Potters Bar, Hertfordshire, UK.
J Thromb Haemost. 2024 Sep;22(9):2449-2459. doi: 10.1016/j.jtha.2024.05.024. Epub 2024 Jun 5.
Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII 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 von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined.
To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition.
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.
This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that 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 von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain.
These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.
血友病 A 是由功能失调或缺乏凝血因子（F）VIII 引起的，导致纤维蛋白凝块形成效率低下和不受控制的出血事件。抗体抑制剂的产生是接受 FVIII 替代治疗的血友病 A 患者的临床并发症。LE2E9 是一种先前从轻度/中度血友病 A 患者中分离出来的抗 C1 结构域抑制剂，可破坏 FVIII 与血管性血友病因子和 FIXa 的相互作用，尽管 LE2E9 介导的 FVIII 中和所依赖的分子间接触尚不清楚。
确定 FVIII 与 LE2E9 复合物的结构并表征其抑制机制。
FVIII 与 LE2E9 的抗原结合片段（Fab）NB2E9 结合，并用低温电子显微镜确定其结构。
本报告介绍了 FVIII 与 NB2E9 结合的 3.46 Å 结构，其表位包括 FVIII 残基 S2040 至 Y2043、K2065 至 W2070 和 R2150 至 H2155。结构分析表明，LE2E9 表位与 2A9（一种源自鼠的抑制剂）的表位部分重叠，这表明这些残基代表 FVIII 小鼠和血友病 A 患者的 C1 结构域上的一个共同抗原区域。此外，FVIII：NB2E9 结构阐明了 LE2E9 聚糖的取向，说明了聚糖如何在空间上阻止 FVIII C1 结构域与血管性血友病因子 D' 结构域之间的相互作用。FVIIIa：FIXa 复合物的假设模型表明，NB2E9 聚糖与 FIXa 轻链之间可能存在冲突。
这些结果描述了 FVIII C1 结构域上的一个抗原“热点”，并为工程具有降低抗原性的 FVIII 替代治疗药物提供了结构基础。
