VWF-Dʹ makes extensive interactions with FVIII; visualization of FVIII-a3 showcases a central role of sulfated Y1680 in binding VWF R816
The structure of BIVV001 provides insights into the regulation of the FVIII-VWF complex and disease-causing mutations
Interaction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF DʹD3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-DʹD3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand Disease (VWD) type 2N. Furthermore, the FVIII-VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, where endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-DʹD3 is covalently linked to an Fc domain of a B domain-deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-DʹD3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII-VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF-Dʹ/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816, which, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.