Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy

Abstract

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.

Clinical Utility of Subcutaneous Factor VIII Replacement Therapies in Hemophilia A: A Review of the Evidence

“…8 BIVV001 is an EHL-FVIII fused to Fc fragment coupled with the FVIII binding D'D3 domain of VWF and two XTEN linkers aiming to reduce degradation and clearance. 9 A recent Phase 1-2a openlabel clinical trial reported a significantly prolonged halflife of BIVV01 that was up to four times the half-life associated with standard recombinant FVIII (rFVIII), allowing an FVIII prophylaxis with a weekly treatment interval. 10 Despite the improvements achieved, the exclusive intravenous route of administration of EHL-FVIII products remains a substantial source of burden for patients and caregivers.…”

Factor VIII/VWF complex caught on camera

“…In this issue of Blood, Fuller et al 1 reveal the first detailed structure of the key hemostatic complex formed between factor VIII (FVIII) and von Willebrand factor (VWF).…”

A Conformational Transition of Von Willebrand Factor’s D’D3 Domain Primes It For Multimerization

“…Mutations within the VWF D'D3 domain that impede this high-affinity binding lead to type 2N von Willebrand disease, a condition characterized by reduced plasma levels of FVIII 19,20 . Structural and biochemical data reveal binding of the FVIII C1 domain to the D' modules of VWF and, additionally, hint at interactions of FVIII with the VWF D3 core 16,17,21,22 . Here, we employ magnetic tweezers (MT) to study the conformational change in the D'D3 domain necessary for multimerization as well as the interaction of the D'D3 domain with FVIII.…”

“…Mutations within the VWF D’D3 domain that impede this high-affinity binding lead to type 2N von Willebrand disease, a condition characterized by reduced plasma levels of FVIII 19,20 . Structural and biochemical data reveal binding of the FVIII C1 domain to the D’ modules of VWF and, additionally, hint at interactions of FVIII with the VWF D3 core 16,17,21,22 .…”

“…However, details of this necessary conformational change are currently unknown. In addition to enabling multimerization, the D'D3 domains serve another function critical for hemostasis: By binding coagulation factor VIII (FVIII), they protect FVIII from rapid clearance 17,18 and transport it to sites of vascular injury. Mutations within the VWF D'D3 domain that impede this high-affinity binding lead to type 2N von Willebrand disease, a condition characterized by reduced plasma levels of FVIII 19,20 .…”