Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII

Abstract

• The VWF D9 domains are flexibly tethered entities projecting outside antiparallel dimers of the VWF D3 domain. • Extensive interactionsbetween the VWF D9 domain and primarily the FVIII C1 domain mediate VWF-FVIII association.Binding to the von Willebrand factor (VWF) D9D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D9D3 alone and in complex with FVIII. The D9D3 dimer ([D9D3] 2 ) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D9. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D9 domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. IntroductionThe strong association of plasma factor VIII (FVIII) with circulating von Willebrand factor (VWF) secures FVIII from rapid clearance in the blood. The VWF-FVIII complex forms through a high-affinity interaction between the FVIII light chain and the VWF D9D3 domains. 1Mutations within VWF that abrogate or abolish this high-affinity binding lead to type 2N von Willebrand disease, a condition characterized by reduced plasma levels of FVIII. 2The tertiary structure of mature VWF, particularly at the N-terminal D9D3 domains, regulates the affinity for FVIII. VWF circulates as a multi-subunit protein comprising repeated domains that distinctly facilitate VWF packaging and hemostasis.3 The VWF propeptide (domains D1and D2) catalyzes the multimerization of VWF via intermolecular disulfide bonds at the D3 domain ( Figure 1A). 4 In the absence of propeptide-dependent posttranslational modifications to the D9D3 domains, VWF binds FVIII with reduced affinity.5 Cleavage of the propeptide by furin facilitates FVIII stabilization in the circulation. 6 We and others have previously reported that VWF fragments are sufficient to bind FVIII and that propeptide processing of these VWF fragments enhances the affinity for FVIII.7-9 Several of these VWF fragments were also sufficient to elevate FVIII levels in VWF-deficient mice. 7To further explore the association between VWF and FVIII, we used single-particle negative-stain electron microscopy (EM) to characterize the architecture of dimeric VWF D9D3 domains ([D9D3] 2 ) alone and in complex with FVIII. Study designProtein expression, purification, and analyses are detailed in supplemental Data available on the Blood Web site. The online version of this article contains a data supplement. Results and discussionThere is an Inside Blood Commentary on this article in this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734. , each monomer appears as an ovoid density along the dimer symmetry axis accompanied by a weaker elongated density, which we term the "handle," in the periphery. The dimensions of the handle are ;20Å ...

The renaissance of complement therapeutics

“…Ra Pharma is developing a , RA101495, that binds C5 with high affinity and allosterically inhibits convertase-mediated cleavage 93 . After receiving orphan drug designation for PNH, Ra advanced RA101495 to phase II trials using a once-daily subcutaneous self-administration scheme 94–96 .…”

Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A

“…These studies have shown that the positively charged VWF-D9 surface is probably a binding site for FVIII-ap. 56,57 More detailed models are on the horizon, as the 3-dimensional structures suggest additional mutagenesis studies to identify specific residues that mediate FVIII-VWF binding.…”

“…48,50 A VWF-D9 solution structure 55 ( Figure 3), as well as modeling based on single-particle electron microscopy of complexes formed by FVIII and VWF-D9D3 domains, 56,57 have allowed further analysis of the FVIII-VWF interface and of VWF-D9 mutations associated with reduced FVIII-VWF affinity and type 2N VWD. These studies have shown that the positively charged VWF-D9 surface is probably a binding site for FVIII-ap.…”

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

“…20,21 Recently negativestain electron microscopy and hydrogen-deuterium exchange mass spectrometry (HDX MS) studies have revealed that VWF primarily interacts with the C1 domain of fVIII. 22,23 VWF binding protects fVIII from endocytosis 24 and the human-derived anti-C1 domain antibody KM33 inhibits fVIII uptake by human monocyte-derived dendritic cells (MDDCs). 25 Moreover, B-domain deleted (BDD) fVIII mutant constructs containing alanine substitutions at exposed C1 residues Arg2090, Lys2092, and Phe2093 alone or in combination showed reduced fVIII uptake by human MDDCs and macrophages and immunogenicity in fVIII knockout mice compared with wild-type BDD fVIII.…”