Solution structure of the major factor VIII binding region on von Willebrand factor

• 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 ...

Section: Resultssupporting

confidence: 90%

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

Yee¹,

Oleskie²,

Dosey³

et al. 2015

“…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.…”

Section: Fviii and Vwf Structural Biologymentioning

confidence: 99%

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

Pipe

Montgomery

Pratt

et al. 2016

185

186

A normal hemostatic response to vascular injury requires both factor VIII (FVIII) and von Willebrand factor (VWF). In plasma, VWF and FVIII normally circulate as a noncovalent complex, and each has a critical function in the maintenance of hemostasis. Furthermore, the interaction between VWF and FVIII plays a crucial role in FVIII function, immunogenicity, and clearance, with VWF essentially serving as a chaperone for FVIII. Several novel recombinant FVIII (rFVIII) therapies for hemophilia A have been in clinical development, which aim to increase the half-life of FVIII (∼12 hours) and reduce dosing frequency by utilizing bioengineering techniques including PEGylation, Fc fusion, and single-chain design. However, these approaches have achieved only moderate increases in halflife of 1.5-to 2-fold compared with marketed FVIII products. Clearance of PEGylated rFVIII, rFVIIIFc, and rVIII-SingleChain is still regulated to a large extent by interaction with VWF. Therefore, the half-life of VWF (∼15 hours) appears to be the limiting factor that has confounded attempts to extend the half-life of rFVIII. A greater understanding of the interaction between FVIII and VWF is required to drive novel bioengineering strategies for products that either prolong the survival of VWF or limit VWF-mediated clearance of FVIII. (Blood.

“…The structure of D9 shows how E' extends the highly dynamic TIL9 module away from the D3 assembly to bind FVIII ( Figure 1J). 7 von Willebrand C (VWC) modules have 2 disulfide-rich subdomains ( Figure 1H). 8,9 The E module of D assemblies is related to the first VWC subdomain.…”

mentioning

confidence: 99%

“…8,9 The E module of D assemblies is related to the first VWC subdomain. 2,7 The 6 tandem VWC modules extend VWF length and give it flexibility (Figure 2). Platelet integrin a IIb b 3 binds an RGD motif in the VWC4 module 2 ( Figure 1A).…”

mentioning

confidence: 99%

von Willebrand factor, Jedi knight of the bloodstream

Springer

2014

383

454

When blood vessels are cut, the forces in the bloodstream increase and change character. The dark side of these forces causes hemorrhage and death. However, von Willebrand factor (VWF), with help from our circulatory system and platelets, harnesses the same forces to form a hemostatic plug. Force and VWF function are so closely intertwined that, like members of the Jedi Order in the movie Star Wars who learn to use “the Force” to do good, VWF may be considered the Jedi knight of the bloodstream. The long length of VWF enables responsiveness to flow. The shape of VWF is predicted to alter from irregularly coiled to extended thread-like in the transition from shear to elongational flow at sites of hemostasis and thrombosis. Elongational force propagated through the length of VWF in its thread-like shape exposes its monomers for multimeric binding to platelets and subendothelium and likely also increases affinity of the A1 domain for platelets. Specialized domains concatenate and compact VWF during biosynthesis. A2 domain unfolding by hydrodynamic force enables postsecretion regulation of VWF length. Mutations in VWF in von Willebrand disease contribute to and are illuminated by VWF biology. I attempt to integrate classic studies on the physiology of hemostatic plug formation into modern molecular understanding, and point out what remains to be learned.