Domain organization of membrane‐bound factor VIII

Stoilova‐McPhie, Svetla; Lynch, Gillian C.; Ludtke, Steven J.; Pettitt, B. Montgomery

doi:10.1002/bip.22199

Cited by 33 publications

(46 citation statements)

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“…We are also showing a new pFVIII-LNT helical reconstruction resolved at 15.5 Å resolution. Fitting of the FVIII-LNT structure within the cryo-EM map resolved at 15.5 Å agrees with the concept that pFVIII associates with the membranes mainly through the C2 domain, as previously demonstrated for the pdFVIII organized in membrane-bound 2D crystals13 and the hFVIII-LC helically organized on LNT37. We can therefore confirm that the membrane-bound pFVIII domain organization on LNT is similar to the one previously resolved from our hFVIII-LC-LNT helical reconstructions37.…”

Section: Discussionsupporting

confidence: 90%

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

Dalm

Galaz-Montoya

Miller

et al. 2015

Sci Rep

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Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

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Section: Discussionsupporting

confidence: 90%

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

Dalm

Galaz-Montoya

Miller

et al. 2015

Sci Rep

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“…The structures and affinities of FVIII binding to its various partners, though informative, must be interpreted with caution, as interactions between isolated components of, for example, the intrinsic tenase complex may differ somewhat from those that occur physiologically, at wound sites and under shear forces ( Figure 2). Therefore, the growing body of FVIII structural studies (crystallographic, [48][49][50] fluorescence resonance energy transfer, 51 cryoelectron microscopic, 52 etc) are best considered a series of "snapshots" from the ensemble of conformations accessible to this multidomain protein.…”

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

Blood

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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.

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“…The mapping of this series of FVIII-C2 domain epitopes will facilitate additional studies to model the domain orientations in FVIII (which may well differ among the solution, vWF-bound, and membrane-bound FVIII structures), 21,22,42 as well as the interactions between FVIIIa and the other components of the intrinsic tenase complex. The high-resolution definition of these physiologically relevant and medically important epitopes also suggests specific sites at which the FVIII sequence could be modified to generate less antigenic FVIII variants.…”

Section: Blood 24 April 2014 X Volume 123 Number 17 B-cell Epitopesmentioning

confidence: 99%

“…on May 12, 2018. by guest www.bloodjournal.org From inhibitors because their identification pointed to a previously underappreciated and important role for the FVIII-C2 domain in proteolytic activation of FVIII. Their localization at a surface distinct from the other types of epitopes, and also on an outer surface of the FVIII protein (ie, not at an interdomain interface), is shown in Figure 3A-B.The mapping of this series of FVIII-C2 domain epitopes will facilitate additional studies to model the domain orientations in FVIII (which may well differ among the solution, vWF-bound, and membrane-bound FVIII structures), 21,22,42 as well as the interactions between FVIIIa and the other components of the intrinsic tenase complex. The high-resolution definition of these physiologically relevant and medically important epitopes also suggests specific sites at which the FVIII sequence could be modified to generate less antigenic FVIII variants.…”

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High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

Nguyen

Lewis

Ettinger

et al. 2014

Blood

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• Amino acid residues comprising B-cell epitopes recognized by neutralizing antifactor VIII antibodies (inhibitors) have been identified. • Amino acids contributing significant antigen-antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins.Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigenantibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients. (Blood. 2014;123(17):2732-2739 IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is a serious complication that may be encountered when FVIII replacement therapy is administered to patients with hemophilia A (HA). It affects 25% to 30% of the treated HA population, with a peak occurrence after ;14 FVIII infusions.1-3 Autoimmune responses to FVIII can also occur, 4 and although this happens only rarely, the resulting bleeding phenotype can be severe. Inhibitors can be difficult and extremely expensive to manage clinically. Interestingly, porcine FVIII has been used effectively in the clinic as a "bypass" therapy; that is, a therapeutic protein that can evade neutralization by anti-FVIII antibodies in many allo-and autoimmune inhibitor patients. [5][6][7] However, some patients have or could develop antibodies that neutralize porcine FVIII as well, 8 because of antigenic cross-reactivity 9 or because regions in which the porcine sequence differs from the human FVIII sequence stimulate effector T cells, leading to antibody production. Identification of the binding sites (B-cell epitopes) on FVIII that are recognized by inhibitors would allow rational design of novel therapeutic FVIII...

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Domain organization of membrane‐bound factor VIII

Cited by 33 publications

References 50 publications

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

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

High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

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