scholarly journals Von Willebrand Factor Interaction with FVIII: Development of Long Acting FVIII Therapies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-8-SCI-8 ◽  
Author(s):  
Peter J Lenting ◽  
Vincent Muczynski ◽  
Gabriel Aymé ◽  
Cecile V. Denis ◽  
Olivier D. Christophe
Keyword(s):  
Complex Formation ◽  
Von Willebrand Factor ◽  
Half Life ◽  
Antibody Fragment ◽  
Coagulation Factor ◽  
Severe Bleeding ◽  
Single Chain ◽  
Long Acting ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) both play a centrol role in hemostasis, illustrated by the severe bleeding disorders associated with their functional absence. Despite their different functionalities in hemostasis and being products from two different genes, both proteins circulate in a tight, non-covalently linked complex. The physiological concequences of complex formation are many, including stabilization of FVIII heterodimeric structure, protection of FVIII from protelytic degradation, and modulation of FVIII immunogenicity. Another relevant issue relates to the chaperone function of VWF, allowing FVIII to survive in the circulation. FVIII levels are markedly reduced in patients with no detectable VWF protein or with a defect in VWF-FVIII complex formation, indicating that VWF prevents FVIII from premature clearance. Moreover, evidence points to FVIII actually being predominantly cleared as part of the VWF-FVIII complex rather than as a separate protein. First, it is possible to predict FVIII half-life fairly accurately by knowing antigen levels of VWF and its propeptide in combination with blood group. Second, when FVIII and VWF are co-injected in Vwf-deficient mice, FVIII is targeted to the same macrophages as is VWF. Since the end of the 1990s, our knowledge on the clearance mechanism of FVIII and VWF has started to emerge, and multiple clearance receptors for both proteins have now been identified. Interestingly, there exists a large overlap in receptor-repertoire between FVIII and VWF. These findings have taught us that it will be difficult to design single-mutant FVIII or VWF variants that have prolonged half-lives. How then to prolong the half-life of FVIII to improve treatment of hemophilia A? Several novel bioengineered FVIII variants have been developed, including PEGylation, Fc fusion and single-chain design, aiming to increase FVIII half-life. These approaches have so far achieved only moderate increases in half-life (1.5- to 2-fold compared to marketed FVIII products), significantly less than when similar modifications are being applied to factor IX. Indeed, it seems as if in designing these FVIII variants, the role of the significant other in the complex has been overlooked, since FVIII clearance is principally determined by VWF. Could we instead use VWF as a tool to prolong half-life of FVIII? This option is actually limited by the nature of the interaction between VWF and FVIII. Although of high affinity, the interaction is characterized by high association- and dissociation-rates. Infusing FVIII in combination with long-acting VWF variants will therefore result in a rapid redistribution of FVIII to endogenous VWF, as has elegantly been shown by the group of Ginsburg. To overcome this limitation, we have designed a FVIII variant (FVIII-KB013bv) in which we have replaced the B-domain by a single-domain, llama-derived antibody fragment (nanobody) that recognizes the D'D3-region of VWF. Consequently, the dissociation-rate of the VWF/FVIII complex is reduced 100-fold. Preliminary studies revealed that FVIII-KB013bv has a two-fold prolonged half-life compared to FVIII, likely due to improved VWF binding properties. Combination of the FVIII-nanobody fusion protein with long-acting VWF variants is anticipated to prolong its half-life well beyond the limit of the current long-acting FVIII variants. Disclosures Lenting: NovoNordisk: Consultancy, Research Funding.

scholarly journals Use of a Von Willebrand Factor/Coagulation Factor VIII Complex for Treatment of Refractory Inherited Platelet Disorders

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Amber Federizo
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Cost Effective ◽  
Severe Bleeding ◽  
Off Label ◽  
Platelet Disorders ◽  
Von Willebrand ◽  
Willebrand Factor

Inherited platelet disorders are recognized as an important cause of mild to severe bleeding in both children and adults. Patients with platelet disorders may present with mucocutaneous bleeding, gastrointestinal bleeding, menorrhagia, postsurgical, and/or excessive bleeding from traumatic injury. Delta storage pool deficiencies (delta-SPD) are among the most frequent platelet disorders, characterized by dysfunctional dense platelet granules. Bernard Soulier syndrome (BSS) is an autosomal recessive platelet disorder caused by mutations in various polypeptides in the GpIb/IX/V complex, which is the principal receptor for von Willebrand factor (VWF). Treatment of platelet disorders is mainly supportive. Normal hemostasis requires VWF and factor VIII (FVIII) to support platelet adhesion and aggregation at sites of vascular injury. von Willebrand factor is a large multimeric glycoprotein present in human plasma as a series of polymers called multimers. Molecular weights for multimers ranges from 500 kDa for the dimer to over 10,000 kDa for the high molecular weight multimers (HMWM) forming the largest known protein present in human plasma. Each multimeric subunit of VWF has binding sites for the receptor GpIb on nonactivated platelets and the receptor GpIIb/IIIa to facilitate platelet adhesion and platelet aggregation, respectively, making the VWF HMWM important for normal platelet function. Desmopressin (DDAVP), which is known to stimulate the release of VWF and FVIII, is commonly used for treatment of platelet disorders. Potentiation of platelet aggregation at high shear rate may be one mechanism by which DDAVP shortens the prolonged bleeding time of patients with congenital platelet defects. For severe bleeding, platelet transfusion may be required, but patients may develop isoantibodies, rendering this therapy ineffective. For this reason, it may be prudent to reserve platelet transfusion in this patient population for emergent situations, such as trauma. Other patients and/or clinical situations may require recombinant active factor VII (rFVIIa), but this therapy is very costly and not always effective and/or available. Antifibrinolytics may also be used but are not always effective. In four (4) patients with platelet disorders (delta-SPD [n=3]; BSS [n=1]), common supportive therapies were not effective, tolerable, and/or available. It was postulated that off-label infusions of a cost-effective von Willebrand factor/coagulation factor VIII (VWF/FVIII) complex (Wilate, Octapharma SA) might be of benefit in these refractory patients (Table 1). The mechanism of action of DDAVP treatment efficacy relies on the release of existing, stored, functional VWF. In refractory patients with suboptimal VWF functionality, it was reasoned that infusion of exogenous, functional VWF and FVIII could potentially encourage platelet adhesion and aggregation. All refractory patients studied were treated successfully with the VWF/FVIII complex with positive clinical outcomes. As mentioned, the adhesive activity of VWF depends on the size of its multimers, and HMWM are the most effective in supporting interaction with collagen and platelet receptors and in facilitating wound healing under conditions of shear stress in the human vascular system. The VWF/FVIII complex utilized in these patients is known to have minimal amounts of the plasma metalloproteinase ADAMTS13. The HMWM of VWF are, under normal conditions, cleaved by ADAMTS13 to smaller, less adhesive multimers. During the manufacturing process, if the ADAMTS13 is not filtered out of the product almost entirely, the VWF in the vial may become highly proteolyzed. Therefore, a reduction or lack of HMWM resulting from inclusion of ADAMTS13 in the manufactured product is believed to reduce product functionality. Multimeric analysis of the VWF/FVIII complex has shown that it exhibits a physiological triplet structure which resembles normal plasma. In addition, the product has a high safety profile and tolerability as protein impurities are eliminated in the manufacturing process. In summary, the use of a VWF/FVIII complex in four (4) patients with inherited platelet disorders, who were refractory to conventional treatments, provided beneficial, cost-effective clinical outcomes with resolution of bleeding. Disclosures Federizo: Octapharma: Consultancy, Honoraria, Other: Publication support, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; American Thrombosis and Hemostasis Netowrk: Research Funding; Aptevo: Consultancy, Speakers Bureau; National Hemophilia Foundation: Consultancy, Honoraria. OffLabel Disclosure: von Willebrand/FVIII concentrate is currently approved for the treatment of Hemophilia A and von Willebrand. This abstract will review the off-label use of this medication in the treatment of inherited platelet dysfunction.

scholarly journals Cryo-EM Structure of BIVV001 Reveals Coagulation Factor VIII-Von Willebrand Factor D'D3 Interaction Mode

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 94-94
Author(s):  
James Fuller ◽  
Joseph Batchelor ◽  
Kevin Knockenhauer ◽  
Hans-Peter Biemann ◽  
Robert Peters
Keyword(s):  
High Resolution ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Single Particle ◽  
Half Life ◽  
Coagulation Factor ◽  
High Affinity ◽  
Affinity Interaction ◽  
Von Willebrand ◽  
Willebrand Factor

Introduction Coagulation Factor VIII (FVIII) is a serine protease cofactor that directly interacts with coagulation factors IXa and X on activated platelets, and enhances FIXa activity toward FX by 105. von Willebrand Factor (VWF), via its D'D3 domains, interacts with FVIII and prevents premature deposition on phospholipids until activation by thrombin. Thrombin cleavage at Arg1689 of FVIII promotes VWF dissociation by disrupting the FVIII a3 high affinity interaction with the VWF D' domain. VWF extends the half-life of circulating FVIII from less than 3 hours to ~11 hours in humans. While crystal structures of FVIII and VWF D'D3 alone have been solved, the atomic details of a formed complex are unknown. We sought to determine the FVIII-VWF D'D3 complex structure by using BIVV001, our investigational new drug currently in clinical trials for the treatment of Hemophilia A. BIVV001 (rFVIIIFc-VWF-XTEN) is a novel fusion protein consisting of single chain B-domain deleted (BDD) human FVIII, the Fc domain of human immunoglobulin G1 (IgG1), the FVIII-binding D'D3 domain of human von Willebrand factor, and 2 XTEN polypeptide linkers. The Fc, VWF, and XTEN linker portions of the molecule are each designed to extend the half-life of FVIII. We anticipated that the tethering of FVIII to D'D3 through the Fc dimer in BIVV001 would stabilize the complex for structural studies. Given the large size of BIVV001, at 312 kDa, we thought it an ideal target for structure determination by single particle cryo-EM. Methods We collected a total of 3955 micrographs of BIVV001 embedded in vitreous ice at 81,000x magnification using a Titan Krios electron microscope equipped with a Gatan BioQuantum K3 energy filter and camera operating in super-resolution mode. Preferential particle orientation was a major challenge that was overcome through a variety of methods. Micrograph movies were motion-corrected and summed, and over 2 million candidate particle coordinates were extracted. Repeated rounds of reference-free 2D classification resulted in a set of 1.2 million particles that generated a reasonable ab initio/de novo 3D model. Initial full 3D refinements of this model produced a map at approximately 5 Å resolution, into which available crystal structures can be readily fit. Subsequent iterative 3D refinement and 3D classification resulted in a final map at high resolution, into which an atomic model was built. Results The structure of BIVV001 was solved by single particle cryo-EM. D' of VWF interacts with the front face of the C1 and A3 domains of FVIII, consistent with a lower resolution, negative stain EM map (Yee et al. 2015. Blood). Interface residues on FVIII identified in an HDX-MS dataset (Chiu et al. 2015. Blood.) largely correspond to this high affinity interaction. D' protrudes upward from the VWF D3 domain, which sits centrally located between the C1 and C2 domains of FVIII at a 45° tilt. By occupying this position, D3 likely sterically blocks the FVIII C domains from binding to membrane. The VWD3 module of the D3 domain contacts the base of the C1 domain, whereas C8-3 binds to the bottom of the C2 domain. The conserved Ca2+ site in VWD3 identified previously (Dong et al. 2019. Blood.) is in the interface with C1. This is consistent with Yee et al., where docking placed D3 below the C domains. In that study, a lack of density between FVIII and VWF D3 in the 3D reconstruction, due to flexibility, prevented the detailed analysis that is possible here. In this study, flexibility in this region is also apparent, as C2 is less well ordered than the rest of FVIII and VWF D3 is the least well-ordered portion of the resolved structure. The XTEN linkers are not visible in the final map and were not apparent in any 2D class averages. The Fc is absent in most 2D class averages, due to a lack of consistent positioning relative to FVIII. In the rare cases where the Fc is visible, it adopts a preferred position on the back side of FVIII below the A3 protrusion. Conclusions The structure of BIVV001 has been solved by cryo-electron microscopy to high resolution. Alignment with previous results and the averaging out of BIVV001 elaborations suggests the structure obtained here likely represents WT FVIII-D'D3. This structure demonstrates how VWF D'D3 prevents premature FVIII deposition on phospholipids. The structural basis of type 2N von Willebrand Disease mutations in D'D3 can be readily interpreted. Next steps include solving a FVIII-D'D3 dimer structure at high resolution. Disclosures Fuller: Sanofi: Employment. Batchelor:Sanofi: Employment. Knockenhauer:Sanofi: Employment. Biemann:Sanofi: Employment. Peters:Sanofi: Employment.

scholarly journals FVIII half-life extension by coadministration of a D′D3 albumin fusion protein in mice, rabbits, rats, and monkeys

2020 ◽  
Vol 4 (9) ◽  
pp. 1870-1880
Author(s):  
Sabine Pestel ◽  
Hans-Wilhelm Beltz ◽  
Philipp Claar ◽  
Holger Lind ◽  
Marcel Mischnik ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Half Life ◽  
Ex Vivo ◽  
Coagulation Factor ◽  
Human Albumin ◽  
Life Extension ◽  
Increased Risk ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D′D3) fused to human albumin (rD′D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD′D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD′D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD′D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Autoantibody Selectively Inhibits Binding of von Willebrand Factor to Glycoprotein Ib. Recognition Site Is Located in the A1 Loop of von Willebrand Factor

1997 ◽  
Vol 77 (04) ◽  
pp. 760-766 ◽  
Author(s):  
Hiroshi Mohri ◽  
Etsuko Yamazaki ◽  
Zekou Suzuki ◽  
Toshikuni Takano ◽  
Shumpei Yokota ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Significant Inhibition ◽  
Von Willebrand Disease ◽  
Recognition Site ◽  
Severe Bleeding ◽  
Bleeding Tendency ◽  
Virtual Absence ◽  
Heavy Chains ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA 20-year-old man with severe von Willebrand disease recently presented a progressive bleeding tendency, characterized recurrent subcutaneous hemorrhages and cerebral hemorrhage. Mixing and infusion studies suggested the presence of an inhibitor directed against vWF:RCo activity of von Willebrand factor (vWF) without significant inhibition of the FVIII:C. The inhibitor was identified as an antibody of IgG class. The inhibitor inhibited the interaction of vWF in the presence of ristocetin and that of asialo-vWF with GPIb while it partially blocked botrocetin-mediated interaction of vWF to GPIb. The inhibitor reacted with native vWF, the 39/34kDa fragment (amino acids [aa] 480/ 481-718) and the recombinant vWF fragment (MalE-rvWF508-704), but not with Fragment III-T2 (heavy chains, aa 273-511; light chains, aa 674-728). A synthetic peptide (aa 514-542) did not inhibit vWF-inhibitor complex formation. We conclude that this is the first autoantibody of class IgG from human origin that recognizes the sequence in the A1 loop of vWF, resulting in a virtual absence of functional vWF and a concomitant severe bleeding tendency although recognition site is different from the residues 514-542 which is crucial for vWF-GPIb interaction.

scholarly journals Regulation of plasma von Willebrand factor

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 96 ◽  
Author(s):  
Karl C Desch
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Vascular Wall ◽  
Healthy Human ◽  
Venous Thromboembolic ◽  
Artery Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a central role in the initiation of blood coagulation. Through interactions between its specific functional domains, the vascular wall, coagulation factor VIII, and platelet receptors, VWF maintains hemostasis by binding to platelets and delivering factor VIII to the sites of vascular injury. In the healthy human population, plasma VWF levels vary widely. The important role of VWF is illustrated by individuals at the extremes of the normal distribution of plasma VWF concentrations where individuals with low VWF levels are more likely to present with mucocutaneous bleeding. Conversely, people with high VWF levels are at higher risk for venous thromboembolic disease, stroke, and coronary artery disease. This report will summarize recent advances in our understanding of environmental influences and the genetic control of VWF plasma variation in healthy and symptomatic populations and will also highlight the unanswered questions that are currently driving this field of study.

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