scholarly journals The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII

1989 ◽  
Vol 257 (3) ◽  
pp. 679-683 ◽  
Author(s):  
A Leyte ◽  
M P Verbeet ◽  
T Brodniewicz-Proba ◽  
J A Van Mourik ◽  
K Mertens
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

The interaction between human Factor VIII and immobilized multimeric von Willebrand Factor (vWF) was characterized. Equilibrium binding studies indicated the presence of multiple classes of Factor VIII-binding sites on vWF. The high-affinity binding (Kd = 2.1 x 10(-10) M) was restricted to only 1-2% of the vWF subunits. Competition studies with monoclonal antibodies with known epitopes demonstrated that the Factor VIII sequence Lys1673-Arg1689 is involved in the high-affinity interaction with vWF.

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.

Factor VIII and Platelets Cooperatively Accelerate Proteolytic Cleavage of Von Willebrand Factor by ADAMTS13 Under Shear Stress

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 590-590
Author(s):  
Christopher G. Skipwith ◽  
Wenjing Cao ◽  
X. Long Zheng
Keyword(s):  
Shear Stress ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Coagulation Factor ◽  
Proteolytic Cleavage ◽  
Cleavage Product ◽  
Shear Stresses ◽  
High Affinity ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 bond of the central A2 domain. Inability to cleave vWF results in thrombotic thrombocytopenic purpura (TTP), which is characterized by profound thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ involvement. Previous studies have demonstrated that coagulation factor VIII (Cao et al, PNAS, 105:7416–21, 2008) or platelets (Shim et al, Blood, 111:651–7, 2008) can accelerate proteolytic cleavage of multimeric vWF in solution by ADAMTS13 under mechanic induced shear stresses. However, the concentrations of factor VIII or platelets required to achieve a detectable increase in the proteolytic cleavage product (350K) were beyond the physiological ranges. Therefore, the physiological significance of these findings remained to be determined. In this study, we assessed whether factor VIII and platelets, both of which bind vWF with high affinity, cooperatively affected the proteolytic cleavage of vWF by ADAMTS13 under high shear stress. All experiments were performed with a fixed concentration of vWF (150 nM) and ADAMTS13 (25 nM), and various concentrations of factor VIII and platelets in a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM CaCl2. The reaction mixtures (total volume 20 μl in a 0.2 ml PCR tube) were subjected to vortexing at 2,500 rpm in MixMate (manufactured by Eppendorf), which generates laminar shear stress of approximately 30 dynes/cm2. We showed that in the absence of factor VIII, lyophilized platelets at the concentration of 1,000×109 per liter did not increase the proteolytic cleavage of plasma-derived vWF by ADAMTS13. An addition of factor VIII (1, 2, and 5 nM) to the reaction mixture markedly accelerated the rate enhancing effect of lyophilized platelets in a concentration-dependent manner. In the presence of physiological concentration of factor VIII (1–2 nM), the proteolytic cleavage product (350K) detected by Western blot under non-reducing conditions reached the maximal intensity at the platelet concentration of 100×109 per liter. B-domain deleted factor VIII (FVIII-SQ) showed the similar cooperativity with platelets accelerating proteolytic cleavage by ADAMTS13. However, a construct with the acidic region (a3) that binds vWF with high affinity removed (FVIII-2RKR) did not show any cooperativity with platelets in vWF proteolysis by ADAMTS13. We conclude that binding of factor VIII and platelets to vWF cooperatively accelerates the proteolytic cleavage of vWF by ADAMTS13 under high (arterial) shear stress. These findings provide novel insight into the regulation of ADAMTS13-mediated vWF proteolysis by a coagulation factor and platelets under physiological conditions. The data also suggest a potential role of factor VIII and platelets in the therapeutic regimen for TTP.

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 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.

scholarly journals Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 823-831 ◽  
Author(s):  
VT Turitto ◽  
HJ Weiss ◽  
TS Zimmerman ◽  
II Sussman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Human Factor ◽  
Rabbit Aorta ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.

Sign in / Sign up

Export Citation Format

Share Document