Von Willebrand Factor Fragments Research Articles

Safety, pharmacokinetics and efficacy of a subcutaneous recombinant FVIII (OCTA101) in adult patients with severe haemophilia A.

Regular, prophylactic intravenous (i.v.) FVIII can be challenging for some patients with haemophilia A. Subcutaneous (s.c.) FVIII administration could provide an alternative treatment option with greater convenience and without the complications associated with venous access. To assess the safety, pharmacokinetics (PK), bioavailability and efficacy of s.c. OCTA101, a recombinant FVIII with a recombinant von Willebrand factor fragment dimer. This was a single-centre, prospective, open-label, phase I/II study (NCT04046848). Previously treated male patients (≥18 years) with severe haemophilia A were eligible for the study. The primary objective of the study was to assess the safety (including immunogenicity) of OCTA101. Secondary objectives included assessments of PK, bioavailability, and the efficacy of prophylaxis. Thirty patients were treated with OCTA101. FVIII inhibitors developed in five (16.7%) patients during daily prophylaxis with 40-60IU/kg (three cases) and 12.5IU/kg (two cases) OCTA101. The trial was therefore terminated. OCTA101 had a 2.5-fold longer terminal half-life compared with i.v. rFVIII, and bioavailability was 16.6%. Efficacy data at study termination indicated that daily prophylaxis with 40-60IU/kg OCTA101 was efficacious in the absence of FVIII inhibitors. Despite promising PK and efficacy results, the trial was terminated due to the incidence of FVIII inhibitors. The occurrence of inhibitors at two dose levels suggests that their development may be related to the subcutaneous route of administration.

Angiopoietin-2 binds to multiple interactive sites within von Willebrand factor

BackgroundBiosynthesis of von Willebrand factor (VWF) in endothelial cells drives the formation of storage-organelles known as Weibel-Palade bodies (WPBs). WPBs also contain several other proteins, including angiopoietin-2 (Ang-2). ObjectivesAt present, the molecular basis of the VWF–Ang-2 interaction is poorly understood. Here, we used immunosorbent-binding assays and specific recombinant VWF fragments to analyze VWF–Ang-2 interactions. ResultsWe found that VWF bound to immobilized Ang-2 most efficiently (half-maximal binding at 0.5 ± 0.1 μg/mL) under conditions of high CaCl2 (10 mM) and slightly acidic pH (6.4-7.0). Interestingly, several isolated recombinant VWF domains (A1/Fc, A2/Fc, D4/Fc, and D’D3-HPC4) displayed dose-dependent binding to immobilized Ang-2. Binding appeared specific, as antibodies against D’D3, A1, and A2 significantly reduced the binding of these domains to Ang-2. Complexes between VWF and Ang-2 in plasma could be detected by immunoprecipitation- and immunosorbent assays. Unexpectedly, control experiments also revealed complexes between VWF and angiopoietin-1 (Ang-1), a protein structurally homologous to Ang-2. Furthermore, direct binding studies showed dose-dependent binding of VWF to immobilized Ang-1 (half-maximal binding at 1.8 ± 1.0 μg/mL). Interestingly, rather than competing for Ang-1 binding, Ang-2 enhanced the binding of VWF to Ang-1 about 3-fold. Competition experiments further revealed that binding to VWF does not prevent Ang-1 and Ang-2 from binding to Tie-2. ConclusionOur data show that both Ang-1 and Ang-2 bind to VWF, seemingly using different interactive sites. Ang-2 modulates the binding of VWF to Ang-1, the (patho)-physiological consequences of which remain to be investigated.

Structural basis of von Willebrand factor multimerization and tubular storage

AbstractThe von Willebrand factor (VWF) propeptide (domains D1D2) is essential for the assembly of VWF multimers and its tubular storage in Weibel-Palade bodies. However, detailed molecular mechanism underlying this propeptide dependence is unclear. Here, we prepared Weibel-Palade body-like tubules using the N-terminal fragment of VWF and solved the cryo-electron microscopy structures of the tubule at atomic resolution. Detailed structural and biochemical analysis indicate that the propeptide forms a homodimer at acidic pH through the D2:D2 binding interface and then recruits 2 D'D3 domains, forming an intertwined D1D2D'D3 homodimer in essence. Stacking of these homodimers by the intermolecular D1:D2 interfaces brings 2 D3 domains face-to-face and facilitates their disulfide linkages and multimerization of VWF. Sequential stacking of these homodimers leads to a right-hand helical tubule for VWF storage. The clinically identified VWF mutations in the propeptide disrupted different steps of the assembling process, leading to diminished VWF multimers in von Willebrand diseases (VWD). Overall, these results indicate that the propeptide serves as a pH-sensing template for VWF multimerization and tubular storage. This sheds light on delivering normal propeptide as a template to rectify the defects in multimerization of VWD mutants.

Autoinhibition of Mouse Von Willebrand Factor: Addressing the Species Compatibility between VWF and Gpibα in Humans and Mice

Introduction: The usage of mouse models to investigate thrombosis and hemostasis has now spanned several decades, with the advent of genetic manipulation elucidating the roles of platelet receptors, secondary messengers, and coagulation factors to name a few. While there are many similarities between humans and mice, the vast differences in physical parameters of blood circulation and coagulation should not be ignored. The incompatibility of mouse platelet receptor glycoprotein (GP)Ibα with human von Willebrand factor (VWF) has been well documented, yet human GPIbα can interact with mouse plasma VWF under shear. In light of the recently discovered autoinhibitory module (AIM) in modulating the activity of human VWF, here we seek to address the role of the AIM in species compatibility between these two interacting proteins.Methods: Recombinant human and mouse VWF fragments containing the A1 domain and either intact or truncated AIM, followed by a poly-histidine tag, were expressed from stably transfected mammalian cells. Recombinant human and mouse GPIbα ligand-binding domain (LBD) (residues 1-290, mature protein numbering) were similarly expressed bearing a biotin acceptor sequence and poly-histidine tag. Stable cell lines co-expressing E. coli biotin ligase were used to express and biotinylate the LBD protein before purification. All proteins were purified by nickel-affinity chromatography followed by size exclusion chromatography. Bio-layer interferometry was performed on an Octet QK e using streptavidin biosensors to capture biotinylated GPIbα LBD fragments. Data was analyzed using a 2:1 heterogeneous ligand binding model for kinetic parameters, and steady state affinity (K D) was calculated either from binding responses at the end of the association phase, or from predicted equilibria from the aforementioned model. Human or mouse washed platelets were isolated by centrifugation, followed by pelleting the PRP in the presence of PGE-1. Platelets were resuspended to 150,000/ μL in modified Tyrode's buffer, and aggregation was monitored in a light transmission aggregometer.Results: Consistent with earlier reports, human AIM-A1 protein (VWF residues 1238-1493), at concentrations below 1 μM, showed no appreciable binding to human GPIbα LBD. Conversely, human truncated AIM-A1 protein (tAIM-A1, residues 1261-1472) exhibited a K D of 348 nM towards the LBD. Mouse tAIM-A1 proteins showed enhanced binding to mouse LBD (K D = 214 nM), but mouse AIM-A1 is able to bind to mouse LBD with a K D of 716 nM, which is considerably higher than the affinity of the human-human pair of tens of μM (Figure 1).Given the increased affinity of mouse AIM-A1 to mouse LBD, we reasoned that the mouse AIM may be less stable than the human counterpart. A chimeric AIM-A1 protein, in which human AIM sequences flank mouse A1 (human residues 1238-1271, mouse 1272-1458, and human 1459-1493), was generated. The chimeric AIM-A1 showed little binding to mouse LBD. However, this protein can still aggregate washed platelets from both species and bind to gain-of-function human GPIbα, indicating it still is a folded domain capable to recognizing its cognate receptor, mouse GPIbα (Figure 2).The isolated mouse AIM-A1 fragments are capable of inducing robust mouse and human platelet aggregation for washed platelets in a dose dependent manner. It had been previously demonstrated that in platelet rich plasma, ristocetin has no effect on mouse VWF. However, washed mouse platelets incubated with 60 nM of mouse AIM-A1 followed by addition of 1.5 mg/mL ristocetin was able to agglutinate the platelets (Figure 2). These results suggest that these proteins may be used to help elucidate the signaling pathways of GPIb-IX-V complex in mice.Conclusion: In agreement with previous reports, we observe that in general mouse AIM-A1 is more active than human AIM-A1 in its propensity to bind to GPIbα. On the aspect that human VWF in incompatible with mouse platelets, we observed that mouse LBD has lower affinity for human tAIM-A1 than human LBD does for mouse tAIM-A1. These findings altogether suggest that there are likely differences in activation forces for each species AIM, and that the GPIbα-A1 interactions, in terms of their mechanical and thermodynamic properties, are somewhat unique, and perhaps optimized, for each species. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetics of Subcutaneously Administered Simoctocog Alfa with Von Willebrand Factor Fragment Candidates in a Minipig Model

Introduction/Objective: The use of the subcutaneous (SC) administration route for hemophilia A therapy is hindered by the very low bioavailability of Factor VIII (FVIII) after SC administration. von Willebrand factor (VWF), the natural chaperone for FVIII, protects FVIII from rapid proteolysis, clearance, binding to phospholipids and uptake by macrophages. These mechanisms most likely contribute to the entrapment and degradation of FVIII in the SC tissue. Therefore, the use of VWF or VWF fragments represents a promising strategy in enabling FVIII to enter the circulation after SC administration. To evaluate this concept, we tested two VWF fragments in a minipig model.Materials/Methods: Two VWF fragments, O™CTA12 and OCTA13, with a C-terminal Strep-tag™ were expressed in a HEK293 cell line and purified by Strep-Tactin™ affinity chromatography. VWF fragment OCTA12 is a dimer containing D'D3 domains engineered to increase the glycosylation level. VWF fragment OCTA13 is a dimer containing D'D3-A1-A2-A3 domains. Both fragments were biochemically characterized by SDS-PAGE, FVIII binding and the ability to inhibit the interaction between FVIII and phospholipids. In addition, the ability of the VWF fragments to interfere with full-length VWF (flVWF) for mediation of platelet adhesion to collagen type III in a flow chamber was examined. Aachener minipigs (three animals per group) were administered with 100 IU/kg BW recombinant human FVIII (rhFVIII; simoctocog alfa) SC either alone or in combination with OCTA12 or OCTA13. As a control the same dose of rhFVIII was administered IV. Blood was taken prior to and 0.5, 1, 2, 4, 8, 24, 32, 48, 72, 96 and 120 h after each administration. The human FVIII antigen concentration and OCTA12 or OCTA13 concentrations were tested in each sample.Results:As confirmed by SDS-PAGE, both VWF fragments were expressed as dimers with minor monomer content. The propeptide was almost entirely cleaved off. The FVIII binding affinity (KD, mean of three measurements) was 0.93 nM for OCTA12 and 1.81 nM for OCTA13. About 90% inhibition of interaction between FVIII and phospholipid monolayer could be achieved for 6-fold molar excess of each fragment. Consequently, this molar ratio was used for the animal studies. No inhibition of full-length VWF activity by OCTA12 or OCTA13 could be observed in the flow chamber assay. The FVIII peak concentrations were as follows: 2 IU/ml at 1 h time point for rhFVIII alone administered IV; 0.02 IU/ml at 8 h time point for rhFVIII alone administered SC; 0.23 IU/ml at 8 h time point for rhFVIII complexed with OCTA12 administered SC. FVIII bioavailability was determined as the area under the curve (AUC) of FVIII:Ag, and the AUC after IV administration was defined as 100%. Bioavailability of FVIII alone after SC administration was 2%. The co-administration of rhFVIII with OCTA12 and OCTA13 increased the bioavailability of FVIII to 41% and 32.9%, respectively. Co-administration of rhFVIII with OCTA12 resulted in 2.3-fold longer time to 1% FVIII trough level compared with IV administration of FVIII at the same dose. Peak levels of 1.17 nM and 0.8 nM VWF fragment could be measured at 8 h for OCTA12 and OCTA13, respectively. At 120 h, 0.14 nM of OCTA13 and a still very high concentration of 0.83 nM of OCTA12 antigen could be measured, indicating a long half-life and/or slow release into the circulation.Conclusions:The two novel VWF fragments OCTA12 and OCTA13 significantly increased the bioavailability of FVIII after SC administration. High levels of VWF fragment OCTA12 could be detected over the entire sampling period, indicating a very long circulatory half-life and/or slow sustainable release of the fragment into the circulation. Importantly, the slow release and/or prolonged circulatory half-life of OCTA12 contributed to significantly prolonged FVIII antigen levels in the circulation after SC administration. In summary, the use of OCTA12 as a FVIII chaperone for SC administration represents a promising strategy to improve hemophilia A therapy in terms of application route and dosing frequency. DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Wuschko:Octapharma Pharmazeutika Produktionsges.m.b.H.: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.

A Novel Von Willebrand Factor Fragment Acts As a Chaperone to Prolong the Half-Life of Recombinant Human FVIII (Simoctocog Alfa) in Hemophilia a Dogs and FVIII/VWF Double Knockout Mice

Introduction/Objective: Subcutaneous (SC) drug delivery represents a convenient method of administration compared to intravenous (IV) delivery. However, due to very low bioavailability this route is not yet applicable for the administration of FVIII as a therapy for hemophilia A (HA). von Willebrand factor (VWF), in particular the D'D3 domains, is known to protect FVIII from proteolytic degradation, rapid clearance and binding to membranes. Therefore VWF represents a promising candidate for a strategy to increase FVIII bioavailability after SC treatment. Different VWF fragments containing at least the D'D3 domains were engineered in order to achieve high FVIII binding affinity, prolonged half-life of the VWF fragment, and consequently high bioavailability of FVIII.Materials/Methods: A modified VWF fragment (OCTA12) or an unmodified D'D3 domain, both with a C-terminal Strep-Tag, was transiently expressed in HEK293 cells and purified by Strep-Tactin® affinity chromatography. Recombinant human FVIII (rhFVIII, simoctocog alfa) complexed with OCTA12, D'D3 or full-length plasma-derived VWF (flVWF, Wilate®) was administered via IV route to FVIII/VWF double knockout (FVIII/VWF DKO) mice at 240 IU FVIII/kg in order to test the ability of the fragments to stabilize FVIII in circulation. Blood samples were taken at 5 minutes and 1, 4, 8, 12, 24, 36 and 48 hours after treatment and analyzed for FVIII activity. Subsequently, IV and SC administration of the complex of rhFVIII and OCTA12 or rhFVIII alone was evaluated in a HA dog model. For this purpose, HA dogs were treated according to the scheme presented in Table 1. Blood samples were taken at 0.5, 1, 2, 4, 8, 12, 24, 32, 48, 72 and 94 hours after treatment and analyzed for FVIII activity.Results: Five minutes after IV administration of rhFVIII complexed with different VWF variants to FVIII/VWF DKO mice, peak activity levels were determined (expressed in % mean ± SEM, n = 15): rhFVIII alone 78.9 ± 6.1; rhFVIII/D'D3 351.2 ± 22.3; rhFVIII/OCTA12 295.9 ± 17.4; rhFVIII/flVWF 404.4 ± 20.5. The FVIII activity levels were significantly higher (statistically evaluated by Student's t-test) when rhFVIII was administered with any VWF variant in comparison to rhFVIII administered alone. The three co-administered VWF species D'D3, OCTA12 or flVWF did not differ significantly in terms of measured FVIII levels. rhFVIII administered alone was rapidly cleared from the circulation (T1/2 = 0.07 h). Co-administration with flVWF or D'D3 resulted in a 4.5- or 6.4-fold prolongation of the half-life respectively (T1/2 = 0.32 h and T1/2 = 0.45 h respectively). The highest half-life prolonging effect (14.7-fold) was measured when rhFVIII was co-administered with OCTA12 (T1/2 = 1.03 h). The IV administration of rhFVIII alone (200 IU/kg) or complexed with OCTA12 (100 IU/kg) in the HA dog resulted in peak FVIII activity level of 215% or 134% respectively at 30 minutes post-infusion (mean of two injections). The SC administration of rhFVIII complexed with OCTA12 resulted in a 10.8% activity level at 30 minutes post-infusion and reached a peak level of 24.6% at 8 h post-infusion (mean of three injections). For calculation of bioavailability, the area under the curve (AUC) of FVIII activity after IV administration of rhFVIII alone was defined as 100%. The AUC of IV-administered rhFVIII/OCTA12 complex at 100 IU/kg (half the dose) was 90%. Bioavailability of FVIII after SC administration of the rhFVIII/OCTA12 complex was 37.7%.Conclusions: The co-administration of rhFVIII with the novel engineered VWF fragment OCTA12 results in high FVIII bioavailability after SC administration in HA dogs. The bioavailability of FVIII was also increased after IV administration of the FVIII/OCTA12 complex in FVIII/VWF DKO mice and HA dogs. Moreover, when complexed with OCTA12, FVIII exhibits prolonged half-life in FVIII/VWF DKO mice. [Display omitted] DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.

Molecular Design and Characterization of a Novel, Vwf Independent Coagulation Factor VIII (LAFATE)

We have known for long that most FVIII is carried by vWF in the blood stream, and began to aware that vWF determines the fate of FVIII whatever modification we make on it for its prolonged circulation. Thus, the consideration that vWF binding property of FVIII is to be conserved is now facing change.The aim of this study is to find out a way to unbind FVIII from endogenous vWF but to stabilize it to generate a novel longer-acting coagulation FVIII. We took a way to shield three major LRP1 binding sites by conjugating PEG and at the same time vWF binding sites by connecting D'D3 domains, the FVIII binding domains of vWF, or its variants using a flexible amino acid linker. We constructed a stable single chain form of FVIII (scFVIII) and employed it as a basic FVIII backbone structure for doing so.We generated FVIII variants according to this strategy, and evaluated their pharmacokinetic property in hemophilia A mice. Unmodified rFVIII (Advate®) was used as the reference. The mice were intravenously injected with the reference and test articles at a dose of 125 IU/kg rFVIII, respectively, and blood was sampled 5 min to 72 h or further after administration. Citrated plasma was prepared and analyzed for FVIII activity (Coamatic®, Chromogenix). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.4 (Pharsight).We first generated three different types of FVIII variants containing vWF D'D3 domains (D'D3 monomer, D'D3 dimer, D'D3-Fc dimer linked to C-terminus of C2 domain or N-terminus of A1 domain of FVIII), and then conjugated a 40kDa PEG to the B-domain of each variant site-specifically. In the subsequent pharmacokinetic study in hemophilia A mice, we observed significant prolongation of half-lives from 1.8- to 3.6-fold comparing with Advate®. The D'D3 monomer linked FVIII exhibited most prolonged half-life, suggesting that a D'D3 monomer is active enough to substantially attenuate binding of endogenous vWF to the chimera.Recently published electron microscopy and hydrogen-deuterium exchange analyses established the C1 domain as the major binding site for the vWF D'D3 domains on FVIII, and noted additional sites located within the a3 acidic peptide and the A3 and C2 domains of FVIII as being implicated in the FVIII-vWF interaction. In the following experiment, we aimed to find out optimal length of the amino acids linker that would secure easy access of D'D3 domains to A3 and C2 domains of FVIII. Considering the distance between these two domains measurable from the structural information of FVIII, we generated D'D3 monomer connected with 60, 120 and 300 amino acids linkers to scFVIII, respectively, PEGylated them and evaluated their pharmacokinetic properties in hemophilia A mice again. These molecules showed 2.9-, 3.5-, and 2.5-fold more prolonged half-lives than Advate®, respectively, and the 120 amino acids linker seemed optimal.Finally we conjugated several PEGs of different type and size, respectively, to the FVIII-D'D3 connected with this optimal linker, and evaluated their PK properties in hemophilia A mice in the next experiment. Consequently, we identified four different PEGs that support best performance, and named these compounds as LAFATE. LAFATE corrected impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner, and stopped acute bleeding as effectively as Advate®, evaluated by the amount of hemoglobin loss for a given time period in a tail-clip bleeding model of hemophilia A mice. LAFATE has a good potential to bring in possible administration of a hemophilia A drug on weekly bases and therefore, warrants further investigation.Related Articles1. The tertiary structure and domain organization of coagulation factor VIII. Betty W. Shen et al., Blood, 20082. A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice. Andrew Yee et al., Blood, 20143. von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends. Peter J. Lenting et al., Blood, 20154. Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry. Po-Lin Chiu et al., Blood, 20155. Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A. Steven W. Pipe et al., Blood, 2016 DisclosuresJo:Mogam Institute for Biomedical Research: Employment. Oh:Mogam Institute for Biomedical Research: Employment. Lee:Mogam Institute for Biomedical Research: Employment. Oh:Green Cross Corporation: Employment. Ryu:Mogam Institute for Biomedical Research: Employment. Kim:Green Cross Corporation: Employment, Equity Ownership. Park:Green Cross Corporation: Employment. Kim:Green Cross Corporation: Employment. Jung:Green Cross Corporation: Employment, Equity Ownership.

Von Willebrand Factor A1 Domain Binds to Clusters II and IV of Low-Density Lipoprotein Receptor-Related Protein 1

Introduction. Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein involved in hemostasis and thrombosis. Genetic and in vivo studies have identified low-density lipoprotein receptor-related protein 1 (LRP) as the major clearance receptor of VWF. The ligand-binding moiety of LRP is represented by complement-type repeats grouped into four clusters, among which clusters II and IV are prominent in ligand binding. In in vitro studies under shear stress conditions, VWF acquires an unfolded conformation and interacts with LRP. More than 30 mutations of VWF have been reported to associate with its increased plasma clearance and result in type 2B von Willebrand disease. The majority of these mutations are located within the VWF A1 domain (A1), which is in agreement with previous reports that isolated A1 binds to LRP. However, the interactive site of LRP has not been identified.Objective. We aim to characterize the LRP binding site for VWF by investigating the interaction of individual LRP clusters II-IV to different A1 variants.Experimental Approach. An A1-containing large N-terminal homo-dimeric proteolytic fragment of VWF (SPIII) was produced upon its proteolytic digestion by V8-protease. Alternatively, we generated three variants of recombinant A1: (i) A1(residues 1261-1478), (ii) a VWD type 2B mutant, A1(V1316M) (residues 1261-1478) and (iii) an N-terminus truncated form, (residues 1269-1478). These proteins were tested for binding to immobilized LRP, purified from human placenta, and recombinant LRP clusters II and IV by surface plasmon resonance.Results. We did not find any interaction between the SPIII proteolytic fragment and LRP, suggesting that this fragment may still preserve a folded conformation of VWF that masked its LRP-binding site. In turn, all three forms of recombinant A1 were found to bind LRP and its clusters II and IV. Notably, A1(V1316M) and A1(1269-1478) had higher affinity for LRP and its clusters than A1. This result is consistent with the enhanced clearance of the VWF(V1316M) in vivo and better exposure of the A1 site in a truncated form of A1 to the platelet receptor GpIb-alpha reported previously.Conclusions. Our data demonstrate that LRP has at least two sites for binding to VWF, which are located within clusters II and IV. This finding is in accordance with previous observations that LRP clusters II and IV can serve as “functional duplicates” for binding selected ligands. DisclosuresNo relevant conflicts of interest to declare.

Clinical significance of measuring ADAMTS-13, its inhibitor and von Willebrand factor in obstetric and gynecological practice

ADAMTS-13 is a crucial metalloproteinase involved in liberating fragments of von Willebrand factor (vWF) into the plasma as well as regulating its activity by cleaving "ultra-large" multimers into smaller and less active counterparts. Many pathological conditions, including those emerged during pregnancy are characterized by increased level of vWF and decreased ADAMTS-13 activity. In this regard, it is necessary to monitor the levels of vWF and ADAMTS-13 activity to prevent thrombotic thrombocytopenic purpura (Moschcowitz disease) as one of the most severe forms of thrombotic microangiopathy.

Recombinant VWF fragments improve bioavailability of subcutaneous factor VIII in hemophilia A mice

AbstractConventional treatment of hemophilia A (HA) requires repetitive IV injection of coagulation factor VIII (FVIII). Subcutaneous administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PLs), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII-stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (1) no significant impact on rhFVIII recovery or half-life after a single IV administration; (2) enhanced bioavailability (up to 18.5%) of rhFVIII after subcutaneous administration; and (3) slow absorption (peak concentration, 6 hours) and prolonged half-life (up to 2.5-fold) of rhFVIII after subcutaneous administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 subcutaneously compared with rhFVIII IV. A single subcutaneous dose of rhFVIII/VWF-12 provided protection in the HA tail-bleeding model for up to 24 hours. In summary, recombinant VWF fragments support FVIII delivery through the subcutaneous space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after subcutaneous administration highlight the potential application of VWF fragments for subcutaneous FVIII prophylaxis in HA.

Decreased RPM reduces von Willebrand factor degradation with the EVAHEART LVAS: implications for device-specific LVAD management.

Continuous-flow left ventricular assist devices (LVADs) produces supraphysiologic shear stress that causes von Willebrand factor (VWF) degradation and a bleeding diathesis. Reduction of revolutions per minute (RPM) with axial-flow LVADs does not decrease shear stress enough to reduce VWF degradation and bleeding. However, it is unknown if RPM reduction with centrifugal flow LVADs may minimize VWF degradation. We tested the hypothesis that RPM reduction preserves VWF multimers in the centrifugal-flow EVAHEART left ventricular assist system (LVAS), which is designed to minimize shear stress and blood trauma. Whole blood samples were collected from humans (n = 28). Blood was circulated in ex vivo mock circulatory loops for 6 hours with an EVAHEART LVAS at 2300 (n = 12), 2100 (n = 8), or 1800 RPM (n = 8). Immunoblotting was used to resolve and quantify VWF multimers and degradation fragments. RPM reduction from 2300 to 2100 to 1800 RPM significantly decreased EVAHEART blood flow from 5.8 ± 0.4 to 4.3 ± 0.6 to 4.1 ± 0.5 L/min (analysis of variance [ANOVA], P = .03). RPM reduction protected VWF from pathologic degradation. At lower RPMs, significantly greater levels of VWF multimers were observed (ANOVA, P = .001). Similarly, at lower RPMs, significantly fewer VWF fragments, a product of VWF degradation, were observed (ANOVA, P = .007). RPM reduction significantly reduced VWF degradation with the centrifugal-flow EVAHEART LVAS, an LVAD specifically designed with low shear stress. Different LVADs have unique hematologic footprints and should be managed with device-specific protocols. Adjustment of RPM to minimize blood trauma while still maintaining physiologic hemodynamics has the potential to decrease complications related to LVAD-associated von Willebrand's disease, such as gastrointestinal bleeding and hemorrhagic stroke.

Binding of Factor VIII to D'D3 and A2 Domains of Von Willebrand Factor Facilitates Mechanical Unraveling of the A2 Domain

Background: Proteolytic cleavage of von Willebrand factor (VWF) by a plasma metalloprotease ADAMTS13 is critical for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (FVIII) through its light chain to VWF significantly accelerates the proteolytic cleavage of VWF by ADAMTS13 under fluidic shear. However, the mechanism underlying the FVIII-cofactor activity enhancing VWF proteolysis by ADAMTS13 under shear is not fully understood. Present study aims to test a hypothesis that FVIII binding to the D'D3 and/or other adjacent domains may facilitate a mechanic-induced conformational change in the central A2 domain of VWF, resulting in an increased accessibility and the cleavage of Tyr1605-Met1606 bond by ADAMTS13. Methods: Recombinant B-domainless FVIII and various VWF fragments (D'D3, D'D3A1, D'D3A1A2, and A2) with an Avi-His-tag on their N-termini and a Spy-tag on their C-termini were expressed and affinity-purified to homogeneity. Laser optical tweezers analysis determined the force-induced conformational changes of VWF fragments in the absence or presence of FVIII. Atomic force microscopy (AFM) and surface plasmon resonance (SPR) determined the binding events and kinetics between FVIII and various VWF domains. Results: Laser optical tweezers analysis demonstrated a Gaussian distribution of unfolding extension events upon pulling the single D'D3A1 fragment with the most probable extension distance of 22.8 ± 1.9 nm (mean ± SEM). Addition of 5 nM FVIII to the D'D3A1 or D'D3 fragment resulted in only a small decrease (~25%) or no change in the pulling extension profile. However, two peaks of Gaussian distribution were detected when pulling the D'D3A1A2 with one peak at ~18 nm and the other at ~40 nm. Addition of 5 nM FVIII to the D'D3A1A2 fragment, only one unfolding event was detected with the most probable unfolding extension distance of 24.5 ± 0.4 nm. Moreover, a single Gaussian distribution of unfolding extension profile was detected when pulling an isolated A2 fragment with the most probable unfolding extension of 31 ± 1 nm. Interestingly, addition of 5 nM FVIII resulted in a significant reduction of the most probable unfolding extension to 16.0 ± 0.4 nm, suggesting that FVIII may directly interact with A2 domain. AFM and SPR demonstrated the binding kinetics between FVIII and various VWF fragments. As shown, the intrinsic off-rate (k0) between FVIII and D'D3 was 0.19 ± 0.9 s-1 and the barrier width (Δx‡) between the naive state and the transition state was 1.5 ± 0.2 Å; the k0 between FVIII and D'D3A1A2 was 2.8 ± 1.0 s-1 and the barrier width (Δx‡) between the naive state and the transitional state was 1.7 ± 0.2 Å. Surprisingly, there was a detectable, but relatively weak binding between FVIII and A2 domain using AFM. SPR experiments confirmed the binding of FVIII to D'D3, D'D3A1A2, and A2 with the dissociation constant (KD) of ~2.9 nM, ~5.2 nM and ~1.6 nM, respectively. These results demonstrate that the specific interaction between FVIII and D'D3 or A2 domain. Conclusions: We have demonstrated for the first time using single-molecule force spectroscopy that binding of FVIII to the D'D3 and/or A2 domain of VWF may facilitate the force-induced conformational changes of the A2 domain, resulting in the enhanced proteolysis by ADAMTS13 under force. Our findings shed new light on how FVIII may regulate VWF proteolysis and function under (patho) physiological conditions. Disclosures Zheng: Shire/Takeda: Research Funding; Alexion: Speakers Bureau; Clotsolution: Other: Co-Founder; Ablynx/Sanofi: Consultancy, Speakers Bureau.

Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease

Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα–adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.

The D' domain of von Willebrand factor requires the presence of the D3 domain for optimal factor VIII binding.

The D'-D3 fragment of von Willebrand factor (VWF) can be divided into TIL'-E'-VWD3-C8_3-TIL3-E3 subdomains of which TIL'-E'-VWD3 comprises the main factor VIII (FVIII)-binding region. Yet, von Willebrand disease (VWD) Type 2 Normandy (2N) mutations, associated with impaired FVIII interaction, have been identified in C8_3-TIL3-E3. We now assessed the role of the VWF (sub)domains for FVIII binding using isolated D', D3 and monomeric C-terminal subdomain truncation variants of D'-D3. Competitive binding assays and surface plasmon resonance analysis revealed that D' requires the presence of D3 for effective interaction with FVIII. The isolated D3 domain, however, did not show any FVIII binding. Results indicated that the E3 subdomain is dispensable for FVIII binding. Subsequent deletion of the other subdomains from D3 resulted in a progressive decrease in FVIII-binding affinity. Chemical footprinting mass spectrometry suggested increased conformational changes at the N-terminal side of D3 upon subsequent subdomain deletions at the C-terminal side of the D3. A D'-D3 variant with a VWD type 2N mutation in VWD3 (D879N) or C8_3 (C1060R) also revealed conformational changes in D3, which were proportional to a decrease in FVIII-binding affinity. A D'-D3 variant with a putative VWD type 2N mutation in the E3 subdomain (C1225G) showed, however, normal binding. This implies that the designation VWD type 2N is incorrect for this variant. Results together imply that a structurally intact D3 in D'-D3 is indispensable for effective interaction between D' and FVIII explaining why specific mutations in D3 can impair FVIII binding.

Clinical and In Vitro Evidence That Left Ventricular Assist Device-Induced von Willebrand Factor Degradation Alters Angiogenesis.

Background Gastrointestinal bleeding from angiodysplasia is a major problem in continuous-flow left ventricular assist device (LVAD) patients. LVAD shear stress causes pathologic degradation of VWF (von Willebrand factor). A mechanistic relationship between VWF degradation and angiodysplasia has not been explored. We tested 2 novel hypotheses: (1) clinical hypothesis: VWF fragments are elevated in LVAD patients that develop angiodysplasia and (2) in vitro hypothesis: VWF fragments generated during LVAD support alter angiogenesis, which may contribute to angiodysplasia. Methods and Results Clinical study: Paired blood samples were collected from continuous-flow LVAD patients (n=35). VWF was quantified with immunoblotting. In vitro experiments: (1) To investigate whether LVAD support alters angiogenesis, human endothelial cells were cultured with LVAD patient plasma (n=11). To investigate mechanism, endothelial cells were cultured with VWF fragments produced by exposing human VWF and ADAMTS-13 (VWF protease) to LVAD-like shear stress (175 dyne/cm2, n=8). Clinical study results: in all patients (n=35, mean support 666±430 days), LVAD support degraded high-molecular-weight VWF multimers ( P<0.0001) into low-molecular-weight VWF multimers ( P<0.0001) and VWF fragments ( P<0.0001). In patients with gastrointestinal bleeding from angiodysplasia (n=7), VWF fragments were elevated ( P=0.02) versus nonbleeders. In contrast, in patients with gastrointestinal bleeding without angiodysplasia, VWF fragments were not elevated versus nonbleeders ( P=0.96). In vitro experiments results: LVAD patient plasma caused abnormal angiogenesis with reduced tubule length ( P=0.04) and migration ( P=0.05). Similarly, endothelial cells grown with VWF degradation fragments exhibited reduced tubule length ( P<0.001) and migration ( P=0.01). Conclusions LVAD patients who bled from angiodysplasia had higher levels of VWF fragments than nonbleeders and gastrointestinal bleeders without angiodysplasia. VWF fragments caused abnormal angiogenesis in vitro. These findings suggest that VWF fragments may be a mechanistic link between LVAD support, abnormal angiogenesis, angiodysplasia, and gastrointestinal bleeding.

A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor

Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

Development of Platforms to Phenotype Variants of Uncertain Significance in VWF

Platelet sequestration at sites of vascular injuries and factor VIII (FVIII) stabilization critically relies on von Willebrand factor (VWF). Specific domains within VWF coordinate these functions and may harbor mutations that result in von Willebrand disease (VWD). To survey all possible mutations that may lead to type 2 VWD, we developed prokaryotic and eukaryotic systems to display VWF fragments that are screened for a specific function and identified by DNA sequencing. We constructed an M13 filamentous phage display library consisting of ~2.8x106 independent clones that express random VWF fragments. Following a single round of selection for platelet binding, bound phage were eluted and analyzed by next generation DNA sequencing, revealing an optimal fragment spanning the A1 domain and a second region of weaker enrichment over the D4 domain. All A1 fragments encompassed C1272-C1458, indicating that intramolecular disulfide bridging of these cysteines optimizes platelet binding. Competitive binding assays between phage displaying a fragment of the D4 domain vs. a control phage resulted in enrichment of the former, suggesting a potential, novel role for the VWF D4 domain. Analogous to our previously reported method for analyzing VWF proteolysis (Kretz CA et al. Proc Natl Acad Sci USA. 2015), we mutagenized and displayed the VWF A1 domain to determine the spectrum of mutations that may alter the interaction between VWF and its platelet receptor, GPIbα. The mutant A1 library comprised ~5x106 independent clones, sufficient to represent all 3,933 single substitutions. Screening this library against platelets in the presence of excess control phage resulted in an enrichment of phage displaying an A1 fragment. DNA sequencing identified mutations located outside the VWF A1/platelet GPIbα interface, indicating allosteric control of this interaction. Screening phage displayed VWF fragments for FVIII binding, however, failed to identify critical residues; thus, we adapted our approach for eukaryotes. When displayed on mammalian cells, the VWF D’D3 domains retained their capacity to bind FVIII. Introduction of the VWD subtype 2N mutation, R816W, into the displayed VWF D’D3 domains abolished FVIII binding, demonstrating feasibility for mutational analyses. Together, these results demonstrate the utility of phage and mammalian display for structure-function analyses and that these platforms provide an efficient method for phenotyping variants of uncertain significance. DisclosuresNo relevant conflicts of interest to declare.

A Short N-Terminal Fragment in Front of the A1 Domain of Von Willebrand Factor Prevents the A1 from Binding the Platelet By Masking a Region Clustered By Type 2B VWD Mutations

Blood glycoprotein von Willebrand Factor (VWF) is constantly circulating in the blood vessel, with its platelet GPIb-IX recognizing unit-the A1 domain-being masked to avoid its adventitious binding and activating platelets. This "self-mask" mechanism, however, remains poorly understood. The impediment in identifying the self-mask ligand is to have the right pair of VWF fragments with opposite binding behavior for comparison. Here we characterize using hydrogen-deuterium exchange mass spec a pair of VWF fragments containing the A1 domain with opposite association to platelets. The results suggest that the short N-terminal region (1238-1260) in front of the A1 domain might involve in protecting the VWF A1 domain by masking a critical region near the binding site for GPIb.We expressed and purified using baby hamster kidney cells two C-terminally His-tagged proteins: both contain the entire A1 domain (1273-1453), with one has more extra N-terminal residues (1238-1472, named lA1) than the other (1261-1472, named sA1). Both proteins seem to be glycosylated and stable without obvious deformation for over one week when stored at 4°C. The analytical ultracentrifugation analysis determined that both proteins are primarily in the monomeric form: the sedimentation coefficient (S) and the derived apparent molecular weight for lA1 are 2.50 S and 36.6 kDa, and those for sA1 are 1.37 S and 27.4 kDa. Interaction of lA1 and sA1 to GPIb, as determined by flow cytometry, is surprisingly opposite: only sA1, but not lA1, efficiently binds washed platelets at the concentration similar to the physiological concentration of vWF in the blood vessel. Increasing lA1 concentration 10 times did not improve its binding. Replacing the platelets with CHO cells that express human GPIb-IX complex exhibited similar result. Implied in these results are that either lA1 adopt a different conformation that disadvantages its binding or the binding site on A1 is masked by the extra amino acids in lA1 compared to sA1. Protein hydrogen-deuterium exchange is a sensitive assay to determine the local conformational change and the existence of the masked region. We acquired hydrogen-deuterium exchange data for both proteins and got nearly 100% sequences coverage from over 150 overlaid peptides for each protein, with on average over 10 redundancy per residue, which allows us to compare the hydrogen-deuterium exchange rate/extent of the A1 domain in lA1 and sA1 in good resolution. The results show that the global hydrogen-deuterium exchange pattern is the same between the two, indicating that the tertiary structures of the two proteins are similar. However, a contiguous region in lA1 is clearly protected from hydrogen-deuterium exchange compared to that in sA1 (indicated by the colorful region in figure 1). The protected region is located at the bottom of the globular A1 domain, including three loop regions (1272-1276; 1304-1310; and 1332-1340) and a part of the a1 helix (1289-1300). Compared to sA1, in this protected region of lA1 the deuterium exchange extents are reduced by 20-60% even after 3-hour incubation. Furthermore, 11 of out of 15 identified "gain-of-function" mutation sites of type 2B VWD are located in this masked region (P1266, H1268, C1272, M1304, R1306, R1308, I1309, S1310, W1313, P1337, R1341); the other 4 are located adjacently (V1314, V1316, L1460, A1461). This good alignment implies that the "gain-of-function" might associate with the detachment of the identified masking ligand in this region.Based on our findings, we propose that this short region in front of the A1 domain may involve in masking the A1 domain from interacting with platelet GPIb-IX. If confirmed, this internal masking ligand might offer new clues on how the mutations on A1 domain affect its role -as "gain-of-function" in type 2B VWD and "loss-of-function" in type 2M VWD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Von Willebrand Factor A1–Collagen III Interaction Is Independent of Conformation and Type 2 Von Willebrand Disease Phenotype

The blood von Willebrand factor (VWF) mediates platelet adhesion to injured vessels by sequestering platelets from blood flow and depositing them to collagen and other exposed subendothelial matrix proteins. This process of capturing platelets to facilitate formation of platelet plugs occurs through transient interactions with platelet glycoprotein Ibα via the VWF A1 domain which also binds collagen. Using a conformationally diverse collection of natively folded and mutation-induced misfolded von Willebrand disease (VWD) variants, we test a recently proposed affinity up-regulation hypothesis which states that collagen binding changes the conformation of the A1 domain to a high-affinity GPIbα binding competent state. With surface plasmon resonance (SPR), we present this diversified collection to collagen and quantify the kinetics of association and dissociation to ascertain the conformational selectivity of collagen. With analytical rheology, we quantify real-time platelet pause times and translocation velocities across a Cu2+ HisTag-chelated and collagen-bound A1 single domain and A1A2A3 tridomain fragment of VWF under shear stress in an ex vivo shear flow microfluidic chamber. In contrast to expected hypothetical outcomes, collagen has limited conformational selectivity for binding A1. A1-collagen binding is independent of gain- or loss-of-function phenotype and under shear stress, platelet translocation pause times on collagen-bound A1A2A3 are either normal or shorter depending on whether A1 is concertedly bound with the A3 domain to collagen. With respect to A1, collagen has an inhibitory role that provides an explanation for the lack of thrombosis in patients with gain-of-function VWD.

High‐affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats

Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.