Full-length Von Willebrand Factor Research Articles

A Mutation in Complement Factor H (W1183R) Enhances VWF Proteolysis By ADAMTS13 Under Shear

Introduction: Complement factor H (CFH) is the primary negative regulator of complement activation in the alternative pathway in both fluidic phase and on cell surface. Loss of function of CFH such as W1183R is associated with atypical hemolytic uremic syndrome (aHUS). Homozygous mutation W1206R in the murine CFH, corresponding to W1183R in human CFH, results in acute aHUS. Interestingly, in these mice, while plasma VWF antigen levels are significantly increased, plasma VWF multimer size is markedly reduced in ADAMTS13-depedent manner. However, the molecular mechanism underlying such a dramatic effect of the mutant CFH (W1206R) on VWF multimer distribution is not known. The goal of this study is to determine how wild type CFH and mutant CFH W1183R affects VWF proteolysis under shear, and the mechanism of action. Methods: Recombinant WT CFH, CFH W1183R, ADAMTS13, and VWF fragments were expressed in mammalian cells and purified to homogeneity. The effects of WT CFH or CFH W1183R on proteolytic cleavage of VWF73 peptide and full-length multimeric VWF were determined. Additionally, the binding and unfolding events were determined by micro scale thermophoresis (MST) and optical tweezer, respectively. Results : WT CFH showed no effect on the cleavage of peptide VWF73 substrate, but CFH W1183R enhanced the proteolytic cleavage of VWF73 by ADAMTS13 in a dose-dependent manner ( Fig. 1). Counterintuitively, WT CFH appeared to inhibit the proteolytic cleavage of multimeric VWF by ADAMTS13 under arterial shear, but CFH W1183R exhibited an opposite effect, modestly enhancing the proteolytic cleavage of multimeric VWF by ADAMTS13 under the same conditions. The MST binding assay demonstrated that both WT CFH and CFH W1183R bind to a short A1, a long AIM-A1, and A1-A2-A3 fragments with a dissociation constant ( K D) of 6.4 x 10 -7, 3.0 x 10 -9 M, and 3.3 x10 -9 mol/L, respectively. These results suggest that CFH may bind to the AIM, A1-A2 junction, and A2 domain. To further assess the impact of CFH binding to A2, we performed optical tweezer experiments. The results showed that both WT CFH (Lc: from 27.4 nm at 12 nM to 40 nm at 1.0 mM) and CFH W1183R (Lc: from 26 nm at 12 nM to 31.8 nm at 1.0 mM) resulted in significantly extended contour length (Lc) of VWF-A2 domain in a concentration-dependent manner ( Fig. 2), suggesting a dramatic conformational change in the A2 domain upon binding by WT CFH or CFH W1183R. Conclusions : Our results demonstrate that WT CFH at physiological concentrations may exhibit a modest inhibitory activity towards cleavage of VWF by ADAMTS13 under shear, but such an inhibitory activity disappears as the result of a point mutation in the C-terminal end of CFH (e.g., W1183R). Instead, the mutant CFH W1183R appears to enhance the VWF cleavage by ADAMTS13, likely through direct binding to the AIM, A1-A2 junction, and A2 domain of VWF, resulting in conformational changes in the central A2 domain.

The Experimental Gene Therapy on Type 2A/Iic VWD Mouse Model with AAV Vector Delivering Only VWF Propeptide Expression cassette4

Type 2A/IIC von Willebrand Disease (VWD) is caused by mutations in the propeptide of von Willebrand Factor (VWF), and characterized by the impaired multimer assembly. The structural analysis revealed that VWF propeptide (D1D2 domain) serves as a pH-sensing template for VWF multimerization and tubular storage, which implies that D1D2 expression "in trans" has the potential to restore the multimerization defect of type 2A/IIC mutatns. The gene therapy of VWD is challenging due to the huge size of VWF gene, which cannot be packed to the virus vectors, particularly recombinant adeno-associated virus (rAAV). In the pilot studies, we had co-transfected AtT20 cell with plasmid encoding full length VWF, containing the Y87S mutation on propeptide region and causing type 2A VWD phenotype, and plasmid expressing only wild type propeptide of VWF. The co-transfection rescued the multimer formation in a concentration dependent manner (Figure 1A )and corrected the functional defects caused by the Y87S mutation (Figure 1B). Inspired by the results of in vitro studies, we thus have designed a novel gene therapy strategy for type 2A VWD utilizing the machinery of VWF multimer assembling. Instead of trying to squeeze the VWF gene into the AAV vector, we had prepared the AAV vector containing the expression cassette of propeptide region directed by endothelial cell specific promoter FLT-1. The mouse model representing natural occurring VWF Y87S mutation identified in type 2A VWD patient was established by criper9 gene editing technique. The AAV vectors was administered via tail vein injection at a dose of 5Ⅹ1011 genome copies to 6 weeks old male mice to deliver the propeptide of VWF gene and the plasma of the treated mice was collected retroorbital pretreatment and at 2- ,4- and 6-weeks post vector injection. The antigen level of VWF was determined by ELISA and the function activity of VWF was measured by collagen binding assay. The status of VWF multimerization was examined by agarose gel electrophoresis. Although the Y87S mutation led to significant reduce of high molecule weight multimer in mice, unlike observed in mutation harboring patient, the mice still had medium and low molecular weight multimer, maintaining ~30% of activity. The expression of propeptide by AAV delivered vector improved the VWF activity significantly to ~70% of wild type control (Figure 1C), and restored the formation of high molecular weight multimer (Figure 1D). The experimental gene therapy conducted on VWD mouse model showed that it would be efficient to express the propeptide alone to correct the type 2A/IIC VWD defect and the preliminary results provide a novel strategy to overcome technique limits on gene therapy of VWD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Type 2M VWD Laboratory Defects More Consistent Than Bleeding Symptoms across Family Members

Background Von Willebrand disease (VWD) is an inherited bleeding disorder characterized by a defect or decrease of von Willebrand factor (VWF). Type 2 VWD results from qualitative defects in VWF. Type 2M VWD is defined by defective platelet binding to GPIb and/or collagen. While the inheritance of type 2M VWD is autosomal dominant, the extent of symptoms across family members has not been well characterized. Methods Subjects included index cases with type 2M VWD and all enrolled family members from a large US study of VWD (Zimmerman Program). Index cases were enrolled due to a prior diagnosis of VWD. All available family members were enrolled if willing. VWF testing performed included VWF antigen (VWF:Ag), VWF platelet binding by either VWF ristocetin cofactor activity (VWF:RCo) or VWF:GPIbM, collagen binding with types I, III or IV collagen (VWF:CB1, VWF:CB3, VWF:CB4) and VWF multimer distribution. Bleeding scores were calculated using the ISTH bleeding assessment tool (BAT). DNA sequencing was performed for the full length VWF gene. Statistics were calculated using GraphPad Prism with a p value of <0.05 considered significant. Family members were defined as affected if they had VWF activity levels consistent with type 2M VWD and presence of genetic variant if identified. Results 24 index cases, 51 affected family members and 40 unaffected family members were available for analysis. The mean VWF:RCo/VWF:Ag ratio for index cases was 0.5 and the mean VWF:CB/VWF:Ag ratio was 1.05. The VWF:RCo/VWF:Ag ratio was similar in affected family members (0.44, p=NS) but higher as expected in unaffected members (0.89, p<0.001 for index case vs. unaffected, p<0.001 for affected vs. unaffected). In contrast, there was no difference in VWF:CB/VWF:Ag ratios between index cases and affected family members (0.94, p=NS). Data for VWF:GPIbM, collagens I and IV were available only on index cases, and showed low VWF:GPIbM/VWF:Ag (mean 0.79), higher VWF:CBI/VWF:Ag (mean 1.31) and lower VWF:CBIV/VWF:Ag (mean 0.85). There was a negative correlation between VWF:GPIbM and VWF:CBIV, with subjects having either low VWF:GPIbM and normal VWF:CBIV or low VWF:CBIV and normal VWF:GPIbM. Similar results were seen for VWF:CB1 although only 1 subject was diagnosed with type 2M VWD and low VWF:CB1. Median bleeding scores were highest in index cases (7.5), intermediate in affected family members (3) and normal in unaffected family members (1). There was a significant difference between index cases and affected family members (<0.015), and between index cases and unaffected family members (p<0.001). One subject had a genetic variant in the VWF A3 domain, one in the VWF D3 domain and the remainder of index cases had a defect in the VWF A1 domain. Conclusions These findings suggest that decreases in platelet and collagen binding activity increase bleeding risk in families with type 2M VWD. Index cases had higher bleeding scores as compared to affected family members, suggesting that diagnosis of family members may require more attention. Precise understanding of these defects may assist in better diagnosis and treatment of von Willebrand disease.

Phage Display Functionally Defines Variants in the Von Willebrand Factor Platelet Binding Domain

Missense variants in the von Willebrand factor (VWF) platelet binding domain, A1, may pathologically hyperactivate or weaken interactions with its platelet receptor, GPIbα, and lead to von Willebrand disease (VWD) subtypes 2B or 2M, respectively. Variants identified in VWD patients and tested as recombinant VWF have supported genotype-phenotype associations and subtyping of VWD by genetic analyses. However, novel variants, most classified as variants of uncertain significance (VUS) are poorly defined. To functionally characterize a large subset of VWF A1 variants (P1254-L1460), we screened a phage display library for binding to a recombinant form of GPIbα used to clinically assess VWF platelet binding activity, GPIbM. Comprised of ~5x10 6 independent clones, the phage display library contained 1,427 unique, missense variants (~36% of all possible single amino acid substitutions) which could be scored for significant enrichment, depletion, or no change following selection for GPIbM binding. The enrichment of phage displayed VWD variants previously classified as VWD subtype 2B significantly segregated from reported 2M variants (mean fold change from preselected phage ~1.06 for 2B vs. ~0.68 for 2M, p < 0.005). To further validate these findings, five depleted, four unchanged, and seven enriched VWF A1 variants were introduced into the full length VWF sequence by site-directed mutagenesis and expressed by transient transfection of HEK293T cells. Conditioned media were collected and analyzed for VWF level (VWF:Ag) and activity (VWF:GPIbM). Of the sixteen variants examined, fourteen (87.5%) exhibited a VWF GPIbM:Ag ratio that was concordant with the phage display findings. Furthermore, the VWF GPIbM:Ag ratios were well correlated with the degree of enrichment by phage display (Pearson R = 0.69, p< 0.01). Taken together, these findings demonstrate phage display as a high content approach to measure and functionally define the platelet-binding activity of genetic variants within the VWF A1 domain. DisclosuresGinsburg: Takeda: Patents & Royalties.

Mapping the Masked Region of the A1 Domain in Full-Length VWF

Von Willebrand factor (VWF) is the largest protein in plasma and promotes the early hemostatic response following vascular injury. The hemostatic response of VWF relies on the exposure of its A1 domain, which recognizes its receptor GPIbα on the platelet surface. To avoid binding and activating platelets in the absence of injury, the A1 domain is dormant in full-length VWF through a mechanism that is not completely understood. We recently characterized two recombinant A1 fragments (longA1, residues 1238-1472; and shortA1, residues 1261-1472) with differential GPIbα-binding affinities by hydrogen-deuterium exchange (HDX) mass spectrometry. We found that N- and C-terminal residues flanking the A1 domain (residues 1238-1271 and 1459-1472) cooperatively constitute an autoinhibitory module (AIM) that masks a region in A1 and impedes its binding to GPIbα[1]. To determine if the A1 domain in full-length VWF is “masked” in the same manner, we have characterized human plasma-derived VWF using the same HDX approach.On-column peptic fragmentation was performed following HDX from 10 to 10,000 sec. We obtained 946 sequenced peptides covering 96.4% of the VWF sequence with an average redundancy of 6 per residue. Among them, 113 peptides covered the A1 domain, allowing HDX analysis of A1 at high resolution. We found that residues around the 1272-1458 disulfide bond, helix α1, loops α1β2 and β3α2 in the A1 domain, which are protected by the AIM in the longA1 fragment (residues 1238-1472), are deeply protected in full-length VWF, suggesting that AIM in full-length VWF likely masks A1 in a similar pattern. Additionally, sheet β3, helix α3, and loops β4α3 in the A1 domain of full-length VWF reported lower HDX rates than their counterparts in the shortA1 fragment. These regions, along with those protected by AIM, form a contiguous surface that significantly overlaps with the GPIbα-binding interface (Fig. 1). Residues in loop β6α6, helix α6 and helix α1 also reported lower HDX rates, but they are separate from the regions protected by AIM and the GPIbα-binding interface.In summary, our results suggest that AIM and likely other domains in full-length VWF are in direct contact with the A1 domain and regulate the hemostatic properties of VWF.Figure 1: Difference in HDX between isolated VWF A1 domains and full length VWF.1 Deng W, Wang Y, Druzak SA, Healey JF, Syed AK, Lollar P, Li R. A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor. Journal of thrombosis and haemostasis : JTH . 2017. 10.1111/jth.13775. [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.

Identification of a VWFA1 Mutation Attenuating Thrombus Growth but Not Platelet Adhesion

The balance between hemostasis and thrombosis is critical to human health. However, intensified thrombosis that breaks this balance is often observed in diseases like hypertension, diabetes and obesity, leading to atherothrombosis in the coronary and cerebral circulations [1]. Current therapeutic strategies to reduce thrombotic risk are efficacious, as they have reduced morbidity and mortality, but still suboptimal, as commercialized drugs carry the risk of excessive bleeding. A new generation of drugs targeted to increase or maintain antithrombotic efficacy while reducing the negative impact on hemostasis is needed.The formation of platelet aggregates over an arterial vascular injury site is a two-step process. Firstly, binding between platelet glycoprotein (GP) Ibα - a component of the GPIb-IX-V receptor complex - and subendothelial von Willebrand Factor (VWF) at its A1 domain (VWFA1) enables recruitment of circulating platelets under high shear force. This represents the first layer of the hemostatic plug initiated by VWFA1-GPIb and consolidated by adhesion of activated integrins. Secondly, adhesion receptors on the up-facing side of the attached platelets engage circulating proteins including VWF, fibrinogen and fibronectin, among others, attracting new platelets into the growing thrombus. Importantly, this step is shared by atherothrombosis as a mechanism of thrombus growth. The GPIbα-VWFA1 interaction operates in both steps, which respectively mediate platelet adhesion and thrombogenesis [2]. Notably, when VWF is immobilized, its association with platelet GPIbα is mediated by two-dimensional (2D) kinetics; in contrast, the attachment of plasma VWF to immobilized platelets in the growing aggregate is governed by three-dimensional (3D) kinetics.We have identified positively charged, GPIbα-non-contact residues in helices α4/α5 of the VWFA1 that partially affect the VWF-GPIbα interaction. A triple mutation, R636/K642/K643A (referred to as M13) (Fig. 1A) drastically reduces the 3D on-rate of binding to GPIbα without significantly affecting the off-rate, which remains comparable to VWFA1-WT as measured by surface plasma resonance. In this assay, purified GPIbα molecules were immobilized on a surface that was then perfused with soluble monomeric VWFA1 at different concentrations. As a consequence of the decreased 3D on-rate, the 3D GPIbα binding affinity of M13 VWFA1 is 20-fold lower than that of WT VWFA1 (Fig. 1B). In agreement with this finding, aggregometry experiments showed that platelet agglutination mediated by dimeric M13 VWFA1 in the presence of ristocetin was greatly attenuated compared to WT (Fig. 1C), and the speed of agglutination was also slowed (Fig. 1D), implying that M13 is weaker in establishing platelet-platelet interactions.In contrast, the M13 mutation does not compromise the adhesion of platelets to immobilized VWFA1. In a flow chamber system, anticoagulated whole blood was perfused over a surface pre-coated with monomeric WT or M13 VWFA1. Under a range of shear rates and with different VWFA1 coating densities, the coverage of platelets over immobilized M13 VWFA1 was never less, if not greater than on WT VWFA1 (Fig. 1E,F, upper panels); accordingly, the rolling velocity of adherent platelets was similar or slower on M13 than WT VWFA1 (Fig. 1E,F, lower panels).We next investigated how the VWF M13 mutation affects in vivo thrombogenesis using a VWF-/- mouse strain in which platelets expresses human GPIbα. Mammalian cell expressed full-length human WT and M13 VWF was infused into these mice in combination with human FVIII to correct for the coagulation associated with VWFdeficiency; a carotid artery lesion was then caused with 9% ferric chloride. Our preliminary results showed that WT VWF restored a complete thrombotic occlusion of the artery, but mutant M13 VWF was without effect (Fig. 1G).Overall, our data suggests that VWFA1 domain can be engineered to support efficient platelet adhesion, which is key to hemostasis, while being less prone to mediate endovascular thrombogenesis. The development of this study should provide new insights into the distinct mechanisms that support VWF contributing to hemostasis or thrombosis, as well as into designing more selective pro-hemostatic VWF molecules with a reduced risk of causing thrombotic complications.

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.

Genotype-Phenotype Relationship and the Role of Alloantibodies in Type 3 VWD in the Zimmerman Program

Type 3 von Willebrand Disease (VWD) is the rarest and most severe form of VWD and is characterized by the complete absence of von Willebrand factor (VWF). Inheritance is autosomal recessive although heterozygous carriers may be affected with type 1 VWD. As part of the Zimmerman Program for the Molecular and Clinical Biology of VWD (Zimmerman Program) we sought to explore the molecular pathogenesis, correlate bleeding phenotype and severity, and determine the presence and impact of alloantibodies in type 3 VWD.57 index cases (IC) with a pre-existing diagnosis of type 3 VWD by their local treatment center were enrolled into the Zimmerman Program. Clinical VWF testing for phenotypic analysis was done in a central laboratory at BloodCenter of Wisconsin and included FVIII, VWF:Ag, VWF:RCo, VWFpp, VWF:CB and multimer analysis. Full-length exonic VWF sequencing was performed, including intron/exon boundaries. If two sequence variants (SV) were not identified, cases were further investigated by array comparative genomic hybridization (aCGH) to identify any large deletions or duplications in the VWF gene. Bleeding symptoms were quantified using the ISTH bleeding score (ISTH BS). The presence of VWF alloantibodies was determined in type 3 index cases and affected family members (AFM) using an ELISA based assay that detected IgM and IgG antibodies.Of the 57 IC with type 3 VWD enrolled, 42 (74%) had central laboratory testing confirming type 3 (absence of VWF), 7 subjects (12%) had detectable levels and were re-classified as type 1-severe, and 8 subjects (14%) had a significantly increased VWFpp/VWF:Ag and were re-classified as type 1C. Sequencing results were available on 40 of the confirmed 42 type 3 IC. SV were identified in 37 of these subjects (93%): 31 subjects had 2 SVs, 6 subjects had only 1, and 3 had none; 68 of the 80 SVs expected were identified (85%). Of the SVs identified, 37% were frameshift, 29% nonsense, 15% missense, 9% splice site, 7% large deletions detected only by aCGH, and 3% intronic. The large deletions included heterozygous deletions of exon 1-3, exon 4-5, exon 18 and exon 35-38 in combination with another SV. One case only had 2 different deletions (exon 1-3 and exon 4-5) on separate alleles.An equal number (21) of females and males comprised the type 3 cohort with a mean age at enrollment of 24 (range 0-61). Median bleeding score in this group was 15, with no difference between males and females (15). Adults (n=25) had a median ISTH BS of 18 that was significantly increased (p<0.0005) compared to a score of 11 in pediatric subjects (<18 years old, n=17). No difference in ISTH BS was observed according to number of SV or between those with or without null alleles. The highest median bleeding subscores reported were hemarthrosis (3), followed by nose bleeds (2), bruising (2), and oral cavity bleeding (2) and bleeding from minor wounds (1).51 type 3 subjects, including 42 IC and 9 AFM, were tested for alloantibodies to VWF. 16 subjects showed positivity on two separate assays to either IgM or IgG using a 3 SD cutoff above the mean of normal pooled plasma: 4 were markedly positive and 12 were slightly positive. Of the 4 markedly positive subjects, 1 subject had IgM antibodies and 3 had IgG. 3 of the subjects with alloantibodies had no SVs (acquired VWD ruled out), while 1 subject had 2 nonsense variants. ISTH BS was not different between the 16 subjects with an alloantibody (16.5) compared to those without (16). Longitudinal studies will determine the correlation between treatment with VWF concentrate and development of alloantibodies in type 3 VWD.In this type 3 VWD cohort, we have found that some type 1-severe and type 1C patients have been misdiagnosed as type 3. We have also observed that not all type 3 subjects have 2 SV and in fact, 15% of type 3 single alleles did not have an identifiable SV. Null alleles including nonsense, frameshift and large deletions account for 73% of the variant alleles in this cohort. Bleeding symptoms are severe in type 3 and are predominantly due to hemarthrosis, nose bleeds, bruising, oral cavity bleeding and minor wound bleeding. While alloantibodies have been previously associated mainly with homozygous large deletions, our data suggest that type 3 VWD, regardless of the genetic cause, carries a risk of alloantibody formation. DisclosuresFlood:CSL Behring: Consultancy; Shire: Consultancy. Gill:Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

Variability in Bleeding Phenotype in Type 3 VWD Families

Type 3 von Willebrand Disease (VWD) is a rare and severe form of VWD characterized by complete absence of von Willebrand factor (VWF). Patients with type 3 VWD typically present with moderate to severe mucocutaneous bleeding as well as muscle hematomas and hemarthroses. While inheritance has classically been considered autosomal recessive, there is increasing evidence for co-dominant inheritance, with heterozygous carriers affected with low VWF levels.We sought to explore the bleeding phenotype in type 3 index cases (IC) and family members (FM) enrolled in the Zimmerman Program study using the ISTH bleeding assessment tool (ISTH-BAT), and to correlate baseline bleeding score (ISTH BS) and sequence variant (SV) location as well as analyze prospective bleeding diatheses over time using an interim bleeding score (iBAT).Analysis included type 3 VWD index cases (IC), type 3 FM, type 1 affected family members (AFM) and healthy controls (HC). Clinical laboratory phenotyping was performed at BloodCenter of Wisconsin and included FVIII, VWF:Ag, VWF:RCo, VWFpp, VWF:CB3 and multimer analysis. Genotyping involved full-length VWF sequencing, array comparative genomic hybridization to detect deletions and Fabric Genomics Opal software to determine pathological variants. Bleeding symptoms were quantified using the ISTH-BAT bleeding score where baseline scores (ISTH BS) included entire history at time of enrollment and interim bleeding scores (iBAT) that represented bleeding occurring since the initial ISTH BS was obtained. Type 3 obligate carriers (OC) were defined as either having offspring with type 3 VWD or being an offspring of an individual with type 3 VWD. Type 1 AFM cohort included subjects with type 1/low VWF who were related to a type 1 IC.The type 3 families consisted of 45 unrelated type 3 IC, 56 OC with type 1/low VWF (Type 3 OC), 23 normal unaffected OC, and 8 unaffected family members (UFM). Clinical phenotyping and study entry ISTH BS are detailed in Table 1. Type 3 OC with type 1/low VWF (n=56) had mean VWF:Ag and VWF:RCo lab values which were not different from type 1/low VWF AFM (n=483) however both FVIII (68 vs 56, p<0.001) and ISTH BS (1 vs 3, p<0.0001)were both significantly different. OC without evidence of VWD (n=23) had mean VWF:Ag (79) and FVIII (122) that was significantly different (p<0.01) from healthy controls, however there was no difference in ISTH BS.The median ISTH BS in the type 3 IC was 15, with no difference between males and females. Adults had a median ISTH BS of 18 that was significantly increased (p<0.0005) compared to a score of 11 in pediatric subjects (<18). Only 1 type 3 IC did not have an abnormal BS which was an 8 year old female.No difference in ISTH BS was observed according to number of SV or between those with or without null alleles. There was also no difference in ISTH BS (13) of IC with SV in the propeptide region compared to SV in other regions of VWF (15).The highest median baseline subscores reported were hemarthorsis (3), followed by nose bleeds (2), bruising (2), and oral cavity bleeding (2) and bleeding from minor wounds (1). 17 type 3 IC had at least one follow-up visit where median total iBAT score was 11 with most bleeding due to nosebleeds, bruising, bleeding from minor wounds and oral cavity bleeding (iBAT subscores=1).Prospective bleeding was analyzed in 12 adult type 3 IC using a baseline ISTH BS cutoff of 10 to evaluate if bleeding score would predict future bleeding. 10 subjects had ISTH BS >10 (mean age 33, BS 22) and 2 had BS<10 (mean age 33, BS 10). Those with a baseline ISTH BS >10 had a median iBAT of 13 compared to those with ISTH BS<10 with an iBAT of 6, however this was not significantly different (p=0.06) most likely due to the small number of subjects.While type 3 carriers may have low VWF levels, their bleeding is mild compared to type 1 affected family members, and not significantly different from OC with normal levels. There is a difference in type 3 index case bleeding subscores from baseline study entry compared to interim bleeding most likely reflecting the effect of treatment. In this small cohort of prospective bleeding data, the baseline bleeding score did not seem to predict future bleeding as measured by the interim bleeding score. Continued prospective studies of these patients should provide valuable insight into the variability and changes in bleeding in type 3 VWD families. [Display omitted] DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

The physical spacing between the von Willebrand factor D'D3 and A1 domains regulates platelet adhesion in vitro and in vivo

Background Previous exvivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF invivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested exvivo and invivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF-/- mice caused profound thrombocytopenia, reduced plasma VWF concentrations, lower multimer distribution, and incessant tail bleeding that is reminiscent of von Willebrand disease type 2B. Platelet plugs were noted in the portal veins and hepatic arteries. An anti-D'D3 mAb DD3.3 that displays enhanced binding to VWF containing the N-terminal mucin insert also exhibited increased binding to wild-type VWF under shear and upon ristocetin addition. Conclusion Conformation changes at the VWF D'D3-A1 interface may be a key regulator of thrombosis invivo. Structural features at the A1-A2 interface are likely of less significance.

A common mechanism by which type 2A von Willebrand disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand factor A2 domain FRET construct.

Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.

Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13

Abstractvon Willebrand factor (VWF) and the metalloprotease a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) are present both within endothelial cells (ECs) and in peripheral blood. Calcium concentrations are lower in intracellular compartments (80-400 μM) compared with the extracellular milieu (∼1.25 mM). Because low calcium favors VWF A2-domain proteolysis by ADAMTS13, the dependence of proteolysis rates on calcium was assayed both within ECs and in blood. Confocal microscopy studies demonstrate partial perinuclear colocalization of VWF with ADAMTS13 in human umbilical vein ECs (HUVECs). Consequently, low levels (5%-10%) of VWF cleavage products were detected in HUVEC lysates and also culture-supernatant following EC stimulation. This proteolysis occurred before disulfide bond formation. Compared with wild-type VWF A2-domain, calcium-binding mutants including the common von Willebrand disease (VWD) type 2A R1597W mutant were expressed in an open conformation in ECs and were highly susceptible to intracellular proteolysis. Fluorescence resonance energy transfer measurements demonstrate strong calcium-dependent VWF-A2 conformation changes at concentrations ≮500 μM, with unfolding rates being fourfold higher for monomeric VWF A2-domain compared with multimeric, full-length VWF. Under shear, physiological levels of ADAMTS13 did not cleave VWF strings on HUVECs, unless platelets were attached to stretch these strings under flow. Further, VWF-platelet string cleavage under shear proceeded with equal efficiency in the absence and presence of calcium at shear stress ≥1 dyn/cm2. Overall, low calcium levels may promote intracellular VWF proteolysis particularly during VWD type 2A disease. Calcium has a negligible effect on VWF-platelet string proteolysis under physiologically relevant fluid shear.

Clinical and laboratory phenotype variability in type 2M von Willebrand disease

Background von Willebrand factor (VWF) is a multimeric coagulation factor that tethers platelets to injured subendothelium. Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in VWF with preserved multimer distribution. Objectives Through the Zimmerman Program for the Molecular and Clinical Biology for VWD, five VWF sequence variations were studied in subjects diagnosed with type 2M VWD. Methods Bleeding phenotype was assessed using the ISTH bleeding assessment tool. Full-length VWF gene sequencing was performed for each subject. Each variant was placed into a recombinant VWF vector using site-directed mutagenesis and expressed in HEK293T cells as homozygous or heterozygous VWF. Variant expression, collagen binding and platelet GPIbα binding were studied through ELISA assays. Multimer analysis was performed by gel electrophoresis. Results Bleeding scores were elevated for all subjects except for the p.P1162L and p.R1374C variants. Although all had reduced VWF ristocetin cofactor activity/VWF antigen ratios on plasma testing, recombinant VWF did not show a classic type 2M phenotype for any of the five variants. Homozygous expression of variants p.D1283Y, p.R1349C, p.R1374C and p.I1453N was consistent with type 2A VWD, although all had normal expression as heterozygous recombinant VWF. Variant p.P1162L had normal VWF expression and function, consistent with the lack of bleeding symptoms. Conclusions Although originally classified as type 2M VWD, these homozygous recombinant VWF variants do not fulfill complete 2M VWD diagnostic criteria. A better classification schema and improved testing for putative type 2M variants is needed in order to effectively diagnose and treat affected patients.

ADAMTS‐13 glycans and conformation‐dependent activity

Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

Identification of Copy Number Variants in Type 1 and Type 3 VWD by aCGH in the Zimmerman Program

von Willebrand Disease (VWD) is a common inherited bleeding disorder caused by a quantitative or qualitative abnormality of von Willebrand Factor (VWF). Genetic analysis for VWD commonly utilizes DNA sequencing to identify sequence variants (SV) in VWF, however this technique cannot detect copy number variants (CNV), including deletions and duplications, which cause approximately 10% of genetic diseases. To determine the role of CNV in VWD, we performed array Comparative Genomic Hybridization (aCGH) on 122 mutation-negative subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD (Zimmerman Program) where full-length VWF sequencing, including 5' and 3' regions and intron/exon boundaries, failed to identify a SV that would explain their phenotype. These subjects included 84 low VWF, 16 type 1, 10 type 1C, 2 type 2A, and 10 type 3. Clinical testing performed at BloodCenter of Wisconsin included FVIII, VWF:Ag, VWF:RCo and VWFpp. Bleeding symptoms were quantified using the ISTH bleeding score (BS). A VWF high-resolution aCGH array was custom designed using long oligonucleotide probes ~60 bp in length to avoid multiple sequence variations, repeated sequences and the pseudogene. Data was manually inspected and analyzed with CytoSure Interpret Software. A multiplex PCR approach was used to map deletion breakpoints and to confirm the presence and zygosity of the deletions in the index case as well as family members (FM).Large deletions were identified in 12% of this cohort (14 subjects) where no other VWF mutations were found. Of these 14 subjects, 5 were type 1, 5 type 1C and 4 type 3, which represents CNV found in 31% of the type 1, 50% of type 1C and 40% of the type 3 tested. No CNV were found in the 84 low VWF subjects or the 2 type 2A patients. Mean VWF:Ag (6 IU/dl) and VWF:RCo (13 IU/dl) in subjects with deletions was statistically different (p<0.0001) compared to those with no large deletion (31 and 33 IU/dl). Subjects with deletions had a median ISTH BS of 4 whereas those with no deletion had a score of 6 which was not significantly different (p=0.61).The most common deletion was the previously described in-frame exon 4-5 (c.221-977_532+7059del) which was identified in 5% of this cohort (4 type 1 subjects and 2 type 3 subjects). In these 6 unrelated families, the FM with the deletion had a lower mean VWF:Ag (25 IU/dl), VWF:RCo (26 IU/dl) and higher ISTH BS (3) than FM without the deletion with VWF:Ag of 74, VWF:RCo of 76 and ISTH BS of 1 (p<0.05). The second most frequent (4%) was an in-frame deletion, exon 33-34 (c.5621-478_5842+2440delinsGCAGCATAAGCATAAAGC) which was the only CNV found in the type 1C group (5/10 subjects). FM with exon 33-34 deletion had mean VWF:Ag of 21, increased VWFpp/VWF:Ag (4.3) and ISTH BS (3), while FM without the deletion had mean VWF:Ag of 107, normal VWFpp/VWF:Ag (1.3) and ISTH BS (1). This 3.5kb deletion results in a loss of 9 cysteines which may be associated with reduced plasma survival of the VWF in these patients. The remaining deletions were found in single subjects, representing 1% of the index cases tested. We identified novel deletions in exon 1-3 and exon 11. Interestingly, the exon 1-3 deletion had a novel breakpoint, c.-30029_220+3487del , and was found in a type 3 index case who was compound heterozygote for the exon 4-5 deletion as well. An out-of-frame deletion, c.2282-809_2442+2811delinsT, was found in exon 18 in 1 type 3 subject. This deletion was reported in the VWF database but the breakpoints had not been mapped. This deletion alone causes low VWF and confers a type 3 phenotype when inherited with a single nucleotide deletion on the other VWF allele. Another novel out-of-frame deletion, exon 35-38 (c.5843-2754_6799-1517del), was found in a type 3 patient where we confirmed that it segregated with FM who had type 1 VWD, but resulted in type 3 when inherited along with a frameshift mutation caused by a single nucleotide duplication.aCGH is a sensitive and powerful method to identify CNV in the VWF gene, narrow the breakpoint locations for mapping, and characterize the different mechanisms for type 1 and type 3 VWD. Both in-frame and out-of-frame deletions may be a common mechanism of type 1 and result in reduced levels of VWF in plasma. While aCGH may not be as beneficial for subjects with "low VWF", it should be considered when attempting to determine the underlying genetic cause of type 1 patients with VWF:Ag levels <30 or type 3 when a SV is not identified by VWF sequencing. DisclosuresFlood:Baxalta: Consultancy; CSL Behring: Consultancy. Friedman:Alexion: Speakers Bureau; NovoNordisk: Consultancy; Shire: Consultancy; CSL Behring: Consultancy.

Long-Term Expression of Von Willebrand Factor Via Piggybac-Mediated Gene Transfer

von Willebrand disease (vWD), caused by a deficiency in von Willebrand factor (vWF), is the most common inherited bleeding disorder, affecting up to 1% of the population; those affected have a varied bleeding phenotype. von Willebrand factor plays two important roles in hemostasis including platelet adhesion and stability of factor VIII (FVIII). As such, patients with von Willebrand disease type 3 have no measurable vWF protein and demonstrate low levels of FVIII. To date, persistent long-term expression of full-length vWF using gene transfer strategies has not been demonstrated, largely due to the immense size of vWF cDNA (8.4 kb). Non-viral vector systems, such as piggyBac, are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a "cut and paste" mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize PB-mediated vWF gene transfer will confer long-term expression and improve the bleeding phenotype in an animal model of vWD. We engineered PB transposon to carry a human von Willebrand factor cDNA (PB vWF). When transfected with PB-vWF and a hyperactive transposase, iPB7, the hepatocarcinoma cell line, HepG2, demonstrated secretion of vWF into supernatants compared to mock transfected controls (p < 0.01). Next, we evaluated the in vivo gene transfer efficiency in von Willebrand deficient mice by hydrodynamic tail-vein injection using PB-vWF driven by the murine albumin enhancer/human alpha anti-trypsin promoter. vWF null mice received 25 or 50 micrograms each of the PB-vWF transposon and iPB7 to determine long-term expression and phenotypic correction. vWF levels were measured prior to injection and then every 4 weeks for up to 24 weeks. Results revealed therapeutic levels (on average 50% normal mice) of vWF post gene transfer with stable expression for 24 weeks in most mice. These data indicate the PB transposon vector system may provide a long-term gene transfer strategy for vWD. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. PB-vWF gene transfer resulted in at least partial bleeding phenotype correction via tail clip. Additionally, treated mice demonstrated a rescue of the FVIII activity. These data show that the PB vector can be used to deliver this large transgene expression cassette to the liver and achieve long-term expression and phenotypic correction in vivo. DisclosuresStaber:Emergent BioSolutions: Honoraria; Baxalta: Honoraria.

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

AbstractEnhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.