Von Willebrand Factor Half-life Research Articles

Multifaceted pathomolecular mechanism of a VWF large deletion involved in the pathogenesis of severe VWD

AbstractAn in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell–cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.

Exome Sequencing Studies Identify Mutations in STAB2 As a Genetic Risk for Venous Thromboembolic Disease

Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, F2 and seven other very low effect size loci are associated with VTE. Rare mutations in the anticoagulant genes PROC (protein C), PROS1 (protein S) and SERPINC1 (antithrombin III) have been linked to highly penetrant VTE risk in family studies but no genome-wide study of rare variants has been reported to date.We performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with well characterized unprovoked VTE and compared results to a previously genome sequenced control cohort of 5784 unrelated Europeans. We then identified single nucleotide variants, small insertion or deletions that were present at allele frequencies less than 0.005% in ExAC and had equal sequencing coverage between groups. Using a dominant model and a collapsing association analysis, we looked for genes that were more likely to harbor “knockout” (frame shift, nonsense, splice site and damaging missense) mutations in cases versus controls. Strikingly, ranked by p-value, among 17,395 genes tested, the top 4 genes were PROS1 (P= 2.01E-09, odds ratio: 11.8), STAB2 (P=2.70E-7, odds ratio 3.37), PROC (P=3.24E-05, odds ratio 11.0) and SERPINC1 (P=1.10E-04, odds ratio 8.5), Figure. These findings suggest that heterozygous deficiency for the STAB2 encoded protein, stabilin-2, confers VTE susceptibility similar to the previously well described phenotypes of heterozygous protein C, protein S and antithrombin III deficiency.Stabilin-2 is a single transmembrane glycoprotein scavenger receptor expressed on venous endothelium of liver sinusoids and spleen. We detected damaging STAB2 mutations in 29 (7%) VTE cases versus 141 (2%) of controls. Common and uncommon variants in STAB2 have been associated with elevated plasma von Willebrand factor (VWF) and coagulation factor VIII levels, which are known risk factors for VTE. Stabilin-2 is implicated as a clearance receptor for VWF, so that haploinsufficiency of stabilin-2 may increase plasma VWF half-life. To test this hypothesis, we examined 1162 individuals in the Twins UK Study (VTE status unknown) with available whole genome sequencing data and measured their plasma VWF levels by AlphaLISA. VWF levels were adjusted by log transformation and ABO blood type tagging SNPs. We identified 38 individuals (3.2%, all heterozygous) with rare damaging variants in STAB2. VWF levels were significantly higher in individuals (N=30) with rare STAB2 variants compared to those without (N=1016) (Mann-Whitney, P= 0.0046).To further examine stabilin-2 function in vivo, we compared Stab2 deficient mice (Stab2 -/-) to wild type (Stab2 +/+) controls. Stab2 -/- mice had elevated plasma levels of factor VIII:C (118%, p=0.0006), but no differences in VWF (p=0.58). Next, we examined thrombosis in a mouse model where venous blood clotting was induced by inferior vena cava stenosis. Thrombi were weighed and longitudinal sections were analyzed by quantitative immunohistochemistry 24 hours post-stenosis. Stab2 -/- mice developed significantly larger thrombi than Stab2 +/+ mice (194%, n=19, p=0.003) but no difference in DVT incidence (64% vs 61%, P= 0.85). Despite equivalent hematocrit (P= 0.91) and white type thrombus (P> 0.05), thrombi from Stab2 -/- mice were comprised of more red type thrombus (185%, P= 0.004).Through an unbiased analysis of whole exome sequencing in 6,157 individuals, this studyconfirms the role of haploinsufficiency of PROC, PROS1 and SERPINC1 for VTE susceptibility and identifies STAB2 deficiency as a potential new risk factor. Analysis of an independent human cohort of 1162 Europeans demonstrates that STAB2 haploinsufficiency is associated with elevated VWF levels, a known risk factor for VTE. Finally, a mouse model of Stab2 deficiency demonstrates enhanced thrombosis, though without an association with elevated VWF levels. Taken together, these results suggest that rare genetic variants in STAB2 collectively play a role in individuals with unprovoked VTE by prolonging the plasma half-life of procoagulant factors such as VWF, adding an important new gene to the short list of known risk factors for this common disease. [Display omitted] DisclosuresReitsma:Leiden University Medical Center; VarmX B.V.: Equity Ownership, Patents & Royalties: co-inventor on a patent application related to this abstract.

The Biological Significance of von Willebrand Factor O-Linked Glycosylation.

Glycosylation is a key posttranslational modification, known to occur on more than half of all secreted proteins in man. As such, the role of N- and O-linked glycan structures in modulating various aspects of protein biology is an area of much research. Given their prevalence, it is perhaps unsurprising that variations in glycan structures have been demonstrated to play critical roles in modulating protein function and have been implicated in the pathophysiology of human diseases. von Willebrand factor (VWF), a plasma glycoprotein that is essential for normal hemostasis, is heavily glycosylated, containing 13 N-linked and 10 O-linked glycans. Together, these carbohydrate chains account for 20% of VWF monomeric mass, and have been shown to modulate VWF structure, function, and half-life. In this review, we focus on the specific role played by O-linked glycans in modulating VWF biology. Specifically, VWF O-linked glycans have been shown to modulate tertiary protein structure, susceptibility to ADAMTS13 proteolysis, platelet tethering, and VWF circulatory half-life.

Von Willebrand disease type Vicenza: In search of a classification for the archetype of reduced von Willebrand factor survival.

Type Vicenza von Willebrand disease (VWD) features a von Willebrand factor (VWF) with a very short half‐life, and is classified as a form of type 1 VWD. To test the appropriateness of type Vicenza VWD classification, the main features of 17 patients from eight unrelated families were analysed. They had low VWF antigen levels and function (always below 20 U/dl); ristocetin‐induced platelet aggregation sometimes normal, sometimes reduced/absent (even in the same patient); normal platelet VWF levels; an increased VWF propeptide to VWF antigen ratio (8.74 ± 1.65 vs. normal 1.04 ± 0.28) and a reduced VWF half‐life. Plasma VWF multimer levels were homogeneously reduced, and unusually large VWF multimers were sometimes present. Recombinant p.R1205H VWF showed a normal synthesis, release, function, and multimer pattern, with no ultra‐large VWF multimers. The mathematical model by Galvanin et al. was used to explore the kinetic changes in VWF after DDAVP. It showed that the release, but especially the proteolysis (k proteol 1.0−3 ± 2.5−3 vs. normal 4.5−4 ± 6.4−4) and elimination (k el 1.0−2 ± 5.2−3 vs. normal 1.1−3 ± 6.8−4) of type Vicenza VWF were significantly higher than normal. The increased elimination is consistent with the short half‐life, while the increased proteolysis was unexpected. As a shorter survival of VWF is wholly responsible for the type Vicenza VWD phenotype (VWF synthesis, structure and function are normal), it might be better to classify it as a type 2 VWD (rather than type 1) to emphasise the greater interaction with clearance receptors as a new VWF functional defect.

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin-mediated clearance.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease.

Acquired von Willebrand Syndrome Hiding Inherited von Willebrand Disease Can Explain Severe Bleeding in Patients With Aortic Stenosis.

Aortic stenosis may be complicated by an acquired von Willebrand syndrome that rarely causes significant bleeding, raising the question of why it does so in a few cases. To seek an explanation, we studied 5 severe bleeder aortic stenosis patients in a cohort of 49 patients, using the flowchart for inherited von Willebrand disease. Approach and Results: All 5 patients were lacking in large and intermediate VWF (von Willebrand factor) multimers, 3 had reduced plasma and platelet VWF levels, and none showed PFA100 closure. Two patients (those with most multimers missing) also had a short VWF half-life. Genetic analyses on the 3 patients with reduced platelet VWF levels revealed that one carried both the c.1164C>G and the c.7880G>A mutations, and another carried the c.3390C>T mutation, while the third had one of the 2 VWF alleles relatively less expressed than the other (25% versus 75%). No genetic alterations emerged in the other 2 patients. Successful replacement of the stenotic aortic valve, performed in the 2 patients with VWF mutations, did not correct their abnormal VWF multimer picture-unlike what happened in the aortic stenosis patients without bleeding symptoms. Our findings suggest that acquired von Willebrand syndrome can develop in patients with hitherto-undiagnosed inherited von Willebrand disease. Since von Willebrand disease is the most common bleeding disorder, this possibility should be considered in aortic stenosis patients-especially those with a more severe bleeding history and more disrupted VWF laboratory patterns-because they risk hemorrhage during aortic valve replacement.

Increased galactose expression and enhanced clearance in patients with low von Willebrand factor

AbstractGlycan determinants on von Willebrand factor (VWF) play critical roles in regulating its susceptibility to proteolysis and clearance. Abnormal glycosylation has been shown to cause von Willebrand disease (VWD) in a number of different mouse models. However, because of the significant technical challenges associated with accurate assessment of VWF glycan composition, the importance of carbohydrates in human VWD pathogenesis remains largely unexplored. To address this, we developed a novel lectin-binding panel to enable human VWF glycan characterization. This methodology was then used to study glycan expression in a cohort of 110 patients with low VWF compared with O blood group-matched healthy controls. Interestingly, significant interindividual heterogeneity in VWF glycan expression was seen in the healthy control population. This variation included terminal sialylation and ABO(H) blood group expression on VWF. Importantly, we also observed evidence of aberrant glycosylation in a subgroup of patients with low VWF. In particular, terminal α(2-6)-linked sialylation was reduced in patients with low VWF, with a secondary increase in galactose (Gal) exposure. Furthermore, an inverse correlation between Gal exposure and estimated VWF half-life was observed in those patients with enhanced VWF clearance. Together, these findings support the hypothesis that loss of terminal sialylation contributes to the pathophysiology underpinning low VWF in at least a subgroup of patients by promoting enhanced clearance. In addition, alterations in VWF carbohydrate expression are likely to contribute to quantitative and qualitative variations in VWF levels in the normal population. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

Site-Directed Pegylation at Specific Sites Significantly Prolongs the Half-Life of A1A2A3-VWF By Markedly Attenuating LRP1-Mediated Clearance

IntroductionDeficiencies of both von Willebrand Factor (VWF) and FVIII are associated with significant bleeding phenotypes. Consequently, patients with VWD or hemophilia A commonly require replacement therapy with coagulation factor concentrates. However, as infused VWF and FVIII have relatively short plasma half-lives, patient therapy generally necessitates frequent re-dosing. Development of a long-acting rVWF therapy thus represents an important unmet clinical need. We and others have previously demonstrated that the A1A2A3 domains of VWF play a critical role in regulating macrophage-mediated clearance of VWF in vivo. Importantly, crystal structures of the A-domains have also well characterized. In this study, we sought to utilize this data to investigate the hypothesis that site-specific PEGylation within the A1A2A3 domains could be used as a novel strategy to inhibit macrophage-mediated clearance, and thereby inform development of a rVWF molecule with extended plasma half-life.MethodologySite-directed mutagenesis was used to engineer novel surface cysteine residues at selected sites within A1A2A3-VWF. Following purification and characterization, individual A1A2A3 cysteine variants were PEGylated using 40kDa PEG maleimide. Clearance of unPEGylated and PEGylated A1A2A3 variants were assessed in VWF-/- mice. VWF-macrophage interactions were quantified in vitro using differentiated THP-1 macrophages. VWF binding to LRP1 clearance receptor was assessed using both immunosorbant assays and Surface Plasmon Resonance.ResultsNovel single cysteine residues were introduced at stringently selected sites within A1A2A3-VWF. These sites spanned all 3 A-domains and included; S1286C, Q1353C, M1545C, L1591C, V1636C, Q1652C, V1803C and S1807C. Interestingly, the introduction of these novel cysteine residues in both the A1 and A3 domains of VWF did not alter the rate of VWF clearance compared to WT A1A2A3-VWF. Conversely however, the A2 domain was less tolerant for the insertion of cysteines, with L1591C and V1636C variants demonstrating a significantly reduced VWF plasma half-life of approx. 1.5 fold versus WT-A1A2A3 (p<0.05).Subsequently, the engineered cysteine residues were modified by covalent attachment of a 40kDa branched PEG molecule. All variants achieved greater than 80% PEG conjugation efficiency, except V1636C which was eliminated from further study. Remarkably, PEG conjugation displayed site-specific effects on the in vivo half-life of A1A2A3-VWF. For example, PEGylation at S1286C within the A1 domain resulted in a marked increased in VWF half-life compared to WT-A1A2A3 VWF (92.4±6 vs 18.3±0.9 mins, respectively, p<0.001). Conversely, PEGylation at the adjacent site in the A1 domain, Q1353C, or downstream at M1545C within A2 had no significant effect on VWF half-life (23.3±1 and 20.8±3 mins, respectively). Interestingly, despite the fact that no previous roles have been described for the A3 domain of VWF in regulating its clearance, we observed a significant extension in VWF half-life for PEGylated variants within the A3 domain, V1803C and S1807C, (93.3±9 mins and 58.0±5 mins, respectively, p<0.05).Macrophage LDL receptor related protein 1 (LRP1) has been implicated as key cellular mediator of VWF clearance in vivo. Interestingly, in keeping with the reduced clearance observed for PEGylated VWF variants S1286C, V1803C and S1807C, binding of these variants to clearance receptor LRP1 cluster II and IV was ablated. Conversely, PEGylated variants which failed to extend VWF half-life (Q1353C and M1545C) displayed LRP1 binding that was comparable to WT-A1A2A3 VWF. Interestingly, PEGylation at specific sites in A2 (L1591C and Q1652C) which served to increased VWF half-life displayed normal binding to LRP1 cluster IV. However, binding of these variants to LRP1 cluster II was reduced by 90% compared to WT-A1A2A3.ConclusionCollectively, our novel data demonstrate that cysteine-directed PEGylation at specific sites within the A1 (S1286C), A2 (L1591C, Q1652C) and A3 (V1803C and S1807C) domains of A1A2A3-VWF inhibits binding to macrophage clearance receptor LRP1 in vitro. Consequently, these PEGylated A1A2A3-VWF variants demonstrate an extended circulatory half-life in vivo compared to wild type A1A2A3-VWF. Taken together, these results support the use of site-specific PEGylation as a potential approach to develop long-acting full length rVWF molecules. DisclosuresCooke:Pfizer: Employment. Terraube:Pfizer: Employment. Cohen:Pfizer: Employment. Pittman:Pfizer: Employment. Cunningham:Pfizer: Employment. Lambert:Pfizer: Employment. O'Donnell:Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; CSL Behring: Consultancy; Octapharma: Speakers Bureau; Leo Pharma: Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Bayer: Research Funding, Speakers Bureau; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.

Characterization of Interaction of Factor VIII with Engineered Variants of a Single-Chain Variable Antibody Fragment

IntroductionReplacement therapy for Hemophilia A requires frequent infusions of Factor VIII (FVIII) due to its relatively short half-life of ~12 h in plasma. Previous attempts to extend this half-life by genetic and chemical modification of FVIII met the barrier of ~20 h, which is a half-life of von Willebrand factor (VWF), a carrier of FVIII in plasma.A single-chain variable antibody fragment (scFv) KM33 was shown to inhibit FVIII activity, and interactions with VWF and the low-density lipoprotein receptor-related protein 1 (LRP), the major clearance receptor of FVIII (Bovenschen et al, 2005, Blood, 106:906-12). A study indicated that scFv KM33 may prolong the half-life of FVIII in mice to the level exceeding that of VWF half-life (Mertens et al, US Patent 2008, 20080219983A1). This would make scFv KM33 a promising tool for new designs of the longer-acting FVIII products.Study objectiveWe aimed to generate a scFv KM33 variant that can delay FVIII clearance but can be removed from FVIII during its activation by thrombin. Such antibody fragment may extend the half-life of FVIII above that of VWF.Experimental designWe generated three scFv KM33 variants with different linkers connecting the subunits VL and VH of the antibody fragment. The linkers contained variants of thrombin cleavage sites identical to those on FVIII. The proteins were expressed using a baculovirus system, purified by Ni-affinity and size exclusion chromatography (SEC), and tested for their properties.ResultsThe engineered scFv variants, along with the unmodified KM33, were tested for binding to FVIII by surface plasmon resonance (SPR). All scFv versions demonstrated similar affinity for FVIII (~1 nM). In addition, a selected variant of scFv inhibited FVIII binding to LRP. These showed that the modifications of scFv did not affect its binding to FVIII.Thrombin treatment of the engineered scFv variants resulted in dissociation of their VL and VH domains, verified by SEC. However, the respective rates of thrombin cleavage were slower than that of FVIII. The preparation of a thrombin-cleaved scFv still inhibited the interaction of FVIII with LRP by SPR, similarly to that observed for the unmodified KM33. All variants of scFv inhibited FVIII activity in a thrombin generation assay suggesting that their moiety remained in complex with FVIII upon its activation.Conclusions•The rate of thrombin cleavage of sites within FVIII is higher than that of the identical sites within the scFv. This suggests that additional determinants of FVIII (e. g. sulfated tyrosines adjacent to the sites) contribute to the higher rate of cleavage.•The cleavage of the linker between the VL and VH subunits of scFv KM33 results in dissociation of the subunits and breakdown of the antibody fragment. This mechanism is likely applicable to any scFv, and may be useful in a broad range of applications involving such ligands.•Both subunits of thrombin-cleaved scFv KM33, most likely, re-assemble on FVIII and form a tertiary complex FVIII/VL/VH. In turn, thrombin cleavage of the scFv, complexed with FVIII, does not result in its dissociation from FVIII. These indicate that in such design, an scFv should have lower affinity for FVIII to ensure its release from the complex. DisclosuresNo relevant conflicts of interest to declare.

BIVV001: The First Investigational Factor VIII Therapy to Break Through the VWF Ceiling in Hemophilia A, with Potential for Extended Protection for One Week or Longer

Introduction: The standard of care for patients with severe hemophilia A is prophylactic factor VIII (FVIII) replacement. Conventional recombinant FVIII products are efficacious but require frequent administration because of their short half-life, which reflects the dependence of FVIII on von Willebrand factor (VWF). Recombinant FVIII Fc fusion protein (rFVIIIFc) provides an extended dosing interval, as well as joint protection and improved quality of life (Oldenburg et al, Haemophilia, 2018; Wang et al, Blood, 2016), with a well-characterized safety profile. While rFVIIIFc reduces the required administration frequency, longer prophylactic dosing intervals that also offer maximum overall protection are still an unmet need for patients with severe hemophilia A. Increasing the half-life of rFVIII is ultimately dependent upon decoupling FVIII and endogenous VWF.BIVV001 (rFVIII-VWF-XTEN) is a novel investigational rFVIII therapy with single-chain FVIII, the Fc domain of human immunoglobulin G1, 2 XTEN polypeptides, and the FVIII-binding D′D3 domain of VWF, designed to circulate in plasma independently of VWF, thereby breaking the VWF half-life ceiling. Here, we present the low-dose cohort results of EXTEN-A, a Phase 1/2a study assessing the safety and tolerability of a single dose of BIVV001, and the pharmacokinetic (PK) characteristics of a single dose of BIVV001 compared with rFVIII.Methods: EXTEN-A (NCT03205163) is an open-label, dose-escalation, multicenter study. Previously treated adult males with severe hemophilia A (<1 IU/dL [<1%] endogenous FVIII activity) with ≥150 exposure days to FVIII products were included. Patients were assigned to either the low-dose cohort (25 IU/kg of rFVIII and 25 IU/kg of BIVV001; n≥6) or the high-dose cohort (65 IU/kg of rFVIII and 65 IU/kg of BIVV001; n≥8). Escalation from the low-dose cohort, and enrolment of patients to the high-dose cohort was undertaken after assessment of available data from the low-dose cohort.After a screening and washout period of up to 28 days, patients received a single dose (25 or 65 IU/kg) of rFVIII. After a 3- to 4-day washout period, patients received a single dose of BIVV001 at the same dose level as rFVIII. Blood samples for PK analysis were collected for 3 days after dosing of rFVIII and up to 14 days after dosing of BIVV001. Inhibitor testing was performed 14 and 28 days following BIVV001 administration. Adverse events, clinical abnormalities in laboratory tests (including inhibitor development), and PK parameters were assessed. An interim analysis is planned, including the first 2 patients of the high-dose cohort.Results: Out of 7 patients enrolled in the low-dose cohort (25 IU/kg), 6 patients were dosed with BIVV001. Patients in this group were primarily white, with 1 patient of Asian descent, and 1 of Hispanic/Latino ethnicity. Patient ages ranged from 19 to 60 years. Low-dose BIVV001 was well tolerated and no inhibitors were detected through 28 days after BIVV001 dosing. Low-dose BIVV001 demonstrated an extended half-life of 37.6 hours, compared with a 12.1-hour half-life for rFVIII. Average FVIII activity post-infusion of BIVV001 was 12.2% at 5 days and 5.3% at 7 days.At least 8 patients will be enrolled in the high-dose cohort (65 IU/kg); preliminary data for the first 2 patients will be reported.Conclusions: BIVV001 was well tolerated in 6 patients with severe hemophilia A who were treated with a single low dose (25 IU/kg). No patient developed an inhibitor to FVIII. Low-dose cohort data demonstrated a breakthrough in the half-life of rFVIII therapy, with BIVV001 providing sustained FVIII levels that could potentially allow for more optimal, extended protection for patients. DisclosuresKonkle:Genentech: Consultancy; Spark: Consultancy, Research Funding; Pfizer: Research Funding; Gilead: Consultancy; CSL Behring: Consultancy; Bioverativ: Research Funding; BioMarin: Consultancy; Sangamo: Research Funding; Shire: Research Funding. Shapiro:Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin: Research Funding; Prometic Life Sciences: Consultancy, Research Funding; Bioverativ, a Sanofi Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo Biosciences: Consultancy; Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer Healthcare: Other: International Network of Pediatric Hemophilia; OPKO: Research Funding; Octapharma: Research Funding; Kedrion Biopharma: Consultancy, Research Funding; Bio Products Laboratory: Consultancy; Daiichi Sankyo: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding. Quon:Bioverativ, a Sanofi Company: Speakers Bureau; Octapharma: Consultancy; Genetech: Consultancy, Speakers Bureau; Bayer: Consultancy; NovoNordisk: Consultancy, Speakers Bureau; Shire: Speakers Bureau. Staber:uniQure: Honoraria; NovoNordisk: Consultancy; Bayer: Honoraria. Suzuki:Chugai Pharmaceutical Co., Ltd: Research Funding, Speakers Bureau. Poloskey:Bioverativ: Employment. Rice:Bioverativ: Employment. Katragadda:Bioverativ: Employment. Rudin:Bioverativ: Employment, Equity Ownership. Fruebis:Bioverativ: Employment, Other: Clinical Development.

Von Willebrand factor clearance - biological mechanisms and clinical significance.

The mechanisms involved in regulating von Willebrand factor (VWF) clearance remain poorly understood. However recent studies have shown that macrophages play a critical role in regulating the half-life of VWF, and have identified specific lectin (including asialoglycoprotein, macrophage galactose-type lectin, Sigec-5 and C-type lectin domain family 4 member M) and scavenger receptors (including low-density lipoprotein receptor-related protein-1, scavenger receptor A1 and stabilin-2) that are involved in VWF clearance. Further studies will be required to determine the relative importance of these individual receptors with respect to physiological and pathological VWF clearance. Nevertheless, recent clinical data have highlighted the importance of enhanced VWF clearance in the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, increased clearance also contributes to reduced VWF levels in many patients with type 2 and type 3 VWD. Improved understanding regarding VWF clearance is not only of direct biological relevance, but may also have important implications for the development of novel therapeutic agents with extended plasma half-lives for the treatment of both VWD and haemophilia A.

The common VWF single nucleotide variants c.2365A>G and c.2385T>C modify VWF biosynthesis and clearance

AbstractPlasma levels of von Willebrand factor (VWF) vary considerably in the general population and this variation has been linked to several genetic and environmental factors. Genetic factors include 2 common single nucleotide variants (SNVs) located in VWF, rs1063856 (c.2365A>G) and rs1063857 (c.2385T>C), although to date the mechanistic basis for their association with VWF level is unknown. Using genotypic/phenotypic information from a European healthy control population, in vitro analyses of recombinant VWF expressing both SNVs, and in vivo murine models, this study determined the precise nature of their association with VWF level and investigated the mechanism(s) involved. Possession of either SNV corresponded with a significant increase in plasma VWF in healthy controls (P < .0001). In vitro expression confirmed this observation and highlighted an independent effect for each SNV (P < .0001 and P < .01, respectively), despite close proximity and strong linkage disequilibrium between them both. The influence of c.2365A>G on VWF levels was also confirmed in vivo. This increase in VWF protein corresponded to an increase in VWF messenger RNA (mRNA) resulting, in part, from prolonged mRNA half-life. In addition, coinheritance of both SNVs was associated with a lower VWF propeptide-to-VWF antigen ratio in healthy controls (P < .05) and a longer VWF half-life in VWF knockout mice (P < .0001). Both SNVs therefore directly increase VWF plasma levels through a combined influence on VWF biosynthesis and clearance, and may have an impact on disease phenotype in both hemostatic and thrombotic disorders.

Von Willebrand factor propeptide to antigen ratio identifies platelet activation and reduced von Willebrand factor survival phenotype in mice

Background Reduced plasma survival of von Willebrand factor (VWF) is characteristic of patients with type 1C von Willebrand disease (VWD). These subjects can be identified by an increased steady-state ratio of plasma VWF propeptide (VWFpp) to VWF antigen (VWF:Ag). A similar phenotype occurs in mice with the Mvwf1 allele. Objectives To (i) determine if the VWFpp/VWF:Ag ratio can be used to identify a 'type 1C' phenotype in mice, (ii) determine the most reliable method for murine blood sampling, and (iii) identify the source of VWF released during problematic blood collection. Methods 'Platelet-VWF' and 'endothelial-VWF' mice were generated by bone marrow transplantation between C57BL/6J and VWF-/- mice. Several blood sampling methods were used and murine VWFpp and VWF:Ag levels determined. Plasma and platelet VWF:Ag and VWFpp, VWF multimers and VWF half-life were examined in mouse strains with and without Mvwf1. Results A single retro-orbital bleed and vena cava collection were found to be the optimal methods of blood collection. Problematic collection resulted in release of VWF from platelets and endothelium. The VWFpp/VWF:Ag ratio identified strains of mice with reduced VWF survival. Conclusion Assay of murine VWFpp and VWF:Ag has utility in determining the acceptability of murine blood samples for coagulation testing and in identification of a reduced VWF survival phenotype in mice.

A Mechanistic Model to Quantify von Willebrand Factor Release, Survival and Proteolysis in Patients with von Willebrand Disease.

A reduced von Willebrand factor (VWF) synthesis or survival, or its increased proteolysis, alone or in combination, contributes to the development of von Willebrand disease (VWD).We describe a new, simple mechanistic model for exploring how VWF behaves in well-defined forms of VWD after its 1-desamino-8-D-arginine vasopressin (DDAVP)-induced release from endothelial cells. We aimed to ascertain whether the model can consistently predict VWF kinetic changes. The study involved 9 patients with VWD types Vicenza (a paradigmatic form with a reduced VWF survival), 8 type 2B, 2 type 2A-I, 1 type 2A-II (associated with an increased VWF proteolysis), and 42 normal controls, whose VWF levels were measured after a 24-hour-long DDAVP test. The rate constants considered were: k0, associated with the VWF release phase; k1, illustrating the phase of conversion from high- to low-molecular-weight VWF multimers; and ke, associated with the VWF elimination phase. The amount of VWF released (D) was also measured. ke and D were significantly higher in O than in non-O blood group controls; k1 was also higher, but less markedly so. All the parameters were accelerated in type Vicenza, especially ke (p < 0.0001), which explains the significant reduction in VWF half-life. In types 2B and 2A-II, k1 was one order of magnitude higher than in controls, which explains their loss of large VWF multimers. All parameters except ke were lower in type 2A-I.The proposed mechanistic model clearly describes the altered biochemical pathways in well-characterized VWD, prompting us to suggest that it might help clarify elusive forms of VWD too.

Type 2B von Willebrand disease with or without large multimers: A distinction of the two sides of the disorder is long overdue.

Most, but not all patients with type 2B von Willebrand disease (VWD)—which features gain-of-function mutations in the A1 domain of von Willebrand factor (VWF)—have no circulating large VWF multimers. Similarities and differences were analysed in 33 type 2B patients, 12 with a normal and 21 with an abnormal multimer pattern, to see whether they should be considered separately. The minimum aggregating dose of ristocetin was similarly reduced in both patient groups, and modulated by their underlying VWF mutations. Platelet VWF content was normal in all patients lacking in large multimers, but sometimes reduced in those with a normal multimer pattern. All the former patients and none of the latter had persistent or transient thrombocytopenia. A short VWF half-life (affecting plasma VWF levels) was seen in both groups, but more pronounced in patients without large multimers. Bleeding scores were also high in all patients, but more so in those without large multimers, apparently regardless of their platelet count. The marked phenotypic heterogeneity of type 2B VWD concerns not only patients’ VWF multimer pattern, but also their bleeding risk, and consequently their appropriate treatment too. Hence the need to clearly distinguish between type 2B VWD with normal or abnormal VWF multimers.

Complex changes in von Willebrand factor-associated parameters are acquired during uncomplicated pregnancy.

BackgroundThe coagulation protein von Willebrand Factor (VWF) is known to be elevated in pregnancy. However, the timing and nature of changes in VWF and associated parameters throughout pregnancy are not well understood.ObjectivesTo better understand the changes in VWF provoked by pregnancy, we studied VWF-associated parameters in samples collected over the course of healthy pregnancies.MethodsWe measured VWF antigen (VWF:Ag), VWF propeptide (VWFpp), Factor VIII (FVIII), and ADAMTS13 activity in samples collected from 46 women during pregnancy and at non-pregnant baseline. We also characterized pregnant vs. non-pregnant VWF multimer structure in 21 pregnancies, and performed isoelectric focusing (IEF) of VWF in two pregnancies which had samples from multiple trimesters.ResultsVWF:Ag and FVIII levels were significantly increased during pregnancy. ADAMTS13 activity was unchanged. VWFpp levels increased much later in pregnancy than VWF:Ag, resulting in a progressive decrease in VWFpp:Ag ratios. FVIII:VWF ratios also decreased in pregnancy. Most pregnancies exhibited a clear loss of larger VWF multimers and altered VWF triplet structure. Further evidence of acquired VWF qualitative changes in pregnancy was found in progressive, reversible shifts in VWF IEF patterns over gestation.ConclusionsThese data support a new view of pregnancy in which VWF can acquire qualitative changes associated with advancing gestational age. Modeling supports a scenario in which both increased VWF production and doubling of the VWF half-life would account for the data observed. We propose that gestation induces a prolongation in VWF survival, which likely contributes to increased total VWF levels and altered VWF structure.

Upstream Mechanisms Causing Type 1C Von Willebrand Disease (VWD): Contribution Of Defective Von Willebrand Factor (VWF) Multimerization, Regulated Storage, and Secretion

One mechanism causing type 1 VWD is the reduced survival of VWF in plasma (type 1C VWD), characterized by markedly decreased VWF:Ag and VWF half-life, essentially normal multimers, increased ratio of VWF propeptide (VWFpp) to VWF:Ag, robust response to DDAVP, and normal ratios of VWF:CB, FVIII, or VWF:RCo to VWF:Ag. We enrolled 502 index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 of the index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of these, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL. Sequence variations were identified in the VWF D3, A1, A2, and D4 domains. Little is known regarding the mechanisms causing type 1C VWD, but it has been assumed that VWF undergoes normal intracellular processing and secretion with rapid clearance upon release into plasma. We hypothesized that defective intracellular processing may contribute to the type 1C phenotype. We studied 10 type 1C variants including C1130Y, W1144G, R1205H, N1231S, R1315C, V1411E, R1527W, N2041S, Y2160C, and S2179F. Variants were expressed alone (homozygously) or with wild-type (WT) VWF (heterozygously) in HEK293T cells and VWF secretion, multimer structure, and binding to collagen (types III and VI), GPIb-alpha, and FVIII was analyzed. To assess regulated storage, variants were expressed homozygously in HEK293 cells where WT VWF forms elongated pseudo-Weibel-Palade bodies (pWPB). Five variants (C1130Y, R1315C, V1411E, N2041S, Y2160C) had severely decreased secretion and defective multimerization when homozygously expressed. These variants did not form pWPB, but appeared to co-localize with the endoplasmic reticulum, consistent with the severely impaired secretion. One variant, W1144G, had mildly reduced secretion, formed only dimeric VWF, and unexpectedly did not form pWPB. These multimer defective variants demonstrated decreased collagen binding and GPIb-alpha binding as would be predicted. The remaining variants (R1205H, N1231S, R1527W, S2179F) were normally secreted, multimerized, stored in pWPB, and had normal binding to FVIII, collagen, and GPIb-alpha. Interestingly, FVIII binding to homozygous VWF D3 variants C1130Y and W1144G was substantially reduced. This result is not entirely unexpected as the FVIII binding region in VWF has been mapped to the D'-D3 region. Co-expression with WT VWF essentially corrected defective secretion, although some variants still had moderately reduced secretion. Multimer structure appeared normal for all heterozygous variants, although staining which discriminates between variant and WT alleles revealed that for some variants, little variant VWF was actually expressed when transfected at a 1:1 ratio with WT. In sum, when variants were homozygously expressed, we observed a constellation of processing and functional defects. Only R1205H, N1231S, R1527W, and S2179F variants demonstrated normal processing and function. Heterozygous expression (consistent with patients) corrected most of the observed defects, although reduced secretion persisted for a subset of variants. We can conclude that while reduced plasma survival of VWF is a major determinant of the type 1C phenotype, additional upstream processing defects may contribute to the severity of the overall VWD phenotype. Disclosures:No relevant conflicts of interest to declare.

Macrophage LRP1 contributes to the clearance of von Willebrand factor

AbstractThe relationship between low-density lipoprotein receptor–related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1−) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1− mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1− mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1− mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1+ mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm2, implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving β2-integrins that binds both VWF and LRP1 also is implicated because inhibition of β2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1− mice (0.08 ± 0.15 U/mL).

Pregnancy-Induced Shifts in Von Willebrand Factor (VWF) Parameters Support a Prolongation in VWF Survival

Abstract 33Levels of the hemostatic protein von Willebrand Factor (VWF) are known to increase markedly during pregnancy. The mechanism by which VWF levels are increased has long been thought to be due to estrogen stimulation of vascular endothelial cells to synthesize and release more VWF. We hypothesized an alternate scenario in which prolongation of VWF survival contributes to the overall increase in VWF levels during pregnancy.To test this hypothesis, we measured VWF parameters in a repository of plasma samples acquired longitudinally over healthy pregnancies. Third trimester and postpartum (≥ 6 weeks) samples were available for 45 pregnancies, while prepregnancy, first, and second trimester samples were also available on a subset of pregnancies. VWF propeptide (VWFpp) and VWF antigen (VWF:Ag) levels were determined by ELISA (GTI Diagnostics), Factor VIII:C (FVIII:C) was performed by a one-stage assay, and ADAMTS13 activity was measured via cleavage of an enzyme-linked peptide substrate. ABO blood group, a known modifier of VWF:Ag levels and VWF survival, was determined by ABO allele-specific PCR (GTI Diagnostics).We found VWFpp, VWF:Ag, and FVIII:C levels were all significantly increased in the third trimester, as expected, while ADAMTS13 activity remained unchanged (see Table 1). Furthermore, the VWFpp:Ag ratio dropped by nearly half, consistent with a prolongation in VWF survival, while the VWFpp also rose, supporting a coexistent increase in VWF production. Unexpectedly, the decrease in the VWFpp:Ag ratio could be detected early during pregnancy, before the second trimester. Also surprisingly, we observed the VWF:Ag/FVIII:C ratio increase significantly during pregnancy. In a subset analysis of the data categorized by ABO blood group (group O, A, or B), the observed differences in VWF parameters between the third trimester and baseline remained statistically significant for all blood types.Table 1VWF parameters (+/− 1SD) in 45 healthy pregnancies.3rd TrimesterPostpartump-valueVWF:Ag (U/dL)192 +/− 7282 +/− 308.3 × 10−15VWFpp (U/dL)160 +/− 42132 +/− 310.00015FVIII:C (% Activity)200% +/− 63%114% +/− 38%6.9 × 10−11ADAMTS13 (% Activity)119 +/− 30122 +/− 330.599VWFpp/Ag Ratio0.91 +/− 0.271.75 +/− 0.631.4 × 10−13VWF/FVIII Ratio1.03 +/− 0.450.77 +/− 0.270.00054p-values determined using Student's two-tailed, paired t-testCollectively, these data point to previously unsuspected and biologically relevant qualitative changes in VWF during pregnancy. In a model accounting for the volume expansion of pregnancy and evidence of increased VWF production (and assuming one compartment, single-order kinetics, and a stable VWFpp half-life), we calculated that an increase in the half-life of VWF (1.9 fold) would account for the observed excess in VWF:Ag in the third trimester. Furthermore, the change in the VWF/FVIII ratio suggests a pregnancy-induced alteration in the VWF:FVIII stoichiometry such that pregnancy-associated VWF carries less FVIII. This new model of pregnancy-induced VWF change has direct relevance to the prothrombotic state of pregnancy, and could relate to the increased risk of venous thromboembolism (VTE), the major cause of maternal mortality in the developed world. This fresh perspective on the shifts in VWF and FVIII levels which occur in healthy pregnancy may also lend new insight into the mechanisms underlying the derangements of VWF which accompany other life-threatening pregnancy-associated complications, such as preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet syndrome). Disclosures:No relevant conflicts of interest to declare.

Inhibition of Von Willebrand Factor (VWF) by ARC1779 Normalizes Von Willebrand Factor/FVIII Levels and Corrects Thrombocytopenia in VWD Type 2b Patients,

Abstract 3377 Introduction:Infusion of the anti-VWF aptamer ARC1779 effectively elevated platelet counts in some patients with congenital thrombocytopenic purpura, and blunted the platelet drop induced by desmopressin in patients with type 2b von Willebrand disease (VWD). Thus, we hypothesized that ARC1779 may increase VWF levels and correct thrombocytopenia by blocking the hyperactive VWF A1 domain in type 2b VWD. Methods:Thrombocytopenic patients suffering from type 2b VWD received intravenous infusions of ARC1779 (4 mcg/kg/min) for 72 hours in a prospective clinical trial. Patients were monitored using previsouly published assays (Thromb Haemost. 2010;104:563–70). Data are provided as median and the range and were tested for significance by repeated measures analysis of variance. Results:All patients had moderate thrombocytopenia, low levels of VWF:RCo, VWF:CBA, FVIII:C, and collagen/ADP closure times >300s (platelet function analyzer PFA-100). ARC1779 was well tolerated and safe even in a patient who suffered from active chronic gastrointestinal bleeding due to angiodysplasias before start of infusion. Plasma concentrations of ARC1779 increased to 76 mcg/mL (59–130) which suppressed free VWF A1 domains in the REAADS assay to a minimum of <3 to 5%. Consistent with the estimated in vivo half-life of VWF, VWF/FVIII levels increased as early as 12h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. That was best exemplified by VWF:RCo levels which increased from 32% (26–45%) 10-fold to 367% (215–418%) 8h after end of infusion, and decreased again to 33% (20–40%) during follow up. Similarly VWF:CBA levels increased 16-fold (8 to 69-fold), VWF: Ag levels 5.2 fold (3 to 27-fold), and FVIII levels 4.3 fold (3 to 6-fold). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40/nL (38–58/nL) to a maximum of 146/nL (107–248/nL). This was observed although patients were suffering from chronic liver disease due to hepatitis C infection or associated immune thrombocytopenia, which could have blunted the platelet response. All changes were statistically significant (p<0.007). Conclusion:This clinical trial underscores the therapeutic efficacy of the VWF A1 antagonist aptamer ARC1779. ARC1779 significantly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2b patients. This could facilitate (i) the collection of autologous platelets for platelet transfusions pre-operatively or (ii) interferon therapy in patients whose HCV therapy could not be initiated or had to be stopped due to thrombocytopenia. As angiodysplasias may be caused by defective or deficient VWF (Blood 2011;117:1071–80), it may also be tempting to further investigate the potential of chronic anti-VWF aptamer therapy to treat angiodysplasia. Disclosures:Knoebl:Archemix Corp.: Consultancy. Pabinger:Archemix Corp.: Consultancy. Gilbert:Archemix Corp.: previous employment. Schaub:Archemix Corp.: Consultancy, previous employment. Jilma:Archemix Corp.: Consultancy, Research Funding.