Von Willebrand Factor Clearance Research Articles

The Endothelial Lectin Receptor CLEC4M Internalizes Factor VIII and Von Willebrand Factor Via a Clathrin-Coated Pit-Dependent Mechanism

Regulation of plasma levels of the coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII) involves a dynamic balance between biosynthesis, secretion, and clearance. Clearance of VWF and FVIII occurs through receptor-mediated endocytosis, with both LDL receptor gene family (eg. LRP1), and lectin receptors (eg. asialoglycoprotein receptor, siglec-5) contributing to this process. We have previously characterized the endothelial lectin CLEC4M as an endocytic receptor for FVIII and VWF. These proteins represent the first endogenous glycoprotein ligands indentified for CLEC4M.Previous studies have characterized CLEC4M as an adhesive receptor capable of facilitating viral infection in trans. Thus, the mechanism by which CLEC4M internalizes VWF and FVIII, and the subsequent fate of these ligands, is uncharacterized. We hypothesize that CLEC4M is able to endocytose VWF and FVIII via a clathrin-coated pit-dependent mechanism. We further hypothesize that endocytosis of FVIII and VWF by CLEC4M-expressing cells targets FVIII and VWF to lysosomes for degradation.As CLEC4M is expressed exclusively on the endothelial cells of the hepatic sinusoids and lymph nodes, and commercially prepared liver sinusoidal endothelial cells do not retain CLEC4M expression, we generated a HEK 293 cell line that stably expresses CLEC4M (>90% positive by flow cytometry). We first inhibited the CLEC4M-mediated endocytic pathways by preincubating cells with methyl-β-cyclodextrin to deplete cell membrane cholesterol, dynasore hydrate to inhibit dynamin GTPase activity, and pitstop-2 to block the N-terminus of clathrin. Cells were then incubated with recombinant human FVIII, or plasma derived VWF-FVIII complex for 1 hour and internalization was visualized by immunofluorescence. A quantitative analysis of VWF or FVIII-positive objects was performed using Image Pro software.CLEC4M-expressing 293 cells internalized both FVIII as well as the VWF-FVIII complex. Binding and internalization of VWF was reduced by methyl-β-cyclodextran (65%, p=0.067), by dynasore hydrate (92%, p=0.055), and by pitstop-2 (83%, p=0.032). Binding and internalization of FVIII was similarly reduced by methyl-β-cyclodextran (50%, p=0.0050), by dynasore hydrate (60%, p=0.0225), and by pitstop-2 (90%, p=0.0086). This suggests that CLEC4M mediates endocytosis of VWF and FVIII via a clathrin-coated pit-dependent mechanism that involves lipid rafts.To visualize the endocytic pathway of VWF and FVIII, CLEC4M-expressing 293 cells were incubated with FVIII or the VWF-FVIII complex for 15, 30, or 60 minutes. Colocalization of FVIII, VWF and/or CLEC4M with markers for early endosomes (early endosomal antigen-1), late endosomes (Rab9), and lysosomes (LAMP1) was observed using immunofluorescence. For all conditions, CLEC4M-negative and isotype controls were performed.We have previously confirmed that FVIII and VWF are internalized by CLEC4M expressing cells, and that VWF colocalizes with a marker for early endosomes. When CLEC4M-expressing cells were exposed to the VWF-FVIII complex, we observed a partial colocalization of VWF and FVIII, confirming that CLEC4M is able to internalize the VWF-FVIII complex. When CLEC4M-expressing cells were exposed to FVIII, we observed colocalization between FVIII and early endosomal antigen 1 within 15 minutes, confirming that upon internalization by CLEC4M, VWF and FVIII are targeted to early endosomes.We next observed the transport of VWF and FVIII to late endosomes and lysosomes. When CLEC4M-expressing cells were incubated with VWF and FVIII for 30 minutes, we observed colocalization of both proteins with Rab9, a marker for late endosomes. When CLEC4M-expressing cells were incubated with FVIII for 1 hour, we observed colocalization of FVIII with LAMP1, a lysosomal marker, suggesting that the internalization of FVIII by CLEC4M leads to lysosome-mediated degradation of FVIII. In contrast, incubation of CLEC4M-expressing cells with VWF for up to 2 hours did not result in co-localization with LAMP1.These studies confirm that, in the context of the stable cell system used in these experiments, the cell surface lectin receptor CLEC4M mediates endocytosis of FVIII and VWF through a clathrin-coated pit-dependent pathway. Internalization of VWF and FVIII by CLEC4M targets these proteins to early and late endosomes; FVIII is subsequently targeted to lysosomes for degradation. Disclosures:James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

Genetic Variants In PLG and SIGLEC14 Contribute To Plasma Plasminogen Level Variation

The conversion of plasminogen (PLG) to plasmin is a critical step in the activation of the fibrinolytic system. Due to its broad substrate specificity, plasmin plays a pleotropic role in the circulation and extravascular space, controlling the degradation of fibrin clots as well as potential functions in regulating cell migration and angiogenesis. PLG activation is regulated through the interaction of tPA or uPA with plasminogen activator inhibitor type one (PAI-1). Genome-wide association studies (GWAS) have identified polymorphisms associated with the plasma levels of tPA and PAI-1, but to date, no studies have directly examined the control of PLG levels.In order to identify genetic determinants of PLG levels we performed GWAS in a cohort of 3244 young healthy individuals (557 sibships) who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS).(Desch KC et al. PNAS 2013, 110:588-593) All subjects provided written informed consent. Details of the genotyping and data cleaning process for these cohorts are as previously described. (Desch KC et al. PNAS 2013, 110:588-593) PLG levels were measured in both cohorts by AlphaLISA (Perkin-Elmer, Waltham, MA). Levels were natural log transformed and adjusted for participant age, gender and BMI (TSS only) prior to use in heritability estimates and GWAS, which were performed using the PLINK, EMMAX, MERLIN, GCTA and METAL software packages.The median PLG levels were 101.7 IU/dL and 104.7 IU/dL in the GABC and TSS cohorts respectively and had a 1.7 fold variation between the 5th and 95th percentiles. The narrow-sense heritability using intra-class correlation was 59.7% based on 1277 siblings in GABC and TSS, which was similar to the estimate of 60.2% based on the genotype data and the MERLIN software package. GWAS for PLG in the European only subset of GABC (n=940) did not reveal any genome-wide significant signals. However, GWAS for PLG levels in TSS (n=2304) revealed 9 significant SNPs on chromosome 6q26 at the PLG (plasminogen) and LPA (lipoprotein A) locus that together explained 7.8% of the variance in PLG levels according to the GCTA method, Figure 1. The top SNP was rs4252129 (p = 3.47E-25) which results in a single amino acid substitution (R523W) in PLG. The next strongest associations were with two SNPs near LPA, rs1084651 and rs783149 (p = 1.60E-13 and 1.07E-12 respectively). These SNPs are located between LPA and PLG (Figure 1). To determine if the LPA SNPs were independent from the top PLG SNP, a conditional analysis was performed using rs4252129 as a covariate. Rs1084651 and rs783149 remained significant (p= 7.73E-16 and 6.48E-15 respectively), verifying that they represent a second, independent association signal. Meta-analysis of both cohorts (using 723,715 common SNPs) confirmed the 6q26 signal and revealed another significantly associated region on 19q13.41, Figure 2. Two SNPs, rs10412972 and rs11084102 (p = 2.16E-09 and 1.14E-08, respectively) are intergenic variants 4.8 and 12.6 Kb downstream from SIGLEC14. The significant SNPs in the meta-analysis, 6 on 6q26 and 2 on 19q13.41, explained 5.8% of the variance for PLG. The top SNP in the TSS GWAS, rs4252129, had a minor allele frequency (MAF) of 1.4% in GABC and was not included in the GABC GWAS or meta-analysis.In summary, our data identify two loci (at PLG/LPA and SIGLEC14) that harbor genetic variants contributing to the variation of plasma PLG levels in healthy young adults. The strongest association was observed for a non-synonymous SNP in the PLG gene itself, with a MAF of 1.3% in the HapMap CEU population and 2.2% in TSS. The minor allele (W523) is associated with a 14.1% decrease in mean plasma PLG level. This low frequency, non-synonymous SNP, or another SNP in LD with it, may be the functional mutation leading to decreased PLG levels through unknown mechanisms. Whether the significant SNPs near LPA affect lipoprotein A or PLG is not clear. However, the association with causal variants at LPA is possible and consistent with previous biochemical studies demonstrating competitive inhibition of PLG binding to endothelial surfaces by lipoprotein A, or an effect of lipoprotein A on endothelial PAI-1 expression or the PAI-1 interaction with tPA/uPA. Finally, these studies identified two significant SNPs near SIGLEC14. This gene encodes a sialic acid lectin type receptor protein, closely related to SIGLEC 5 recently implicated in the clearance of von Willebrand factor. [Display omitted] [Display omitted] Disclosures:Ginsburg:Shire plc: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaeuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Merck pharmaceuticals: Consultancy; Boston Children's Hospital: From Baxter to Children's Hosp (VWF), From Baxter to Children's Hosp (VWF) Patents & Royalties; University of Michigan: From Baxter to University of Michigan (ADAMTS13) Patents & Royalties.

CHANGES OF VON WILLEBRAND FACTOR DURING PREGNANCY IN WOMEN WITHOUT AND WITH VON WILLEBRAND DISEASE

Delivery in von Willebrand disease (VWD) represents a significant hemostatic challenge because of the variable pattern of changes observed during pregnancy of von Willebrand factor (VWF) and factor VIII (FVIII), the protein carried by VWF. Since a wide heterogeneity of phenotypes and of the underlying pathophysiological mechanisms is associated with this disorder, a prompt and careful evaluation of pregnant women with VWD is requested in order to plan the most appropriate treatment at time of parturition. VWF and FVIII increase significantly during pregnancy in normal women, already within the first trimester, reaching levels by far >100 U/dL by the time of parturition. Women with VWD, levels at baseline of VWF and FVIII >30 U/dL have us a high likelihood to achieve normal levels at the end of pregnancy; thus specific anti-hemorrhagic prophylaxis is seldom required. Women with basal level <20 U/dL usually have a poor increase since most of these women carry mutations associated with increased VWF clearance or are compound heterozygous for different VWF mutations; that prevent the achievement of satisfactory hemostatic levels. While women with mutations associated with increased clearance show a full, albeit transitory correction of their hemostatic deficiency after desmopressin administration, compound heterozygous need replacement therapy because they do not respond well to this agent. Patients with abnormal VWF:RCo/VWF:Ag ratio at baseline (e.g. <0.6), typically associated with type 2 VWD, maintain the abnormality throughout pregnancy and VWF:RCo usually does not attain safe levels ≥50 U/dL. These women require replacement therapy with VWF-FVIII concentrates. Delayed post-partum bleeding may occur when replacement therapy is not continued for some days. Tranexamic acid may be useful at discharge to avoid excessive lochia.

VWF propeptide and ratios between VWF, VWF propeptide, and FVIII in the characterization of type 1 von Willebrand disease

AbstractDuring posttranslational modifications of von Willebrand factor (VWF), the VWF propeptide (VWFpp) is cleaved. The ratio between VWFpp and VWF antigen (VWF:Ag) and the ratio between factor VIII (FVIII:C) and VWF:Ag may be used to assess synthesis and clearance of VWF. We analyzed the contribution of VWFpp and ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag in the pathophysiological characterization of type 1 von Willebrand disease (VWD) in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD) study. The VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were increased among patients compared with unaffected family members and healthy controls. The VWFpp/VWF:Ag ratio was higher in individuals heterozygous for missense mutations than in those heterozygous for null alleles. In contrast, the FVIII:C/VWF:Ag ratio was highest among heterozygotes for VWF null alleles. The ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag indicate that the pathophysiological mechanisms of type 1 VWD include reduced production and accelerated clearance of VWF, but that often a combination of both mechanisms is implicated.

Characterizing polymorphisms and allelic diversity of von Willebrand factor gene in the 1000 Genomes

The von Willebrand factor (VWF) gene is highly polymorphic, with variants correlated with VWF antigen levels, adhesion activity, clearance and factor VIII binding. VWF mutations are detected in patients with von Willebrand disease (VWD), whereas polymorphic variants could be associated with thrombosis. However, information on the ethnic diversity of VWF variants and their association with diseases is limited. To characterize novel VWF variants from different ethnicities in the general population. We analyzed samples from 1092 subjects of 14 ethnicities available in the 1000 Genomes database for VWF variants and their potential functional impacts. We identified 2728 SNPs and 91 insertions and deletions that had a high level of ethnic diversity, with Africans having the highest number of variants. The highest level of diversity was found in the D' and D2 domains. Among 94 non-synonymous variants, 31 were predicted to be deleterious, including 19 that were previously associated with VWD. Most of these 'VWD variants' had allele frequencies consistent with disease incidence in European subjects, but some had a significantly higher frequency in other ethnicities. The mutations R2185Q, H817Q and M740I associated with type 1 and type 2N VWD were present in more than 13% of African subjects. These results highlight the complexity of VWF variations in different ethnic groups and emphasize the importance of interrogating variations on multiple ethnic backgrounds for associations with bleeding and thrombosis.

Acquired von Willebrand Syndrome Associated with Hashimoto's Thyroiditis and Subcutaneous Mucosa-associated Lymphoid Tissue Lymphoma

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those of congenital von Willebrand disease. We herein report a case of AVWS associated with Hashimoto's thyroiditis and subcutaneous mucosa-associated lymphoid tissue lymphoma. An IgG autoantibody against von Willebrand factor (VWF) was detected. The antibody bound to VWF but did not inhibit VWF activity. Rapid clearance of VWF seemed to be the cause of AVWS in the present case. VWF-containing concentrates stopped the bleeding. Even if such a complication is rare, for AVWS patients, prompt recognition of the underlying mechanism can save lives.

Genetic Variability of the CLEC4M Endothelial Lectin Receptor Modulates Binding and Internalization of Von Willebrand Factor and Contributes to Variance in Plasma VWF Levels

Abstract 16Type 1 von Willebrand’s Disease (VWD) can result from decreased synthesis or accelerated clearance of von Willebrand Factor (VWF), resulting in partial quantitative deficiency. Approximately 35% of individuals with Type 1 VWD do not have a putative mutation in their VWF gene, suggesting that genes other than VWF may contribute to the pathophysiology of this disease. Recently, the CHARGE GWAS meta-analysis identified single nucleotide polymorphisms in the gene encoding the C-type lectin domain family 4 member M (CLEC4M) as being significantly associated with plasma VWF levels in normal individuals. CLEC4M is a pathogen recognition receptor with a polymorphic extracellular neck region consisting of a variable number of tandem repeats (VNTR) (3 – 9 repeats). We hypothesize that CLEC4M binds to and clears VWF from the circulation, and that different CLEC4M VNTR alleles may contribute to differences in plasma levels of VWF in normal subjects and patients with Type 1 VWD.Previously, genotyping of 555 subjects (196 cases with type 1 VWD, and 362 family members) for the CLEC4M VNTR number showed that the most frequently documented alleles were VNTR 5 (29%), 6 (15%), and 7 (53%). Family-based association analysis on kindreds with type I VWD has demonstrated a significant excess transmission of VNTR 6 to the type I VWD phenotype (p=0.005) and an association of this VNTR allele with VWF:RCo (p=0.037). In the present studies, we have complemented this genetic association data with experiments to directly evaluate the ability of CLEC4M to bind and internalize VWF. Further, we characterized the ability of different CLEC4M VNTR alleles to facilitate VWF clearance.Binding of VWF to CLEC4M was assessed with a modified ELISA using a recombinant CLEC4M-Fc chimera. CLEC4M-Fc bound to Humate P (plasma-derived VWF-FVIII) in a dose-dependent manner. CLEC4M-Fc also bound to recombinant human VWF, and factor VIII-free plasma-derived VWF. CLEC4M-Fc demonstrated a 70% increase in binding to de-O-glycosylated Humate P (p=0.041), and a 75% decrease in binding to de-N-glycosylated Humate P (p=0.046) relative to controls. Additionally, pre-incubation of CLEC4M-Fc with the polysaccharide mannan attenuated binding to all VWF preparations by approximately 50%.Binding and internalization of VWF by HEK 293 cells stably expressing CLEC4M (VNTR allele 7) was assessed with immunofluorescence and ELISA. Binding of VWF co-localized with CLEC4M expression on HEK 293 cells. CLEC4M and VWF co-localized with early endosomal antigen-1, suggesting that CLEC4M participates in receptor-mediated endocytosis of VWF. CLEC4M-expressing cells bound and internalized VWF in a dose- and time-dependent manner relative to controls. Preincubation of CLEC4M expressing cells with mannan inhibited VWF binding and internalization by 50% (p=0.0088).The contribution of CLEC4M genetic variability to VWF binding and internalization was measured using HEK 293 cells expressing CLEC4M with 4, 6, 7, and 9 tandem repeats. Decreased binding and internalization of VWF was observed with cells expressing CLEC4M 4 and 9 tandem repeat constructs as compared to CLEC4M with 7 tandem repeats (CLEC4M 4 – 60% reduction, p < 0.001; CLEC4M 9 – 45% reduction, p=0.006). Cells expressing the CLEC4M VNTR combination 4 and 9, had a 55% decrease in binding and internalization of VWF relative to cells expressing CLEC4M with 7 VNTRs (p < 0.001). These VNTR associated differences in VWF binding/internalization were not accounted for by variances in the CLEC4M expression levels in the transfected HEK 293 cells.These studies demonstrate that the C-type lectin CLEC4M binds to and internalizes VWF through an N-glycan-dependent mechanism. Additionally, it provides further evidence that polymorphisms in the CLEC4M gene contribute to quantitative VWF deficiency. Disclosures:Montgomery:Gen-Probe/GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

Von Willebrand disease biology

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Use of a mouse model to elucidate the phenotypic effects of the von Willebrand factor cleavage mutants, Y1605A/M1606A and R1597W

von Willebrand Factor (VWF) is tightly regulated by the metalloproteinase ADAMTS13, which cleaves VWF to reduce VWF multimer size and binding affinity for collagen and platelets. This study examines two VWF mutations, R1597W (enhanced cleavage) and Y1605A-M1606A (decreased cleavage), to determine their impact on VWF, in addition to ADAMTS13-mediated cleavage. In vitro mouse ADAMTS13 digestions were performed on recombinant proteins. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA, following which VWF antigen, multimer profile and VWF propeptide levels were determined. A ferric chloride injury model of thrombosis was also evaluated. In vitro ADAMTS13 digestion of full-length mouse VWF required > 97-fold higher ADAMTS13 levels for Y1605A/M1606A, and 68% lower ADAMTS13 levels for R1597W compared with wild type. In vivo, R1597W had reduced VWF:Ag and both mutations exhibited increased VWF propeptide/VWF:Ag ratios. R1597W multimers show a lower molecular weight profile compared with wild type and Y1605A/M1606A mice. When co-injected with Adamts13 cDNA, Y1605A/M1606A multimers were larger compared with wild type, and R1597W showed only a single multimer band and decreased clearance via VWFpp/VWF:Ag ratio. R1597W was associated with reduced thrombus formation but normal platelet accumulation in a ferric chloride injury model while Y1605A/M1606A had a loss of occlusive thrombi but increased platelet accumulation compared with wild type. This study demonstrates that mutations that alter ADAMTS13 cleavage also can affect VWF clearance, VWF antigen level, multimer structure and thrombotic potential in the VWF knockout hydrodynamic injection model.

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).

Detection of Non Inhibitory Binding Antibodies to Von Willebrand Factor Affecting the Clearance of VWF:Ag in Von Willebrand Disease

Abstract▪2275▪This icon denotes a clinically relevant abstractVon Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF, the largest multimeric plasma glycoprotein, facilitates platelet aggregation and stabilizes FVIII in the circulation. Inhibitory antibodies to VWF have been reported in 10% – 15% of type 3 VWD patients repetitively treated with plasma-derived VWF/FVIII concentrates. The clinical impact of non-inhibitory antibodies to VWF has not been previously reported. To investigate the immunogenicity of a novel recombinant human VWF (rhVWF), both total binding anti-VWF antibodies and inhibitory anti-VWF antibodies (VWF:Ristocetin Cofactor Activity [RCo], VWF:Collagen Binding Activity [CB], VWF:FVIII Binding [FVIIIB]) were assessed in a Phase 1 multicenter clinical study. Total binding anti-VWF antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) employing polyclonal anti-human IgG antibodies. Plasma samples were analyzed in two steps employing a screening assay determining the titer of the binding antibodies and in a second step, confirming the specificity of the antibody in a competition assay. Samples were considered positive, when a sample was at least two titer steps lower than the antibody titer detected in the screening assay necessitating a screening titer of at least 1:80. Neutralizing antibodies to the key functional activities of VWF, such as VWF:RCo, VWF:CB and VWF:FVIIIB, were measured by assays based on the Bethesda assay established for quantitative analysis of FVIII inhibitors (Nijmegen modification of the Bethesda assay). To exclude false positive results, the detection limit for anti-VWF inhibitors was set to 1 BU/mL for all 3 assays. Three of 39 subjects screened had a pre-existing high titer non-neutralizing binding antibody (all 1/1280) to VWF. One of these 3 subjects was excluded from the study due to the presence of an inhibitory antibody to VWF:CB (1.3 BU/mL) at screening. Two of the 3 subjects were enrolled and treated with rVWF and/or pdVWF concentrate as part of the safety, tolerability and PK assessments. The high titer binding anti-VWF antibodies were associated with a significant decreased VWF:Ag activity post infusion of either pdVWF or rVWF and consequential decreased activity of VWF:RCo, VWF:CB and FVIII:C. For example, one of the subject had a reduced VWF:Ag incremental recovery of 1.1 IU/dL/(U VWF:RCo/kg) (mean of cohort 1.6 IU/dL/[U VWF:RCo/kg]) and a reduced VWF:Ag t1/2 2.4 hours (mean of cohort t1/2 25.3 hours) post infusion of rhVWF dosed at 50 IU VWF:RCo/kg. Relevant data of all PK assessments and antibody titers will be presented. None of the study subjects developed an inhibitory antibody to either VWF or FVIII and there was no impact on the subsequent treatment of bleeding episodes with commercial pdFVIII/VWF concentrates noted by the treating physician. The clinical relevance of non-neutralizing antibodies to VWF requires additional investigation. Disclosures:Suiter:Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment. Varadi:Baxter Innovations GmbH: Employment. Chapman:Baxter Innovations GmbH: Employment. Engl:Baxter Innovations GmbH: Employment. Wong:Baxter Healthcare Corp: Employment. Ewenstein:Baxter Healthcare Corp: Employment.

A Critical Role for N- and O-Linked Carbohydrates In Modulating Von Willebrand Factor Clearance In Vivo

Abstract 382▪FN2▪This icon denotes a clinically relevant abstractVWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF−/− mice.Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p<0.01). Digestion of pdVWF with α2–3 neuraminidase to remove predominantly O-linked sialic acid (which constitutes less than 20% total VWF sialylation) was also sufficient to markedly enhance VWF clearance (t1/2 = 13.1 min; p<0.05). In the presence of the asialoglycoprotein receptor (ASGPR)-antagonist ASOR, the mean residence time of Neu-VWF was identical to that of pd-VWF. Recent studies have shown that macrophages may be important in VWF clearance. Since the ASGPR is expressed on both hepatocytes and macrophages, the effect of macrophage depletion on VWF clearance was assessed. Pre-treatment with liposome-encapsulated clodronate depleted F4/80+CD11b+ murine macrophages by 75%, and significantly prolonged Neu-VWF survival. However Neu-VWF survival was not corrected to that observed in the presence of ASOR. For example, plasma Neu-VWF survival after 5 mins was corrected from 30±6% to 92±7% in the presence of ASOR, compared to 78±10% following clodronate macrophage-depletion. Cumulatively, these findings demonstrate that both N- and O-linked sialylation are critical in protecting VWF against ASGPR-mediated clearance. Moreover, ASGPR-modulated clearance is at least in part macrophage-dependent. ß-galactose residues exposed following removal of capping sialic acid are recognised by the ASGPR. To further define the role of specific sugars in regulating VWF clearance, the effect of terminal sialic acid and sub-terminal galactose removal by sequential neuraminidase and galactosidase digestions was studied. Surprisingly, VWF exposed to sequential neuraminidase and galactosidase digestions (NeuGal-VWF) was cleared rapidly from the plasma in a monophasic fashion (t1/2 = 4.8 min). Moreover, treatment with PNGase F to completely remove N-linked carbohydrate structures also markedly decreased the plasma half-life (PNG-VWF; t1/2 = 2.1 min). In keeping with their lack of exposed galactose residues, the enhanced clearance of NeuGal-VWF and PNG-VWF were not mediated via the ASGPR (ASOR had no significant effect). In contrast, macrophage depletion by liposomal clodronate significantly inhibited the enhanced clearance of both NeuGal-VWF and PNG-VWF respectively. These data suggest that the ASGPR is not the only macrophage receptor involved in modulating VWF clearance, which is consistent with the relatively minor prolongation in VWF survival previously reported in Asgpr1−/− mice.These novel data demonstrate that variation in the N- or O-linked carbohydrate structures significantly modulate VWF half-life in vivo. Moreover, VWF clearance is not mediated solely through the ASGPR, but may also require additional as yet unidentified macrophage receptors for full clearance. Therefore, qualitative and quantitative variation in VWF glycosylation represents a key regulator of VWF clearance, and as such is likely to be of direct pathophysiological significance. Disclosures:No relevant conflicts of interest to declare.

Expanded phenotype–genotype correlations in a pediatric population with type 1 von Willebrand disease

Recent phenotype-genotype studies have provided valuable insights into the pathophysiology of type 1 von Willebrand disease (VWD); however, no study has examined an exclusively pediatric cohort. To describe phenotype-genotype correlations in a selected pediatric cohort with a historical diagnosis of type 1 VWD, using first-degree family members as controls. Comprehensive phenotypic assessment included standard assays of von Willebrand factor (VWF) level and function, bleeding score, desmopressin response, VWF propeptide (VWFpp) level, and platelet-derived VWF mRNA level. Fourteen VWF mutations were identified in 17 of 23 index cases (ICs) (aged 5-17 years), including four that were previously unreported (L60P, nt1658 insT, Q1388X, and C2237F). VWFpp levels were lower in ICs than in unaffected controls (median 49 vs. 86 U dL(-1) , P < 0.0001). A VWFpp/VWF antigen ratio of > 1.6 was observed in eight of nine ICs with a suboptimal response to desmopressin, including four of four with the R1205H (Vicenza) mutation (median 7.9), and three of four IC with the R1315C mutation (median 1.9). The R1315C mutation was also associated with a reduced absolute VWFpp level (median 32 U dL(-1) ), a previously unreported finding. The amount of platelet-derived VWF mRNA was significantly reduced in individuals with nonsense mutations. Increased VWF clearance and intracellular retention are important mechanisms underlying type 1 VWD in pediatric patients, concordant with the observations of larger, predominantly adult, cohort studies. Additionally, in some patients, nonsense-mediated decay of mutant mRNA transcripts may be contributory. Several mechanisms underlie the variable phenotype associated with the R1315C mutation. The potential utility of VWFpp as an independent marker of VWF biosynthesis and release warrants further research.

Assessment ofvon Willebrand Factor Propeptide Improves the Diagnosis ofvon Willebrand Disease

One of the more recent findings concerning Von Willebrand disease (VWD) is that a shorter Von Willebrand factor (VWF) survival either decides or modulates the VWD phenotype by downregulating circulating VWF levels. VWF survival is currently investigated with the desmopressin (DDAVP) test, a time-consuming strategy enabling the main pharmacokinetic parameters (e.g., VWF half-life elimination time and clearance) to be defined. An alternative now available involves assaying the VWF propeptide (VWFpp) in single steady-state blood samples, which reportedly increases as VWF survival decreases. This article demonstrates how measuring VWFpp and calculating the VWFpp-to-VWF:antigen ratio (VWFpp ratio) are good alternatives to DDAVP for investigating VWF survival. In type 1 VWD, the VWFpp ratio has been found normal in patients with pure quantitative VWF defects, markedly increased in cases with an isolated decline in VWF survival, and more or less increased in patients with both quantitative defects and a shorter VWF survival. The same applies to type 2B VWD, which is characterized by an increased VWFpp ratio and a shorter VWF survival, with values that appear inversely related. Exploring VWF half-life by assaying VWFpp is useful not only for the more precise characterization of VWD but also for defining its most appropriate treatment.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Bleeding tendency and efficacy of anti-haemorrhagic treatments in patients with type 1 von Willebrand disease and increased von Willebrand factor clearance

Accelerated clearance of von Willebrand factor (VWF) has been recently identified as a major pathophysiologic mechanism inducing low VWF in some patients with von Willebrand disease (VWD). The frequency of bleeding and the best treatment of these patients have never been evaluated prospectively in large series of patients. It was the aim of the present study to prospectively evaluate clinical events of 60 heterozygous patients with VWD Vicenza (VWD-VI) carrying R1205H VWF mutation and 23 with C1130F mutation, both characterised by markedly increased VWF clearance. During 71 months of follow-up, 65% of patients with VWD-VI and 61% with C1130F required treatment. The rate of spontaneous bleeding requiring consultation/treatment was 7.5/100 patients-year in patients with C1130F mutation vs. 1.9/100 patients-year in those with R1205H (p=0.004). This difference persisted also by multivariate analysis adjusted for sex, age and blood group (hazard ratio [HR]=3.3 for C1130F, 95% confidence interval [CI] 1.16-9.27) and females were at greater risk of bleeding (HR=3, 95%CI 1.01-9.93) because of menorrhagia. Only 3/15 (20 %) women in fertile age with VWD-VI compared to 8/9 (89 %) with C1130F mutation required consultation/treatment for menorrhagia (iron supplementation, combined oral contraceptives, tranexamic acid). Almost all dental extractions, minor surgeries and deliveries occurring during follow-up were successfully managed with desmopressin. Major surgery required factor VIII/VWF concentrates, but a few cases benefited from desmopressin. In conclusion, similar to patients with type 1 VWD, also in patients with increased VWF clearance desmopressin maintains a major therapeutic role.

Genetic Determinants of Plasma Von Willebrand Factor Antigen Levels: A Target Gene SNP and Haplotype Analysis of the ARIC Cohort

AbstractAbstract 4310 Background and Objectives:Von Willebrand factor (VWF) is an essential component of hemostasis. It is known that multimer size and the amount of circulating VWF impact its hemostatic function. Genetic factors play a major role in regulating VWF synthesis and clearance and are reported to contribute to 66% of the variation in plasma VWF antigen level. We have analyzed 78 SNPs distributed throughout the VWF gene and haplotypes constructed from those SNPs for an association with VWF antigen level in 7,856 subjects of European descent in the Atherosclerosis Risk in Communities cohort. Methods and Results: Blood was drawn after an 8 hour fasting period and VWF antigen level was determined by a commercial ELISA kit. All subjects underwent analysis for 78 SNPs available from Affymetrix 6.0 chip in the region encompassing the VWF gene. We used the fastPHASE 1.2 program to resolve haplotypes from the unphased SNP genotype data. Using Haploview we determined SNPs in strong linkage disequilibrium (LD), their co-segregation rates and underlying haplotypes. Linear models were used to evaluate the association of actual or log VWF antigen levels with each SNP and haplotypes derived from those SNPs. Eighteen (16 are intronic) of the 78 SNPs were found to significantly associate with levels of VWF antigen (Table 1). All are clustered in a 50 kb region of the VWF gene in spite of the fact that the 78 SNPs studied are distributed throughout the VWF gene without apparent clusters. All 78 SNPs are co-segregated in 9 LD haplotype blocks, but a majority of positive SNPs (88.9%) are located in Block 5 and 6, which significantly correlate with VWF antigen level. The O blood type contributes to ∼ 15% of variation in VWF antigen levels. The association for SNPs and Haplotypes grows stronger after ABO effects are removed. Among non-genomic factors, age and BMI are the most significant factors and contribute to 4.8% and 1.4% of variation in VWF antigen level. Conclusions: We have found a strongly positive association between VWF level and SNPs and haplotypes from a strikingly concentrated region of the VWF gene. This region encodes the D2, D' and D3 domains of VWF, including the propeptide and multimerization and Factor VIII binding sites. The physiological significance of these clustered SNPs on VWF synthesis or clearance remains to be further investigated. Our data suggest that this region plays an important role in regulating VWF antigen level and support the idea that combinations of SNPs in LD can provide additive affects. Disclosures:No relevant conflicts of interest to declare.

Von Willebrand’s disease diagnosis and laboratory issues

In this paper, the recent developments in the diagnosis and laboratory issues of von Willebrand's disease (VWD) are presented. Dr. Castaman reviews the functional tests available for the diagnosis of VWD and their pathophysiological significance, focusing on which tests are best used in the diagnosis and classification of VWD. Dr Montgomery reviews an emerging issue that is accelerated clearance of von Willebrand factor (VWF) occurring in some variants of VWD. This phenotype can be suspected by the presence of an increased ratio between the VWF propeptide and the VWF antigen. These patients have typically a robust, but short-lived increase of FVIII and VWF after desmopressin. Dr Meschengieser reviews the determinants of bleeding after surgery in patients with VWD, emphasizing the role of bleeding history in predicting this risk.

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

AbstractType 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbα binding. These “gain of function” mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF−/− mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride–induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride–induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

Von Willebrand factor variant p.Arg924Gln marks an allele associated with reduced von Willebrand factor and factor VIII levels.

von Willebrand factor (VWF) variant c.2771G>A; p.R924Q has been described as a benign polymorphism or a possible marker for a null allele and been associated with mild bleeding phenotypes. It was identified in several patients in recent type 1 von Willebrand disease (VWD) studies. To determine whether the p.R924Q allele contributes to reduced VWF levels and type 1 VWD. One thousand one hundred and fifteen healthy controls and 148 index cases from the MCMDM-1VWD study were genotyped for c.2771G>A; VWF and FVIII levels were analyzed in ABO blood group stratified individuals and the p.R924Q variant was expressed in 293 EBNA cells. c.2771G>A was present in six index cases, five of whom had a second VWF variant which probably contributed to the phenotype. A common core haplotype identified in families, which included the rare G allele of c.5843-8C>G, was present in the majority of 35 c.2771G>A heterozygous controls. c.2771G>A contributed about 10% variance in VWF and FVIII levels in controls and 35% variance when co-inherited with blood group O. Recombinant p.R924Q VWF had no effect on in vitro expression and heterozygous family members had normal VWF-FVIII binding and normal clearance of VWF and FVIII. The allele bearing c.2771A leads to reductions in VWF and FVIII levels particularly in combination with blood group O. Its inheritance alone may be insufficient for VWD diagnosis, but it appears to be associated with a further VWF level reduction in individuals with a second VWF mutation and it contributes to population variance in VWF and FVIII levels.