Pathogenesis Of Von Willebrand Disease Research Articles

Von Willebrand disease and von Willebrand factor.

In the past several decades, knowledge of the underlying pathogenesis of von Willebrand disease (VWD) has increased tremendously, thanks in no small part to detailed genetic mapping of the von Willebrand Factor (VWF) gene and advances in genetic and bioinformatic technology. However, these advances do not always easily translate into improved management for patients with VWD and low-VWF levels. For example, the treatment of pregnant women with VWD both pre- and postpartum can be complicated. While knowledge of the VWF genotype at some amino acid positions can aid in knowledge of who may be at increased risk of thrombocytopenia or insufficient increase in VWF levels during pregnancy, in many cases, VWF levels and bleeding severity is highly heterogeneous, making monitoring recommended during pregnancy to optimize treatment strategies. VWF AND COVID-19: New challenges related to the consequences of dysregulation of hemostasis continue to be discovered. The ongoing COVID-19 pandemic has highlighted that VWF has additional biological roles in the regulation of inflammatory disorders and angiogenesis, disruption of which may contribute to COVID-19 induced vasculopathy. Increased endothelial cell activation and Weibel-Palade body exocytosis in severe COVID-19 lead to markedly increased plasma VWF levels. Coupled with impairment of normal ADAMTS13 multimer regulation, these data suggest a role for VWF in the pathogenesis underlying pulmonary microvascular angiopathy in severe COVID-19. With the increased affordability and availability of next-generation sequencing techniques, as well as a push towards a multi-omic approach and personalized medicine in human genetics, there is hope that translational research will improve VWD patient outcomes.

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.

New treatment approaches to von Willebrand disease.

von Willebrand disease (VWD) is the commonest inherited bleeding disorder and results from either a quantitative or qualitative deficiency in the plasma glycoprotein von Willebrand factor (VWF). Recent large cohort studies have significantly enhanced our understanding of the molecular mechanisms involved in the pathogenesis of VWD. In contrast, however, there have been relatively few advances in the therapeutic options available for the treatment of bleeding in patients with VWD. Established treatment options include tranexamic acid, 1-deamino-8-d-arginine vasopressin (DDAVP), and plasma-derived VWF concentrates. In addition, a recombinant VWF has also recently been developed. In this review, we focus on how recent insights into the clinical and molecular aspects underpinning VWD are already beginning to influence treatment in the clinic. For example, a number of different bleeding assessment tools (BATs) have been developed to objectively assess bleeding symptoms in patients with VWD. Interestingly, however, these BAT scores may also have an important role to play in predicting bleeding risk in VWD. Furthermore, recent studies have demonstrated that enhanced VWF clearance plays a critical role in the etiology of both type 1 and type 2 VWD. These findings have direct translational relevance with respect to the use of DDAVP in patients with VWD. As understanding of the mechanisms involved in VWD pathogenesis continues to advance, novel treatment options are likely to emerge. Critically, however, large adequately powered and stratified clinical trials will be required to address the outstanding questions that remain regarding VWD treatment optimization.

Biophysical Characterization of Mechanosensors within the Plasma Protein von Willebrand Factor and its Receptor Platelet Glycoprotein Ib-IX

The large, multimeric plasma protein von Willebrand Factor (VWF) plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting. Platelet binding is achieved by interaction between the A1 domain of the Iba chain of platelet surface receptor Glycoprotein (GP) Ib-IX. Both GPIB-IX and VWF are capable of sensing and reacting to tensile forces. Using the single-molecule force measurement approach, we have recently identified a quasi-stable mechano-sensitive domain (MSD) of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIba subunit. The MSD unfolds at 5-20 pN when subjected to mechanical stretch by the engaged A1. Unfolding of the MSD triggers receptor signaling and stimulates platelets. Similarly, earlier studies by the Springer group have identified the mechanosensor within VWF, the A2 domain (i.e., the domain adjacent to A1). Tensile force at the level of 10-20 pN unfolds the A2 structure, leading to A2's subsequent cleavage by plasma enzyme ADAMTS-13 (Zhang, X. et al. Science, 324:1330-4). Since both MSD and A2 unfold at a similar force level, simultaneous activation of both mechanosensors may occur given that the interacting VWF and GPIb-IX are stretched to provide sufficient force. Herein, we show that optical tweezer-controlled pulling of the full-length of VWF on GPIb-IX indeed induced unfolding of both MSD and A2. Additional studies using recombinant wildtype and mutant VWF suggest that the unfolding of MSD and A2 is dependent on the mechanical strengths of the VWF-GPIba and VWF-collagen interactions, as well as on the activation state of the VWF. Together, the study has revealed the biomechanical properties of VWF and GPIb-IX, with significant implications for the pathogenesis of Von Willebrand Disease and GPIb-IX- related blood diseases.

Genetic Analysis of Von Willebrand Disease By Using the Exome Sequencing Approach

Abstract Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder characterized by quantitative and/or qualitative von Willebrand factor (VWF) abnormalities. VWF gene is a large gene consists of 52 exons, wheras its pseudogene corresponds to exons 23-34 that makes the analysis much complicated. In this study, we performed genetic analyses of the VWF gene from 20 Japanese VWD patients, aimed to find the causative genetic mutations by using various techniques including the exome sequencing approach, and to explore the diversity of yet unknown VWD pathogenetic mechanisms. Methods: Patients' samples were collected after the written informed consent has been obtained. First, we analyzed exon 28 of VWF genes from peripheral blood genomic DNA of the patients by PCR using allele-specific primers, and analyzed DNA sequences of the patients by Sanger sequencing method. If patients show the type 3 phenotype, the possibility of deletion or insertion of the gene was taken into consideration, then MLPA method was selected. In cases the gene mutation was not detected by exon 28 sequencing or MLPA, we analyzed all exons of VWF by Sanger method. In selected patients, the exome sequencing was employed by using the next-generation sequencer Hi-Seq 2500 (Illumina) and exomes were captured using SureSelect XT Human All Exon V5+UTRs kit (Agilent Technologies). Results: Twenty VWD cases were studied. As a result of the VWF exon 28 sequencing, we identified causative gene mutation in 12 cases (10 cases were type 2, 2 cases were type 1). Gene mutations found in exon 28 were p.R1308C, p.R1315C, p.V1316M, p.R1334W, p.A1461D, p.L1503R, p.S1506L, p.R1597Q, p.G1609R. In one case and her sister of type 3, MLPA could not detect the large deletions or insertions. Sanger sequencing showed the they were compound heterozygous for an p.E2341X (c.7021G&gt;T) mutation in exon 41 and a p.Y2631X (c.7892-7893insA) mutation in exon 48. In another case of type 3, we used exome sequencing and found a novel homozygous gene mutation p.G2752S (c.8254G&gt;A) in exon 52. The mutation was confirmed by Sanger sequencing and the propositus was homozygote for the mutation, while the parents and two sons had the same mutation in the heterozygote. In the multimer analysis of plasma, multimers larger than the dimer in size were hardly visible, but there was a faint monomer band was present in the plasma. Multimeric patterns of his family were normal. Discussion: We analyzed the VWF genes of 20 VWD cases and identified causative gene mutations in 14 cases including three novel mutations (p.E2341X, p.Y2631X, p.G2752S). We previously demonstrated BOECs established from the type 3 patient (p.E2341X and p.Y2631X) reproduced the phenotype of the disease (ASH 2014). Meanwhile, G2752S mutation is located in CTCK domain, therefore possibly it affects the VWF dimer formation. However, the causative gene mutations were not detected in 6 cases (5 cases were type 1, 1 case was type 2) by exome sequencing. Although previous observations have pointed out that causative VWF gene mutations are not detectable in about 30 % of entire type 1 cases, a newer approach would be needed to elucidate molecular pathogenesis of VWD. Disclosures Suzuki: Baxalta: Honoraria; Novo Nordisk Pharma: Honoraria; Bayer Healthcare: Honoraria. Matsushita:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Asahi Kasei Pharma: Honoraria, Research Funding, Speakers Bureau; Kaketsuken: Honoraria, Research Funding, Speakers Bureau; Seamens: Speakers Bureau; Japan Blood Products Organization: Honoraria, Research Funding; Kyowa-Kirin: Honoraria, Research Funding; Novartis Pharma: Honoraria, Speakers Bureau; Sysmex: Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novo Nordisk Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eisai: Research Funding; Nihon Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Octapharma AG: Honoraria; CLS-Behling: Research Funding.

Establishment of Blood Outgrowth Endothelial Cells from a Type 3 Von Willebrand Disease (VWD) Patient with a Neutralizing Anti-VWF Inhibitors

Background: Type 3 von Willebrand disease (VWD) is characterized by a complete loss type of von Willebrand Factor (VWF) with the rarest disease frequency and hemorrhagic symptom is the most severe among other VWD types. The development of alloantibodies directed against VWF occurs in approximately 10% of patients with type 3 VWD. In this study, we found the VWF gene alterations and established blood outgrowth endothelial cells (BOECs) a from a Japanese type 3 VWD patient with an anti-VWF inhibitors.Case: A 5-year-old woman suffered from epistaxis, purpura and easy bruising and has been diagnosed as VWD. Her plasma level of FVIII:C was 1.8% and VWF:Ag and VWF:RCo was <5% and <10%, respectively. Her plasma lacked VWF multimers, indicating that she had type 3 VWD. To treat her frequent muco-cutaneous bleeding, purified plasma-derived FVIII/VWF concentrates were administrated followed by inhibitor development. After about 20ED of FVIII/VWF concentrates, an anaphylactic symptom comprised of cough, dyspnea, and wheezing developed at the time of dosing. An high titer inhibitory antibody against VWF was confirmed by a Bethesda assay based on a assay of VWF:RCo. Her younger sister also had type 3 VWD.Methods: Patients samples were collected after the written informed consent has bee obtained. We performed MLPA for genetic deletion or insertion. Then we amplified the all exons including the exon/intron boundaries of the VWF gene by PCR using allele-specific primers, and analyzed DNA sequences of the patient. Peripheral blood mononuclear cells were obtained from the patient and BOECs were then established. Briefly, buffy coat mononuclear cells were isolated and seeded onto a 6-well tissue culture plate precoated with type 1 rat tail collagen at 37°C, 5% CO2, in a humidified incubator. Medium was changed daily for 7 days and then every other day until the first passage. The endothelial identity of the BOECs was confirmed by flow cytometric and immunofluorescence analyses using endothelial markers with antibodies to CD31, VE-Cadherin (CD144) and VEGFR-2 (CD309).Results: The large deletion and insertion of VWF gene were not found. Direct sequencing showed the propositus and her sister were compound heterozygous for an E2341X (c.7021G>T) mutation in exon41 and a Y2631X (c.7892-7893insA) mutation in exon48. These were therefore two novel nonsense mutations and normal VWF polypeptides could not be translated. Flow cytometric analysis indicated that established BOECs expressed cell surface CD31 and CD309, whereas CD34 was not detected. By immunofluorescence analysis of fixed BOECs, VWF signal was remarkably reduced from the patient, in spite of normal VE-Cadherin expression. VWF specific ELISA was performed for both cell supernatant and lysates of patient’s BOECs but immunodetectable VWF was not secreted.Discussion: We identified two novel nonsense mutations causing type3 VWD. BOECs established from the patient reproduced the phenotype of the disease, suggesting that BOEC analysis is useful for studying the molecular pathogenesis of von Willebrand disease. DisclosuresNo relevant conflicts of interest to declare.

Mutations in the D1 Domain Inhibit Propeptide-Dependent Multimerization, Trafficking and Regulated Secretion of Von Willebrand Factor in VWD

Gene mutations play an important role in the pathogenesis of von Willebrand disease (VWD), resulting in the qualitative defect or quantitative deficit of von Willebrand factor (VWF). VWF propeptide is composed of D1 and D2 domain, which acts as a covalent oxidoreductase in the multimerization. In addition, the propeptide is necessary for the transport from endoplasmic reticulum (ER) to Golgi-apparatus, basal secretion and regulated secretion of VWF. However, the mechanisms of the mutation in D1 domain impairing VWF multimerization and causing low VWF levels in patients remain unknown. Herein we identified four mutations in the D1 domain from VWD patients, and assessed the effect of these mutations on the function of propeptide.We identified p.G39R, p.D141N, p.K157E, and p.C379G in three VWD patients. To characterize the roles of four mutations in propeptide-depend multimerization, we generated several truncated-VWFs, D1D2D¡¯D3 (residues 1-1241), including G39R, K157E, D141N, C379G and wide type (WT). These constructs were then expressed in HEK 293 cells, and were evaluated the D¡¯D3-dimer formation of mutations and WT. Full-length VWF comprising mutations and WT were also restructured and were transfected in HEK 293 cells. We then analyzed the VWF multimer distribution and VWF antigen in the cell supernatant and cell lysate. We also assessed VWF retention in ER and the stimulation secretion by phorbol-12-myristate-13- acetate (PMA) of mutations and those of WT.Two type 3 and 1 type 1 patients were enrolled in our study. VWF antigen were 3, 1 and 8 IU/dL, and VWF:Rco were 2.1, 2.3 and 5.6 IU/dL respectively in three patients. VWF multimer distribution exhibited none in two type 3 patients and normal-like multimer pattern in the type 1. Sequence analysis of VWF gene showed two heterozygous mutations (p.G39R and D141N) in D1 domain of one type 3 patient, and a heterozygous K157E in propeptide and a heterozygous C1165R in D3 domain of the other patient with type 3. Type 1 patients had a heterozygous C379G mutation. Among these four mutations in D1 domain, p.G39R, p.K157E, and p.C379G were novel.In the supernatant of transfected cell, dimerization of D¡¯D3 was absent in truncated-G39R. Compared with that of truncated-WT, decreased but detectable dimerizations were detected in K157E, D141N, and C379G. Similar results were also observed in multimerization of full-length constructs. The multimer assembly was too low to visualize in G39R, whereas decreased medium and absent large VWF multimers were seen in K157E, D141N, and C379G.By immunofluorescence imaging, all full-length VWF variants were detained in ER in different degrees (fig 1). The basal secretions of G39R, K157E, D141N, and C379G were (2.7¡À0. 3)%, (2.5¡À0.2)%, (26.0¡À4.1)%, and (22.4¡À3.8)% of WT respectively. However, VWF antigens in the lysate of transfected cells were (116.5¡À5.4)%, (90.9¡À3.0)%, (91.7¡À0.4)% and (113.8¡À2.9)% of WT for G39R, K157E, D141N, and C379G. No detectable secretion increases of mutant VWF induced by PMA were observed in the transfected cells, while WT-VWF with PMA exhibited increased secretion from 0.69% to 1.66%. [Display omitted] These four mutations in D1 domain downgraded the activity of propeptide as the covalent oxidoreductase, and impaired the muiltimeriztion induced by propeptide. They also interfered with VWF transport from ER to Golgi-apparatus and caused the VWF retentions in ER. Therefore, they further reduced the basal secretion and regulated secretion of mature VWF, which could explain the possible pathogenesis of quantitative deficit of VWF in VWD. DisclosuresNo relevant conflicts of interest to declare.

Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort

Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17-18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.

Comparative Evaluation of Five Different Assays for Ristocetin Cofactor Activity Determination,

Abstract 3317 Introduction:Despite significant advances in the comprehension of the molecular pathogenesis of Von Willebrand Disease (VWD), the diagnosis of this condition remains one of the greatest challenges of daily hematology practice, especially in settings of low prevalence such as primary care services. Ristocetin cofactor activity (VWF:RCo) is one of the pillars of the diagnosis of VWD and is also essential for its management. However, reproducibility of tests available to measure VWF:RCo is still a major issue, as evidenced by coefficient of variations (CV) as high as 30%, 45% and 27% in the ECAT, NEQAS and PALQ external quality assessment program. Classical methods to measure VWF:RCo include light-transmission platelet agregometry (LPA) and visual agglutination with formaldehyde fixed human platelet (VA), but alternative methods continue to evolve. An automated method (BC) has been recently shown superior to LPA and VA. Other methods for which limited comparative precision studies are lacking include a new LIA-based automated assay (LIA) and flow citometry (FC), using fluorescence-marked platelets and ristocetin. Methods:Here we evaluate five different VWF:RCo assays, including classical LPA and VA, two different commercially available automated coagulometer-based methods (BC and LIA), and FC. Reference ranges for the each method were established in twenty healthy adults. Plasma samples from thirty-three patients with VWD (type 1: n=26; type 2A or 2M: n=4; type 3: n=3) followed at our comprehensive care center (IHTC-Campinas, Brazil) were assayed for the comparative studies. Diagnosis of VWD was based on historical levels of VWF:Ag (assayed by ELISA) and VWF:RCo (assayed by both LTA and VA) obtained from medical records, and from bleeding history (evaluated by VWD bleeding score). Imprecision studies (intra-assay variability), were performed in different samples: commercial lyophilized normal and abnormal plasma, as well as plasma from two additional VWD patients with borderline results. All analyses were performed in the same samples, in an external quality assessment program-participating university laboratory. Results:Despite using similar conditions than those described in an original report, and repeated experiments, the flow cytometry-based assay (FC) was not reproducible in our hands, yielding extremely high CV that prevented its use in a clinical laboratory. Mean and reference ranges for VWF: RCo assays levels in healthy adults were: VA: 0.84 IU/ml (0.62–1.07); LTA: 1.13 IU/ml (0.92–1.35); BC: 0.93 IU/ml (0.75–1.12). The automated BC method yielded the lower intra-assay variability, in the range of 1.13% to 5.46% in different samples, compared to values as high as 29.4% for LTA. Correlation coefficient (r) ranged from 0.93 between BC reagent and LTA to 0.85 between VA and BC reagent. Mean VWF: RCo levels in patients ranged from 22.99 ± 17.80 with BC to 22.60 ± 18.50 with LTA and 20.11± 14.50 with VA. In twenty-six type I VWD patients, mean VWF:RCo/VW:Ag ratio ranged from was 0.7 (range 0.4–1.5) with the VA method to 0.8 (range 0.8–1.3) with the BC method. In four type 2 VWD patients, mean VWF:RCo/VW:Ag ratio was 0.2 (range 0.3–0.4) using VA method to 0.4 (range 0.4–0.5) with the BC method. Concordance, evaluated using the Bland-Altman plot, was higher between LTA and BC method. Conclusion:In our study, LTA and VA performance was consistent with historical data, presenting very high CV, but good correlation and concordance with new automated methods. Of note, VA, a simple, inexpensive and equipment-independent method, presented better CV than LTA and should be regarded as a good alternative the diagnosis of VWD in areas with limited resources. Unfortunately, flow cytometry-based VWF:RCo methods apparently suffer from the same problems of LTA and VA regarding standardization. New automated assay present enhanced precision compared to VA and LTA, with the added benefits of automation in the setting of a Hemostasis laboratory. Ongoing improvements in protocols of these new assays, leading to even higher precision, could translate into more confident limits of normal and pathological VWF:RCo levels, enabling critical changes in the diagnosis, management and even in the definition of VWD. Disclosures:No relevant conflicts of interest to declare.