Variant Of Von Willebrand Disease Research Articles

The severe von Willebrand Disease variant p.M771V leads to impaired anterograde trafficking of Von Willebrand factor in patient-derived and base-edited ECFCs

The severe von Willebrand Disease variant p.M771V leads to impaired anterograde trafficking of Von Willebrand factor in patient-derived and base-edited ECFCs

PB0830 Inspecting Underlying Pathogenesis Mechanisms of Von Willebrand Disease Variants Sited in A Domains of the von Willebrand Factor

PB0830 Inspecting Underlying Pathogenesis Mechanisms of Von Willebrand Disease Variants Sited in A Domains of the von Willebrand Factor

Hemophilia: The Past, the Present, and the Future.

Hemophilia: The Past, the Present, and the Future.

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

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

Obstacles to Early Diagnosis and Treatment of Inherited von Willebrand Disease: Current Perspectives.

Von Willebrand disease (VWD), the most common inherited bleeding disorder, is highly heterogeneous, and its early diagnosis may be difficult, especially for mild cases and in qualitative von Willebrand factor (VWF) defects. Appropriate VWD diagnosis requires the combination of personal and/or family history of bleeding and abnormal VWF laboratory testing. The use of bleeding assessment tools has been helpful in standardizing bleeding history collection and quantification of bleeding symptoms to select patients who may benefit of further hemostatic testing. Type 1 and 3 VWD which represent quantitative VWD variants are relatively easy to diagnose. The diagnosis of type 2 VWD requires multiple assessments to evaluate the effects induced by the responsible abnormality on the heterogeneous functions of VWF. Sensitive and reproducible tests are needed to evaluate different VWF activities, starting from measuring VWF-platelet interaction. In the recent years, several increasingly sensitive, rapid and automated assays have been developed, but they are not widely available so far. Genetic testing for VWD diagnosis is not a common practice because VWF gene is very large and highly polymorphic and therefore it is used only in specific cases. It is evident that the early and correct VWD diagnosis allows optimal management of bleeding and situations at risk. Tranexamic acid, desmopressin, replacement therapy with plasma-derived concentrates with a variable content of VWF and FVIII, or the new recombinant VWF are the different therapeutic options available. Careful VWD classification guides treatment because desmopressin is widely used in type 1 while replacement therapy is the cornerstone of treatment for type 2 and 3 variants.

Laboratory variability in the diagnosis of type 2 VWD variants

Unlabelled Box AbstractIntroductionType 2 von Willebrand disease (VWD) refers to patients with a qualitative defect in von Willebrand factor. Accurate diagnosis of type 2 VWD subtypes can be challenging. Aim of the studyTo compare the historical diagnosis of type 2 VWD with current laboratory testing. MethodsSubjects were enrolled in the Zimmerman Program either because of a preexisting diagnosis of VWD (retrospective cohort) or from evaluation for bleeding symptoms or suspected VWD (prospective cohort). Original diagnosis was assigned by the local center and central diagnosis was based on central laboratory testing. ResultsTwo hundred and seventeen index cases in the retrospective cohort and 35 subjects in the prospective cohort carried a local diagnosis of type 2 VWD (29% and 6% of enrolled index cases, respectively). In the retrospective cohort, the diagnosis was confirmed in 66% of cases with a preexisting diagnosis of 2A, 77% 2B, 54% 2M, and 72% 2N. In the prospective cohort, 31% were confirmed 2A, 60% 2B, 23% 2M, and 100% 2N. Several genetic variants were repeatedly implicated in subjects with changed diagnosis: p.M1304R, p.R1315C, p.R1374C, and p.R1374H. ConclusionsBoth the prospective and retrospective cohorts demonstrated consistent variation in subjects whose diagnosis changed between 2A, 2B, and 2M. The importance of accurately diagnosing type 2 VWD may be most significant in the 2B subtype given potential concerns with the use of desmopressin in type 2B VWD. Some genetic variants appear in multiple types of VWD, making specific diagnoses challenging.

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

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

What have we learned from large population studies of von Willebrand disease?

Von Willebrand factor (VWF) is a critical regulator of hemostatic processes, including collagen binding, platelet adhesion, and platelet aggregation. It also serves as a carrier protein to normalize plasma factor VIII synthesis, release, and survival. While VWF protein measurements by immunoassay are reasonably comparable between institutions, the measurement of VWF ristocetin cofactor activity (VWF:RCo) has significant variability. Other tests of VWF function, including collagen binding or platelet glycoprotein IIb-IIIa binding, are not universally available, yet these functional defects may cause major bleeding even with normal VWF antigen (VWF:Ag) and VWF:RCo assays. This results in both the overdiagnosis and underdiagnosis of VWD. Newer assays of VWF function (using recombinant glycoprotein Ib rather than whole platelets) have been developed that may improve interlaboratory variability. Some of these tests are not uniformly available and may not be licensed in the United States. Large longitudinal studies of VWF in von Willebrand disease (VWD) patients are not available. Patients are sometimes diagnosed with a single diagnostic VWF panel. Plasma VWF levels increase with age, but it is not clear if this results in less bleeding or whether different normal ranges should be used to identify age-related decreases in VWF. In order to quantitatively compare bleeding symptoms in VWD patients and normal individuals, recent studies in the European Union, Canada, United Kingdom, Holland, and the United States have used semiquantitative bleeding assessment tools (BATs). Even with careful centralized testing, including functional assays of VWF, addition of a BAT does not solve all of the problems with VWD diagnosis. No matter where the line is drawn for diagnosis of VWD, VWF is still a continuous variable. Thus, VWD can be a severe hemorrhagic disease requiring frequent treatment or a mild condition that may not be clinically relevant. As will be discussed by Dr. Goodeve in her presentation, genetics has helped us to diagnose type 2 functional variants of VWD but has not been helpful for the many patients who are at the interface of normal and low VWF and carry the possible diagnosis of type 1 VWD. The hematologist's management of patients with reduced levels of VWF still requires both the art and science of clinical medicine.

Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami

Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.

Rapid discrimination of the phenotypic variants of von Willebrand disease

AbstractApproximately 20% to 25% of patients with von Willebrand disease (VWD) have a qualitative defect of the von Willebrand factor (VWF) protein activities. Variant VWD typically is classified as type 1C, 2A, 2B, 2M, or 2N depending on the VWF activity defect. Traditionally, diagnosis has relied on multiple clinical laboratory assays to assign VWD phenotype. We developed an enzyme-linked immunosorbent assay (ELISA) to measure the various activities of VWF on a single plate and evaluated 160 patient samples enrolled in the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease with type 2 VWD. Using linear discriminate analysis (LDA), this assay was able to identify type 1C, 2A, 2B, 2M, or 2N VWD with an overall accuracy of 92.5% in the patient study cohort. LDA jackknife analysis, a statistical resampling technique, identified variant VWD with an overall accuracy of 88.1%, which predicts the assay's performance in the general population. In addition, this assay demonstrated correlation with traditional clinical laboratory VWF assays. The VWF multiplex activity assay may be useful as a same-day screening assay when considering the diagnosis of variant VWD in an individual patient.

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination

Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA isthe requirement of fresh blood sample. In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines. Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays. The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P<0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio. Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.

A Von Willebrand Factor (VWF) Functional Screening Assay and Its Correlation with Conventional Clinical Assays of VWF Function

The diagnosis of variant von Willebrand Disease (VWD) currently involves evaluating multiple aspects of von Willebrand Factor (VWF) functions using separate, individual assays. This process can be time consuming and may delay definitive diagnosis of variant VWD. To address this issue, we have previously described a novel, rapid ELISA-based VWF functional screening assay that allows for variant VWD phenotype assignment and can provide same-day results. When considering application of this assay as a screening test, it is important to investigate its correlation with conventional clinical assays of VWF function. Therefore, the objective of our study was to investigate the relationship of our qualitative and semi-quantitative assay with traditional quantitative assays of VWF functions.161 plasma samples from the Zimmerman PPG were analyzed on a novel ELISA-based platform, which measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:FVIIIB (binding to FVIII), VWF:CBIII (binding to collagen III), and VWFpp (propeptide). Samples were compared to a 30% standard control plasma by a ratio of each unique VWF functional activity over the VWF:Ag. Data were compared to available quantitative data from the BloodCenter of Wisconsin clinical laboratory on the same patient plasma samples. Plasma samples included 21 type 1, 30 type 1C, 23 type 2A, 23 type 2B, 20 type 2M, 17 type 2N, 5 type 2N carriers (heterozygote), and 22 potential hemophilia A (HA) subjects. Data was analyzed by linear regression and power function. In all VWD samples analyzed by power function, VWF:Ag by our assay correlated with the clinical assay with an R2 of 0.794. For VWFpp from 111 VWD subjects, the power function revealed our assay correlated with the clinical assay with an R2 of 0.630, and sub-analysis of 51 type 1 and 1C VWD subjects showed an R2 of 0.673. VWF:CBIII analysis of all type 2A, 2B, and 2M subjects through linear regression revealed an R2 of 0.675. VWF:IbCo analysis was separated into type 2B and non-type 2B VWD subjects due to the significant VWF:IbCo enhancement seen in our assay because of augmented binding of type 2B VWF to glycoprotein Ib. While correlation of our VWF:RCo with the clinical VWF:RCo was poor, the VWF:IbCo was able to discriminate type 2B from non-type 2B. VWF:FVIIIB analysis showed complete discrimination of type 2N and potential HA subjects. In addition, there was discrimination of type 2N VWD subjects from type 2N carriers; however these data were not fit into the statistical model because there were too few subjects in each category.In summary, our study shows that each individual component of the VWF functional screening assay shows correlation with traditional quantitative assays of VWF functions. The best correlation was seen with the VWF:Ag, and there was also discrimination of type 2B, type 2N, type 2N carriers, and HA subjects demonstrated in our analysis. These data indicate the potential use of our assay as a laboratory screening test to expedite diagnosis of variant VWD. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Type 2N Von Willebrand Disease Mutations Using Ιn Vitro and Ιn Vivo Mouse Models

Introduction: Von Willebrand factor (VWF) is a multimeric glycoprotein that serves as the carrier for the essential coagulation cofactor, factor VIII (FVIII). Both plasma levels of VWF and its FVIII-binding ability can influence plasma levels of FVIII. Type 2N von Willebrand disease (VWD) is associated with a reduced binding affinity of VWF for FVIII, resulting in accelerated proteolysis and clearance of FVIII (plasma levels 5 – 30% of normal). Type 2N VWD is a recessive trait and patients are either homozygous or compound heterozygous for 2N alleles. We hypothesize that type 2N VWD mutations can alter the expression and FVIII-binding ability of VWF. In these studies, we characterize three type 2N VWD mutations in vitro and in a murine model. R854Q (20-30% FVIII) is the most common 2N allele and is associated with a mild phenotype, while R816W (<10% FVIII) is associated with a severe phenotype. The R763A mutation inhibits propeptide cleavage that likely sterically interferes with the FVIII-binding ability of VWF.Methods: Type 2N VWD mutations were generated in the murine VWF cDNA. Heterologous VWF synthesis/secretion was characterized in vitro using HEK 293T cells and in vivo using hydrodynamic gene transfer of the murine VWF cDNA into VWF deficient mice. Binding of FVIII to type 2N variants was assessed in vitro using a solid phase binding assay and in vivo in VWF deficient mice by a FVIII chromogenic activity assay.Results: In HEK 293 T cells, biosynthesis of type 2N VWD variants was not significantly different from wild type VWF while secretion of all type 2N VWD variants was decreased relative to wild type: R763A (66%, p=0.0043), R816W (53%, p=0.0004), R854Q (4%, p<0.0001). Immunofluorescent staining of transfected HEK 293 cells demonstrated impaired pseudo-Weibel Palade body formation for the R854Q variant. Western blot analysis under denaturing conditions demonstrated that approximately 50% of the secreted R763A protein remained attached to the propeptide. Multimeric profiles of plasma-derived type 2N VWD mutants were normal. In vitro binding of plasma-derived murine type 2N VWD mutants to recombinant human FVIII was reduced relative to wild type VWF: R763A (56%, p=0.0009), R816W (10%, p<0.0001), R854Q (46%, p=0.0002).Type 2N VWD mutants were expressed alone or in a compound heterozygous state (R816W/R854Q) in VWF deficient mice. A trend of lower VWF:Ag levels were observed for type 2N VWD mutants relative to wild type (average 4.8 U/mL) after 14 days: R763A (35.7%), R816W (53.1%), R854Q (21.3%), except for compound heterozygous condition R816W/R854Q (103%).Plasma levels of FVIII:C are significantly reduced in VWF deficient mice (15-20% of normal). We measured the ability of hydrodynamically expressed type 2N VWD mutants to stabilize endogenous FVIII:C in VWF deficient mice. Hepatic expression of wild type VWF stabilized endogenous plasma FVIII:C, resulting in a significant increase in FVIII:C after 14 days (7.7-fold increase above baseline, p=0.0002). For the type 2N VWD mutants, variable partial stabilization of endogenous FVIII:C was observed relative to baseline: R763A (4.7-fold increase, p=0.01), R816W (1.2-fold decrease, p=0.04), R816W/R854Q (4.8-fold increase, p<0.0001), R854Q (2.1-fold increase, p=0.06).The correlation coefficient between VWF:Ag and FVIII:C was assessed for samples with VWF:Ag between 0.5-10 U/mL. Correlation between wild type VWF expression and FVIII:C was highly positive (r2=0.85, slope=189.5 ± 15.7, p<0.0001). Correlation between VWF:Ag and FVIII:C for mice expressing type 2N VWD mutants was variable: R763A (r2=0.89, slope=235.3 ± 18.15, p<0.0001), R816W (r2=0.591, slope=0.96 ± 2.8, p=0.7433), R816W/854Q (r2=0.72, slope=91.32 ± 10.64, p<0.0001) and R854Q (r2=0.705, slope=156.7 ± 24.4, p=0.0002). The slopes for R816W (p<0.0001) and R816W/R854Q (p=0.009) mutants were significantly different from wild type, suggesting impaired FVIII-stabilization in vivo.Conclusion: Expression of the type 2N VWD severe mutant R816W or the compound heterozygous R816W/R854Q mutant can recapitulate type 2N VWD in a murine model. Type 2N VWD mutations are associated with impaired secretion of VWF and/or decreased binding and stabilization of endogenous FVIII. DisclosuresNo relevant conflicts of interest to declare.

Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.

A Novel Rapid Screening Assay for the Diagnosis of the Phenotypic Variants of Von Willebrand Disease (VWD)

▪ Backgroundvon Willebrand disease (VWD) is a very common inherited bleeding disorder. The current phenotypic classification of VWD variants includes disorders of both quantitative and qualitative defects in von Willebrand Factor (VWF) that determines optimal treatment of patients. Current phenotype determination is multimodal, cumbersome, performed by only a few specialized laboratories, and may delay the definitive diagnosis necessary in proper selection of therapy. We have developed an ELISA-based strip assay that is capable of rapid determination of relative qualitative and quantitative VWF functionality to correctly assign phenotypic variants of VWD. Methods136 VWD plasma samples from the Zimmerman PPG were analyzed on a new ELISA based platform. In single, individual wells this assay measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:F8B (binding to FVIII), VWF:CB3 (binding to collagen III), and VWF:pp (propeptide) in comparison to a 30% normal control standard in a single ELISA strip assay. The study included 22 type 1 VWD, 32 type 1C VWD, 18 type 2A VWD, 23 type 2B VWD, 20 type 2M VWD, 7 type 2N VWD, 4 type 3 VWD, and 10 hemophilia A subjects. Each sample was run in single wells for each assay and optical densities (OD) were compared to the OD of a 30% standard control plasma and a 100% VWF:Ag control. The standard ELISA plate read time was 30 minutes and full assay can be accomplished in 3 hours. Two of the coauthors were blinded as to the Zimmerman PPG VWD phenotypes of test samples. Using the ELISA strip results, phenotype assignment was determined and then compared to the unblinded Zimmerman PPG VWD diagnosis. Further statistical analysis of VWF functional profile relationships was performed using the Mann-Whitney test and ROC analysis, and can quantify the ability to identify these phenotypes. ResultsVWF functional profiles based on visually observed ratio relationships correctly assigned VWD phenotypic variant on first attempt in 122 of 136 subjects (89.7%). Repeat testing of the 14 incorrectly assigned subjects along with 11 random, correctly assigned subjects for a validation check, accurately re-assigned 9 of 14 previously incorrect phenotypes, suggesting initial plate to plate variability since all ELISA plates were made fresh for each run. Previously correctly assigned subjects, 11 of 11, remained correctly assigned. Comparing specific phenotypes revealed VWF:IbCo/VWF:Ag is good at separating type 1C from 2A; ROC area under the curve 0.875, with an optimal ratio threshold 0.649 (sensitivity 0.969, specificity 0.611, p<0.001) and excellent at separating type 2B from type 1; ROC area under the curve 0.978 with a ratio threshold 1.268 (sensitivity 1.000, specificity 0.864, p<0.001), but was poor at separating type 2A from 2M; ROC area under the curve 0.622. VWF:F8B/VWF:Ag was excellent at separating hemophilia A from type 2N; ROC area under the curve 1.000 with a ratio threshold 0.865 (sensitivity 1.000, specificity 1.000, p<0.001). VWF:CB3/VWF:Ag was excellent at separating type 2M from 2A; ROC area under the curve 0.961 with a ratio threshold 0.757 (sensitivity 0.950, specificity 0.889, p<0.001) and excellent at separating type 1C from 2A; ROC area under the curve 0.922 with a ratio threshold 0.597 (sensitivity 1.000, specificity 0.778, p<0.001). ConclusionsThe rapid VWF functional screening assay is able to discriminate VWD phenotypic variants with good sensitivity and specificity, and may facilitate the rapid laboratory assignment of VWD phenotypes once a subject with low VWF is identified. Disclosures:No relevant conflicts of interest to declare.

VWF Interaction With Type IV Collagen Is Mediated Through Critical VWF A1 Domain Residues

Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium via binding sites for platelet glycoprotein Ib and collagen. The binding sites for types I and III collagen in the VWF A3 domain are well characterized, and defects in this region have been implicated in von Willebrand disease (VWD). Additional collagens present in the vasculature may also be involved in interactions with VWF. A VWF A1 sequence variation, p.R1399H, has been associated with decreased binding to type VI collagen, but the clinical significance of this observation remains unclear. Type IV collagen is a common component of the basement membrane and as such may be an important ligand for VWF. While some VWD testing utilizes types I or III collagen, current clinical testing does not include collagen IV or VI.To characterize the role of the VWF A1 domain in VWF-type IV collagen interactions, we generated several A1 domain variant human and/or murine recombinant VWF (rVWF) constructs including R1399H and several type 2M VWD variants localized to the same region (S1387I, Q1402P, and an 11 amino acid deletion mutant encompassing amino acids 1392-1402). These constructs were then expressed in HEK 293T cells. To further assess the role of the A1 domain, scanning alanine mutagenesis (SAM) of residues 1387 through 1412 was conducted. VWF antigen levels (VWF:Ag), collagen binding with type III (VWF:CB3), IV (VWF:CB4), or VI (VWF:CB6) collagen were determined, and multimer distribution was assessed for all recombinant VWF variants.The role of R1399H in the context of human rVWF was characterized initially. Although VWF:Ag, VWF:CB3, and multimer distribution were normal for R1399H compared to wild-type (WT VWF), VWF:CB4 was undetectable. To examine this effect in a mouse model, the R1399H variant was expressed in the context of murine rVWF and collagen binding was determined. Similar to the human variant, murine R1399H rVWF demonstrated significantly reduced binding to murine type IV collagen, at only 7% of the binding seen with WT murine rVWF. In order to examine the behavior of R1399H under shear conditions, either WT or R1399H murine rVWF DNA was hydrodynamically injected into the tail veins of VWF -/- mice to induce expression of the proteins; blood was drawn from the vena cava 24 hours later and then examined on the VenaFlux flow apparatus. VWF expression levels and multimer distribution were similar for the R1399H- and WT-injected mice. Under static conditions, the murine plasma-derived R1399H demonstrated decreased VWF:CB4, at only 16% of the levels seen with WT VWF. No defect was seen in VWF:CB3. Furthermore, when binding to type IV collagen was assessed under flow conditions by VenaFlux, platelet adhesion was significantly decreased in mice expressing R1399H VWF as compared to mice expressing WT VWF.When examining other A1 domain variants, Q1402P and del1392-1402 demonstrated absent VWF:CB4 while S1387I demonstrated a significant reduction in VWF:CB4 compared to WT VWF. All SAM VWF A1 domain variants demonstrated normal expression, multimerization, and VWF:CB3. However, type IV collagen binding was absent for R1392A, R1395A, R1399A, and K1406A and was reduced to less than 50% of WT VWF for Q1402A, K1405A, and K1407A. These residues map to an outside face of the VWF A1 domain crystal structure, and are likely the critical residues for VWF binding to type IV collagen.Taken together, these data demonstrate that the type IV collagen binding site localizes to a specific region of the VWF A1 domain. Mutations in this region of VWF may be clinically significant due to a defect in the ability of VWF to attract platelets to exposed type IV collagen which may contribute to bleeding symptoms seen in VWD. Disclosures:No relevant conflicts of interest to declare.

Binding Of VWF To Type IV Collagen: An Additional Collagen Binding Mechanism Beyond Types I, III, and VI Collagen?

Von Willebrand disease (VWD) is a common bleeding disorder characterized by defects in the interaction of von Willebrand factor (VWF) with its ligands factor VIII, platelet glycoprotein Ib, and/or collagen. Nearly all assessment of collagen binding is performed with types I or III collagen. Type VI collagen, which is formed as a tight triple helix, has also been reported to interact with VWF. In contrast, type IV collagen lacks this tight helical structure and is located in the basal lamina. Type IV collagen may therefore serve as another important ligand for VWF. We examined this interaction in samples from the Zimmerman Program for the Molecular and Clinical Biology of VWD, a large US study of subjects with all types of VWD.Subjects were enrolled based on a pre-existing diagnosis of VWD and compared to a cohort of healthy controls. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and type III collagen binding using 1 mcg/mL human placental type III collagen (VWF:CB3) were performed in the clinical laboratory at the BloodCenter of Wisconsin. Type IV and type VI collagen binding (VWF:CB4 and VWF:CB6) were performed in the research laboratory by ELISA using 1 mcg/mL human placental type IV or type VI collagen captured on amine binding plates. DNA sequencing was performed on all subjects to identify VWF sequence variations. Bleeding scores were calculated according to the scoring system used in the MCMDM-1VWD study.For this analysis, 251 healthy controls, 535 type 1, 49 type 2A, 16 type 2B, 18 type 2M, 11 type 2N, and 18 type 3 VWD were included. The mean VWF:CB4/VWF:Ag ratio for the healthy controls was 0.96, with a standard deviation of 0.24. This was similar to the mean VWF:CB6/VWF:Ag ratio (mean 0.95, p=NS), but slightly lower than the mean VWF:CB3/VWF:Ag ratio (mean 1.05, p<0.01). The mean VWF:CB4/VWF:Ag ratio was 0.87 for the type 1 VWD subjects. The mean VWF:CB4/VWF:Ag ratio was significantly lower in type 1 subjects as compared to healthy controls (p<0.01). This was attributed in part to the presence of several A1 domain sequence variations, including p.R1399H, p.F1369I, and p.L1382P. While the latter sequence variations were isolated findings in single subjects, p.R1399H was present in 1% of the healthy controls, 1% of the type 1, and 6% of the type 2M VWD subjects.Mean VWF:CB4/VWF:Ag ratios were low for both type 2A and type 2B VWD subjects, at 0.54 and 0.52 respectively. This was not significantly different from the VWF:CB3/VWF:Ag data (p=NS for both type 2A and 2B VWD), consistent with the loss of high molecular weight multimers in these subjects and the concomitant reduction in collagen binding. As expected, VWF:CB4/VWF:Ag ratios were normal in the type 2N subjects, with a mean of 1.04. The type 2M subjects, however, displayed a significant reduction in type IV collagen binding, with a mean VWF:CB4/VWF:Ag ratio of 0.79. One subject with an 11 amino acid deletion (from p.R1392 through p.Q1402) had absent binding to type IV collagen yet normal binding to type III collagen with a VWF:CB3/VWF:Ag ratio of 0.82. Another subject with p.I1425F and p.R1399H had a VWF:CB4/VWF:Ag ratio of 0.48, and VWF:CB3/VWF:Ag ratio of 1.10. Type IV collagen binding did not correlate with VWF:RCo (R squared less than 0.1). The type 2M subjects with a mean VWF:CB4/VWF:Ag ratio less than 0.7 had higher bleeding scores, with a mean bleeding score of 9 compared to a mean of 6 for type 2M subjects with a mean VWF:CB4/VWF:Ag ratio greater than 0.7. Not surprisingly, the subjects with type 3 VWD and undetectable VWF:Ag also had undetectable VWF:CB4.These data suggest that type IV collagen binding reflects an independent function of VWF apart from platelet binding, and distinct from binding to type III collagen. In our study, more variation was seen between type IV and type III collagen than between type IV and type VI collagen. Neither VWF:CB4 nor VWF:CB6 are currently tested as part of the clinical workup of VWD. Measurement of this function may assist in identification of variant VWD, particularly in the subgroup of patients with type 2M VWD. Disclosures:No relevant conflicts of interest to declare.

Collagen Binding Provides a Sensitive Screen for Variant von Willebrand Disease

von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test. Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between individuals with multimer group results within (controls) and outside the reference intervals were assessed with Mann-Whitney tests. The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with multimer distribution within the reference intervals and 0.51 for those with multimer distribution outside the reference intervals (P < 0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for patients with type 1, and 100% for patients with type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all patients with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD. These findings suggest that VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate the clinical utility of VWF:CB.

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.

Biogenesis and Exocytosis of Weibel-Palade Bodies Is Affected by Naturally Occurring Von Willebrand Disease Variants within the A1-A3 Domains of VWF

AbstractAbstract 1072 Background:Von Willebrand Factor (VWF) is synthesized in endothelial cells and megakaryocytes and is either secreted constitutively into plasma or stored in specific organelles; Weibel-Palade bodies (WPB) in endothelial cells or α -granules in megakaryocytes and platelets. Release of von Willebrand factor from WPB in response to desmopressin (DDAVP), an agonist of WPB exocytosis, is clinically applied to raise VWF plasma levels in patients with von Willebrand disease (VWD). A subset of patients with VWD type 1, a quantitative defect, shows a reduced response to DDAVP. This reduced response suggests the absence of recruitable WPB. The VWF propeptide (D1-D2 domains) together with the D’D3 domains are necessary for the tubular assembly of VWF. The assembly of VWF into tubules drives WPB formation. Some variants in the VWF A1 to A3 domains have been linked to reduced DDAVP responsiveness. This suggests that determinants for WPB formation may reside outside the D1-D2-D’-D3 domains. We hypothesize that a reduced tendency to assemble into VWF tubules underlies the defective DDAVP response in VWD type 1 patients with mutations in the A domains of VWF. Methods:Human Embryonic Kidney (HEK) 293 cells were transiently transfected with plasmids containing full-length wild-type VWF or 6 naturally occurring VWF variants located throughout the A domains of VWF. The following mutations, originally identified in type 1 VWD patients, were studied: p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His (A1 domain), p.Tyr1584Cys, p.Arg1583Trp (A2 domain) and p.Val1822Gly (A3 domain). Medium and lysates of transfected cells were collected to measure basal VWF secretion. Transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the regulated VWF secretion. Confocal- and Transmission electron microscopy (TEM) were used to study the formation of WPB. Results:Cells transfected with WT-VWF or VWF p.Arg1583Trp formed numerous elongated pseudo-WPB as evidenced by confocal microscopy. p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His, p.Tyr1584Cys and p.Val1822Gly were able to form these organelles, but in most cases the pseudo-WPB were shorter and more round compared to those formed by WT-VWF. Retention of VWF in the ER was present in about 50% of the cells expressing p.Leu1307Pro and 25–35% of the cells expressing p.Ser1285Pro and p.Val1822Gly as compared to 10% in WT-VWF. The ER retention in p.Arg1374His, p.Tyr1584Cys and p.Arg1583Trp variants was comparable with WT-VWF. Upon co-transfection with WT-VWF the defective elongation of pseudo-WPB was partly corrected. WPB formation was further studied using TEM. In WT-VWF transfected cells, elongated and electron dense pseudo-WPB were observed. p.Arg1583Trp showed cigar-shaped pseudo-WPB with typical VWF striations. Shorter and more round pseudo-WPB were found in cells expressing the VWF variants p.Ser1285Pro, p.Arg1374His, p.Tyr1584Cys. The p.Val1822Gly variant showed some elongated pseudo-WPB, although most of the structures were round. The round organelles are, however, recognizable as pseudo-WPB as they contain tubular structures indicating storage of VWF tubules. The p.Leu1307Pro and p.Val1822Gly showed reduced VWF basal secretion, even in the heterozygous state. Both mutations, together with p.Ser1285Pro, also showed impaired regulated secretion of VWF in both single and co-transfections with wt-VWF. Conclusion:Our data shows that naturally occurring VWD variants within the A domains of VWF can cause defects in both WPB formation and (regulated) secretion of VWF. This is in contrast with previous data which suggests that only the propeptide and D1'-A1 domain of VWF are essential for normal WPB formation. In our study however, we found that two mutations located within the A2 and A3 domain (p.Tyr1584Cys and p.Val1822Gly) also interfere with the formation of elongated WPB as evidenced by round storage organelles containing VWF tubules. Defects in WPB formation and regulated secretion of VWF may be the underlying cause of the poor response to DDAVP infusion seen in a subset of VWD type 1 patients. Disclosures:No relevant conflicts of interest to declare.