Von Willebrand Disease Mutations Research Articles

Phenotypic and genotypic (exon 28) characterization of patients diagnosed with von Willebrand disease type 1 in Eastern Saudi Arabia.

Von Willebrand factor (VWF) is a plasma glycoprotein that plays a key role in hemostasis. Mutations in this protein can result in von Willebrand disease (VWD), the most common form of bleeding disorder in humans. Patients with type 1 VWD have a quantitative plasmatic deficiency of normal structural and functional VWF. Our study aimed to investigate the phenotypic and genotypic characteristics of VWD type 1 patients in eastern Saudi Arabia, focusing on exon 28. We included patients previously diagnosed with WWD type 1 at the King Fahad teaching hospital in Al Khobar and their family members. The correlations between various phenotypic data and genotypic (exon 28) were analyzed using statistical software (SPSS) version 21. While these variants were generally considered benign with minor clinical effects, our analysis did identify two pathogenic variants that could lead to severe VWD symptoms. Specifically, we found these two pathogenic variants in three VWD patients from Saudi Arabia, providing essential insights into pathogenic VWD mutations in this population. Our study, therefore, sheds light on the prevalence of VWF variants in the eastern province of the Kingdom and highlights the need for continued research into the genetic causes of VWD in this region.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain

AbstractType 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain’s thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.

Novel gene mutation causing type 3 Von Willebrand disease: a case report and review of literature

Von Willebrand disease (VWD) Type 3 is an uncommon bleeding disorder, resulting from the near absence of Von Willebrand factor (VWF) and extremely low factor-VIII levels. It is a close differential diagnosis of hemophilia. A wide heterogeneity of VWD mutations are reported in the literature. We report a 16-year-old girl with hemarthrosis, finally diagnosed with Type 3 VWD. Clinical exome sequencing confirmed the diagnosis, revealing a homozygous mutation c.4387G>T (p. Glu1463Ter) in exon 28 of the VWF gene, a unique mutation not yet reported in the literature.

CLINICAL AND HEMORRHAGIC PROFILES OF PATIENTS WITH VON WILLEBRAND DISEASE

The von Willebrand factor (VWF) gene is located on Chr12p12, and deleterious mutations in this gene cause von Willebrand disease (VWD). VWD is an inherited coagulopathy that affect FvW both quantitatively and qualitatively. There are three main types of the disease (types 1, 2, and 3) and a total of 6 subgroups. In type 1, patients have a lower concentration of circulating FVW. In type 2, a lower activity of circulating FVW is observed, and in type 3, there is virtually no circulating FVW. VWD is the most common and least diagnosed coagulation disorder. The aim of this study was to analyze the clinical and hemorrhagic profiles of patients with VWD treated at Fundação Hemominas. Patients were invited to participate when they visited the treatment center. The 30 patients answered two questionnaires: 1) Bleeding Assessment Score - ISTH (BAT-ISTH); and 2) Arthralgia Assessment. Among female patients (n = 17) the mean (MED) age was 38.5 years (IQR 34.5-43.8) and the MED bleeding score was 13 (IQR 8.8-16.5). The most common bleeding events occurring were: cutaneous bleeding (n = 15 – 88.2%), bleeding from the oral cavity (n = 14 – 82.4%), menorrhagia (n = 13 – 74.5%), bleeding in small wounds (n = 11 – 64.7%) and dental extractions (n = 11 – 64.7%). In addition, eight patients (47%) presented shoulder and knee pain. Among male patients (n = 13) the MED age was 42.5 years (IQR 24-54.8) and the bleeding score MED was 13 (IQR 8-17.8). The most common bleeding events occurring were: bleeding from the oral cavity (10 – 76.9%), epistaxis (8 – 61.5%), cutaneous bleeding (7 – 53.8%), dental extractions (7 – 53.8%), and other types of bleeding (7 – 53.8%). Shoulder pain was related by five patients (38.5%). Based on the clinical hemorrhagic profile of patients, it was found a statistical difference (p = 0.044) between male and female regarding epistaxis (61.5% in male and 29.4% female patients). This study was limited by aggregation of patients in one group independently of VWD type and no patients were genotyped. Furthermore, this is an ongoing project and the number of participants needs to be increased to draw conclusions. We thank our participants, Fapemig, Hemominas, and the CGSH of the Ministry of Health.

Analysis of von Willebrand Disease in the "Heart of Europe".

Background von Willebrand disease (VWD) is a genetic bleeding disorder caused by defects of von Willebrand factor (VWF), quantitative (type 1 and 3) or qualitative (type 2). The laboratory phenotyping is heterogenic making diagnosis difficult. Objectives Complete laboratory analysis of VWD as an expansion of the previously reported cross-sectional family-based VWD study in the Czech Republic (BRNO-VWD) and Slovakia (BRA-VWD) under the name “Heart of Europe,” in order to improve the understanding of laboratory phenotype/genotype correlation. Patients and Methods In total, 227 suspected VWD patients were identified from historical records. Complete laboratory analysis was established using all available assays, including VWF multimers and genetic analysis. Results A total of 191 patients (from 119 families) were confirmed as having VWD. The majority was characterized as a type 1 VWD, followed by type 2. Multimeric patterns concordant with laboratory phenotypes were found in approximately 83% of all cases. A phenotype/genotype correlation was present in 84% (77% type 1, 99% type 2, and 61% type 3) of all patients. Another 45 candidate mutations (23 novel variations), not found in the initial study, could be identified (missense 75% and truncating 24%). An exon 1–3 gene deletion was identified in 14 patients where no mutation was found by direct DNA sequencing, increasing the linkage up to 92%, overall. Conclusion This study provides a cross-sectional overview of the VWD population in a part of Central Europe. It is an addition to the previously published BRNO-VWD study, and provides important data to the International Society of Thrombosis and Haemostasis/European Association for Haemophilia and Allied Disorders VWD mutation database with identification of novel causal mutations.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

Comparison of von Willebrand factor platelet‐binding activity assays: ELISA overreads type 2B with loss of HMW multimers

BackgroundA number of new assays with different measuring principles are available to measure von Willebrand factor (VWF) glycoprotein Ib (GPIb)‐binding activity, but little is known about how these assays might behave differently for subtypes of von Willebrand disease (VWD). ObjectivesThe Comparison of Assays to Measure VWF Activity (COMPASS‐VWF) study was designed to compare all available VWF GPIb‐binding activity assays for VWF. We specifically searched for particular assay behavior differences. Patients/MethodsTo sort out random differences from systematic assay behavior deviations, all assays were performed in different laboratories on the same samples in a blinded fashion. Samples from 53 normal controls and 42 well‐characterized VWD patients were reanalyzed in this study to dissect assay‐specific discrepancies. ResultsNo assay behavior differences were found for 53 normal controls. For VWD patients, we found the following systematic assay behavior patterns: (a) All ELISA assays for VWF:GPIbR as well as VWF:GPIbM are insensitive to detect the low VWF activity of VWD type 2B patients with loss of high molecular weight multimers; (b) VWF:Ab assay reports higher activity for the p.V1665E mutation than all other assays; and (c) all ristocetin‐based assays (including VWF:RCo using fixed platelets) but the AcuStar assay report discrepantly low VWF activity for the p.P1467S polymorphism. No systematic assay‐specific difference was observed for either the particle agglutination VWF:GPIbM assay or the AcuStar assay using magnetic beads. ConclusionsDifferent assay principles may lead to discrepant results for certain VWD types or mutations. Therefore, a more extensive study for a large number of patients is needed to better characterize the incidence and relevance of such assay‐specific differences.

Platelet‐type von Willebrand disease: Local disorder of the platelet GPIbα β‐switch drives high‐affinity binding to von Willebrand factor

BackgroundMutations in the β‐switch of GPIbα cause gain‐of‐function in the platelet‐type von Willebrand disease. Structures of free and A1‐bound GPIbα suggest that the β‐switch undergoes a conformational change from a coil to a β‐hairpin. ObjectivesPlatelet‐type von Willebrand disease (VWD) mutations have been proposed to stabilize the β‐switch by shifting the equilibrium in favor of the β‐hairpin, a hypothesis predicated on the assumption that the complex crystal structure between A1 and GPIbα is the high‐affinity state. MethodsHydrogen‐deuterium exchange mass spectrometry is employed to test this hypothesis using G233V, M239V, G233V/M239V, W230L, and D235Y disease variants of GPIbα. If true, the expectation is a decrease in hydrogen‐deuterium exchange within the β‐switch as a result of newly formed hydrogen bonds between the β‐strands of the β‐hairpin. ResultsHydrogen—exchange is enhanced, indicating that the β‐switch favors the disordered loop conformation. Hydrogen—exchange is corroborated by differential scanning calorimetry, which confirms that these mutations destabilize GPIbα by allowing the β‐switch to dissociate from the leucine‐rich‐repeat (LRR) domain. The stability of GPIbα and its A1 binding affinity, determined by surface plasmon resonance, are correlated to the extent of hydrogen exchange in the β‐switch. ConclusionThese studies demonstrate that GPIbα with a disordered loop is binding‐competent and support a mechanism in which local disorder in the β‐switch exposes the LRR—domain of GPIbα enabling high‐affinity interactions with the A1 domain.

Cryo-EM Structure of BIVV001 Reveals Coagulation Factor VIII-Von Willebrand Factor D'D3 Interaction Mode

Introduction Coagulation Factor VIII (FVIII) is a serine protease cofactor that directly interacts with coagulation factors IXa and X on activated platelets, and enhances FIXa activity toward FX by 105. von Willebrand Factor (VWF), via its D'D3 domains, interacts with FVIII and prevents premature deposition on phospholipids until activation by thrombin. Thrombin cleavage at Arg1689 of FVIII promotes VWF dissociation by disrupting the FVIII a3 high affinity interaction with the VWF D' domain. VWF extends the half-life of circulating FVIII from less than 3 hours to ~11 hours in humans. While crystal structures of FVIII and VWF D'D3 alone have been solved, the atomic details of a formed complex are unknown. We sought to determine the FVIII-VWF D'D3 complex structure by using BIVV001, our investigational new drug currently in clinical trials for the treatment of Hemophilia A. BIVV001 (rFVIIIFc-VWF-XTEN) is a novel fusion protein consisting of single chain B-domain deleted (BDD) human FVIII, the Fc domain of human immunoglobulin G1 (IgG1), the FVIII-binding D'D3 domain of human von Willebrand factor, and 2 XTEN polypeptide linkers. The Fc, VWF, and XTEN linker portions of the molecule are each designed to extend the half-life of FVIII. We anticipated that the tethering of FVIII to D'D3 through the Fc dimer in BIVV001 would stabilize the complex for structural studies. Given the large size of BIVV001, at 312 kDa, we thought it an ideal target for structure determination by single particle cryo-EM. Methods We collected a total of 3955 micrographs of BIVV001 embedded in vitreous ice at 81,000x magnification using a Titan Krios electron microscope equipped with a Gatan BioQuantum K3 energy filter and camera operating in super-resolution mode. Preferential particle orientation was a major challenge that was overcome through a variety of methods. Micrograph movies were motion-corrected and summed, and over 2 million candidate particle coordinates were extracted. Repeated rounds of reference-free 2D classification resulted in a set of 1.2 million particles that generated a reasonable ab initio/de novo 3D model. Initial full 3D refinements of this model produced a map at approximately 5 Å resolution, into which available crystal structures can be readily fit. Subsequent iterative 3D refinement and 3D classification resulted in a final map at high resolution, into which an atomic model was built. Results The structure of BIVV001 was solved by single particle cryo-EM. D' of VWF interacts with the front face of the C1 and A3 domains of FVIII, consistent with a lower resolution, negative stain EM map (Yee et al. 2015. Blood). Interface residues on FVIII identified in an HDX-MS dataset (Chiu et al. 2015. Blood.) largely correspond to this high affinity interaction. D' protrudes upward from the VWF D3 domain, which sits centrally located between the C1 and C2 domains of FVIII at a 45° tilt. By occupying this position, D3 likely sterically blocks the FVIII C domains from binding to membrane. The VWD3 module of the D3 domain contacts the base of the C1 domain, whereas C8-3 binds to the bottom of the C2 domain. The conserved Ca2+ site in VWD3 identified previously (Dong et al. 2019. Blood.) is in the interface with C1. This is consistent with Yee et al., where docking placed D3 below the C domains. In that study, a lack of density between FVIII and VWF D3 in the 3D reconstruction, due to flexibility, prevented the detailed analysis that is possible here. In this study, flexibility in this region is also apparent, as C2 is less well ordered than the rest of FVIII and VWF D3 is the least well-ordered portion of the resolved structure. The XTEN linkers are not visible in the final map and were not apparent in any 2D class averages. The Fc is absent in most 2D class averages, due to a lack of consistent positioning relative to FVIII. In the rare cases where the Fc is visible, it adopts a preferred position on the back side of FVIII below the A3 protrusion. Conclusions The structure of BIVV001 has been solved by cryo-electron microscopy to high resolution. Alignment with previous results and the averaging out of BIVV001 elaborations suggests the structure obtained here likely represents WT FVIII-D'D3. This structure demonstrates how VWF D'D3 prevents premature FVIII deposition on phospholipids. The structural basis of type 2N von Willebrand Disease mutations in D'D3 can be readily interpreted. Next steps include solving a FVIII-D'D3 dimer structure at high resolution. Disclosures Fuller: Sanofi: Employment. Batchelor:Sanofi: Employment. Knockenhauer:Sanofi: Employment. Biemann:Sanofi: Employment. Peters:Sanofi: Employment.

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.

The von Willebrand factor D′D3 assembly and structural principles for factor VIII binding and concatemer biogenesis

AbstractD assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D′D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D′D3 forms the binding site for factor VIII. The crystal structure of monomeric D′D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D′ with its TIL′ and E′ modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL′ domain of D′ distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

Analysis of von Willebrand Disease in the South Moravian Population (Czech Republic): Results from the BRNO-VWD Study.

von Willebrand disease (VWD) is an inherited bleeding disorder caused by a quantitative (type 1 and 3) or qualitative (type 2) defect of von Willebrand factor (VWF). The heterogeneity of laboratory phenotyping makes diagnosing difficult. A cross-sectional, family-based VWD study in a collaboration between University Hospital Brno (Czech Republic) and Antwerp University Hospital (Belgium) to improve the understanding of laboratory phenotype/genotype correlation. A total of 205 patients with suspected VWD were identified from historical records. Complete laboratory analysis was established using all available VWD assays including VWF multimers and genetic analysis. Based on the current International Society of Thrombosis and Haemostasis (ISTH) - Scientific and Standardization Committee VWD classification and type 2A sub-division into 2A/IIA, IID, IIC and IIE, the majority was characterized as a type 1 VWD, followed by type 2. Proposed laboratory phenotypes were confirmed by their multimeric pattern within 98% of this cohort. All type 2, 3 and 75% of type 1 VWD patients were confirmed by underlying causative mutations. Forty-six different causal mutations (117 not previously described in the literature) could be identified. Fifty per cent of all cases was represented by eight individual mutations, mainly p.Pro812ArgfsX31. Thirteen patients had a large heterozygous gene alteration. Although an extensive panel of tests was used, VWD classification and (sub)typing remains difficult and fluid. This study provides a cross-sectional overview of the VWD population in the Czech Republic and provides important data to the ISTH/European Association for Haemophilia and Allied Disorders VWD mutation database in linking causal mutations with unique VWD (sub)types. It also identifies new, as not previously described in the literature, causal mutations.

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

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

A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor

Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

A novel platelet-type von Willebrand disease mutation (GP1BA p.Met255Ile) associated with type 2B “Malmö/New York” von Willebrand disease

Interaction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also result in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GP1BA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting the presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT- with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.

On-Rate Switching under Force Increases the Binding of von Willebrand Factor A1 to GPIbα

Alterations in flow during bleeding trigger the von Willebrand factor (VWF) A1 domain to bind to glycoprotein Ibα (GPIbα) on platelets, initiating blood clotting. Using a laser tweezers and a Receptor and Ligand in a Single Molecule (ReaLiSM) construct, we find that force can switch A1 and/or GPIbα to a different state with a faster on-rate; this provides an additional mechanism for activating VWF binding to platelets. Mutations in VWF induce von Willebrand disease (VWD), a common human bleeding disorder. Our results show that force escalates the effects of VWD mutations with respect to on-and off-rates, explaining pathophysiology. Using the effective concentration of receptor and ligand in ReaLiSM to convert single molecule kon (units of s−1) to bulk phase kon (units of s−1M−1) allows the calculation of an equivalent binding affinity. We find that using extrapolated, zero-force kon and koff values from the low-force state of the bond to calculate the binding affinity provides remarkably good agreement with bulk phase measurements.

The co-influence of VWD type 2B/2M mutations in the A1 domain and platelet GPIbα on the rate of cleavage to VWF by ADAMTS13

IntroductionIn plasma, the size of the von Willebrand factor (VWF) multimer is down-regulated by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13). The binding of platelets or glycoprotein (GP) Ibα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to VWF. Both type 2B and type 2M von Willebrand disease (VWD) result in bleeding disorders with the diathesis of increased and decreased binding affinity between GPIbα and VWF, respectively. However, the influence of 2B/2M VWD mutations in the A1 domain and GPIbα on cleavage by ADAMTS13 to VWF needs further study. Materials and MethodsDifferent types of full-length human recombinant VWF (rVWF) were expressed, including three type 2B mutations (P1337L, H1268D, and R1308C), one type 2M mutation (D1302G), and wild type (WT). The three characterized types of rVWF were digested by ADAMTS13 under static conditions or high-shear stress. The interaction of rVWF and ADAMTS13 was also tested by plate-binding assays. ResultsUnder static (natured) conditions or high-shear stress, type 2B mutants exhibited a higher susceptibility to ADAMTS13 than rVWF-WT, whereas type 2M mutant was normal. While under static (denatured) conditions or high-shear stress (with GPIbα fragment) rVWF-WT showed an even higher susceptibility to ADAMTS13 than the two type 2B mutants studied. ConclusionType 2B mutations localized in the A1 domain could enhance the sensitivity to ADAMTS13-mediated proteolysis. When GPIbα participated, there was a dramatically increased proteolytic cleavage of VWF by ADAMTS13 to rVWF-WT, excluding some type 2B mutants.

Structural Origins of Misfolding Propensity in the Platelet Adhesive Von Willebrand Factor A1 Domain

The von Willebrand factor (VWF) A1 and A3 domains are structurally isomorphic yet exhibit distinct mechanisms of unfolding. The A1 domain, responsible for platelet adhesion to VWF in hemostasis, unfolds through a molten globule intermediate in an apparent three-state mechanism, while A3 unfolds by a classical two-state mechanism. Inspection of the sequences or structures alone does not elucidate the source of this thermodynamic conundrum; however, the three-state character of the A1 domain suggests that it has more than one cooperative substructure yielding two separate unfolding transitions not present in A3. We investigate the extent to which structural elements contributing to intermediate conformations can be identified using a residue-specific implementation of the structure-energy-equivalence-of-domains algorithm (SEED), which parses proteins of known structure into their constituent thermodynamically cooperative components using protein-group-specific, transfer free energies. The structural elements computed to contribute to the non-two-state character coincide with regions where Von Willebrand disease mutations induce misfolded molten globule conformations of the A1 domain. This suggests a mechanism for the regulation of rheological platelet adhesion to A1 based on cooperative flexibility of the α2 and α3 helices flanking the platelet GPIbα receptor binding interface.

Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases

Mutations in the ultralong vascular protein von Willebrand factor (VWF) cause the common human bleeding disorder, von Willebrand disease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding. Gain-of-function mutations in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster on-rate, providing a mechanism for activating VWF binding to platelets. Switching occurs near 10 pN, a force that also induces a state of the receptor-ligand complex with slower off-rate. Force greatly increases the effects of VWD mutations, explaining pathophysiology. Conversion of single molecule kon (s(-1)) to bulk phase kon (s(-1)M(-1)) and the kon and koff values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.

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.