Mutations In Glycoprotein Research Articles

Adaptation potential of H3N8 canine influenza virus in human respiratory cells

In 2004, the equine-origin H3N8 canine influenza virus (CIV) first caused an outbreak with lethal cases in racing greyhounds in Florida, USA, and then spread to domestic dogs nationwide. Although transmission of this canine virus to humans has not been reported, it is important to evaluate its zoonotic potential because of the high contact opportunities between companion dogs and humans. To gain insight into the interspecies transmissibility of H3N8 CIV, we tested its adaptability to human respiratory A549 cells through successive passages. We found that CIV acquired high growth properties in these cells mainly through mutations in surface glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). Our reverse genetics approach revealed that HA2-K82E, HA2-R163K, and NA-S18L mutations were responsible for the increased growth of CIV in human cells. Molecular analyses revealed that both HA2 mutations altered the optimum pH for HA membrane fusion activity and that the NA mutation changed the HA-NA functional balance. These findings suggest that H3N8 CIV could evolve into a human pathogen with pandemic potential through a small number of mutations, thereby posing a threat to public health in the future.

An Insight into the Roles of Zona Pellucida in Growth and Development of Mammalian Ocytes and Embryos: Changes of Age-related and Cryopreservation: A Review

The zona pellucida (ZP) is a extracellular transparent coat surrounding the oocyte and preimplantation embryo in mammals. The ZP separates between the surrounding cumulus cells and oocyte. It plays a pivotal role in oocyte growth and fertilization and early embryo development. Zona pellucida formation and glycoproteins, roles of ZP on female fertility and infertility, ZP micromanipulation on embryo development and changes of ZP upon vitrification of oocytes is discussed. Zona pellucida is formed of glycoproteins during ovarian follicle growth and development. It protects the oocyte from physical or chemical damage, initiates fertilization, prevents polyspermy and supports early embryonic development. The ZP is responsible for recognition between oocytes and sperm. Mutations in glycoproteins cause female infertility characterized by empty follicle syndrome and oocyte degeneration. The ZP removal is detrimental for embryo implantation. Furthermore, there is a biophysical role of ZP during vitrification of oocytes. Hence, the current review article was designed to collect, discuss and highlight the characters of zona pellucida, roles in female fertility and infertility, ZP micromanipulation on embryo development and changes of ZP upon vitrification.

On the in silico application of the center-of-mass distance method

This study aims to protocolize the utilization of the center-of-mass (CoM) distance method in GROMACS MD simulation software as a useful method for evaluating the binding affinity change in heterodimeric protein due to induced changes in one of the units. The hypothesis underlines the basic principles in biophysics, that an increase of the binding affinity is expected to reduce the relative CoM distance between monomers, while the opposite is expected to increase the relative CoM distance. However, it has been found that the CoM distance analysis must be strictly preformed during the convergent phase of systems’ dynamics, once the monomers enter mutually stable conformation — a limitation which has usually been overlooked. The method was used to study the impact of K417Y severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface glycoprotein (S-protein) mutation. It has been found that the K417Y mutation favors reduced binding affinity between SARS-CoV-2 S-protein and human angiotensin-converting enzyme 2 (hACE2) receptor, which is due to the loss of the permanent K417-D30 salt bridge in favor of a temporary Y417-D30 hydrogen bond. The destabilizing impact of K417Y mutation on S-protein–hACE2 complex was confirmed by radius of gyration analysis.

Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Analysis of highly frequent point mutations in glycoprotein C, glycoprotein N, and nucleoprotein of CCHFV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is classified among top 10 priority pathogens by World Health Organization. CCHFV belongs to Bunyaviridae family and negative sense ssRNA genome composed of three RNA segments: L, M, and S. RNA viruses show higher mutation rate as compared to DNA viruses. To gain deeper understanding of impact of point mutations in CCHFV M and S segment, mutation profiling, homology modeling, and molecular dynamic (MD) simulation were performed. Structural glycoproteins (glycoprotein C [Gc] and glycoprotein N [Gn]) of CCHFV are important for host-virus interaction and genome packaging, whereas CCHFV nucleoprotein (NP) is crucial for viral replication. Hence, current study is focused on evaluation of eight mutations in structural glycoproteins (Gc: 7 and Gn: 1) of M segment and seven mutations in NP of S segment. All these mutations were highly frequent, with mutation frequency between 0.81 and 1.0 and found to be persistent in the recent strains of CCHFV. Solubility analysis predicted that selected point mutations reduce solubility of Gc protein and increase solubility of Gn and NP proteins. MD simulation study deciphered that A1046V and G1158E in Gc protein, I778T in Gn protein, and H195R in NP protein displayed large deviation and fluctuation, and affected intramolecular interactions. In conclusion, we observed that point mutations could impact structure, stability, and host-virus interaction of protein, and might lead to evolution of new strains for better survival and drug resistance.

A Computational Workflow to Predict Biological Target Mutations: The Spike Glycoprotein Case Study.

The biological target identification process, a pivotal phase in the drug discovery workflow, becomes particularly challenging when mutations affect proteins' mechanisms of action. COVID-19 Spike glycoprotein mutations are known to modify the affinity toward the human angiotensin-converting enzyme ACE2 and several antibodies, compromising their neutralizing effect. Predicting new possible mutations would be an efficient way to develop specific and efficacious drugs, vaccines, and antibodies. In this work, we developed and applied a computational procedure, combining constrained logic programming and careful structural analysis based on the Structural Activity Relationship (SAR) approach, to predict and determine the structure and behavior of new future mutants. "Mutations rules" that would track statistical and functional types of substitutions for each residue or combination of residues were extracted from the GISAID database and used to define constraints for our software, having control of the process step by step. A careful molecular dynamics analysis of the predicted mutated structures was carried out after an energy evaluation of the intermolecular and intramolecular interactions using the HINT (Hydrophatic INTeraction) force field. Our approach successfully predicted, among others, known Spike mutants.

Comparison of genome replication fidelity between SARS-CoV-2 and influenza A virus in cell culture

Since the emergence of COVID-19, several SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) variants have emerged and spread widely. These variants are produced through replication errors of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Seasonal epidemics of influenza are also known to occur because of new variants of influenza A virus (IAV), which are generated by the introduction of mutations by viral RdRp with low fidelity. Variants with different antigenicities appear because of mutations in envelope glycoproteins. In this study, we calculated and compared the mutation rates in genome replication of IAV and SARS-CoV-2. Average mutation rates per passage were 9.01 × 10–5 and 3.76 × 10–6 substitutions/site for IAV and SARS-CoV-2, respectively. The mutation rate of SARS-CoV-2 was 23.9-fold lower than that of IAV because of the proofreading activity of the SARS-CoV-2 RdRp complex. Our data could be useful in establishing effective countermeasures against COVID-19.

Mpropred: A machine learning (ML) driven Web-App for bioactivity prediction of SARS-CoV-2 main protease (Mpro) antagonists.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic emerged in 2019 and still requiring treatments with fast clinical translatability. Frequent occurrence of mutations in spike glycoprotein of SARS-CoV-2 led the consideration of an alternative therapeutic target to combat the ongoing pandemic. The main protease (Mpro) is such an attractive drug target due to its importance in maturating several polyproteins during the replication process. In the present study, we used a classification structure-activity relationship (CSAR) model to find substructures that leads to to anti-Mpro activities among 758 non-redundant compounds. A set of 12 fingerprints were used to describe Mpro inhibitors, and the random forest approach was used to build prediction models from 100 distinct data splits. The data set's modelability (MODI index) was found to be robust, with a value of 0.79 above the 0.65 threshold. The accuracy (89%), sensitivity (89%), specificity (73%), and Matthews correlation coefficient (79%) used to calculate the prediction performance, was also found to be statistically robust. An extensive analysis of the top significant descriptors unveiled the significance of methyl side chains, aromatic ring and halogen groups for Mpro inhibition. Finally, the predictive model is made publicly accessible as a web-app named Mpropred in order to allow users to predict the bioactivity of compounds against SARS-CoV-2 Mpro. Later, CMNPD, a marine compound database was screened by our app to predict bioactivity of all the compounds and results revealed significant correlation with their binding affinity to Mpro. Molecular dynamics (MD) simulation and molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) analysis showed improved properties of the complexes. Thus, the knowledge and web-app shown herein can be used to develop more effective and specific inhibitors against the SARS-CoV-2 Mpro. The web-app can be accessed from https://share.streamlit.io/nadimfrds/mpropred/Mpropred_app.py.

Myricetin possesses the potency against SARS-CoV-2 infection through blocking viral-entry facilitators and suppressing inflammation in rats and mice

Myricetin (3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) is a common flavonol extracted from many natural plants and Chinese herb medicines and has been demonstrated to have multiple pharmacological activities, such as anti-microbial, anti-thrombotic, neuroprotective, and anti-inflammatory effects. Previously, myricetin was reported to target Mpro and 3CL-Pro-enzymatic activity to SARS-CoV-2. However, the protective value of myricetin on SARS-Cov-2 infection through viral-entry facilitators has not yet been comprehensively understood. The aim of the current study was to evaluate the pharmacological efficacy and the mechanisms of action of myricetin against SARS-CoV-2 infection both in vitro and in vivo. The inhibitory effects of myricetin on SARS-CoV-2 infection and replication were assessed on Vero E6 cells. Molecular docking analysis and bilayer interferometry (BLI) assays, immunocytochemistry (ICC), and pseudoviruses assays were performed to evaluate the roles of myricetin in the intermolecular interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and angiotensin-converting enzyme 2 (ACE2). The anti-inflammatory potency and mechanisms of myricetin were examined in THP1 macrophages in vitro, as well as in carrageenan-induced paw edema, delayed-type hypersensitivity (DTH) induced auricle edema, and LPS-induced acute lung injury (ALI) animal models. The results showed that myricetin was able to inhibit binding between the RBD of the SARS-CoV-2 S protein and ACE2 through molecular docking analysis and BLI assay, demonstrating its potential as a viral-entry facilitator blocker. Myricetin could also significantly inhibit SASR-CoV-2 infection and replication in Vero E6 cells (EC50 55.18μM), which was further validated with pseudoviruses containing the RBD (wild-type, N501Y, N439K, Y453F) and an S1 glycoprotein mutant (S-D614G). Moreover, myricetin exhibited a marked suppressive action on the receptor-interacting serine/threonine protein kinase 1 (RIPK1)-driven inflammation and NF-kappa B signaling in THP1 macrophages. In animal model studies, myricetin notably ameliorated carrageenan-induced paw edema in rats, DTH induced auricle edema in mice, and LPS-induced ALI in mice. Our findings showed that myricetin inhibited HCoV-229E and SARS-CoV-2 replication in vitro, blocked SARS-CoV-2 virus entry facilitators and relieved inflammation through the RIPK1/NF-κB pathway, suggesting that this flavonol has the potential to be developed as a therapeutic agent against COVID-19.

Detection of live SARS-CoV-2 virus and its variants by specially designed SERS-active substrates and spectroscopic analyses

A method using label-free surface enhanced Raman spectroscopy (SERS) based on substrate design is provided for an early detection and differentiation of spike glycoprotein mutation sites in live SARS-CoV-2 variants. Two SERS-active substrates, Au nanocavities (Au NCs) and Au NPs on porous ZrO2 (Au NPs/pZrO2), were used to identify specific peaks of A.3, Alpha, and Delta variants at different concentrations and demonstrated the ability to provide their SERS spectra with detection limits of 0.1–1.0% (or 104−5 copies/mL). Variant identification can be achieved by cross-examining reference spectra and analyzing the substrate-analyte relationship between the suitability of the analyte upon the hotspot(s) formed at high concentrations and the effective detection distance at low concentrations. Mutation sites on the S1 chain of the spike glycoprotein for each variant may be related and distinguishable. This method does not require sample preprocessing and therefore allows for fast screening, which is of high value for more comprehensive and specific studies to distinguish upcoming variants.

Amplicon-Based Nanopore Sequencing of Patients Infected by the SARS-CoV-2 Omicron (B.1.1.529) Variant in India.

We report the sequencing of SARS-CoV-2 Omicron variants from 75 patients, using nanopore long-read sequencing chemistry. These data show a range of mutations in spike glycoprotein that are both unique and common to other populations.

Pseudotyped Viruses for Marburgvirus and Ebolavirus.

Marburg virus (MARV) and Ebola virus (EBOV) of the Filoviridae family are the most lethal viruses in terms of mortality rate. However, the development of antiviral treatment is hampered by the requirement for biosafety level-4 (BSL-4) containment. The establishment of BSL-2 pseudotyped viruses can provide important tools for the study of filoviruses. This chapter summarizes general information on the filoviruses and then focuses on the construction of replication-deficient pseudotyped MARV and EBOV (e.g., lentivirus system and vesicular stomatitis virus system). It also details the potential applications of the pseudotyped viruses, including neutralization antibody detection, the study of infection mechanisms, the evaluation of antibody-dependent enhancement, virus entry inhibitor screening, and glycoprotein mutation analysis.

The free fatty acid-binding pocket is a conserved hallmark in pathogenic β-coronavirus spike proteins from SARS-CoV to Omicron.

As coronavirus disease 2019 (COVID-19) persists, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic β-coronaviruses (β-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation, while the open, infectious conformation is devoid of LA. Electron tomography of SARS-CoV-2-infected cells reveals that LA treatment inhibits viral replication, resulting in fewer deformed virions. Our results establish FFA binding as a hallmark of pathogenic β-CoV infection and replication, setting the stage for FFA-based antiviral strategies to overcome COVID-19.

Association of Clinical Features with Spike Glycoprotein Mutations in Iranian COVID-19 Patients.

Mutations in spike glycoprotein, a critical protein of SARS-CoV-2, could directly impact pathogenicity and virulence. The D614G mutation, a non-synonymous mutation at position 614 of the spike glycoprotein, is a predominant variant circulating worldwide. This study investigated the occurrence of mutations in the crucial zone of the spike gene and the association of clinical symptoms with spike mutations in isolated viruses from Iranian patients infected with SARS-CoV-2 during the second and third waves of the COVID-19 epidemic in Isfahan, the third-largest city in Iran. The extracted RNA from 60 nasopharyngeal samples of COVID-19 patients were subjected to cDNA synthesis and RT-PCR (in three overlapping fragments). Each patient's reverse transcriptase polymerase chain reaction (RT-PCR) products were assembled and sequenced. Information and clinical features of all sixty patients were collected, summarized, and analyzed using the GENMOD procedure of SAS 9.4. Analysis of 60 assembled sequences identified nine nonsynonymous mutations. The D614G mutation has the highest frequency among the amino acid changes. In our study, in 31 patients (51.66%), D614G mutation was determined. For all the studied symptoms, no significant relationship was observed with the incidence of D614G mutation. D614G, a common mutation among several of the variants of SARS-CoV-2, had the highest frequency among the studied sequences and its frequency increased significantly in the samples of the third wave compared to the samples of the second wave of the disease.

Influence of SARS-CoV-2 variants’ spike glycoprotein and RNA-dependent RNA polymerase (nsp12) mutations on remdesivir docking residues

Rapid emergence and evolution of novel SARS-CoV-2 variants has raised concerns about their potential impact on efficiency of currently available vaccines. Among the most significant target mutations in the virus are those of the spike glycoprotein. Remdesivir, which inhibits the polymerase activity of the RNAdependent RNA polymerase RdRp, is the only medicine approved by FDA for treatment of COVID-19 (nsp12). The docking features of the flexible ligand (remdesivir) with the stiff receptors was investigated in the present study (S protein and RdRp interaction). In various studies, the spike glycoprotein and RdRp mutations were found to have a significant influence upon viral behaviour and, as a result, affect human health. The docking position of remdesivir with the S and RdRp proteins was shown to be unaffected by mutations in the missing loops. The remdesivir can only bind the B and C chains of S protein. Some mutations can be transferred between variations, without changing the type of amino acid, such as K417N, L452R, N501Y, D614G, T716I, and S982A.

Identification of Surface Glycoprotein Mutations of SARS-CoV-2 in Isolated Strains from Iraq

Background: The global pandemic of coronavirus disease is a societal, economic, and publichealth crisis that is still underway. The spike glycoprotein of SARS-CoV-2 is one of the primary ingredients for virulence, tissue tropism, and host areas. Aim: This study aimed to determine mutations in the S protein of the Iraqi COVID-19 isolates.Full genome sequences of Iraqi strains were obtained from GISAID. Using statistical saturation mutagenesis and other informatics methods, we investigated 20 sequences of SARS-CoV-2 S protein missense mutation isolates in Iraq selected from NCBI.The following mutations were detected for all the strains under study compared to the wild type: L452R, A522V, E583D and D614G. The number of mutations in the strains was different depending on the location of the state from which the sample was collected The D614G mutation was found in 19 strains. One strain had three mutations, while the other was a wild form strain. The structure of the mutant protein changes dramatically, as does the energy of the atoms concerning the docking position, affecting the protein's stability.The mutation sites would improve the S protein's stability. Molecular docking of RBD-ACE2 is affected differently by residues L452R and A522V.

A Bibliometric Analysis of COVID-19 Researches: Omicron Variant

The aims of this research were to analyse the omicron variant COVID-19 literature published during the COVID- 19 pandemic and to guide future research relating to this variant. From 2021, November 26th to 2022, January 31th , the Pubmed (pubmed.gov) all databases collection was searched for publications related to omicron variant of COVID-19. The keywords used during this search were (omicron sars cov and (omicron covid-19). Exported Microsoft Excel files, Bibliometrics indicator analysis was performed using Bibliometric/Biblioshiny, a web interface application accessed using R-Studio. In total, 262 publications related to omicron varian of COVID- 19 were identified. In these publications, the most active author, journal, country and organisation were Wang L, Nature, China and University Of Oxford, respectively. The most commonly used keywords were humans, sars-cov-2, covid-19, mutation, covid-19 vaccines, spike glycoprotein coronavirus, adult, animals, covid-19/epidemiology/virology and antibodies viral/immunology. This Bibliometric analysis is helpful for mapping studies related to the omicron variant of COVID- 19. This article provides an overview of further researches related to omicron variant of COVID- 19 For example, by searching for keywords, this variant is associated with gender, spike glycoprotein coronavirus and mutation.

The impacts of 13 novel mutations of SARS-CoV-2 on protein dynamics: In silico analysis from Turkey

SARS-CoV-2 inherits a high rate of mutations making it better suited to the host since its fundamental role in evolution is to provide diversity into the genome. This research aims to identify variations in Turkish isolates and predict their impacts on proteins. To identify novel variations and predict their impacts on protein dynamics, in silico methodology was used. The 411 sequences from Turkey were analysed. Secondary structure prediction by Garnier-Osguthorpe-Robson (GOR) was used. To find the effects of identified Spike mutations on protein dynamics, the SARS-CoV-2 structures (PDB:6VYB, 6M0J) were uploaded and predicted by Cutoff Scanning Matrix (mCSM), DynaMut and MutaBind2. To understand the effects of these mutations on Spike protein molecular dynamics (MD) simulation was employed. Turkish sequences were aligned with sequences worldwide by MUSCLE, and phylogenetic analysis was performed via MegaX. The 13 novel mutations were identified, and six of them belong to spike glycoprotein. Ten of these variations revealed alteration in the secondary structure of the protein. Differences of free energy between the reference sequence and six mutants were found below zero for each of six isolates, demonstrating these variations have stabilizing effects on protein structure. Differences in vibrational entropy calculation revealed that three variants have rigidification, while the other three have a flexibility effect. MD simulation revealed that point mutations in spike glycoprotein and RBD:ACE-2 complex cause changes in protein dynamics compared to the wild-type, suggesting possible alterations in binding affinity. The phylogenetic analysis showed Turkish sequences distributed throughout the tree, revealing multiple entrances to Turkey.

Omicron: Understanding the Latest Variant of SARS-CoV-2 and Strategies for Tackling the Infection.

The new variant of concern of SARS‐CoV‐2, namely Omicron, has triggered global fear recently. To date, our knowledge of Omicron, particularly of how S glycoprotein mutations affect the infectivity of the virus and the severity of the infection, is far from complete. This hinders our ability to treat the disease and to predict the future state of SARS‐CoV‐2 threats to well‐being and economic stability. Despite this, efforts have been made to unveil the routes of transmission and the efficiency of existing vaccines in tackling Omicron. This article reviews the latest understanding of Omicron and the current status of the use of vaccines and drugs for infection control. It is hoped that this article can offer insights into the development of more effective measures to tackle the pandemic.

Next-generation sequencing of von Willebrand factor and coagulation factor VIII genes: a cross-sectional study in Croatian adult patients diagnosed with von Willebrand disease

AimTo identify the von Willebrand factor (VWF) gene variant status in Croatian adult patients diagnosed with von Willebrand disease (VWD), provide differential diagnosis of VWD subtypes, and identify patients with mild hemophilia A (HA) who were earlier misdiagnosed as VWD.MethodsCoagulation testing included determination of VWF gain-of-function mutant glycoprotein Ib binding activity (VWF:GPIbM), VWF antigen, VWF collagen-binding activity, and multimeric analysis. Genetic analysis of VWF and FVIII genes was performed with next-generation sequencing (NGS).ResultsThe study enrolled 50 patients (72% women; median age 37 years, range 18-75) from 44 unrelated families. Fourteen patients were heterozygous for VWF gene variants compatible with type-1 VWD. Twelve had variants associated with type 2, of whom seven were classified as type 2A, four as type 2B, and one as type 2N. Six type-3 VWD patients were either homozygotes for null variants or combined heterozygotes. Eleven variants within the VWF gene were novel. Three female patients had variants within the FVIII gene, and were re-classified as mild-HA carriers, of whom one had causative novel variants both within VWF and FVIII genes. Fifteen patients remained without a defined genetic cause of their disorder, of whom five had VWF:GPIbM levels below 50%.ConclusionCroatian adult patients with VWD have considerable genetic heterogeneity. NGS of both VWF and FVIII genes provided accurate differential diagnosis of VWD subtypes and distinction of VWD from mild HA.