Von Willebrand Factor Function Research Articles

Carbamylation-A Pathologic Posttranslational Modification Affecting Platelet and Von Willebrand Factor Function during Uremic Kidney Disease.

Carbamylation-A Pathologic Posttranslational Modification Affecting Platelet and Von Willebrand Factor Function during Uremic Kidney Disease.

An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays.

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst. Key features • Automated pre-dilution setup for von Willebrand factor concentrates of various natures. • Combination of a liquid handling system (CyBio FeliX) with a coagulation analyzer (BCS-XP). • Simplifies method transfer to other laboratories. • Basic training for CyBio FeliX and BCS-XP is required. Graphical overview VWF:RCo assay principle and measurement setup. Platelets (yellow ellipsoids) with negative surface charge (- - -) are treated with formaldehyde, which partly denatures the cell surface and thus stabilizes platelets for use as assay reagents. Stabilized platelets (dark-yellow-framed yellow ellipsoids) are then brought in contact with ristocetin A (chemical structure shown; black dots), which binds to the platelet surface and facilitates binding of VWF (green circles). The graphs show an example of quantitative determination of platelet agglutination by measurement of light transmission, where increasing amounts of VWF increase light transmission over time. The photo in the left-bottom corner shows the CyBio FeliX setup for VWF sample dilution and the photo in the right-bottom corner displays the BCS-XP system, which is used for VWF:RCo measurements.

Improved prevention of bleeding episodes with emicizumab in 3 patients with concomitant hemophilia A and von Willebrand disease.

The typical phenotype of hemophilia A (HA) is that of frequent bleeding episodes, up to several per month, unless prophylactic factor VIII (FVIII) replacement or alternatives are given. Related bleeding may be heightened in severity or frequency by co-morbid bleeding disorders. Based on the reported prevalence of von Willebrand disease (VWD) of up to 1% of the general population, the co-existence of HA and VWD occurs, but is likely less recognized. Prophylactic FVIII replacement may or may not adequately prevent bleeding in persons with HA and mild VWD, and plasma-derived concentrates containing FVIII and von Willebrand factor (VWF) may be used for more successful bleeding prophylaxis. However, therapy adherence remains problematic for many reasons, one being treatment via intravenous access. Emicizumab is a nonfactor subcutaneous prophylactic therapy for HA that may overcome this concern. We describe three patients, with severe HA and VWD, for whom regular FVIII/VWF prophylaxis was deemed inadequate. FVIII/VWF prophylaxis was replaced with weekly prophylactic injections of the bispecific monoclonal antibody, emicizumab. When the patients were transitioned to emicizumab, all experienced a significant reduction in their annualized bleed rate (ABR). Although the mechanism of action does not directly affect or replace VWF function, emicizumab may be an effective prophylaxis alternative to FVIII/VWF concentrate in patients with concomitant severe HA and VWD.

Carbamylation Is Instrumental in End-Stage Kidney Disease Coagulopathies: The Impact on von Willebrand Factor and Platelet Functionality.

Chronic kidney disease (CKD) is a progressive, irreversible, and incurable condition characterized by high morbidity and mortality, affecting approximately one-tenth of the global population. Rise of urea-derived cyanate levels in CKD patients, severalfold higher in comparison to those found in healthy individuals, leads to an increased rate of carbamylation of lysine residues of proteins and peptides. This posttranslational modification plays an important role in the progression of kidney failure but also in the onset of CKD-related complications, including previously reported coagulopathies. In this study, we have explored the impact of carbamylation on the functionality of von Willebrand factor (vWF), a pivotal player in hemostasis, and its implications for platelet adhesion. We have explored carbamylated vWF's interactions with its partner proteins via ELISA. Mass spectrometry was employed to identify modified lysine residues. Blood platelets isolated from healthy donors were carbamylated, and their activation, binding to endothelium and thromboxane release were evaluated using flow cytometry, adhesion assays and ELISA, respectively. Using mass spectrometry we detected the vWF's lysine residue smost susceptible to carbamylation. This modification has in turn affected vWF's interactions with its key binding partners: decreased binding to collagen types I/III but increased the affinity to factor FVIII, while its binding to fibrinogen remained unchanged. Carbamylation of vWF impeded vWF-blood platelet binding, but carbamylation of platelets led to their increased thrombin-dependent activation as observed by enhanced phosphatidylserine exposure, improved their binding to vascular endothelium, at the same time decreasing the production of the prothrombotic mediator, thromboxane A2. Our findings highlight the multifaceted impact of carbamylation on vWF and platelets, disturbing the delicate balance of coagulation cascade. These alterations could contribute to the complex hemostatic imbalance in ESKD, underscoring the need for further research to fully understand these mechanisms and their clinical implications.

Analytical and Clinical Validation of a Non-Ristocetin Based VWF Assay on 2 Automated Analyzers in a Large Reference Laboratory.

Historically, von Willebrand factor (VWF) activity assays utilized ristocetin despite limitations including poor limits of detection and high imprecision. Newer VWF activity assays such as the INNOVANCE® VWF Ac assay, however, do not rely on ristocetin to measure platelet-dependent VWF function. The purpose of this study was to evaluate the analytical and clinical performance of the Siemens Healthineers INNOVANCE VWF Ac Assay on the Siemens BCS® XP and the Sysmex® CS-2500 systems in a large reference laboratory setting. Performance indicators for the INNOVANCE VWF Ac assay were the limit of quantitation (LoQ), precision, and method comparison. Method comparison studies were performed using remnant plasma patient samples from routine coagulation tests and analyzed using both the INNOVANCE VWF Ac assay and the Siemens Healthineers ristocetin-dependent BC von Willebrand Reagent. Evaluation of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems demonstrated good precision and a lower LoQ compared to the BC von Willebrand Reagent. Method comparisons support the use of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems to measure platelet-dependent VWF function. The INNOVANCE VWF Ac assay was able to further assist in von Willebrand disease classification in 6/7 (86%) samples when the result was below the LoQ for the BC von Willebrand Reagent (ristocetin cofactor activity). These data are consistent with the 2021 American Society of Hematology/International Society on Thrombosis and Haemostasis/National Hemophilia Foundation/World Federation of Hemophilia von Willebrand disease guidelines that suggest using newer assays such as the INNOVANCE VWF Ac assay in place of ristocetin cofactor activity assays.

Cryptic Extensibility in von Willebrand Factor Revealed by Molecular Nanodissection.

Von Willebrand factor (VWF) is a multimer with a variable number of protomers, each of which is a head-to-head dimer of two multi-domain monomers. VWF responds to shear through the unfolding and extension of distinct domains, thereby mediating platelet adhesion and aggregation to the injured blood vessel wall. VWF's C1-6 segment uncoils and then the A2 domain unfolds and extends in a hierarchical and sequential manner. However, it is unclear whether there is any reservoir of further extensibility. Here, we explored the presence of cryptic extensibility in VWF by nanodissecting individual, pre-stretched multimers with atomic force microscopy (AFM). The AFM cantilever tip was pressed into the surface and moved in a direction perpendicular to the VWF axis. It was possible to pull out protein loops from VWF, which resulted in a mean contour length gain of 217 nm. In some cases, the loop became cleaved, and a gap was present along the contour. Frequently, small nodules appeared in the loops, indicating that parts of the nanodissected VWF segment remained folded. After analyzing the nodal structure, we conclude that the cryptic extensibility lies within the C1-6 and A1-3 regions. Cryptic extensibility may play a role in maintaining VWF's functionality in extreme shear conditions.

A comprehensive exploration of chronic kidney disease and dialysis in Canada's Indigenous population: from epidemiology to genetic influences.

This study aims to review the escalating prevalence of chronic kidney disease (CKD) and end-stage renal disease (ESRD) among Canada's Indigenous population, focusing on risk factors, hospitalization and mortality rates, and disparities in kidney transplantation. The study explores how these factors contribute to the health outcomes of this population and examines the influence of genetic variations on CKD progression. The review synthesizes data on prevalence rates, hospitalization and mortality statistics, and transplantation disparities among Indigenous individuals. It also delves into the complexities of healthcare access, including geographical, socioeconomic, and psychological barriers. Additionally, the manuscript investigates the impact of racial factors on blood characteristics relevant to dialysis treatment and the genetic predispositions influencing disease progression in Indigenous populations. Indigenous individuals exhibit a higher prevalence of CKD and ESRD risk factors such as diabetes and obesity, particularly in regions like Saskatchewan. These patients face a 77% higher risk of death compared to their non-Indigenous counterparts and are less likely to receive kidney transplants. Genetic analyses reveal significant associations between CKD and specific genomic variations. Through analyses, we found that healthy Indigenous individuals may have higher levels of circulating inflammatory markers, which could become further elevated for those with CKD. In particular, they may have higher levels of C-reactive protein (CRP) fibrinogen, as well as genomic variations that affect IL-6 production and the function of von Willebrand Factor (vWF) which has critical potential influence on the compatibility with dialysis membranes contributing to complications in dialysis. Indigenous people in Canada are disproportionately burdened by CKD and ESRD due to socioeconomic factors and potential genetic predispositions. While significant efforts have been made to assess the socioeconomic conditions of the Indigenous population, the genetic factors and their potential critical influence on compatibility with dialysis membranes, contributing to treatment complications, remain understudied. Further investigation into these genetic predispositions is essential.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Von Willebrand factor and hematogenous cancer metastasis under flow.

Hematogenous metastasis involves cancer cell migration to different locations from the primary tumor through the blood circulation. Von Willebrand factor (VWF) has been shown to play an important role in tumor cell adhesion to and extravasation from the endothelial cell lining of blood vessel walls during cancer metastasis. VWF may contribute to this process by interacting with tumor cells, endothelial cells, and platelets through various cell membrane receptors, such as platelet glycoprotein (GP)Ibα, P-selectin, ανβ3 and αIIbβ3 integrins, and glycocalyx. Blood flow can mechanically extend and activate VWF to bind platelets and associate intermolecularly with other VWF molecules in plasma or on the surface of endothelial cells, cancer cells, or platelets. This suggests a mechanoregulatory role of VWF in mediating the interactions between VWF and these cells to promote cancer cell adhesion to blood vessels. In this review, we will summarize the current knowledge of VWF function and the role of hydrodynamic forces in hematogenous cancer metastasis.

Discrepant low von Willebrand factor activity results on the ACL TOP analyzer are frequent in unselected patients with myeloproliferative neoplasms and show no correlation with high-molecular-weight multimer loss or bleeding phenotype

BackgroundBleeding complications are common in patients with myeloproliferative neoplasms (MPNs), with a subset developing acquired von Willebrand disease. Despite this association, a wide spectrum of von Willebrand factor (VWF) abnormalities are described, and the performance of modern assays remains unclear. ObjectivesTo comprehensively describe the pattern of VWF laboratory abnormalities in the MPN population. MethodsWe collected samples from 74 unselected clinic patients with MPNs to evaluate VWF quantitatively and qualitatively via multiple methods, correlating findings with a retrospective analysis of clinical bleeding data. VWF assays were performed on both ACL TOP (Instrumentation Laboratory) and Acustar (Instrumentation Laboratory) analyzers using HemosIL reagents (Instrumentation Laboratory), along with multimer analysis by gel electrophoresis. ResultsFunctional VWF measurements were not concordant between assays, with a median ACL TOP VWF glycoprotein IbR to antigen ratio (VWF:GPIbR/VWF:Ag) of 0.57 (IQR, 0.43-0.71) compared to a median Acustar VWF:GPIbR/VWF:Ag of 0.91 (IQR: 0.82-1.03;P < .001). The ACL TOP showed disproportionately lower results, with 73% of patients having a ratio <0.7. Despite this, no patient experienced loss of high-molecular-weight multimers by gel electrophoresis. An inverse relationship was observed between platelet count and functional ratios on both ACL TOP (R2 = 0.20; P < .001) and Acustar (R2 = 0.18; P = .0011) analyzers. While clinically significant bleeding events were relatively common (11% patients), there was no association with VWF assay abnormalities, and generally, an alternate cause(s) was identified. ConclusionDiscrepancies in functional VWF assays are common in patients with MPN, particularly by ACL TOP VWF:GPIbR. Based on our limited series, a VWF functional to an antigenic ratio of <0.7 (“type 2 pattern”) alone is poorly predictive of bleeding risk.

Functional Assessment of VWD Type 2N Variants Using Mammalian Cell Displayed Surface-Tethered Von Willebrand Factor

Background von Willebrand factor (VWF) is a large plasma protein critical in hemostasis and thrombosis. Defects in VWF cause the inherited autosomal bleeding disorder von Willebrand disease (VWD). One critical function of VWF is to bind circulating coagulation factor VIII (FVIII) and protect it from rapid clearance. Type 2N VWD is a recessive form of VWD characterized by low FVIII levels due to impairment in VWF-FVIII binding. Type 2N VWD clinically is similar to mild-to-moderate hemophilia A, an X-linked bleeding disorder caused by deficiency in FVIII. It is critical to distinguish type 2N VWD from hemophilia A because the treatments are different. VWF genotyping can inform accurate diagnoses, treatment, and testing in relatives. With the rapid expansion of DNA sequencing, a growing number of DNA variants are being identified in the VWF gene. However, the interpretation of significance of these variants is often challenged by insufficient evidence to inform confident classification regarding pathogenicity. In vitro functional assays provide an avenue to develop the evidence needed to accurately assign functional significance to DNA variants. We adapted multiplexed surface tethering of extracellular proteins (MultiSTEP), our mammalian cell surface display system for secreted proteins, to display VWF and test the functional impact of VWF variants on VWF-FVIII binding. Methods We constructed a VWF cDNA of common VWF coding sequences by mutating rare variants to the more common nucleotides. The regions encoding the VWF propeptide were deleted (∆Pro-VWF). ∆Pro-VWF was cloned into our MultiSTEP cassette that provides a C-terminal flexible linker, Strep II tag, and CD28 transmembrane domain. Three VWF missense variants implicated in type 2N VWD (Table 1) that had varying levels of evidence to support classification of pathogenicity were chosen for study - VWF c.2561 G&amp;gt;A (p.R854Q), c.2572 T&amp;gt;A (p.C858S), and c.2635 G&amp;gt;A (p.D879N). Variants were subcloned into our ∆Pro-VWF cassette and then recombined into the genomically integrated MultiSTEP landing pad in HEK293 Freestyle cells. Following induction of expression, VWF-displaying cells were incubated with recombinant FVIII. VWF expression was measured using a polyclonal anti-VWF antibody, and VWF-FVIII binding was detected with two different monoclonal antibodies to FVIII. Cells were incubated with fluorescently-conjugated secondary antibodies and cell surface VWF and FVIII abundance determined by flow cytometry. Results We show that ∆Pro-VWF robustly displays in our MultiSTEP system and binds FVIII. All three VWF missense variants express VWF at levels similar to ∆Pro-VWF. Relative to ∆Pro-VWF all three variants show marked impairment in VWF-FVIII binding (Figure 1), providing strong in vitro evidence for pathogenicity in type 2N VWD useful in variant reinterpretation (bold column in Table 1). Conclusions We have developed an in vitro mammalian cell surface display system for the interrogation of VWF variants and adapted it to study VWF-FVIII binding. The Pathogenic variant p.R854Q shows decreased VWF-FVIII binding in this assay, as expected. The Likely Pathogenic variant p.D879N and the Variant of Uncertain Significance p.C858S both exhibit a profound loss of VWF-FVIII binding in this assay. Our results provide evidence for reclassification of p.C858S to Likely Pathogenic for type 2N VWD. We expect our system can be used at scale to similarly interrogate the functional significance of a multiplexed library of VWF variants and improve variant interpretation in VWD.

Regulation of von Willebrand factor by ADAMTS13 ameliorates lipopolysaccharide-induced lung injury in mice.

The relationship between von Willebrand factor (VWF) and inflammation has attracted considerable attention in recent years. VWF, which is stored in the Weibel-Palade bodies (WPBs) of endothelial cells (ECs), is released from WPBs in response to inflammatory stimuli and is thought to contribute to inflammation by promoting leukocyte extravasation. In this study, lung injury model mice were produced by intratracheal injection with lipopolysaccharides. The severity of lung inflammation was evaluated in mice with different genotypes (wild-type, Vwf-/-, Adamts13-/-) and mice treated with drugs that inhibit VWF function. Lung inflammation was significantly ameliorated in Vwf-/- mice compared with wild-type mice. Furthermore, inflammation was significantly suppressed in wild-type mice treated with anti-VWF A1 antibody or recombinant human ADAMTS13 compared with the untreated control group. The underlying mechanism appears to be an increased VWF/ADAMTS13 ratio at the site of inflammation and the interaction between blood cell components, such as leukocytes and platelets, and the VWF A1 domain, which promotes leukocyte infiltration into the lung. This study suggested that ADAMTS13 protein and other VWF-targeting agents may be a novel therapeutic option for treatment of pulmonary inflammatory diseases.

The interplay between adsorption and aggregation of von Willebrand factor chains in shear flows

Von Willebrand factor (VWF) is a giant extracellular glycoprotein that carries out a key adhesive function during primary hemostasis. Upon vascular injury and triggered by the shear of flowing blood, VWF establishes specific interactions with several molecular partners in order to anchor platelets to collagen on the exposed subendothelial surface. VWF also interacts with itself to form aggregates that, adsorbed on the surface, provide more anchor sites for the platelets. However, the interplay between elongation and subsequent exposure of cryptic binding sites, self-association, and adsorption on the surface remained unclear for VWF. In particular, the role of shear flow in these three processes is not well understood. In this study, we address these questions by using Brownian dynamics simulations at a coarse-grained level of resolution. We considered a system consisting of multiple VWF-like self-interacting chains that also interact with a surface under a shear flow. By a systematic analysis, we reveal that chain-chain and chain-surface interactions coexist nontrivially to modulate the spontaneous adsorption of VWF and the posterior immobilization of secondary tethered chains. Accordingly, these interactions tune VWF’s extension and its propensity to form shear-assisted functional adsorbed aggregates. Our data highlight the collective behavior VWF self-interacting chains have when bound to the surface, distinct from that of isolated or flowing chains. Furthermore, we show that the extension and the exposure to solvent have a similar dependence on shear flow, at a VWF-monomer level of resolution. Overall, our results highlight the complex interplay that exists between adsorption, cohesion, and shear forces and their relevance for the adhesive hemostatic function of VWF.

Impact of transcatheter aortic valve implantation on circulating von Willebrand factor in patients with severe aortic stenosis.

Severe aortic stenosis (AS) can lead to degradation of high molecular weight (HMW) von Willebrand factor (VWF) which can result in haemostatic abnormalities. While studies have explored changes in VWF profiles before and after surgical aortic valve replacement (SAVR), the longer-term changes in VWF profiles pre- and post-transcatheter aortic valve implantation (TAVI) are less understood. Our primary objective was to identify differences in VWF multimer profiles and VWF function pre-TAVI and 1-month post-TAVI. Our secondary objective was to correlate VWF markers with measures of AS severity. Adult patients with severe AS referred for TAVI at our institution were prospectively enrolled in this cohort study. Blood samples were collected for plasma analysis at three time points for all patients: 1day pre-TAVI, 3days post-TAVI, and 1-month post-TAVI. VWF antigen, activity, propeptide, collagen binding, multimers, and factor VIII coagulant activity were determined at each time point. Correlations between VWF parameters and severity of AS were assessed. Twenty participants (15 males, five females) with severe AS were recruited for the study. There was a significant increase in HMW VWF between pre-procedure and 1-month post-TAVI (p<.05). There was a transient increase in VWF antigen levels and activity at 3-days post TAVI that decreased to pre-TAVI levels at 1-month. There were no statistically significant correlations between VWF markers and AS severity. This is the first study to elucidate longer-term (>1week) improvements in HMW VWF after a TAVI procedure in severe AS patients.

Von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel–Palade bodies

BackgroundThe von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel–Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. MethodsStatic-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. ResultsStatic-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF–Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. ConclusionsTogether, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction.

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.

Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

Abstract TMP4: BB-031 Reduces Platelet Reactivity And Attenuates Von Willebrand Factor Levels In A Large Vessel Occlusion Stroke Model

Introduction: Acute ischemic strokes (AIS) are a leading cause of death and long-term disability worldwide. Von Willebrand Factor (VWF) is an attractive target for anti-thrombotic therapeutics as it plays a pivotal role in platelet adhesion, activation, and aggregation with large vessel occlusion (LVO) stroke. VWF is stored in an ultra large form in platelet α-granules and Weibel-Palade bodies of endothelial cells from where it is released during injury or inflammation. BB-031 is an RNA aptamer that binds and inhibits VWF, and both prevents and lyses an occlusive thrombus. Hypothesis: We hypothesize BB-031 treatment will inhibit VWF thereby reducing platelet reactivity and attenuating the increase in VWF levels in a canine model of LVO stroke. Methods: A canine embolic MCAO model was used to assess BB-031 efficacy of VWF inhibition and platelet activity compared to placebo. After 6 hours of LVO with an autologous clot, intravenous treatment was initiated with placebo (n=7), or 0.5 (n=8), 1.0 (n=4), and 5.0 (n=4) mg/kg BB-031. Whole blood was collected at baseline, 6 hours after occlusion then at 10, 15, 20, 25, 30, 60 min after treatment and lastly, at time of sacrifice 9 hours after MCAO induction. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating VWF and platelet function analysis (PFA-100 Siemens) was utilized to determine platelet reactivity. Results: VWF levels immediately after LVO in vehicle control were significantly attenuated within 10 minutes of administration with all BB-031 concentrations (p&lt;0.05). Within 15 minutes after vehicle treatment, VWF levels increased by 3.102 ± .382 ng/ml whereas BB-031 administration of 0.5, 1.0, and 5.0 mg/kg reduced VWF levels by 0.223 ± .154 ng/ml, 0.269 ± .304 ng/ml and 1.053 ± .267 ng/ml respectively. Platelet reactivity to ADP/Collagen (PFA-100) revealed significantly increased platelet closing time in all BB-031 treated canines from immediately after administration through 60 minutes. (p&lt;0.05). Conclusion: BB-031 treatment demonstrated significant VWF inhibition and reduced platelet reactivity in a canine AIS LVO model compared to placebo. These findings indicate that BB-031 may offer a superior therapeutic treatment paradigm for ischemic stroke.

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Removal of the vicinal disulfide enhances the platelet-capturing function of von Willebrand factor

AbstractA redox autoinhibitory mechanism has previously been proposed, in which the reduced state of the vicinal disulfide bond in the von Willebrand factor (VWF) A2 domain allows A2 to bind to A1 and inhibit platelet adhesion to the A1 domain. The VWF A1A2A3 tridomain was expressed with and without the vicinal disulfide in A2 (C1669S/C1670S) via the atomic replacement of sulfur for oxygen to test the relevance of the vicinal disulfide to the physiological platelet function of VWF under shear flow. A comparative study of the shear-dependent platelet translocation dynamics on these tridomain variants reveals that the reduction of the vicinal disulfide moderately increases the platelet-capturing function of A1, an observation counter to the proposed hypothesis. Surface plasmon resonance spectroscopy confirms that C1669S/C1670S slightly increases the affinity of A1A2A3 binding to glycoprotein Ibα (GPIbα). Differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry demonstrate that reduction of the vicinal disulfide destabilizes the A2 domain, which consequently disrupts interactions between the A1, A2, and A3 domains and enhances the conformational dynamics of A1-domain secondary structures known to regulate the strength of platelet adhesion to VWF. This study clarifies that the reduced state of the A2 vicinal disulfide is not inhibitory but rather slightly activating.