vWF A1 Domain Research Articles

COVID-19 microthrombosis: unusually large VWF multimers are a platform for activation of the alternative complement pathway under cytokine storm.

ADAMTS13, a metalloproteinase, specifically cleaves unusually large multimers of von Willebrand factor (VWF), newly released from vascular endothelial cells. The ratio of ADAMTS13 activity to VWF antigen (ADAMTS13/VWF) and indicators of the alternative complement pathway (C3a and sC5b-9) are both related to the severity of COVID-19. The ADAMTS13/VWF ratio is generally moderately decreased (0.18–0.35) in patients with severe COVID-19. When these patients experience cytokine storms, both interleukin-8 and TNFα stimulate VWF release from vascular endothelial cells, while interleukin-6 inhibits both production of ADAMTS13 and its interaction with VWF, resulting in localized severe deficiency of ADAMTS13 activity. Platelet factor 4 and thrombospondin-1, both released upon platelet activation, bind to the VWF-A2 domain and enhance the blockade of ADAMTS13 function. Thus, the released unusually-large VWF multimers remain associated with the vascular endothelial cell surface, via anchoring with syndecan-1 in the glycocalyx. Unfolding of the VWF-A2 domain, which has high sequence homology with complement factor B, allows the domain to bind to activated complement C3b, providing a platform for complement activation of the alternative pathway. The resultant C3a and C5a generate tissue factor-rich neutrophil extracellular traps (NETs), which induce the mixed immunothrombosis, fibrin clots and platelet aggregates typically seen in patients with severe COVID-19.

The N-terminal autoinhibitory module of the A1 domain in von Willebrand factor stabilizes the mechanosensor catch bond.

The von Willebrand factor (VWF), by interacting with the circulatory system and platelets, harnesses hemodynamic forces to form hemostatic plugs or occlusive thrombi. The autoinhibitory modules (AIMs) flanking the VWF-A1 domain were found to contribute to its biomechanical activation. However, how AIM sequences regulate the VWF-A1 binding behavior is controversial and incompletely understood as their structures are currently unsolvable by crystallography. To address this, we first performed molecular dynamics simulations to predict the N-terminal AIM (N-AIM; residues Q1238–E1260) structure. Excitingly, we found that N-AIM could cooperate with C-AIM to form a joint Rotini-like structure, thereby partially autoinhibiting the VWF-A1–GPIbα interaction. Furthermore, we used biomembrane force probe (BFP) assays to experimentally demonstrate that the VWF-A1 containing long N-AIM sequence (1238-A1) exhibited catch-bond behavior as the force first decelerated (catch) and then accelerated (slip) the dissociation. Conversely, VWF-A1 with short N-AIM (1261-A1) displayed bi-variable behaviors with either catch (1261H-A1) or slip bonds (1261L-A1). Notably, such bi-variable transition happened at low temperatures or high pH levels, whereas Q1238–E1260 stabilized the 1238-A1 catch bond regardless of the environmental factors. The physiological study was complemented by platelet perfusion assays using microfluidics. Taken together, these studies provide new mechanobiology on how N-AIM serves as a mechano-regulator of VWF activity, which inspires future VWF-A1 dependent antithrombotic approaches.

Staphylococcus aureus iron-regulated surface determinant B (IsdB) protein interacts with von Willebrand factor and promotes adherence to endothelial cells

Staphylococcus aureus is the cause of a spectrum of diseases in humans and animals. The molecular basis of this pathogenicity lies in the expression of a variety of virulence factors, including proteins that mediate adherence to the host plasma and extracellular matrix proteins. In this study, we discovered that the iron-regulated surface determinant B (IsdB) protein, besides being involved in iron transport and vitronectin binding, interacts with von Willebrand Factor (vWF). IsdB-expressing bacteria bound to both soluble and immobilized vWF. The binding of recombinant IsdB to vWF was blocked by heparin and reduced at high ionic strength. Furthermore, treatment with ristocetin, an allosteric agent that promotes the exposure of the A1 domain of vWF, potentiates the binding of IsdB to vWF. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound recombinant A1 domain with KD values in the micromolar range. The binding of IsdB and adhesion of S. aureus expressing IsdB to monolayers of activated endothelial cells was significantly inhibited by a monoclonal antibody against the A1 domain and by IsdB reactive IgG from patients with staphylococcal endocarditis. This suggests the importance of IsdB in adherence of S. aureus to the endothelium colonization and as potential therapeutic target.

Mutated VWA8 Is Associated With Developmental Delay, Microcephaly, and Scoliosis and Plays a Novel Role in Early Development and Skeletal Morphogenesis in Zebrafish.

Von Willebrand A domain-containing protein 8 (VWA8), also named KIAA0564, is a poorly characterized, mitochondrial matrix-targeted protein having a putative ATPase activity. VWA8 is comprising of ATPase-associated domains and a VWFA domain associated with ATPase activity inside the cell. In the present study, we describe a large consanguineous family of Saudi origin segregating a complex developmental syndrome in an autosomal recessive fashion. All the affected individuals exhibited severe developmental disorders. DNA from three patients was subjected to whole-exome sequencing followed by Sanger sequencing. VWA8 knock-down zebrafish morpholinos were used to study the phenotypic effect of this gene on zebrafish development. A homozygous missense variant [c.947A > G; p.(Asp316Gly)] was identified in exon 8 of the VWA8 gene, which perfectly segregated with the disease phenotype. Using zebrafish morpholino, we observed delayed development at an early stage, lack of movement, light sensitivity, severe skeletal deformity such as scoliosis, and facial dysmorphism. This is the first homozygous variant identified in the VWA8 gene underlying global developmental delay, microcephaly, scoliosis, limbs, and cardiovascular malformations in humans. We provide genetic and molecular evidence using zebrafish morpholino for a homozygous variant in the VWA8 gene, associated with such a complex developmental syndrome in humans.

COCH-related autosomal dominant nonsyndromic hearing loss: a phenotype-genotype study.

This phenotype-genotype study aimed to investigate the extent of audioprofile variability related to cochlin major domains and to identify potential ethnic-specific differences associated with COCH-related hearing loss. Eight Korean families (26 cases) were diagnosed with COCH-related hearing loss by exome sequencing. Audiometric test results were combined with those from nine published East Asian families (20 cases) and compared with those from 38 European-descent families (277 cases). Audioprofiles were created by grouping audiometric test results into age ranges by age at testing and then averaging hearing loss thresholds by frequency within age ranges. The functional impact of the identified variants was assessed in vitro by examining the intracellular trafficking, secretion, and cleavage of cochlin. In both East Asian and European-descent families segregating COCH-related hearing loss, deafness-associated variants in non-LCCL domains of cochlin were associated with hearing loss that was more severe earlier in life than hearing loss caused by variants in the LCCL domain. Consistent with this phenotypic difference, functional studies demonstrated distinct pathogenic mechanisms for COCH variants in a domain-dependent manner; specifically, a cytotoxic effect was observed for the p.Phe230Leu variant, which is located in the vWFA1 domain. No ethnic-specific differences in hearing loss progression were observed, except for those attributable to an overrepresentation of presymptomatic cases in the European-descent cohort.

AFM Identifies a Protein Complex Involved in Pathogen Adhesion Which Ruptures at Three Nanonewtons.

Staphylococci bind to the blood protein von Willebrand Factor (vWF), thereby causing endovascular infections. Whether and how this interaction occurs with the medically important pathogen Staphylococcus epidermidis is unknown. Using single-molecule experiments, we demonstrate that the S. epidermidis protein Aap binds vWF via an ultrastrong force, ∼3 nN, the strongest noncovalent biological bond ever reported, and we show that this interaction is activated by tensile loading, suggesting a catch-bond behavior. Aap-vWF binding involves exclusively the A1 domain of vWF but requires both the A and B domains of Aap, as revealed by inhibition assays using specific monoclonal antibodies. Collectively, our results point to a mechanism where force-induced unfolding of the B repeats activates the A domain of Aap, shifting it from a weak- to a strong-binding state, which then engages into an ultrastrong interaction with vWF A1. This shear-dependent function of Aap offers promise for innovative antistaphylococcal therapies.

Backbone resonance assignments of the A2 domain of mouse von Willebrand factor.

von Willebrand factor (vWF) is an adhesive plasma protein that is important for platelet adhesion in normal hemostasis in response to vascular injury. Although large vWF multimers are released from storage granules of platelets and (sub-)endothelial cells in response to hemostatic stimuli, for normal physiological function, vWF multimers are required to be cleaved into smaller multimeric forms. The plasma metalloproteinase ADAMTS13 specifically cleaves the peptide bond located in the middle of the A2 domain of vWF (vWF-A2), but the cleavage site is buried inside the structure of vWF and is difficult to access in the absence of elevated flow shear stress. On the other hand, in the presence of high vascular shear stress, the structure of vWF-A2 is supposed to be unfolded, thereby becoming accessible for proteolysis by ADAMTS13. However, the atomic-level mechanism underlying shear-induced structural changes of vWF-A2 remains unclear and to date no solution NMR information is available. In this study, we present the backbone 1H, 13C, and 15N resonance assignments of mouse vWF-A2; side chain assignments of 13Cβ are also provided. Secondary structure propensity analysis based on the assigned chemical shifts showed that mouse vWF-A2 forms similar secondary structures in solution to the previously determined crystal structure of human vWF-A2. The obtained NMR assignment data will contribute to an atomic-level characterization of shear-induced unfolding of vWF-A2 in solution.

Characterization of a Nanobody That Binds Von Willebrand Factor and Prevents Von Willebrand Factor Cleavage By Plasmin

Introduction: Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adherence to exposed subendothelial collagen at sites of vascular injury. To prevent spontaneous platelet aggregation, accumulation of high molecular weight vWF multimers is prevented by ADAMTS13-mediated cleavage in the vWF A2 domain. Moreover, emerging data demonstrates plasmin-mediated cleavage of vWF in its open conformation in the A1-A2 linker, suggesting a role for plasmin in regulation of vWF multimer composition as a back up for ADAMTS13.Aim: We recently identified a llama-derived anti-vWF nanobody recognizing vWF in a conformation sensitive to plasmin cleavage. Our aim was to characterize this nanobody in terms of binding to- and interference with vWF functionality.Methods and results: The nanobody was produced by immunization of llamas with wildtype vWF, followed by preparation of cDNA from isolated peripheral blood lymphocyte-derived RNA and selection of the vWF A1 domain specific nanobody via phage display. To study recognition of vWF by the nanobody and possible modulating factors we developed an ELISA assay based on a nanobody-coated microtiter plate. Plasmin pre-treatment of normal pooled plasma (NPP) significantly increased nanobody recognition of vWF, but only after subjecting the plasma to shear by vortexing. Ristocetin also increased recognition by the nanobody; however, addition of plasmin to ristocetin-opened vWF in NPP did not further enhance nanobody binding. This suggests that, in contrast to ristocetin, vortexing was not sufficient to fully expose all vWF A1 domains, but the remaining hidden A1 domains were exposed by plasmin cleavage, allowing additional vWF to bind to the nanobody.Ellipsometry was used to assess competition of the nanobody and plasmin binding to vWF. Polyclonal anti-human vWF antibodies were coated to silicon slides, followed by addition of vWF concentrate (Haemate) or recombinant vWF R1306W, plasmin and the nanobody in varying order. We found that plasmin binds much more efficiently to R1306W vWF than to globular vWF. If the nanobody was bound to the R1306W vWF first, plasmin could not bind anymore. Conversely, after plasmin binding, only a small proportion of the nanobody still recognizes the plasmin-sensitive conformation of vWF, suggesting plasmin occupied the majority but not all binding sites and a possible difference in affinity for this conformation of vWF.A flow-cytometry based platelet-activation assay was performed to study the effect of plasmin on the ability of vWF in its plasmin-sensitive conformation to bind to washed platelets. Fluorescent detection of vWF binding was mediated by FITC-labeled monoclonal anti-vWF antibodies or biotin-conjugated labeled nanobody in combination with streptavidin-FITC. Addition of plasmin increased ristocetin-modified vWF binding to platelets by 3.7-fold. This effect of plasmin was abrogated in the presence of heparin, consistent with work by Brophy et al. (2017) showing that heparin prevents cleavage of vWF by plasmin. Hence, it appears that plasmin-cleaved vWF has increased binding affinity to platelets. Of note, a similar pattern in ristocetin-activated vWF to platelet binding upon addition of plasmin (and heparin) was detected by the nanobody. This demonstrates that the nanobody does not interfere with platelet binding of vWF. Moreover, binding of plasmin-cleaved vWF resulted in reduced platelet activation, as determined from P-selectin expression.Discussion: In conclusion, we have identified an anti-vWF nanobody capable of blocking plasmin binding to- and subsequent cleavage of vWF. This nanobody is unable to bind globular vWF in solution or vWF immobilized by polyclonal anti-vWF antibodies, but efficiently interacts with shear- and ristocetin-activated vWF and recombinant vWF comprising the type 2B R1306W mutation. The nanobody epitope on vWF is expected to overlap with- or lie in close proximity to the plasmin binding site in the A1-A2 linker, possibly resulting in steric hindrance. Current studies focus on providing more direct evidence of the vWF binding site of the nanobody. From a clinical perspective, the sensitivity of R1306W vWF to plasmin-mediated cleavage may explain the bleeding diathesis in VWD type 2B patients with this mutation. DisclosuresNo relevant conflicts of interest to declare.

Identification of a VWFA1 Mutation Attenuating Thrombus Growth but Not Platelet Adhesion

The balance between hemostasis and thrombosis is critical to human health. However, intensified thrombosis that breaks this balance is often observed in diseases like hypertension, diabetes and obesity, leading to atherothrombosis in the coronary and cerebral circulations [1]. Current therapeutic strategies to reduce thrombotic risk are efficacious, as they have reduced morbidity and mortality, but still suboptimal, as commercialized drugs carry the risk of excessive bleeding. A new generation of drugs targeted to increase or maintain antithrombotic efficacy while reducing the negative impact on hemostasis is needed.The formation of platelet aggregates over an arterial vascular injury site is a two-step process. Firstly, binding between platelet glycoprotein (GP) Ibα - a component of the GPIb-IX-V receptor complex - and subendothelial von Willebrand Factor (VWF) at its A1 domain (VWFA1) enables recruitment of circulating platelets under high shear force. This represents the first layer of the hemostatic plug initiated by VWFA1-GPIb and consolidated by adhesion of activated integrins. Secondly, adhesion receptors on the up-facing side of the attached platelets engage circulating proteins including VWF, fibrinogen and fibronectin, among others, attracting new platelets into the growing thrombus. Importantly, this step is shared by atherothrombosis as a mechanism of thrombus growth. The GPIbα-VWFA1 interaction operates in both steps, which respectively mediate platelet adhesion and thrombogenesis [2]. Notably, when VWF is immobilized, its association with platelet GPIbα is mediated by two-dimensional (2D) kinetics; in contrast, the attachment of plasma VWF to immobilized platelets in the growing aggregate is governed by three-dimensional (3D) kinetics.We have identified positively charged, GPIbα-non-contact residues in helices α4/α5 of the VWFA1 that partially affect the VWF-GPIbα interaction. A triple mutation, R636/K642/K643A (referred to as M13) (Fig. 1A) drastically reduces the 3D on-rate of binding to GPIbα without significantly affecting the off-rate, which remains comparable to VWFA1-WT as measured by surface plasma resonance. In this assay, purified GPIbα molecules were immobilized on a surface that was then perfused with soluble monomeric VWFA1 at different concentrations. As a consequence of the decreased 3D on-rate, the 3D GPIbα binding affinity of M13 VWFA1 is 20-fold lower than that of WT VWFA1 (Fig. 1B). In agreement with this finding, aggregometry experiments showed that platelet agglutination mediated by dimeric M13 VWFA1 in the presence of ristocetin was greatly attenuated compared to WT (Fig. 1C), and the speed of agglutination was also slowed (Fig. 1D), implying that M13 is weaker in establishing platelet-platelet interactions.In contrast, the M13 mutation does not compromise the adhesion of platelets to immobilized VWFA1. In a flow chamber system, anticoagulated whole blood was perfused over a surface pre-coated with monomeric WT or M13 VWFA1. Under a range of shear rates and with different VWFA1 coating densities, the coverage of platelets over immobilized M13 VWFA1 was never less, if not greater than on WT VWFA1 (Fig. 1E,F, upper panels); accordingly, the rolling velocity of adherent platelets was similar or slower on M13 than WT VWFA1 (Fig. 1E,F, lower panels).We next investigated how the VWF M13 mutation affects in vivo thrombogenesis using a VWF-/- mouse strain in which platelets expresses human GPIbα. Mammalian cell expressed full-length human WT and M13 VWF was infused into these mice in combination with human FVIII to correct for the coagulation associated with VWFdeficiency; a carotid artery lesion was then caused with 9% ferric chloride. Our preliminary results showed that WT VWF restored a complete thrombotic occlusion of the artery, but mutant M13 VWF was without effect (Fig. 1G).Overall, our data suggests that VWFA1 domain can be engineered to support efficient platelet adhesion, which is key to hemostasis, while being less prone to mediate endovascular thrombogenesis. The development of this study should provide new insights into the distinct mechanisms that support VWF contributing to hemostasis or thrombosis, as well as into designing more selective pro-hemostatic VWF molecules with a reduced risk of causing thrombotic complications.

High-resolution structure of the vWF A1 domain in complex with caplacizumab, the first nanobody-based medicine for treating acquired TTP

von Willebrand factor (vWF) is a huge oligomeric glycoprotein involved in blood homeostasis. However, this protein is also implicated in acquired thrombotic thrombocytopenic purpura (TTP). The blocking of its binding with platelets has been recognized as an attractive therapeutic strategy for treating acquired TTP. Caplacizumab, a bivalent single-domain antibody (VHH), is the first FDA-approved nanobody drug against vWF for the treatment of acquired TTP. Here, we describe the crystal structure of the A1 domain of vWF in complex with the caplacizumab nanobody at the resolution of 1.60 Å. This structure elucidates the precise epitope and binding mode of caplacizumab. Unexpectedly, caplacizumab binds to the bottom face of the vWF A1 domain and does not create any steric clash with platelet-receptor glycoprotein Ib (GPIb) bound to vWF. However, its binding can stabilize the different conformation within the N-terminus and α1β2 loop from the GPIb bound structure, suggesting that the mechanisms of caplacizumab would not be the direct competition of GPIb binding to vWF A1 domain but the conformational arrestment of vWF in an inappropriate state to platelet adhesion. This high-resolution structure would provide helpful information for the design of improved anti-vWF therapeutics for the treatment of acquired TTP.

Type 2A and 2M von Willebrand Disease: Differences in Phenotypic Parameters According to the Affected Domain by Disease-Causing Variants and Assessment of Pathophysiological Mechanisms.

Type 2A and 2M von Willebrand disease (VWD) broadly show similar phenotypic parameters, but involve different pathophysiological mechanisms. This report presents the clinical and laboratory profiles of type 2A and type 2M patients genotypically diagnosed at one large center. Higher bleeding score values and a higher incidence of major bleeding episodes were observed in type 2A compared with type 2M, potentially reflective of the absence of large and intermediate von Willebrand factor (VWF) multimers in 2A. In type 2A, most of disease-causing variants (DCVs) appeared to be responsible for increased VWF clearance and DCV clustered in the VWF-A1 domain resulted in more severe clinical profiles. In type 2M, DCV in the VWF-A1 domain showed different laboratory patterns, related to either reduced synthesis or shortened VWF survival, and DCV in the VWF-A2 domain showed patterns related mainly to shortened survival. VWF-type 1 collagen binding/Ag (C1B/Ag) showed different patterns according to DCV location: in type 2A VWD, C1B/Ag was much lower when DCVs were located in the VWF-A2 domain. In type 2M with DCV in the VWF-A1domain, C1B/Ag was normal, but with DCV in the VWF-A2 domain, C1B/Ag was low. The higher frequency of major bleeding in VWD 2M patients with DCV in the VWF-A2 domain than that with DCV in the VWF-A1 domain could be a summative effect of abnormal C1B/Ag, on top of the reduced VWF-GPIb binding. In silico modeling suggests that DCV impairing the VWF-A2 domain somehow modulates collagen binding to the VWF-A3 domain. Concomitant normal FVIII:C/Ag and VWFpp/Ag, mainly in type 2M VWD, suggest that other nonidentified pathophysiological mechanisms, neither related to synthesis/retention nor survival of VWF, would be responsible for the presenting phenotype.

Shear Stress Accumulation Enhances von Willebrand Factor-Induced Platelet P-Selectin Translocation in a PI3K/Akt Pathway-Dependent Manner.

Platelet adhesion and activation through the interaction of von Willebrand factor (VWF) with platelet glycoprotein (GP) Ibα are the early key events in hemostasis and thrombosis especially under high blood shear stress. P-selectin translocation from α granule to the cell surface is a typical platelet function phenotype, which makes the platelet-induced inflammatory response of flowing leukocytes possible and can be induced by either chemical agonists (thrombin, ADP, etc.) or high blood shear stress, but regulations of VWF mutation and blood shear stress on VWF-induced P-selectin translocation remain unclear. With flow cytometry, parallel plate flow chamber, and immunofluorescence staining techniques, we examined the P-selectin translocation of platelets on immobilized wild-type (WT) VWF-A1 domain and its two mutants, the gain-of-function (GOF) mutant R1308L and the loss-of-function (LOF) mutant G1324S, respectively. The results showed that the VWF-A1-induced platelet P-selectin translocation was triggered, accelerated, and enhanced by fluid shear stress and could be correlated with shear stress accumulation (SSA, the product of fluid shear stress and mechanical stimulus time), and the PI3K/Akt axis was involved in the platelet P-selectin translocation. The force-triggered P-selectin translocation occurred quickly on partial platelet surface first and then extended gradually to the whole platelet surface as SSA increased. The P-selectin translocation process would be promoted by the GOF mutation (R1308L) but slowed down by the LOF mutation (G1324S). These findings demonstrated a force-enhanced regulation mechanism for the VWF-induced platelet P-selectin translocation through the PI3K/Akt pathway and provided a novel insight into the mechano-chemical regulation mechanism for the key events, such as platelet activation and functional phenotype change in hemostasis and thrombosis.

Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study

BackgroundVon Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous...

The VWF/LRP4/αVβ3-axis represents a novel pathway regulating proliferation of human vascular smooth muscle cells.

Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvβ3 signalling abolished proliferation. However, VWF did not bind to αvβ3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvβ3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.

The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

A Novel COCH Mutation Affects the vWFA2 Domain and Leads to a Relatively Mild DFNA9 Phenotype.

To study the genotype and phenotype of a Dutch family with autosomal dominantly inherited hearing loss. Genotype-phenotype correlation study. Genetic analysis consisted of linkage analysis, variable number of tandem repeats analysis, and Sanger sequencing. Audiovestibular function was examined. Regression analysis was performed on pure tone audiometry and speech recognition scores and correlated with the age and/or level of hearing loss. Tertiary referral center. A large Dutch family presenting with sensorineural hearing loss. Identification of the underlying genetic defect of the hearing loss in this family. Results of pure tone and speech audiometry, onset age, progression of hearing loss and vestibular (dys)function. A novel mutation in COCH, c.1312C > T p.(Arg438Cys), cosegregates with hearing loss and a variable degree of vestibular (dys)function in this family. The reported mean age of onset of hearing loss is 33 years (range, 18-49 yr). Hearing loss primarily affects higher frequencies and its progression is relatively mild (0.8 dB/yr). Speech perception is remarkably well preserved in affected family members when compared with other DFNA9 families with different COCH mutations. These findings expand the genotypic and phenotypic spectrum of DFNA9. The c.1312C > T mutation, which affects the vWFA2 domain, causes a relatively mild audiovestibular phenotype when compared with other COCH mutations.

Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion

BackgroundThrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis.MethodsThe 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays.ResultsThree members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro.ConclusionsWe have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina.Graphical

Binding of Gpibα to VWF A2 Domain Alters Mechanical Unraveling of the A2 Domain

Background: Von Willebrand factor (VWF), is a large, multimeric plasma glycoprotein that plays a critical role in hemostasis. VWF is synthesized and secreted as ultra large (UL) multimers that contain up to 100 protomers. If not processed by ADAMTS13, a plasma metalloprotease, ULVWF may initiate the spontaneous formation of life-threatening thrombi, as seen in thrombotic thrombocytopenic purpura (TTP). The cleavage site is buried under the central β-sheet within the VWF-A2 domain and tensile force is required to expose the cleavage site for ADAMTS13. Our prior studies demonstrate that several VWF-binding proteins, including coagulation factor VIII, apoB100/LDL, as well as the ectodomains of platelet glycoprotein Iba (GPIba), appear to function as cofactors that facilitate the proteolytic cleavage of VWF by ADAMTS13 under shear. However, the mechanism underlying GPIba enhancing effect on ADAMTS13-mediated VWF proteolysis is yet to be determined. Methods: Recombinant human GPIbα was purchased from Sino Biological. The recombinant VWF-A2 domain fragment with a SpyTag on the N-terminus and an AviTag-HisTag on the C-terminus was expressed in the HEK 293T cells and affinity-purified by the Ni-NTA affinity chromatography. Biotinylation was performed in vitro using a biotin-labeling kit. The binding between VWF-A2 and GPIbα was studied by a custom-built atomic force microscope (AFM) using our established single-molecule binding study protocol. MicroScale Thermophoresis was conducted by a Monolith device to detect the binding affinity between the VWF-A2 and GPIbα. A dual-beam mini-tweezers instrument was utilized to characterize the force-induced conformational changes of the A2 domain in the absence and presence of GPIbα with a pulling speed of 200 nm/s. Results: AFM results indicated that specific binding interactions occurred between GPIbα and VWF-A2. Monolith MST assay revealed a strong binding affinity (Kd of ~20 nM) between GPIbα and the VWF-A2 fragment. In the optical tweezer study, pulling on a single VWF-A2 resulted in an unfolding event at 10-30 pN with an extension ranging from 30 to 40 nm (Fig. 1A). Gaussian fits of the unfolding extension distributions revealed a most probable force-induced extension of 34.87 ± 2.2 nm (mean ± SEM) (Fig. 1B). Addition of 100 nM of GPIbα led to a noticeable decrease in both unfolding force and extension of VWF-A2 (Fig. 1A). The most probable unfolding extension reduced to 16.05 ± 0.2 nm in the presence of 100 nM of GPIbα (Fig. 1B), indicating the binding of GPIbα may influence mechanical unfolding of VWF-A2. Further, the unfolding results were analyzed by a worm-like chain model fit, which yielded a contour length for the initially folded structure of VWF-A2 at 58.83 ± 2.0 nm (mean ± SEM) and 24.53 ± 0.2 nm in the absence and presence of GPIbα, respectively (Fig. 1C), indicating that the specific interactions between GPIbα and A2 domain may partially unfold the A2 domain. Conclusions: These results demonstrate for the first time that binding of GPIbα to VWF-A2 may alter the force-induced conformational changes in the A2 domain. Under physiological conditions, the glycocalicin (or soluble GP1bα) may bind the VWF-A2 and cause A2 partial unfolding, which may result in excessive cleavage of VWF by ADAMTS13, thus regulating hemostasis. Figure legend: Fig.1. GPIbα influences the mechanical unfolding of the A2 domain of VWF. (A) Typical optical tweezer pulling traces of A2 domain in the absence (red) and presence (blue) of GPIbα (100 nM). The arrows point to the unfolding events. The pulling speed is 200 nm/s. (B) The histograms of the unfolding extension of pulling VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα at 200 nm/s. Solid lines are Gaussian fits to the distributions. (C) The relationship between unfolding force (pN) and unfolding extension (nm) of pulling the VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα. The data are fitted to the worm-like chain model (solid lines). Horizontal and vertical error bars are one standard deviation for force and half width of the half bin width for extension, respectively. Figure Disclosures Cao: Ivygen: Consultancy; Bayer: Research Funding. Zheng:Sanofi: Consultancy, Speakers Bureau; Clotsolution: Other: Co-Founder; Alexion: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses.

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA–vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA–vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics—guiding DNA to a specific protein binding site—as the main driving force defining DNA–vWF recognition. The molecular picture of a key shear-mediated DNA–protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses.