Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module.
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SUBMITTER: Arce NA
PROVIDER: S-EPMC8060278 | biostudies-literature | 2021 Apr
REPOSITORIES: biostudies-literature
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Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module.
Arce Nicholas A NA Cao Wenpeng W Brown Alexander K AK Legan Emily R ER Wilson Moriah S MS Xu Emma-Ruoqi ER Berndt Michael C MC Emsley Jonas J Zhang X Frank XF Li Renhao R
Nature communications 20210421 1
Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM ...[more]
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Project description:Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.
| S-EPMC5585049 | biostudies-literature
Project description:Essentials The self-inhibitory mechanism of von Willebrand factor (VWF) remains unclear. Residues flanking the A1 domain of VWF form a discontinuous autoinhibitory module (AIM). rVWF1238-1493 exhibited greater thermostability and inactivity than its shorter counterparts. The cooperative coupling between the N- and C- AIM regions are required for inhibiting A1.SummaryBackground The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF1238-1493 ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF1238-1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238-1461 , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271-1493 , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.
| S-EPMC6173635 | biostudies-literature
Project description:BackgroundVon Willebrand factor (VWF) is a multimeric plasma protein that bridges the gap between vessel injury and platelet capture at high shear rates. Under high shear or tension, VWF can become activated upon the unfolding of its autoinhibitory module (AIM). AIM unfolding exposes the A1 domain, allowing for binding to platelet glycoprotein (GP)Ibα to initiate primary hemostasis. The characteristics of the AIM and its inhibitory properties within mouse VWF are unknown.ObjectivesTo determine and characterize the autoinhibitory properties of mouse VWF.MethodsRecombinant mouse VWF A1 fragments containing or lacking the flanking regions around the A1 domain were generated. We tested the ability of these fragments to bind to human or mouse GPIbα and platelets. We compared the unfolding of mouse AIM-A1 to human AIM-A1 by single-molecule force spectroscopy.ResultsRecombinant mouse AIM-A1 binds with higher affinity to GPIbα than its human counterpart. Recombinant mouse proteins lacking part of the AIM show increased binding to GPIbα. Activated A1 fragments lacking the AIM can effectively agglutinate platelets across the species barrier. Using single-molecule force spectroscopy, we determined that the mouse AIM unfolds under forces similar to the human AIM. Additionally, the human AIM paired with mouse A1 largely recapitulates the behavior of human AIM-A1.ConclusionsOur results suggest that the regulation of VWF-GPIbα binding has been specifically tuned to work optimally in different rheological architectures. Differences in the AIM sequence may contribute to the difference in VWF shear response between human and mice.
| S-EPMC9588639 | biostudies-literature
Project description:BackgroundThe binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O-glycans in VWF are located around the A1 domain. Removing certain O-glycans in the flanking sequences by site-directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals.ObjectivesTo understand if and how desialylation of O-glycans in the flanking sequences increases A1 activity.MethodsA recombinant AIM-A1 fragment encompassing VWF residues 1238-1493 and only O-glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment.ResultsAsialo-AIM-A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen-deuterium exchange rates at residues near the secondary GPIbα binding site and the N-terminal flanking sequence. Asialo-AIM-A1 is less mechanically stable than sialo-AIM-A1, with its unstressed unfolding rate approximately 3-fold greater than the latter.ConclusionsDesialylation of O-glycans around A1 increases its activity by destabilizing the AIM.
| S-EPMC9134874 | biostudies-literature
Project description:von Willebrand Factor (vWF) is a large multimeric protein that binds to platelets and collagen in blood clotting. vWF A2 domain hosts a proteolytic site for ADAMTS13 (A Disintegrin and Metalloprotease with a ThromboSpondin type 1 motif, member 13) to regulate the size of vWF multimers. This regulation process is highly sensitive to force conditions and protein-glycan interactions as the process occurs in flowing blood. There are two sites on A2 domain (N1515 and N1574) bearing various N-linked glycan structures. In this study, we used molecular dynamics (MD) simulation to study the force-induced unfolding of A2 domain with and without a single N-linked glycan type on each site. The sequential pullout of β-strands was used to represent a characteristic unfolding sequence of A2. This unfolding sequence varied due to protein-glycan interactions. The force-extension and total energy-extension profiles also show differences in magnitude but similar characteristic shapes between the systems with and without glycans. Systems with N-linked glycans encountered higher energy barriers for full unfolding and even for unfolding up to the point of ADAMTS13 cleavage site exposure. Interestingly, there is not much difference observed for A2 domain structure itself with and without glycans from standard MD simulations, suggesting roles of N-glycans in A2 unfolding through long-ranged protein-glycan interactions.
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Project description: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.
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Project description:von Willebrand Factor is a mechano-sensitive protein circulating in blood that mediates platelet adhesion to subendothelial collagen and platelet aggregation at high shear rates. Its hemostatic function and thrombogenic effect, as well as susceptibility to enzymatic cleavage, are regulated by a conformational change from a collapsed globular state to a stretched state. Therefore, it is essential to account for the conformation of the vWF multimers when modeling vWF-mediated thrombosis or vWF degradation. We introduce a continuum model of vWF unfolding that is developed within the framework of our multi-constituent model of platelet-mediated thrombosis. The model considers two interconvertible vWF species corresponding to the collapsed and stretched conformational states. vWF unfolding takes place via two regimes: tumbling in simple shear and strong unfolding in flows with dominant extensional component. These two regimes were demonstrated in a Couette flow between parallel plates and an extensional flow in a cross-slot geometry. The vWF unfolding model was then verified in several microfluidic systems designed for inducing high-shear vWF-mediated thrombosis and screening for von Willebrand Disease. The model predicted high concentration of stretched vWF in key regions where occlusive thrombosis was observed experimentally. Strong unfolding caused by the extensional flow was limited to the center axis or middle plane of the channels, whereas vWF unfolding near the channel walls relied upon the shear tumbling mechanism. The continuum model of vWF unfolding presented in this work can be employed in numerical simulations of vWF-mediated thrombosis or vWF degradation in complex geometries. However, extending the model to 3-D arbitrary flows and turbulent flows will pose considerable challenges.
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Project description:Progress in both basic and translational research into the molecular mechanisms of VWD can be seen in multiple fields.Genetics of vwdIn the past several decades, knowledge of the underlying pathogenesis of von Willebrand disease (VWD) has increased tremendously, thanks in no small part to detailed genetic mapping of the von Willebrand Factor (VWF) gene and advances in genetic and bioinformatic technology. However, these advances do not always easily translate into improved management for patients with VWD and low-VWF levels.Vwd and pregnancyFor example, the treatment of pregnant women with VWD both pre- and postpartum can be complicated. While knowledge of the VWF genotype at some amino acid positions can aid in knowledge of who may be at increased risk of thrombocytopenia or insufficient increase in VWF levels during pregnancy, in many cases, VWF levels and bleeding severity is highly heterogeneous, making monitoring recommended during pregnancy to optimize treatment strategies. VWF AND COVID-19: New challenges related to the consequences of dysregulation of hemostasis continue to be discovered. The ongoing COVID-19 pandemic has highlighted that VWF has additional biological roles in the regulation of inflammatory disorders and angiogenesis, disruption of which may contribute to COVID-19 induced vasculopathy. Increased endothelial cell activation and Weibel-Palade body exocytosis in severe COVID-19 lead to markedly increased plasma VWF levels. Coupled with impairment of normal ADAMTS13 multimer regulation, these data suggest a role for VWF in the pathogenesis underlying pulmonary microvascular angiopathy in severe COVID-19.ConclusionWith the increased affordability and availability of next-generation sequencing techniques, as well as a push towards a multi-omic approach and personalized medicine in human genetics, there is hope that translational research will improve VWD patient outcomes.
| S-EPMC9094051 | biostudies-literature
Project description:Shear-induced conformational changes of von Willebrand factor (VWF) play an important role in platelet activation. A novel approach describing VWF unfolding on the platelet surface under dynamic shear stress is proposed. Cumulative effect of dynamic shear on platelet activation via conformational changes of VWF is analysed. The critical condition of shear-induced platelet activation is formulated. The explicit expression for the threshold value of cumulative shear stress as a function of VWF multimer size is derived. The results open novel prospects for pharmacological regulation of shear-induced platelet activation through control of VWF multimers size distribution.
| S-EPMC7289367 | biostudies-literature
Project description:Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)3 that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of approximately 21 pN and approximately 63 nm, respectively. Stretching of (A1A2A3)3 yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.
| S-EPMC2856187 | biostudies-literature