Biophysical investigation of interactions between SARS-CoV-2 spike protein and neuropilin-1.

Recent studies have suggested that neuropilin-1 (NRP1) may serve as a potential receptor in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the biophysical characteristics of interactions between NRP1 and SARS-CoV-2 remain unclear. In this study, we examined the interactions between NRP1 and various SARS-CoV-2 spike (S) fragments, including the receptor-binding domain (RBD) and the S protein trimer in a soluble form or expressed on pseudovirions, using atomic force microscopy and structural modeling. Our measurements shows that NRP1 interacts with the RBD and trimer at a higher binding frequency (BF) compared to ACE2. This NRP1-RBD interaction has also been predicted and simulated via AlphaFold2 and molecular dynamics simulations, and the results indicate that their binding patterns are very similar to RBD-ACE2 interactions. Additionally, under similar loading rates, the most probable unbinding forces between NRP1 and S trimer (both soluble form and on pseudovirions) are larger than the forces between NRP1 and RBD and between trimer and ACE2. Further analysis indicates that NRP1 has a stronger binding affinity to the SARS-CoV-2 S trimer with a dissociation rate of 0.87 s-1 , four times lower than the dissociation rate of 3.65 s-1 between NRP1 and RBD. Moreover, additional experiments show that RBD-neutralizing antibodies can significantly reduce the BF for both ACE2 and NRP1. Together, the study suggests that NRP1 can be an alternative receptor for SARS-CoV-2 attachment to human cells, and the neutralizing antibodies targeting SARS-CoV-2 RBD can reduce the binding between SARS-CoV-2 and NRP1.

Penetration of Cell Surface Glycocalyx by Enveloped Viruses Is Aided by Weak Multivalent Adhesive Interaction.

Viral infection usually begins with adhesion between the viral particle and viral receptors displayed on the cell membrane. The exterior surface of the cell membrane is typically coated with a brush-like layer of molecules, the glycocalyx, that the viruses need to penetrate. Although there is extensive literature on the biomechanics of virus-cell adhesion, much of it is based on continuum-level models that do not address the question of how virus/cell-membrane adhesion occurs through the glycocalyx. In this work, we present a simulation study of the penetration mechanism. Using a coarse-grained molecular model, we study the force-driven and diffusive penetration of a brush-like glycocalyx by viral particles. For force-driven penetration, we find that viral particles smaller than the spacing of molecules in the brush reach the membrane surface readily. For a given maximum force, viral particles larger than the minimum spacing of brush molecules arrest at some distance from the membrane, governed by the balance of elastic and applied forces. For the diffusive case, we find that weak but multivalent attraction between the glycocalyx molecules and the virus effectively leads to its engulfment by the glycocalyx. Our finding provides potential guidance for developing glycocalyx-targeting drugs and therapies by understanding how virus-cell adhesion works.

Binding of human ACE2 and RBD of Omicron enhanced by unique interaction patterns among SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor-binding domain (RBD) of the spike protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD-ACE2 interface through N501Y, Q498R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Among the Q493K and Q493R, we report that Q493R shows stronger binding to ACE2 than Q493K due to increased interactions. Our MST data confirmed that the Omicron mutations in RBD are associated with a five-fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our results could help explain the Omicron variant's prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity.

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

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

Autoinhibitory module underlies species difference in shear activation of von Willebrand factor.

BACKGROUND: Von 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. OBJECTIVES: To determine and characterize the autoinhibitory properties of mouse VWF. METHODS: Recombinant 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. RESULTS: Recombinant 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. CONCLUSIONS: Our 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.

Binding of Human ACE2 and RBD of Omicron Enhanced by Unique Interaction Patterns Among SARS-CoV-2 Variants of Concern.

The 2019 coronavirus disease (COVID-19) pandemic has had devastating impacts on our global health. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has now overtaken the Delta variant and become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor-binding domain (RBD) of the spike protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD-ACE2 interface through N501Y, Q493K/R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Our MST data confirmed that the Omicron mutations in RBD are associated with a five-fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our result could help explain the Omicron variant’s prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity.

Contribution of the von Willebrand factor/ADAMTS13 imbalance to COVID-19 coagulopathy.

The 2019 coronavirus disease (COVID-19) is the disease caused by SARS-CoV-2 infection. Although this infection has been shown to affect the respiratory system, a high incidence of thrombotic events has been observed in severe cases of COVID-19 and in a significant portion of COVID-19 nonsurvivors. Although prior literature has reported on both the coagulopathy and hypercoagulability of COVID-19, the specifics of coagulation have not been fully investigated. Observations of microthrombosis in patients with COVID-19 have brought attention to potential inflammatory endothelial injury. Von Willebrand factor (VWF) and its protease, A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), play an important homeostatic role in responding to endothelial injury. This report provides an overview of the literature investigating the role the VWF/ADAMTS13 axis may have in COVID-19 thrombotic events and suggests potential therapeutic strategies to prevent the progression of coagulopathy in patients with COVID-19.

Biophysical characterization of lynx-nicotinic receptor interactions using atomic force microscopy.

Nicotinic acetylcholine receptors (nAChRs) are broadly expressed in the central and peripheral nervous systems, playing essential roles in cholinergic neurotransmission. The lynx family proteins, a subset of the Ly6/uPAR superfamily expressed in multiple brain regions, have been shown to bind to nAChRs and modulate their function via allosteric regulation. The binding interactions between lynx and nAChRs, however, have not been systematically quantified and compared. In this work, we characterized the interactions between lynx1 or lynx2 and α3ß4- or α7-nAChRs using single-molecule atomic force microscopy (AFM). The AFM technique allows the quantification of the off-rate of lynx-nAChR binding and of the energetic barrier width between the bound state and transition state, providing a biophysical means to compare the selectivity of lynx proteins for nAChR subtypes. Results indicate that lynx1 has a marginal preference for α7- over α3ß4-nAChRs. Strikingly, lynx2 exhibits a two order of magnitude stronger affinity for α3ß4- compared to α7-nAChRs. Together, the AFM assay serves as a valuable tool for the biophysical characterization of lynx-nAChR binding affinities. Revealing the differential affinities of lynx proteins for nAChR subtypes will help elucidate how lynx regulates nAChR-dependent functions in the brain, including nicotine addiction and other critical pathways.

Flow-regulated nucleation protrusion theory for collapsed polymers.

The globular-stretch transition of a collapsed polymer in low strain rate elongational flow is studied using polymeric protrusion kinetics scaling laws and numerical simulation. Results demonstrate the influence of fluid flow on the occurrence probability of long-length thermally nucleated polymeric protrusions, which regulate collapsed polymer unfolding in low strain rate flows. Further, we estimate that the globular-stretch transition rate (k_{s}) in low strain rate (∈[over ̇]) elongational flows varies as k_{s}∼e^{-α∈[over ̇]^{-1}}. Results here reveal that the existing approach of neglecting the effects of fluid flow on thermally nucleated protrusions distribution is not valid for analyzing polymer unfolding behavior in low strain rate flows. Neglecting such an effect overestimates the constant α in the scaling law of transition rate (k_{s}∼e^{-α∈[over ̇]^{-1}}) by a factor of 2.

Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. It is known that the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 interacts with the human angiotensin-converting enzyme 2 (ACE2) receptor, initiating the entry of SARS-CoV-2. Since its emergence, a number of SARS-CoV-2 variants have been reported, and the variants that show high infectivity are classified as variants of concern according to the United States Centers for Disease Control and Prevention. In this study, we performed both all-atom steered molecular dynamics (SMD) simulations and microscale thermophoresis (MST) experiments to characterize the binding interactions between ACE2 and RBD of all current variants of concern (Alpha, Beta, Gamma, and Delta) and two variants of interest (Epsilon and Kappa). We report that RBD of the Alpha (N501Y) variant requires the highest amount of force initially to be detached from ACE2 due to the N501Y mutation in addition to the role of N90-glycan, followed by Beta/Gamma (K417N/T, E484 K, and N501Y) or Delta (L452R and T478 K) variants. Among all variants investigated in this work, RBD of the Epsilon (L452R) variant is relatively easily detached from ACE2. Our results from both SMD simulations and MST experiments indicate what makes each variant more contagious in terms of RBD and ACE2 interactions. This study could shed light on developing new drugs to inhibit SARS-CoV-2 entry effectively.

Differential Interactions Between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. It is known that the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 interacts with the human angiotensin-converting enzyme 2 (ACE2) receptor, initiating the entry of SARS-CoV-2. Since its emergence, a number of SARS-CoV-2 variants have been reported, and the variants that show high infectivity are classified as the variants of concern according to the US CDC. In this study, we performed both all-atom steered molecular dynamics (SMD) simulations and microscale thermophoresis (MST) experiments to characterize the binding interactions between ACE2 and RBD of all current variants of concern (Alpha, Beta, Gamma, and Delta) and two variants of interest (Epsilon and Kappa). We report that the RBD of the Alpha (N501Y) variant requires the highest amount of force initially to be detached from ACE2 due to the N501Y mutation in addition to the role of N90-glycan, followed by Beta/Gamma (K417N/T, E484K, and N501Y) or Delta (L452R and T478K) variant. Among all variants investigated in this work, the RBD of the Epsilon (L452R) variant is relatively easily detached from ACE2. Our results combined SMD simulations and MST experiments indicate what makes each variant more contagious in terms of RBD and ACE2 interactions. This study could help develop new drugs to inhibit SARS-CoV-2 entry effectively.

Binding kinetics of liposome conjugated E-selectin and P-selectin glycoprotein ligand-1 measured with atomic force microscopy.

Various ligand-functionalized liposomes have been developed for targeted therapies. Typically, the binding properties of the ligands and targeted proteins are measured with surface plasmon resonance (SPR), where the proteins are immobilized on a rigid surface. However, the difference of protein-ligand binding kinetics between liposome-conjugated protein and rigid surface-conjugated protein is not fully understood. In this work, the binding kinetics of P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin conjugated on liposome and on rigid surfaces are investigated with Atomic Force Microscopy (AFM). The results suggest that protein orientation and diffusion on liposomal membrane can alter the binding kinetics of the protein-ligand interaction. Specifically, the association and dissociation rate constant of AFM probe-conjugated E-selectin and glass-conjugated PSGL-1 are measured as 9.32 × 104 M-1s-1 and 1.54 s-1, respectively. While for the liposome-conjugated E-selectin and glass-conjugated PSGL-1, the kinetic constants are measured as 5.00 × 107 M-1s-1 and 2.76 s-1, respectively. Thus, there is an order's magnitude increase of binding affinity (from kd = 16.51 µM to kd = 0.06 µM) when protein is attached to liposome compared to attached to a rigid surface. The results might provide better understanding and pave the way for the future design of the ligand-targeted liposomes.

Recent Developments in Nanomaterial-Based Shear-Sensitive Drug Delivery Systems.

Nanomaterial-based drug delivery systems (DDSs) increase the efficacy of various therapeutics, and shear stress has been shown to be a robust modulator of payload release. In the past few decades, a deeper understanding has been gained of the effects of flow in the body and its alteration in pathological microenvironments. More recently, shear-responsive nanomaterial DDSs have been developed. Studies on this subject mainly from the last decade are reviewed here, focusing on innovations of the material design and mechanisms of the shear response. The two most popular shear-controlled drug carriers distinguished by different release mechanisms, that is, shear-deformable nanoparticles (NPs) and shear-dissociated NP aggregates (NPAs), are surveyed. The influence of material structures on their properties such as drug loading, circulation time, and shear sensitivity are discussed. The drug development stages, therapeutic effects, limitations, and potential of these DDSs are further inspected. The reviewed research emphasizes the advantages and significance of nanomaterial-based shear-sensitive DDSs in the field of targeted drug delivery. It is also believed that efforts to rationally design nanomaterial DDSs responsive to shear may prompt a new class of diagnostics and therapeutics for signaling and rectifying pathological flows in the body.

Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module.

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 to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.

Predicting pathological von Willebrand factor unraveling in elongational flow.

The globular-to-unraveled conformation transition of von Willebrand factor (vWF), a large polymeric glycoprotein in human blood plasma, is a crucial step in the process of clotting at sites of vascular injury. However, unraveling of vWF multimers in uninjured vasculature can lead to pathology (i.e., thrombus formation or degradation of vWF proteins by enzyme ADAMTS13, making them nonfunctional). To identify blood flow conditions that might induce pathological unraveling of vWF multimers, here we have computed the globular-to-unraveled transition rate of vWF multimers subjected to varying strain rate elongational flow by employing an enhanced sampling technique, the weighted ensemble method. Weighted ensemble sampling was employed instead of standard brute-force simulations because pathological blood flow conditions can induce undesired vWF unraveling on timescales potentially inaccessible to standard simulation methods. Results here indicate that brief but periodic exposure of vWF to the elongational flow of strain rate greater than or equal to 2500 s-1 represents a source of possible pathology caused by the undesired unraveling of vWF multimers.

Biomechanical characterization of SARS-CoV-2 spike RBD and human ACE2 protein-protein interaction.

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with SARS-CoV-2 RBD decreases to a similar level of the SARS-CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.

Desialylation of O-glycans on glycoprotein Ibα drives receptor signaling and platelet clearance.

During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelets following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelets.

Structure, Dynamics, and Interactions of GPI-Anchored Human Glypican-1 with Heparan Sulfates in a Membrane.

Glypican-1 and its heparan sulfate (HS) chains play important roles in modulating many biological processes including growth factor signaling. Glypican-1 is bound to a membrane surface via a glycosylphosphatidylinositol (GPI)-anchor. In this study, we used all-atom molecular modeling and simulation to explore the structure, dynamics, and interactions of GPI-anchored glypican-1, three HS chains, membranes, and ions. The folded glypican-1 core structure is stable, but has substantial degrees of freedom in terms of movement and orientation with respect to the membrane due to the long unstructured C-terminal region linking the core to the GPI-anchor. With unique structural features depending on the extent of sulfation, high flexibility of HS chains can promote multi-site interactions with surrounding molecules near and above the membrane. This study is a first step toward all-atom molecular modeling and simulation of the glycocalyx, as well as its modulation of interactions between growth factors and their receptors.

Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress.

BACKGROUND: Von Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C-domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined. METHODS: VWF free thiols were blocked with N-ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF-C4 domain. RESULTS: Blockade of VWF free thiols reduced VWF-mediated platelet capture to collagen in a shear-dependent manner, with platelet capture virtually abolished above 5000 s-1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen-bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol-blockade reduced the lifetime and strength of the VWF-collagen bond. Pulling simulations of the VWF-C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free-thiol exchange. CONCLUSIONS: We conclude that free thiols in the C-domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.