Blood Protein Von Willebrand Factor Research Articles

A continuum model for the elongation and orientation of Von Willebrand factor with applications in arterial flow.

The blood protein Von Willebrand factor (VWF) is critical in facilitating arterial thrombosis. At pathologically high shear rates, the protein unfolds and binds to the arterial wall, enabling the rapid deposition of platelets from the blood. We present a novel continuum model for VWF dynamics in flow based on a modified viscoelastic fluid model that incorporates a single constitutive relation to describe the propensity of VWF to unfold as a function of the scalar shear rate. Using experimental data of VWF unfolding in pure shear flow, we fix the parameters for VWF's unfolding propensity and the maximum VWF length, so that the protein is half unfolded at a shear rate of approximately . We then use the theoretical model to predict VWF's behaviour in two complex flows where experimental data are challenging to obtain: pure elongational flow and stenotic arterial flow. In pure elongational flow, our model predicts that VWF is 50% unfolded at approximately , matching the established hypothesis that VWF unfolds at lower shear rates in elongational flow than in shear flow. We demonstrate the sensitivity of this elongational flow prediction to the value of maximum VWF length used in the model, which varies significantly across experimental studies, predicting that VWF can unfold between depending on the selected value. Finally, we examine VWF dynamics in a range of idealised arterial stenoses, predicting the relative extension of VWF in elongational flow structures in the centre of the artery compared to high shear regions near the arterial walls.

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.

Oxidation shuts down an auto-inhibitory mechanism of von Willebrand factor.

The blood protein von Willebrand factor (VWF) is a key link between inflammation and pathological thrombus formation. In particular, oxidation of methionine residues in specific domains of VWF due to the release of oxidants in inflammatory conditions has been linked to an increased platelet-binding activity. However, the atomistic details of how methionine oxidation activates VWF have not been elucidated to date. Yet understanding the activation mechanism of VWF under oxidizing conditions can lead to the development of novel therapeutics that target VWF selectively under inflammatory conditions in order to reduce its thrombotic activity while maintaining its haemostatic function. In this manuscript, we used a combination of a dynamic flow assay and molecular dynamics (MD) simulations to investigate how methionine oxidation removes an auto-inhibitory mechanism of VWF. Results from the dynamic flow assay revealed that oxidation does not directly activate the A1 domain, which is the domain in VWF that contains the binding site to the platelet surface receptor glycoprotein Ibα (GpIbα), but rather removes the inhibitory function of the neighboring A2 and A3 domains. Furthermore, the MD simulations combined with free energy perturbation calculations suggested that methionine oxidation may destabilize the binding interface between the A1 and A2 domains leading to unmasking of the GpIbα-binding site in the A1 domain.

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.

Abstract 1553: Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies

Abstract Cancer immunotherapy with immune checkpoint inhibitors (CPI), interleukin (IL)-2, and IL-12 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-CTLA4 (αCTLA4) + anti-PD-L1 (αPD-L1) and the cytokines IL-2 and IL-12 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokines) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, with 57% of total injected dose depositing in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2/IL-12. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. CBD-fusion to IL-12 significantly decreased hepatotoxicity and systemic IFNg concentration. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. CBD-IL-12 therapy induced remission of B16F10 melanoma and EMT6 breast cancer, along with increased intratumoral IFNg concentration compared to normal IL-12 therapy. Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokines) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. Unlike other active tumor-targeting approaches, CBD-based targeting is novel in a sense that it exploits abnormal tumor vessel structure; this characteristic affords binding to the ubiquitous extracellular matrix protein in the tumor, while limiting exposure to other tissues. CBD-immunotherapeutics may efficiently activate tumor-antigen-specific T cells and increase intratumoral downstream cytokines while maintaining systemic immune homeostasis by avoiding influencing non-tumor antigen specific T cells and systemic cytokine release. Citation Format: Jun Ishihara, Ako Ishihara, Aslan Mansurov, Koichi Sasaki, Steve S. Lee, John-Michael Williford, Lambert Potin, Peyman Hosseinchi, Laura T. Gray, Kiyomitsu Katsumata, Stephen J. Kron, Melody A. Swartz, Jeffrey A. Hubbell. Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1553.

Collagen affinity improves efficacy and safety of antibody and cytokine cancer immunotherapy.

e14232 Background: Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Methods: We addressed this need by targeting both the CPI antibodies anti-CTLA4 (aCTLA4) + anti-PD-L1 (aPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. Results: We show that intravenously (i.v.) administered CBD protein accumulated in tumors in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both aCTLA4+aPD-L1 combination therapy and IL-2. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. Conclusions: Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokine) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms.

Abstract IA07: Using matrix protein affinity to modulate the tumor microenvironment

Abstract Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T-lymphocyte antigen 4 antibody (CTLA4) + anti-programmed death-ligand 1 antibody (PD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, for example 57% of total injected dose depositing in an orthotopic breast tumor model. The CBD was observed to localize throughout the tumor stroma, not merely in the subendothelial space. CBD conjugation or fusion decreased the systemic toxicity of both CTLA4+PD-L1 combination therapy and IL-2, for example eliminating hepatotoxicity with the CPI molecules and ameliorating capillary leak syndrome and pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. Thus, the A3 domain of von Willebrand factor can be used to engineer immunotherapies with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. The targeting approach exploits vascular permeability in the tumor to render the ubiquitous extracellular matrix protein accessible in the tumor, while sparing most other tissues. Citation Format: Jun Ishihara, Ako Ishihara, Koichi Saskai, Steve Seung-Young Lee, Mariko Yasui, Hiroyuki Abe, Lambert Potin, Peyman Hosseinchi, Kazuto Fukunaga, Michal M. Raczy, Laura T. Gray, John-Michael Williford, Masashi Fukayama, Tiffany M. Marchell, Aslan Mansurov, Aaron T. Alpar, Stephen J. Kron, Melody Swartz, Jeffrey A. Hubbell. Using matrix protein affinity to modulate the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA07.

Entrapment of Autologous von Willebrand Factor on Polystyrene/Poly(methyl methacrylate) Demixed Surfaces.

Human platelets play a vital role in haemostasis, pathological bleeding and thrombosis. The haemostatic mechanism is concerned with the control of bleeding from injured blood vessels, whereby platelets interact with the damaged inner vessel wall to form a clot (thrombus) at the site of injury. This adhesion of platelets and their subsequent aggregation is dependent on the presence of the blood protein von Willebrand Factor (vWF). It is proposed here that the entrapment of vWF on a substrate surface offers the opportunity to assess an individual’s platelet function in a clinical diagnostic context. Spin coating from demixed solutions of polystyrene (PS) and poly(methyl methacrylate) (PMMA) onto glass slides has been shown previously to support platelet adhesion but the mechanism by which this interaction occurs, including the role of vWF, is not fully understood. In this work, we report a study of the interaction of platelets in whole blood with surfaces produced by spin coating from a solution of a weight/weight mixture of a 25% PS and 75% PMMA (25PS/75PMMA) in chloroform in the context of the properties required for their use as a Dynamic Platelet Function Assay (DPFA) substrate. Atomic Force Microscopy (AFM) indicates the presence of topographical features on the polymer demixed surfaces in the sub-micron to nanometer range. X-ray Photoelectron Spectroscopy (XPS) analysis confirms that the uppermost surface chemistry of the coatings is solely that of PMMA. The deliberate addition of various amounts of 50 μm diameter PS microspheres to the 25PS/75PMMA system has been shown to maintain the PMMA chemistry, but to significantly change the surface topography and to subsequently effect the scale of the resultant platelet interactions. By blocking specific platelet binding sites, it has been shown that their interaction with these surfaces is a consequence of the entrapment and build-up of vWF from the same whole blood sample.

Allosteric activation of ADAMTS13 by von Willebrand factor.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.

Mechanoregulation in Nanoscale Biology: From Hemostasis to Single-Molecule Centrifugation

Many aspects of biological function and structure are governed by weak non-covalent bonds between and within single molecules. Mechanical force plays a key role in modulating the kinetics of these interactions, which in turn can regulate more complex biological processes. In this talk, I will focus on how forces in the circulatory system regulate hemostasis by acting on the blood protein von Willebrand factor (VWF). Using optical tweezers, we have demonstrated that force acts as a cofactor the enzymatic cleavage of VWF, which in turn down regulates hemostatic potential [1].

Von Willebrand factor self-association on platelet GpIbα under hydrodynamic shear: effect on shear-induced platelet activation

AbstractThe function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ibα (GpIbα) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488–conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce ΔA1-488. Although ΔA1-488 alone did not bind platelets under shear, this protein bound GpIbα on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than ∼ 60 to 70 dyne/cm2. ΔA1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ΔA1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-µm beads bearing either immobilized VWF or anti-GpIbα mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm2. Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIbα via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.

Application of Fluorescence Spectroscopy to Quantify Shear-Induced Protein Conformation Change

Rapid and robust methods are required to quantify the effect of hydrodynamic shear on protein conformation change. We evaluated such strategies in this work and found that the binding of the fluorescent probe 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) to hydrophobic pockets in the blood protein von Willebrand factor (VWF) is enhanced upon the application of fluid shear to the isolated protein. Significant structural changes were observed when the protein was sheared at shear rates ≥ 6000/s for ∼3.5 min. The binding of bis-ANS to multimeric VWF, but not dimeric VWF or control protein bovine serum albumin, was enhanced upon fluid shear application. Thus, high-molecular-weight VWF is more susceptible to conformation change upon tensile loading. Although bis-ANS itself did not alter the conformation of VWF, it stabilized protein conformation once it bound the sheared molecule. Bis-ANS binding to VWF was reduced when the sheared protein was allowed to relax before dye addition. Taken together with functional data in the literature, our results suggest that shear-induced conformation changes in VWF reported by bis-ANS correlate well with the normal function of the protein under physiological/pathological fluid flow conditions. Further, this study introduces the fluorescent dye bis-ANS as a tool that may be useful in studies of shear-induced protein conformation change.

Solution structure of human blood protein Von Willebrand factor

The binding of multimeric blood plasma protein von Willebrand factor (vWF) to platelet receptor GpIb under high shear stress is an important step regulating atherothrombosis. We coupled light scattering with small-angle neutron scattering (SANS) to study the solution conformation of vWF. vWF resembled a prolate ellipsoid with a minor axis of length ~50nm and a major axis of ~240nm. Analysis using the unified equation and high-resolution shape fitting revealed repeating structures within vWF at different length scales, which were indicative of the multimeric protein (120nm), monomer unit (20.1nm) and individual domains (3.1nm). Treatment of vWF with guanidine-HCl, which increases the susceptibility of vWF for proteolysis by ADAMTS-13, caused local structural changes at length scales below ~8nm without altering protein radius of gyration. The results suggested that vWF adopts a compact conformation in solution with non-covalent interactions between individual domains. This prediction was verified in western blot runs where vWF was randomly crosslinked with homobifunctional linker BS3 prior to dissociation of disulfide linkages between vWF monomers using dithiothreitol (DTT). Here, electrophoresis of BS3 and DTT treated samples resulted in a pattern of dimer and trimer units that suggest intramolecular interaction between protein sub-domains. Overall, our studies reveal the key structural features of vWF in solution and suggest mechanisms by which this protein may undergo conformation change and self-association under fluid shear. Supported by grants NIH HL63014 and HL76211