Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers.

Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. In this study, we combine fluorescence correlation spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate γ˙1/2 = 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysfunction.

Ratchet effect for two-dimensional nanoparticle motion in a corrugated oscillating channel.

The motion of a single rigid or elastic particle inside a corrugated narrow channel is investigated by means of Brownian dynamics simulations. Periodic oscillations of one of the asymmetric channel surfaces induce directed particle transport. For different surface structures of the resting channel surface, we determine optimal transport properties in terms of the driving frequency, particle size, and corrugation amplitude. The transport direction is changed when switching from perpendicular motion of the oscillating surface to parallel motion with respect to the resting surface, which can be rationalized by a transition from a flashing to a pushing ratchet effect. We also study the diffusion behavior and find strongly enhanced diffusion for parallel oscillatory motion with a diffusivity significantly larger than for free diffusion. Elastic large particles exhibit suppressed transport with increasing rigidity. In contrast, for small particles, increasing rigidity enhances the particle transport both in terms of particle velocity and diffusivity.

Internal tension in a collapsed polymer under shear flow and the connection to enzymatic cleavage of von Willebrand factor.

By means of Brownian hydrodynamics simulations we show that the tension distribution along the contour of a single collapsed polymer in shear flow is inhomogeneous and above a threshold shear rate exhibits a double-peak structure when hydrodynamic interactions are taken into account. We argue that the tension maxima close to the termini of the polymer chain reflect the presence of polymeric protrusions. We establish the connection to shear-induced globule unfolding and determine the scaling behavior of the maximal tensile forces and the average protrusion length as a function of shear rate, globule size, and cohesive strength. A quasi-equilibrium theory is employed in order to describe the simulation results. Our results are used to explain experimental data for the shear-sensitive enzymatic degradation of von Willebrand factor.

Shear-enhanced adsorption of a homopolymeric globule mediated by surface catch bonds.

The adsorption of a single collapsed homopolymer onto a planar smooth surface in shear flow is investigated by means of Brownian hydrodynamics simulation. While cohesive intra-polymer forces are modeled by Lennard-Jones potentials, surface-monomer interactions are described by stochastic bonds whose two-state kinetics is characterized by three parameters: bond formation rate, bond dissociation rate and an effective catch bond parameter that describes how the force acting on a surface-monomer bond influences the dissociation rate. We construct adsorption state diagrams as a function of shear rate and all three surface-monomer bond parameters. We find shear-induced adsorption in a small range of parameters for low dissociation and association rates and only when the surface-monomer bond is near the transition between slip and catch bond behavior. By mapping on a simple surface-monomer interaction model with conservative pair potentials we try to estimate the conservative potential parameters necessary to observe shear-induced surface adsorption phenomena.

Shear-induced dynamics of polymeric globules at adsorbing homogeneous and inhomogeneous surfaces.

The dynamics and adsorption behavior of a single collapsed homopolymer on a surface in shear flow is investigated by means of Brownian hydrodynamics simulations. We study different homogeneous and inhomogeneous surface models and determine dynamic state diagrams as a function of the cohesive strength, the adhesive strength, and the shear rate. We find distinct dynamical adsorbed states that are classified into rolling and slipping states, globular and coil-like states, as well as isotropic and prolate states. We identify two different cyclic processes based on trajectories of the polymer stretching and the polymer separation from the surface. For adsorption on an inhomogeneous surface consisting of discrete binding sites, we observe stick-roll motion for highly corrugated surface potentials. Although the resulting high surface friction leads to low drift velocities and reduced hydrodynamic lift forces on such inhomogeneous surfaces, a shear-induced adsorption is not found in the presence of full hydrodynamic interactions. A hydrodynamically stagnant surface model is introduced for which shear-induced adsorption is observed in the absence of hydrodynamic interactions.

Ratchet effect for nanoparticle transport in hair follicles.

The motion of a single rigid nanoparticle inside a hair follicle is investigated by means of Brownian dynamics simulations. The cuticular hair structure is modeled as a periodic asymmetric ratchet-shaped surface. Induced by oscillating radial hair motion we find directed nanoparticle transport into the hair follicle with maximal velocity at a specific optimal frequency and an optimal particle size. We observe flow reversal when switching from radial to axial oscillatory hair motion. We also study the diffusion behavior and find strongly enhanced diffusion for axial motion with a diffusivity significantly larger than for free diffusion.

Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor.

We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, "Mutual A domain interactions in the force sensing protein von Willebrand Factor" (Posch et al., 2016) [1].