Exposure of Von Willebrand Factor Cleavage Site in A1A2A3-Fragment under Extreme Hydrodynamic Shear

Abstract

Von Willebrand Factor (vWf) is a giant multimeric extracellular blood plasma involved in hemostasis. In this work we present multi-scale simulations of its three-domains fragment A1A2A3. These three domains are essential for the functional regulation of vWf. Namely the A2 domain hosts the site where the protease ADAMTS13 cleavages the multimeric vWf allowing for its length control that prevents thrombotic conditions. The exposure of the cleavage site follows the elongation/unfolding of the domain that is caused by an increased shear stress in blood. By deploying Lattice Boltzmann molecular dynamics simulations based on the OPEP coarse-grained model for proteins, we investigated at molecular level the unfolding of the A2 domain under the action of a perturbing shear flow. We described the structural steps of this unfolding that mainly concerns the β-strand structures of the domain, and we compared the process occurring under shear with that produced by the action of a directional pulling force, a typical condition of single molecule experiments. We observe, that under the action of shear flow, the competition among the elongational and rotational components of the fluid field leads to a complex behaviour of the domain, where elongated structures can be followed by partially collapsed melted globule structures with a very different degree of exposure of the cleavage site. Our simulations pose the base for the development of a multi-scale in-silico description of vWf dynamics and functionality in physiological conditions, including high resolution details for molecular relevant events, e.g., the binding to platelets and collagen during coagulation or thrombosis.

Molecular interpretation of single-molecule force spectroscopy experiments with computational approaches

“…Modelling shear flow in all-atom MD with explicit solvent is not straightforward, 18 and we used a coarse-grained representations of the protein in implicit solvents with inclusion of hydrodynamic interactions in a lattice-Boltzmann scheme. 39–41 While we could show that a shear rate of ≈10 9 s −1 would lead to protein unfolding on a timescale similar to that of 300 pN directional force, the unfolding mechanisms were markedly different. The unfolding pathways under shear have strong similarities with those observed for thermal unfolding, which is not the case of a directional mechanical force which imposes unfolding along the end-to-end distance.…”

Recent Advances and Emerging Challenges in the Molecular Modeling of Mechanobiological Processes

“…Second, the sampling problem and the limited time scale of these simulations are similar to that observed when running SMDs at experimental pulling forces. For these reasons, coarse-grained biomolecular representations in implicit solvent offer a more direct implementation of hydrodynamic effects and they grant access to longer simulation time scales. ,, Significant progress has been made in the recent years, such as the use of a lattice–Boltzmann scheme for solvent–solute hydrodynamic interactions, and improved coarse-grained models. ,, However, important challenges remain, because even long simulation time scales do not allow to observe significant conformational changes at experimental shear flows. , Employing enhanced sampling techniques, that are usually deployed in a rigorous thermodynamic framework, is not trivial when using these nonequilibrium techniques, but some interesting attempts can be noticed …”

“…As protein conformations evolve under shear, the exposure of protein regions to the solvent changes, and the propagation of the mechanical constraints along the biomolecular structure also varies . Recent simulations have suggested that the force felt by the free end of a protein subject to a constant shear flow significantly varies along time …”

“…Additionally, experimental studies have revealed that the interaction between the A1 domains and the platelet glycoproteins is enhanced by force, which is the signature of a catch-bond system that will be discussed in more details below. While earlier simulations focused on the mechanism of the A1/glycoprotein interaction upon force, ,, more recently, larger scale all-atom MD simulations as well as coarse-grained approaches ,,, have shed light on the molecular mechanisms of the shear effect on domain separation and unfolding upon shear. These questions are not limited to the specific case of the vWf: the peculiar behavior of fibrin (another protein involved in hemostatis) under mechanical load has also recently been investigated using MD simulations …”

“…59,62,63 Significant progress has been made in the recent years, such as the use of a lattice−Boltzmann scheme for solvent−solute hydrodynamic interactions, and improved coarse-grained models. 33,64,65 However, important challenges remain, because even long simulation time scales do not allow to observe significant conformational changes at experimental shear flows. 33,61 Figure 3.…”