Studies of the natural force-induced unfolding of Von Willebrand Factor

When a blood vessel is damaged, cells that are normally below the inner surface of the vessel are exposed to blood. The blood protein, Von Willebrand factor (VWF), then binds to exposed collagen in the vessel and undergoes a conformational change to reveal platelet binding sites. VWF (in large multimeric forms) binds several platelets, which aggregate forming a platelet plug / eventually leading to the formation of a blood clot. Within this process a second VWF cleaving protein (ADAMTS-13) is required to cleave the VWF strands, to prevent the multimers becoming too large and adhesive. Absence of this enzyme is known to lead to diseases such as purpurea. It has been shown that tensile force, caused by rapidly flowing blood helps the cleavage of VWF by ADAMTS-13, believed to be due to unfolding of VWF with consequent exposure of the cleavage site. Understanding how force unfolds VWF is therefore essential to understanding its natural biological function. In this project we propose to investigate the force-induced unfolding of the VWF system by exploiting recent developments in ultra-sensitive force measurements techniques, such as the Biomembrane Force Probe (BFP). The use of force as a denaturant has given new insight into the role of force in a range of biological processes, and as a consequence, researchers have begun to consider force as an important parameter to include in studies of biomolecular structure and function. The atomic force microscope (AFM) has been used for most of these studies, but it is fundamentally limited in this application (by factors such as cantilever stiffness and hydrodynamics) and thus different instrumentation is required to study force-induced unfolding in greater detail. The biomembrane force probe (BFP) is the only instrument capable of spanning a sufficiently positioned and large range of force loading rates, and thus able to overcome these limitations. We at Nottingham have the only BFP of its type outside of the laboratories of its pioneer, Evan Evans, and are the only people to have undertaken dynamic force spectroscopy (DFS) of a protein using a BFP. The project builds on our preliminary experimental studies that have demonstrated that the BFP can provide new insight into protein unfolding under force. Within this project an improved understanding of protein unfolding and the affect of force will emanate, leading to a greater understanding of Nature's design and evolution of unfolding/folding energy landscapes. The project will make a significant impact in our understanding of the properties of VWF in particular, but also on the folding and unfolding of proteins in general in a physiological environment of force.

The blood protein, Von Willebrand factor (VWF), then binds to exposed collagen in damaged blood vessels and a conformational change occurs, exposing platelet binding sites in the A1 and C1 domains. VWF (in large multimeric forms) binds several platelets, eventually leading to the formation of a blood clot. The VWF cleaving protein (ADAMTS-13) cleaves the VWF strands at a cleavage site buried within the A2 domain, and vitally prevents the VWF multimers from becoming too large and adhesive. It has been shown that tensile force caused by rapidly flowing blood facilitates the cleavage of VWF by ADAMTS-13, believed to be due to unfolding the A2 domain and consequent exposure of the cleavage site. Understanding how force unfolds VWF is therefore absolutely essential to understand its natural biological function. Here we propose to investigate the force-induced unfolding of the VWF system by exploiting recent developments in ultra-sensitive force measurements techniques, such as the Biomembrane Force Probe (BFP). Recent studies have demonstrated that force as a denaturant can provide new insight into the structure and function of proteins. Almost exclusively the atomic force microscope (AFM) has been employed for such studies but it is far from ideal in this application (fundamentally limited by factors such as cantilever stiffness and hydrodynamics). The biomembrane force probe (BFP) is the only instrument capable of spanning a sufficiently positioned and large range of force loading rates, and thus overcoming these limitations, and providing significantly more detail. The project will thus provide an improved understanding of protein unfolding and its effect by force, leading to a greater understanding of Nature's design and evolution of unfolding/folding energy landscapes. In particular, the project will make a significant impact in our understanding of the properties of VWF.