Force- and antibiotic-dependence of von Willebrand factor

Von Willebrand factor (VWF) is a molecule in the blood circulation that makes platelets coagulate to stop bleeding from a cut or other injuries. In vessels were blood flow is very fast coagulation has to be efficient to prevent substantial blood loss, but on the other hand at sites of lower flow platelets should not form dangerous clots. Thus, the required regulation is provided by the ultra-large ball-of- yarn-like molecule von Willebrand factor, which becomes unraveled at high flow. The unraveled linear yarn has different biological functions than a compact coiled one. These changes will be screened in aim 1. The scenario of von Willebrand factor A1 domain binding to a platelet is comparable to a safety belt of a car, the stronger VWF is pulled by the blood flow, the stronger it is held by platelets. In aim 2, I study how this mechanism works in a single molecule. The abrupt increase of platelet binding, which is caused by flow can also be induced by an antibiotic named ristocetin. In the 3rd aim I study if ristocetin has the same effect as flow-forces and how this molecule participates in blood clotting. VWF has multiple functions along the chain and has been shown to serve several roles in different processes beyond blood clotting and has been described as molecular bus, a vehicle that transports the bodys own drugs. It turns out to be dependent on the unraveling of the ball-of-yarn shape by flow-forces. Convinced that we still have not seen the bottom, I want to screen the whole sequence of force- extended VWF with a method that yields structural information, in order to pinpoint all regions where deformations occur, which will indicate force dependent functions. VWF is like a fishing line that captures fast flowing platelets, while most binding reactions between large proteins are relatively slow. I will shed light onto the working principle of the fishing hook and also question why ristocetin literally acts as the lure. I will simulate the blood flow in microscopically small channels and detect light emitting dyes attached to the proteins using a microscope. I will pioneer a method were hydrogen atoms are exchanged by heavy hydrogen during deformation of the proteins. This allows detection of flow-induced changes of protein structures. VWF has a structure-function relationship that directly responds to hydrodynamic forces, which makes it ideal for exploring mechano-biology. The inherent discrepancy between in-vitro experiments and nature might be explained by the dynamic force-environment to which many proteins are exposed in living tissues. Here, I make a first attempt to unveil the invisible influence of forces on biomolecules.