Mechanical and biophysical properties of VWF in single molecule experiments

VWF is known to be a shear force sensitive molecule and designed to perform his encoded functions by conformational changes according to the actual shear situation in the blood stream. Even though extensive studies on blood platelet aggregation under shear flow and biochemical approaches elucidated the importance of these conformational changes of VWF for platelet adhesion, direct experimental investigations on the detailed mechanism and its dynamics are largely missing. In fact, there is a need of biophysical investigations of VWF for (i) the mechano-elastic characterization (e.g. load-extension response, relaxation times etc.), and (ii) the molecular recognition properties with respect to adhesion strength and localization of adhesion sites (e.g. platelets). Since interactions between VWF and collagen are a key initial step of hemostasis, VWF/collagen binding studies should be performed. Utilizing atomic force microscopy (AFM) the forces and dynamics of specific VWF- domain/collagen interactions on the single molecular level can directly be probed. Our specialized subgroup in Linz, will specifically focus our AFM expertise on the various interactions of VWF with cells and tissue components. In the first funding period we applied AFM as a new approach to probe the dynamics of binding of selected VWF domains to other VWF domains and to collagen III and VI. For the second funding period we will gain a detailed understanding of the biophysical functionalities of the VWF protein on the single domain level by establishing a unique combination of nano- mechanics and computer simulation studies with Netz (B4), and Gräter/Baldauf (C1). According to the main task as to exploring the links between the mechanical properties of the mechano-sensitive VWF molecule and its function in the interplay with blood components, we will study the formation of VWF-platelet networks and the impact of pathologic VWF mutations in close collaboration with groups Schneppenheim (A1), Wilmanns (C3) Schneider (A2), Wixforth (B1), Rädler (B3). We will thus extend our investigations to collagen type I and IV, to integrin mediated adhesion to platelets under inflammatory conditions. We expect that the clinical relevance of our findings will aid detecting disease and improve the treatment of VWF patients.

Inconspicuous in its inactive state, a protein called von Willebrand factor (VWF) evolves to a real lifesaver in case of an injury. Blood clotting is responsible for stopping a wound from bleeding, but how does this work? As one of our main findings, together with our project partners from all over Germany, we suggested a novel model for the role of VWF in wound closure, which reads as follows: VWF plays an important role in this process: In a normal state without any injury, it drifts around in the blood, coiled like a loose ball of wool (A). When we hurt ourselves (B), however, the situation changes dramatically. In the area of an injury, collagen is exposed and the VWF ball connects to this collagen with a loose end and uncoils in the blood stream. Now, it can capture circulating blood platelets and direct them to the damaged vessel wall (C). Once caught by VWF, the platelet changes its shape dramatically, turning from a non-sticky sphere to a very sticky disc. The platelet further develops small arms (D) to hold other platelets, which were similarly captured by VWF. As a result, a net of many VWF strings and sticky blood platelets is formed and the wound is closed by this sticky plug (E).As helpful this mechanism is in the case of an injury, it can lead to unwanted and dangerous effects, such as thrombosis or stroke, when triggered unwantedly without vessel damage. Therefore it is important to understand what is going on within the VWF while being activated. To explain this process, we used an Atomic Force Microscope (AFM), which can visualize biological structures in the size of only a few millionths of a millimeter and detect unimaginable low forces (trillionth Newton) between molecules. In preliminary studies we succeeded in generating images of single blood platelets. Later, we went one step further into detail. With the help of an AFM we can hang a protein or parts of a protein to a kind of fishing line to study interactions between two proteins. With this, we discovered why the von Willebrand factor does not catch blood platelets when it is coiled. The stronger blood flow in an area of injury uncovers a little sticky area (binding site) of the VWF which then catches the blood platelets. We described this mechanism in great detail and computer simulations confirmed our findings with AFM. We are only at the beginning of VWF research, but once we fully understand VWF and learn how to control it, we could prevent life threatening events like thrombosis or stroke and immediately stop bleeding of big injuries. Star Trek healing devices would then be just a little step away.