Mechanical properties of VWF in single molecule and cell adhesion force experiments using AFM

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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. endothelial cells, platelets etc.). 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. As two specialized subgroups in Linz and Munich, we 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 platelet adhesion to VWF, the size distribution of VWF, the unfolding of VWF-dimers, and 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, 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 of VWF to platelets, and to endothelial cells under inflammatory conditions. Furthermore, we will directly measure changes in the force signature by stretching VWF and VWF mutants under inflammatory conditions. We expect that the clinical relevance of our findings will aid to detect disease and improve the treatment of VWF patients.