Unraveling the Mechano-Regulation of Von Willebrand Factor

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Von Willebrand Factor (VWF) is a large glycoprotein critically involved in hemostasis. VWF senses shear flow irregularities in the blood stream: at sites of vascular injury, where hydrodynamic forces are increased, VWF extends and subsequently promotes platelet adhesion. Since formation of a platelet plug is essential for primary hemostasis, defects in or deficiency of VWF can lead to severe bleeding disorders, known as von Willebrand disease. In the blood, VWF exists in the form of linear multimers comprising a variable number of dimeric subunits, which form the smallest repeating subunits and are linked via N-terminal disulfide bonds. Under static conditions, VWF adopts collapsed conformations; under elevated hydrodynamic forces, the multimers lengthen through opening of intra- and intermolecular interactions and unfolding of the VWF A2 domain. As hydrodynamic peak forces within the molecule strongly correlate with its effective length, lengthening can trigger opening of further interactions at even higher forces. We and others have elucidated some of the critical events in VWF force sensing by single-molecule force spectroscopy, in particular A2 domain unfolding at ~15 pN and the dissociation of a strong intermonomer D4 domain interaction above ~50 pN. Despite these recent advances many open questions remain. Simulations suggest much lower critical forces for initial VWF elongation (~ 1 pN). Additionally, indirect experimental evidence points to loss of interactions, in particular loss of intermonomer C-domain and weak D4 domain interactions at low pH, at forces <5 pN, below the resolution limit of AFM force spectroscopy. Finally, there is currently no mechanistic understanding of the D4 domain mediated intermonomer interactions due to a lack of high-resolution structures.We propose to combine novel force spectroscopy approaches with high-resolution structural information to unravel the force-induced activation and regulation of VWF.Using novel molecular attachment strategies we want to probe VWF force-induced transitions in particular using magnetic tweezers, which can resolve forces down to the femto-Newton range and enable stable measurements for long periods of time (~min to hours) to investigate refolding and rebinding kinetics. Force spectroscopy of wildtype VWF and several mutant and deletion constructs will be combined with high- and low-resolution structure determination, in particular using crystallography, small-angle X-ray scattering, and AFM imaging. Structural knowledge will allow us to interpret the findings from force spectroscopy and guide the design of additional experiments. Structural information combined with direct measurements of the critical initial steps of VWF elongation and regulation will improve our mechanistic understanding of its role in hemostasis and have the potential to direct therapeutic approaches.