Single-Molecule Imaging of Von Willebrand Factor Activation by Flow

Abstract

Von Willebrand Factor (VWF) is a long multimeric blood protein that plays a central role in platelet adhesion at sites of elevated hydrodynamic stresses, such as in injured or stenotic vessels. The function of VWF is closely related to thrombogenesis in cardiovascular diseases and the most common hereditary bleeding disorder von Willebrand disease. The effectiveness of the current therapies is limited partly due to the poor understanding of the activation mechanism of VWF, which is regulated by the hydrodynamic stresses in the blood stream. VWF only binds platelets under high shear or elongational flow. Moreover, the length of VWF is regulated by the ADAMTS13 protease cleavage under high shear flow. Studies suggest a hypothesis that the elevated hydrodynamic stresses exert tensile forces on VWF to unfold the tertiary and secondary structures and expose the binding sites for the platelet membrane receptor GP1b and the protease ADAMTS13.This study elucidates the mechanism of VWF function regulation at single-molecule level under tethered shear flow and elongational flow, which are more effective at activating VWF than pure shear flow according to molecular dynamics simulation. We infuse a FRET tension sensing module in each VWF monomer to report the shape and the tensile force of the multimers, and image them under flow with a microscope system that simultaneously records the length and the fluorescence spectrum of a single VWF multimer. For elongational flow, real-time feedback is applied to trap the fast moving single VWF molecules within the high hydrodynamic stress region and the field of view. By adding fluorescently labeled GP1b and ADAMTS13, the rates of binding and cleavage activities in VWF is also measured. These results provide experimental basis for quantitative modeling of the intra-molecular interactions that are responsible for VWF function regulation.