Single-Molecule Investigations of Von Willebrand Factor in Hydrodynamic Flow

Abstract

Von Willebrand factor (VWF) is a multimeric blood glycoprotein that plays a pivotal role in hemostasis - it serves as a ligand for platelet adhesion and aggregation forming a hemostatic platelet plug. We are studying how hydrodynamic forces that arise in the circulation can regulate hemostasis. For example, the binding of the VWF A1 domain to the platelet protein GPIbα is correlated to conditions of strong hydrodynamic flow. We are testing the hypothesis that quaternary unfolding of VWF under hydrodynamic stress activates the VWF adhesive function by exposing A1 domains for binding to the platelet glycoprotein GPIb, and are studying the dynamics of this process.To accomplish this, we are developing powerful new techniques in single-molecule manipulation and detection to investigate how fluid forces generated in the bloodstream affect the structural conformation and function of VWF. We are creating various hydrodynamic flows to mimic a wide range of blood flow conditions, while directly visualizing the conformational dynamics of single VWF molecules in these environments. First, we have utilized a microfluidic device with a cross-slot geometry to study VWF under elongational flow. To expand these studies, we are also utilizing a microfluidic four-roll mill device that has a stagnation point that will allow prolonged observation of VWF under all flow conditions including elongational, shear, and rotational flow. In addition to the microfluidic devices, we are developing a custom “shear wheel” microscope to study fluorescently labeled VWF under shear flow. These novel techniques will enable us to understand how hydrodynamic forces in the bloodstream act on VWF to cause quaternary unfolding, and how this in turn regulates adhesive activity. These findings should also lead to a better understanding of bleeding disorders that result from abnormalities in flow conditions of the circulatory system.