Elucidating Hydrodynamic Force Regulation of Von Willebrand Factor in Hemostasis

Abstract

Listen

Translate article

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a pivotal role in hemostasis - it serves as a ligand for platelet adhesion and aggregation forming a hemostatic plug. The hemostatic potential of VWF is regulated by hydrodynamic forces arising in the circulation. Hydrodynamic shear at sites of bleeding activate the binding of VWF to platelet receptor GpIb. Hydrodynamic forces in blood flow also attenuated thrombosis promoting ultralarge VWF by activating cleavage in the VWF A2 domain by the metalloprotease ADAMTS13. Here, we investigated how mechanical forces, which occur in the circulation, regulate the structural conformation and function of VWF at the molecular level using a combination single-molecule manipulation and computational modeling. We created various hydrodynamic flows to mimic a wide range of blood flow conditions, while directly visualizing the conformational dynamics of single VWF molecules. First, we developed a assay that uses a microfluidic device with a cross-slot geometry to study VWF under elongational flow and developed a custom “shear wheel” microscope to study VWF under shear flow. These 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. Second, we developed a kinetic model of force-induced enzymatic cleavage of A2 by ADAMTS13. Our simulations, using parameters from single-molecule force experiments, predict the known healthy size distribution of VWF. We also found that calcium ions protects health-sized VWF from cleavage, and that A2 mutations related to the heritable bleeding disorder von Willebrand disease (VWD) shift hemostatic potential by destabilizing the domain structure under force. These newly developed experimental techniques and findings will lead to a better understanding of bleeding disorders and help progress the therapeutic development of VWD.