The large multimeric plasma protein von Willebrand Factor (VWF) plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting. The VWF effectively senses blood flow, changing conformation in rapid or elongated flow from an inactive, compact globule to a more elongated shape that allows the VWF to interact with both platelets and damaged vascular walls. Although the basic biological properties of the VWF have been elucidated, less well understood is the VWF’s biomechanical properties that allow the VWF to sense and respond to different flow environments. The A2 domain of the VWF unfolds in response to tensile force and subsequently exposes its Tyr1605-Met1606 scissile bond for cleavage by ADAMTS13, a metalloprotease in the circulating blood. This process converts the highly thrombogenic, newly secreted, ultra-large VWF multimers into smaller multimeric forms and consequently prevents the formation of thrombus. This discovery has enhanced the understanding of the VWF size regulation in the vasculature, with important implications for both VWF biology and pathophysiology.
Our cross functional team which consists of Dr. Xuanhong Cheng, Dr. Alp Oztekin, and Dr. Edmund Webb, developed combined single-molecule force spectroscopy, microfluidic imaging, and coarse-grained molecular modeling approaches to understand how the biomechanical aspects of the VWF regulate its biological functions. Single-molecule atomic force microscopy and optical tweezers were used to reveal the intra- and intermolecular forces that control the conformation of the VWF under tensile force or flow. Using the biophysical parameters obtained from experiments, we developed a mechanically informed molecular model to describe the VWF’s conformation under varying flow conditions, including when VWF molecules are attached to collagen functionalized surfaces.
Our research also explore experimental tools for single-molecule studies. We utilized the SpyTag technology, first developed by Dr. Mark Howarth at Oxford, to couple proteins with DNA handles for single-molecule studies. This new approach does not need to introduce disulfide bonds between a protein sample and DNA handles. This is particularly important for the VWF, as the VWF has native disulfides in all of its domains, thus making it impossible to implement the conventional disulfide-bonded handle attachment without disrupting the sample’s structure. This new method was applied to our recent study of the intramolecular interactions of VWF fragments.