Molecular scale studies of von Willebrand factor: Surface and shear dependent structure and intra -molecular binding sites

von Willebrand factor (vWf) is a large, multimeric, plasma glycoprotein critical in thrombus formation and normal hemostasis. vWf is indispensable in recruiting platelets to sites of thrombosis (e.g. non-endothelial surfaces) at high fluid shear. Molecular level information about the surface/shear dependent structure, adhesion, interactions and function of critical plasma proteins, such as vWf, is necessary to further molecular understanding of hemostasis and thrombosis.;The aims of this work were: to determine the surface dependent structure of vWf under physiologic pH and ionic strength; to determine the shear dependent structure and adhesion of vWf as related to multimer size; and to determine the accessibility of intra-molecular binding sites on surface adsorbed vWf.;I designed and built a laminar flow cell system which was used with fluid atomic force microscopy (AFM), novel surfaces and purified proteins. Sources of vWf and storage conditions were examined and reliable protocols for the purification and optimal storage of human vWf were implemented. Model hydrophilic and hydrophobic surfaces were used to determine the surface dependent intra-molecular structure of vWf (improving previously published structural detail on vWf). The surface adsorbed intra-molecular structure of vWf on a hydrophilic surface was determined for the first time.;The shear dependent adhesion of vWf dimer, fibrinogen and vWf multimers was determined on hydrophobic and hydrophilic surfaces. Distinct differences in the effect of fluid shear upon the structures of these proteins were observed and correlated to differences in adhesion. These data indicate the contribution of multimerization to the adhesion of vWf. Structures of vWf layers, collagen VI layers and the structure of individual vWf and vWf layers on collagen VI surfaces, were imaged for the first time.;An immuno-gold/AFM technique to determine the accessibility of intra-molecular binding sites on surface adsorbed vWf was established. This is a direct method of correlating molecular scale changes in the structure of vWf (any protein) with the accessibility of functional domains within this structure. Using a panel of monoclonal antibodies against functional domains on vWf, we identified the differential accessibility of specific functional domains within vWf adsorbed on the hydrophobic surface and on the collagen VI surface.