Date

2015

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Mechanical Engineering

First Adviser

Zhang, Xiaohui (Frank)

Other advisers/committee members

Oztekin, Alparslan; Cheng, Xuanhong; Webb III, Edmund; Zhou, Chao

Abstract

von Willebrand Factor (VWF) is a polymeric plasma glycoprotein which is very important for the hemostasis of bleeding blood vessels. When blood vessels are injured, the hydrodynamic force in the bloodstream experiences a sharp increase and the stability of the flow field is disturbed simultaneously. However, von Willebrand factor (VWF), by bridging over platelets and exposed collagen, forms hemostatic plugs to stop bleeding. Responding to the high shear rate in the blood stream, the multimeric VWF wisely alters its conformation from the original compact-like coil to a thread-like shape and exposes as many functional domains as possible, to secure increased binding strength with collagen and higher capturing efficiency with platelets. During the entire process of hemostasis and thrombosis, the A-domains, including A1, A2 and A3, behave as the most influential function group within VWF. A single-molecule study is an experiment that investigates the properties of individual molecules. It has been increasingly utilized into biological studies since late 1980s. The main reasons that single-molecule study can be implemented into biological applications are as follows: first, it is a very direct method that performs precise measurement on the most fundamental parameters (e.g. force, strength, stiffness) of the biological sample; second, the single-molecule study is conducted in real time, hence it enables simultaneous observation that perfectly fulfills demands to record certain biological phenomena (e.g. morphology, stimulation, conformational change); third, it can achieve outstanding resolution and sensitivity, which make single-molecule studiesan ideal method to characterize both the structural and functional properties for biomolecules such as cells and tissue both intermolecularly and intramolecularly. Within this dissertation, two single-molecule devices: atomic force microscope and optical tweezers are employed to study the A-domains of von Willebrand factor (VWF). First, the interaction between VWF and collagen has been comprehensively characterized in domain, monomer and multimer phases. Meanwhile, a quantitative comparison has been given to identify the functional defects of different mutations of von Willebrand Disease (VWD). Second, the adhesiveness between VWF and glycoprotein1 (GP1) receptor on the platelet membrane has been studied carefully to unveil the mechanism of hemostasis and thrombosis. Similarly, the mutant VWF with defects in platelet binding has been examined. Third, based on the first two experiments, another set of experiments has been done on multimeric VWF protein. A new assay method by using two different VWF antibodies to measure intra-molecular interactions within the VWF multimer on the platform of optical tweezers was developed. Throughout the entire study, the A1, A2 and A3 domains within VWF are discussed comprehensively from both functional and structural perspectives. Meanwhile, the data from wild type samples are always compared with the ones from mutations. Therefore, the structural defects are connected with the functional ineffectiveness. Finally, based on the result of this dissertation, several diagnostic solutions are proposed.

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