Date

5-1-2018

Document Type

Thesis

Degree

Master of Science

Department

Bioengineering

First Adviser

Zhang, Xiaohui (Frank)

Abstract

Von Willebrand Factor (vWF) is a large plasma glycoprotein involved in hemostasis by forming platelet plugs at damaged vessel walls. vWF mutation leads to von Willebrand Disease (vWD), a bleeding disorder which impacts one in every 1000 individuals. In the blood stream vWF exists as a multimer and unravels to expose binding sites for platelet and collagen adherence. Furthermore, the size of vWF is essential to normal hemostasis and is controlled by the ADAMTS13 enzyme cleavage at A2 domain of vWF. The project aims to explore the A2 domain and multimeric structural and mechanical properties of vWF wild-type (WT) monomer, mutant N1515Q, N1574Q, and Q1541R.Molecular dynamic simulations were performed to compare the structural differences of WT vWF with and without sugar, mutants Q1541R, N1515Q, and N1574Q. Simulation systems were generated with CHARMM-GUI and the simulation was run in CHARMM. Qualitative analysis was acquired through visualization software and quantitative analysis of hydrogen bonds, protein to protein interaction, thermal fluctuations, and RMSD were obtained through visual molecular dynamics (VMD) and scripts provided by Dr. Wonpil Im. Enzymatic cleavage assay was performed to understand the effect of ADAMTS13 on WT vWF A2 as well as mutants Q1541R, N1515Q and N1574Q. The cleaved portion was estimated by image analysis using Image J. Preliminary assay results indicated lower threshold for ADAMTS13 cleavage susceptible for N1541Q and N1574Q. In addition, single-molecule force optical tweezer experiments were used to characterize A2 and multimeric vWF unfolding. A SpyTag-SpyCatcher system was employed in single-molecule experiments. By characterizing the isolated A2 domain, we can better understand how this particular domain is able to undergo conformational changes as a result of shear force. Later, multimeric vWF is explored to understand how domain-domain interactions affect unfolding which reflects realistic in-vivo vWF A2 domain. The precise small force applied onto vWF reflects and simulates the shear force caused by shear flow in the blood stream that induces conformational change of vWF.

Available for download on Wednesday, August 14, 2019

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