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Date Permissions Signed

8-18-2021

Date of Award

Summer 2021

Document Type

Masters Thesis

Department or Program Affiliation

Department of Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Spiegel, P. Clint

Second Advisor

Amacher, Jeanine

Third Advisor

Smirnov, Sergey L.

Abstract

Hemophilia A is an X-linked disorder that results in uncontrolled bleeding, which is caused by a lack of activity for blood coagulation factor VIII, an essential protein cofactor in the clotting cascade. Factor VIII consists of multiple domains, and binding disruptions between factor VIII and its circulatory partner, von Willebrand Factor, may cause von Willebrand disease. Von Willebrand Disease type 2N is an autosomal recessive disease, and it is caused by binding disruptions between the D’ domain (also known as TIL’E’) of von Willebrand Factor and a3 domain of factor VIII. A 2.9Å Cryoelectron microscopy structure of the FVIII:vWF complex was recently published, and this crystal structure further described the interactions between FVIII and vWF. To further understand the most severe types of von Willebrand Disease type 2N, site-directed mutagenesis of vWF was employed to enhance the understanding of binding disruptions between vWF and FVIII. The designed mutants were transformed, expressed, and purified for further experimental studies. Binding assays were conducted between the mutants against our bioengineered chimeric structure factor VIII, ET3i, via pull-down assays, sedimentation assays, as well as quantitative studies via biolayer interferometry. The results of this present study included the investigation of binding studies between the D’ domain of von Willebrand factor and the a3 domain of factor VIII. The results obtained in this study were interpreted, compared, and concluded to be consistent with the newest cryo-EM structure.

Type

Text

Keywords

Factor VIII, von Willebrand Factor, FVIII, vWF, vWD, hemophilia A

Publisher

Western Washington University

OCLC Number

1264683501

Subject – LCSH

Von Willebrand factor; Von Willebrand disease; Hemophilia; Blood coagulation factor VIII

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

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