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Date Permissions Signed

7-22-2022

Date of Award

Summer 2022

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Spiegel, P. Clint

Second Advisor

Antos, John M.

Third Advisor

Amacher, Jeanine

Abstract

Blood coagulation factor VIII (FVIII) is a crucial protein cofactor within the blood coagulation cascade and facilitates the proteolytic activation of factor X by activated factor IX. During coagulation FVIII is activated and binds, via its C1 and C2 domains, to activated platelet membranes coordinated by interactions with exposed phosphatidylserine on the membrane surface. A deficiency of functional FVIII within a patient's bloodstream leads to the blood disorder hemophilia A, which results in prolonged bleeding episodes. Current treatment for hemophilia A relies on FVIII replacement therapy via the injection of exogenous FVIII. The main complication which arises from FVIII replacement therapy is the development of pathogenic anti-FVIII inhibitory antibodies, which bind to various regions of FVIII disrupting FVIII’s ability to properly interact with its key binding partners, thereby nullifying treatment.

This thesis reports determination of low resolution SAXS envelopes and progress towards obtaining atomic resolution molecular structures of full length FVIII in complex with five different anti-A2 inhibitors, and a human derived anti-C1 inhibitor, NB2E9, in complex with the lone C1 domain. Solving the structure of one of these complexes would provide insight into the mechanism of inhibition for the A2 domain that would be invaluable when designing bioengineered hemophilia A therapeutics. This thesis further reports mutational studies of FVIII’s C1C2 domains, yielding progress towards identifying docking and undocking conformations, represented by ~35-45° rotation of the C2 hydrophobic loops. This utilized designing mutant constructs to form a disulfide bond which locks C1C2 in the proposed undocking conformation to compare lipid binding affinities, to build our understanding of the mechanism of lipid association and dissociation.

Type

Text

Keywords

X-ray Crystallography, Biochemistry, Hemophilia A, Blood Coagulation Factor VIII (FVIII), Structural Biology, Lipid Binding

Publisher

Western Washington University

OCLC Number

1341861740

Subject – LCSH

Blood coagulation factor VIII; Hemophilia--Complications; Blood platelet--Aggregation; Blood platelets--Activation; X-ray crystallography; Mutation (Biology)

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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