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Date Permissions Signed

11-24-2021

Date of Award

Fall 2021

Document Type

Masters Thesis

Department or Program Affiliation

College of Science and Engineering

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Spiegel, P. Clint

Second Advisor

Amacher, Jeanine

Third Advisor

Antos, John M.

Abstract

Blood coagulation factor VIII (fVIII) functions as a cofactor in the blood coagulation cascade for proteolytic activation of factor X by factor IXa. During coagulation, fVIII is activated and subsequently binds to activated platelet surfaces by coordination of the fVIII C1 and C2 domains to the exposed phosphatidylserine of activated platelet membranes. Structural and mutational studies have suggested that both hydrophobic and electrostatic interactions occur between the two tandem C domains and activated lipid surfaces, but models of C domain phospholipid binding propose conflicting regions that directly interact with the membrane surface. This thesis reports the determination of the molecular structure of an isolated fVIII porcine C2 domain in the presence of the phosphatidylserine headgroup (OPLS) at 1.3 Å. The OPLS molecule makes direct contact with Q2213, N2217, S2289, and R2320. This structure represents the first deposited structure of fVIII C domains in the presence of a lipid headgroup moiety. Furthermore, phospholipid binding characteristics of basic residues within the proposed phospholipid binding regions were investigated by mutagenesis. Specifically, mutations at R2163, R2320, and a double mutant of R2163/R2320 caused almost complete abrogation of lipid binding to soluble lipid nanodiscs. Using these findings, an updated model of fVIII lipid binding is proposed using structural information from C2 domain inhibitors, previous literature, and newly defined interactions between C2 and OPLS. Together, this study proposes that R2163 and R2320 are the center of a conserved phospholipid binding motif that extends across homologous blood clotting proteins.

Type

Text

Keywords

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

Publisher

Western Washington University

OCLC Number

1287101005

Subject – LCSH

Blood coagulation factor VIII, Phospholipid antibodies, 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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