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Date Permissions Signed

2-17-2012

Date of Award

2012

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Spiegel, P. Clint

Second Advisor

Anthony-Cahill, Spencer J.

Third Advisor

Smirnov, Sergey L.

Abstract

Normal blood clotting is regulated by blood plasma protein factors in a blood coagulation cascade. Deficiency in one of these proteins, Factor VIII (FVIII), causes the disease hemophilia A, in which the patient is unable to form blood clots. Treatment of hemophilia A involves the infusion of FVIII, and the most common complication in this treatment is the development of antibodies against the therapeutic infusions. Two mouse monoclonal anti-human antibodies, 3E6 and G99, were used as models of inhibitory action against FVIII by binding to the C-terminal C2 domain. A classical antibody, 3E6, blocks the ability of FVIII to bind von Willebrand Factor, a circulatory partner. The classical antibody thereby reduces the stability of FVIII in plasma. A nonclassical antibody, G99, blocks the activation of FVIII, inhibiting the role of FVIII in the blood coagulation cascade. In order to study the inhibitory action of classical and nonclassical antibodies, 3 complexes were prepared, C2:3E6, C2:G99, and 3E6:C2:G99. Small angle X-ray scattering was used to study the structures of the C2 domain in complex with the antigen binding fragment (Fab) of each antibody and in complex with both Fabs simultaneously. Reproducible, low-resolution models of the complexes were generated from scattering data using a software suite from the European Molecular Biology Laboratory. The scattering data for 3E6:C2:G99 was used to generate an elongated shape with antibodies on opposite sides of the C2 domain. Extensive crystal trials were undertaken to prepare for future X-ray crystallography studies of the complexes.

Type

Text

DOI

https://doi.org/10.25710/qree-nh08

Publisher

Western Washington University

OCLC Number

779476950

Subject – LCSH

Blood coagulation factor VIII antibodies; Blood coagulation factor VIII antibodies--Animal models

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 thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

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