Structural studies of blood coagulation factor VIII in complexes with inhibitory antibodies

107 Pages·2016·2.73 MB·English

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WWU Graduate School Collection WWU Graduate and Undergraduate Scholarship
2012
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Rachel Werther
Western Washington University
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Werther, Rachel, "Structural studies of blood coagulation factor VIII in complexes with inhibitory
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Structural Studies of Blood Coagulation Factor VIII
in Complexes with Inhibitory Antibodies

By

Rachel Werther

Accepted in Partial Completion
Of the Requirements for the Degree
Master of Science

Moheb A. Ghali, Dean of the Graduate School

ADVISORY COMMITTEE

Chair, Dr. P. Clint Spiegel
Dr. Spencer Anthony-Cahill
Dr. Serge Smirnov
MASTER’S THESIS

In presenting this thesis in partial fulfillment of the requirements for a master’s degree
at Western Washington University, I grant to Western Washington University the
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not allowed without my written permission.

Rachel Werther
February 17, 2012
Structural Studies of Blood Coagulation Factor VIII in Complexes with
Inhibitory Antibodies

A Thesis
Presented to
The Faculty of
Western Washington University

In Partial Fulfillment
Of the Requirements for the Degree
Master of Science

by
Rachel Werther
February 2012
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 (F ) of each antibody and in complex with
ab
both F s simultaneously. Reproducible, low-resolution models of the complexes were
ab
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.
iv
Acknowledgements

This study was dependent upon the gift of mouse monoclonal hybridoma lines from
Pete Lollar’s lab at Emory University in Atlanta, Georgia. Barry Stoddard’s lab at Fred
Hutchison Cancer Research Center in Seattle, Washington generously allowed the use of
instrumentation for crystal screening, and SAXS data were collected using Stoddard Lab
access to the Advanced Light Source in Berkeley, California. I thank Betty Shen and
Lindsey Doyle of the Stoddard Lab for assistance with SAXS and crystal trials,
respectively. SAXS data was collected at the SIBYLS Beamline of the Advanced Light
Source at Lawrence Berkeley National Lab thanks to the Department of Energy
Integrated Diffraction Analysis grant contract # DE-AC02-05CH11231.

This study was funded by the National Institutes of Health (Grant # 1 R15 HL103518-01),
and it was completed in collaboration with Justin Walter of the Spiegel Lab. I would like
to thank Justin Walter for a great deal of the work described in this thesis, as well as Erin
Macri, Clint Burgess, Masha Polozova and Caileen Brison for assistance in various stages
of the project.

I am grateful to my thesis committee, Spencer Anthony-Cahill, Serge Smirnov, and P.
Clint Spiegel. I am additionally grateful to Spencer Anthony-Cahill and P. Clint Spiegel
for the methods I’ve learned in their labs and for their time and encouragement.
v
Table of Contents
Abstract ……………………………………………………………………………………………………… iv
Acknowledgements ……………………………………………………………………….…………….. v
Abbreviations …………………………………………………………………………..…………...... viii
List of Figures ………………………………………………………………….………………………….. x
Background ………………………………………………………..……………………………………….. 1
History of Hemophilia A …………………………..………………………………………… 1
Blood Coagulation Cascade ……………………………………………………………... 3
Intrinsic Pathway ……………………………………………………………………………… 6
Extrinsic Pathway ……………………………………………………………………………… 6
Intrinsic Tenase Complex …………………………………………………………………… 7
Factor VIII ……………………………………………………………………………………………. 8
Factor VIII and von Willebrand Factor ……………………………………………. 11
Factor VIII Activation and Inactivation …………………………………………….. 12
Inhibitors to Factor VIII ……………………………………………………………………… 14
Structural Determination Using X-Rays …………………………………………… 18
X-Ray Crystallography ………………………………………………………………………. 19
Small Angle X-Ray Scattering ……………………………………………………………… 21
Materials and Methods …………………………………………………………………………… 24
Buffers ……………………………………………………………………………………………. 24
Protein Expression ………………………………………………………………………….. 26
vi
Protein Purification ……………………………………………………………………… 27
Antigen Binding Fragment Purification ……………………………………………. 28
Electrophoresis ………………………………………………………………………………… 29
Binding Assays ………………………………………………………………………………… 30
Complex Purification ……………………………………………………………………… 31
Mass Spectrometry ………………………………………………………………………… 31
Crystal Trials ………………………………………………………………………………….. 32
Small Angle X-Ray Scattering …………………………………………………………… 33
Results and Discussion ………………………………………………………………………………… 35
Preparation of Pure C2 ………………………………………………………………… 35
Preparation of Pure F s ………………………………………………………………….. 36
ab
Mass Spectrometry ………………………………………………………………………. 38
Formation of Complexes ……………………………………………………………….. 39
Binding Assay ……………………………………………………………………………….. 42
SAXS Data …………………………………………………………………………………….. 43
Conclusions …………………………………………………………………………………… 58
Appendix 1: Instructions for Processing SAXS Data …………………………………… 60
References …………………………………………………………………………………………………… 87
vii
Abbreviations

Ab - antibody
alm – spatial information computer file format
BME - β-mercaptoethanol
BIOISIS - BIOlogically Integrated Structures In Solution (website)
dat – data computer file format
D – maximum dimension (of a macromolecule)
max
EDTA - ethylenediaminetetraacetic acid
EMBL – European Molecular Biology Laboratory
F - Antigen binding fragment (of an antibody)
ab
F – Constant fragment (of an antibody)
c
I(0) – intensity of diffracted X-ray at 0 momentum transfer
IgG - Immunoglobulin G
IPTG - isopropyl β-D-1-thiogalactopyranoside
LBNL – Lawrence Berkeley National Laboratory
LB – Luria-Bertani
NaCl – sodium chloride
PBS - phosphate buffered saline
pdb – Protein Data Bank atomic coordinate computer file format
PDB – Protein Data Bank
viii
PMSF - phenylmethanesulfonyl fluoride
P(r) – paired distribution of distances
R – radius of gyration
G
rmsd – root mean square deviation
rpm – rotations per minute
S – sulfate (-containing ion-exchange column)
SAXS - Small angle X-ray Scattering
SDS - sodium dodecyl sulfate
SDS-PAGE - sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEC – size exclusion chromatography
SIBYLS - Structurally Integrated BiologY for Life Sciences
Tris - 2-amino-2-hydroxymethyl-1,3-propanediol
Tris-HCL - 2-amino-2-hydroxymethyl-1,3-propanediol-hydrochloride
v/v – volume/volume
vWF – von Willebrand Factor
w/v – weight/volume
ix