single-molecue study on gpib-alpha and von willebrand factor mediated platelet adhesion and ...

174 Pages·2013·24.96 MB·English

by  Arnold Ju

SINGLE-MOLECUE STUDY ON GPIB-ALPHA AND VON
WILLEBRAND FACTOR MEDIATED PLATELET ADHESION AND
SIGNAL TRIGGERING

A Dissertation
Presented to
The Academic Faculty

by

LINING JU

In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy in the
School of Biomedical Engineering

Georgia Institute of Technology
December 2013

COPYRIGHT ©LINING JU 2013
SINGLE-MOLECUE STUDY ON GPIB-ALPHA AND VON
WILLEBRAND FACTOR MEDIATED PLATELET ADHESION AND
SIGNAL TRIGGERING

Approved by:

Dr. Cheng Zhu, Advisor Dr. Larry V. McIntire
Wallace H. Coulter Department of Wallace H. Coulter Department of
Biomedical Engineering Biomedical Engineering
Georgia Institute of Technology Georgia Institute of Technology
and Emory University School of Medicine and Emory University School of Medicine

Dr. W. Robert Taylor Dr. Jennifer E. Curtis
Wallace H. Coulter Department of School of Physics
Biomedical Engineering Georgia Institute of Technology
Georgia Institute of Technology
and Emory University School of Medicine

Dr. Jing-fei Dong
Hematology Division, Department of
Medicine
University of Washington, Seattle

Date Approved: November 11, 2013
To my dearest family.
I dedicate this thesis
to

My parents

I am forever indebted to my parents, 居礼 (Li Ju) and 孙淑 (Shu Sun). I would not have
been able to finish my Ph.D. thesis without their priceless love, endless patience and
unconditional supports.

My girlfriend

I am so grateful to my girlfriend, Danmei Fan, who was always there to support, comfort
and encourage me to go through hardship.
ACKNOWLEDGEMENTS

I wish to express my deep gratitude to many people who have given me support
and guidance for the work described in this dissertation. I would first like to thank my
advisor Dr. Cheng Zhu for creating a supportive and liberal research environment, which
has encouraged my creativity and imagination in science. I also appreciate his knowledge
and wisdom guiding me though every challenge and difficulty happening during my Ph.D.
study.
I deeply appreciate the invaluable contribution from the collaborators of this
project and the rest of my thesis committee. In particular I wish to thank Dr. Jing-fei
Dong for his enthusiastic advice with constant patience and constructive debate
encouraging me to think deeper and further. I also wish to thank following fellow
collaborators: Dr. Wei Chen, a lab brother and a fellow colleague, for his inspiration in
setting up the biomembrane force probe that I used as the major weapon to finish my
Ph.D. work (Chapter 3). Yunfeng Chen, a lab brother who collaborated with me and
made breakthrough on platelet triggering project (Chapter 6). Haiwei Qiu, a good friend
who helped me take off in engineering research and Vince Fiore, a friend from Thomas
Barker’s lab who discussed science with me for the entire Ph.D. journey and collaborated
on Thy-1 project (Chapter 8). In addition I want to thank Dr. Miguel Cruz, Dr. Matthew
Auton, Dr. Renhao Li, Dr. Zaverio Ruggeri, Dr. Heyu Ni and Dr. Shaun Jackson for their
timely support and joining in with reagents, enabling the project to go in depth. Finally I
II
want to thank Dr. Rodger McEver. It’s a great honor of mine to visit your lab in
Okalahoma City and receive the education in using the flow chamber. The collaborative
efforts between labs have pushed my scientific thinkings from good to great.
I would like to thank the technical supporting staffs in the Institute of
Bioscience and Bioengineering and the Biomedical Engineering at Georgia Tech and
Emory University. I would like to thank Mr. Louis Boulangerand and his co-op students
in ME’s machine shop for taking so many efforts on manufacturing my designs for the
BFP system. I would also like to thank Mr. Matt Winfree from Nikon for supporting the
microscope related equipments.
Special thanks to my colleagues in from labs, particularly Dr. Tadayuki Yago
(McEver lab); Dr. Jizhong Lou and his student Qinghua Ji, Dr. Fang Kong, Dr. Jin Qian,
Dr. Xue Xiang (Zhu lab alumni); Dr. Zhenhai Li, Dr. Baoyu Liu, Jin-sung Hong,
Chenghao Ge, Kaitao Li and Prithiviraj Jothikumar (Zhu lab members); Dr. Leslie
Coburn (McIntire lab alumnus); Dr. Wilbur Lam, Yumiko Sakurai, Dr. David Myers and
other Lam lab people their technical support, helpful discussions and friendship.

III
TABLE OF CONTENTS
ACKNOWLEDGEMENTS............................................................................................II

LIST OF FIGURES.....................................................................................................VIII

LIST OF ABBREVIATIONS........................................................................................XI

SUMMARY..................................................................................................................XIII

CHAPTER 1: INTRODUCTION....................................................................................1

1.1
  Specific aims................................................................................................................1

1.2
  Background and significance.......................................................................................6

1.2.1
  Molecular interactions that mediate platelet adhesion to subendothelial surfaces6

1.2.2
  Kinetic and mechanical properties of VWF–GPIbα interaction.......................11

1.2.3
  Flow-enhanced platelet adhesion to the vessel wall..........................................12

1.2.4
  Structures of A1–GPIbαN complex...................................................................15

CHAPTER 2: MATERIALS AND METHODS...........................................................17

2.1
  Proteins......................................................................................................................17

2.2
  Red blood cells and platelets......................................................................................18

2.3
  GPIb-IX expressing CHO cell lines (CHOαβ9)........................................................19

2.4
  GPIbα from Platelet and CHOαβ9 cell lysates..........................................................20

2.5
  Single-bond experiments...........................................................................................20

2.5.1
  System setup......................................................................................................20

2.5.2
  Protein functionalization....................................................................................22

2.5.3
  Force spectroscopy.............................................................................................23

2.5.4
  Experiments under 37°C....................................................................................26

2.6
  Kinetics analysis........................................................................................................27

2.6.1
  Analyze first-order kinetics from single bond measurements...........................27

2.6.2
  Analyze lifetime distributions with a two-state model......................................28

IV
2.7
  Platelet calcium imaging............................................................................................29

2.7.1
  Ca2+ ratio analysis..............................................................................................30

2.8
  Measurement of molecular site density.....................................................................31

2.8.1
  Fluorescence staining.........................................................................................31

2.8.2
  Data acquisition.................................................................................................31

2.8.3
  Site density determination..................................................................................32

2.9
  Parallel flow chamber assays.....................................................................................34

2.9.1
  Rolling velocity..................................................................................................35

2.9.2
  Tether pause time...............................................................................................36

2.9.3
  Tether rate..........................................................................................................36

2.9.4
  Whole blood functional assay............................................................................37

2.10
 Direct binding assay by ELISA.................................................................................37

2.11
 Statistical testing........................................................................................................38

CHAPTER 3: BUILD A BIOMEMBRANE FORCE PROBE (BFP)........................39

3.1
  Introduction................................................................................................................39

3.2
  The second generation BFP system (BFP-2).............................................................40

3.2.1
  The hardware system.........................................................................................40

3.2.2
  The software system..........................................................................................44

3.3
  BFP experiment procedures.......................................................................................46

3.4
  Fluorescence BFP (fBFP)..........................................................................................49

CHAPTER 4: THE INTERPLAY OF FORCE AND THE A1 DOMAIN N-
TERMINAL FLANKING REGION ON REGULATING THE VWF–GPIB-
ΑLPHA CATCH BOND.................................................................................................52

4.1
  Introduction................................................................................................................52

4.2
  Results........................................................................................................................55

4.2.1
  GPIbα dissociates from 1238-A1 as a triphasic slip-catch-slip bond, 1261-A1 as
a monophasic slip-only bond, and full-length VWF as a biphasic catch-slip bond.......56

4.2.2
  Platelet rolling velocities on 1238-A1, 1261-A1, and VWF were governed by
their respective slip-catch-slip, slip-only, and catch-slip bonds with GPIbα.................60

4.2.3
  Polypeptide Q1238-E1260 binds GPIbα and 1261-A1 and inhibits platelet
adhesion.........................................................................................................................61

V
4.2.4
  Soluble Q1238-E1260 reduces GPIbα bond lifetimes with VWF and 1238-A1
but rescues the catch bond with 1261-A1......................................................................62

4.2.5
  Effects of different forms of WT GPIbα............................................................64

4.2.6
  Effects of VWD mutants....................................................................................65

4.2.7
  Characterization of A1–GPIbα dissociation by a two-state model....................67

4.2.8
  Comparison with previous measurements by different methods.......................70

4.3
  Discussion..................................................................................................................73

CHAPTER 5: TRANSPORT REGULATION OF 2D KINETICS OF VWF–GPIB-
ΑLPHA ASSOCIATION................................................................................................81

5.1
  Introduction................................................................................................................81

5.2
  Results........................................................................................................................83

5.2.1
  2D kinetics dependence of separation distance.................................................85

5.2.2
  2D kinetics dependence of Brownian motion....................................................88

5.2.3
  2D kinetics dependence of diffusion..................................................................89

5.3
  Discussion..................................................................................................................91

CHAPTER 6: VWF–GPIB-ΑLPHA CATCH BOND TRIGGERS PLATELET
SIGNALING BY FORCE PROLONGED BOND LIFETIMES................................94

6.1
  Introduction................................................................................................................94

6.2
  Results........................................................................................................................96

6.2.1
  Platelet GPIbα forms catch bonds with VWF–A1.............................................98

6.2.2
  Optimal force triggers Ca2+ by prolonging A1–GPIbα lifetime via catch bond100

6.2.3
  Ca2+ triggering amplitude correlates with duration of A1 binding events.......105

6.2.4
  Ca2+ triggering amplitude is affected by GPIbα bonding location..................107

6.2.5
  Ca2+ triggering efficiency is affected by GPIbα bonding valency...................108

6.2.6
  Platelet GPIbα triggered signal primes β integrin to an intermediate affinity
3
state 108

6.3
  Discussion................................................................................................................112

CHAPTER 7: CONCLUSIONS...................................................................................119

7.1
  Summary..................................................................................................................119

7.2
  Future studies...........................................................................................................121

VI
CHAPTER 8: MISCELLANEOUS PROJECTS........................................................123

8.1
  A cortical tension study on megakaryocytes by micropipette aspiration................123

8.1.1
  Measure cell cortical tension by micropipette aspiration................................123

8.1.2
  Effect of CIP4 knockdown on cell cortical tension.........................................125

8.1.3
  Characterize the CHRF cells blebbing.............................................................126

8.2
  Investigate the β integrin mediated thymocyte adhesion and its regulation by
1
plexinD1-sema3E pathway..............................................................................................128

8.3
  Force-stiffened synergistic binding of syndecan-4 and β integrin to thy-1............130

1
8.3.1
  Thy-1–K562 adhesions are primarily mediated by Thy-1–α β bonds............133

5 1
8.3.2
  Bond stiffening phenotype gives rise to Thy-1–K562 catch bond..................135

8.3.3
  Syndecan-4 is a co-receptor for Thy-1 required for DBS and lifetime
prolongation.................................................................................................................137

REFERENCES...............................................................................................................140

VII
LIST OF FIGURES
Figure 1-1. A model of platelet adhesion cascade in hemostasis and thrombosis. .....7

Figure 1-2. The latest model of the GPIb-IX-V complex largely drawn as ribbon
diagrams. .................................................................................................................10

Figure 1-3. The VWF domain organization. ...............................................................11

Figure 1-4. Structures of A1–GPIbαN complex...........................................................15

Figure 2-1. The scheme of a biomembrane force probe (BFP). .................................21

Figure 2-2. BFP functionalization with purified molecules. ......................................23

Figure 2-3. Sample raw data of BFP assays. ...............................................................25

Figure 2-4. Sample flow cytometry data.......................................................................33

Figure 3-1. The scheme of BFP-2 instrumentation......................................................41

Figure 3-2. BFP-2 assembly. .........................................................................................44

Figure 3-3. Imaging tracking region and intensity profile for the BFP.....................46

Figure 3-4. The rationale of BFP force calculation......................................................48

Figure 3-5. The BFP-2 test cycle....................................................................................49

Figure 3-6. Schematic of home-built fluorescence biomembrane force probe (fBFP).
...................................................................................................................................50

Figure 4-1. Binding specificity. .....................................................................................56

Figure 4-2. The role of Q1238-E1260 in cis on GPIbα interactions with VWF and
1238-A1. ..................................................................................................................59

Figure 4-3. The effect of Q1238-E1260 on GPIbα interactions with VWF and two
forms of A1 when it is in solution. ........................................................................64

Figure 4-4. Force-dependent lifetimes of WT A1 bonds with different GPIbα
preparations. ...........................................................................................................65

Figure 4-5. Force-dependent lifetimes of A1–GPIbα bonds with VWD mutantations.
...................................................................................................................................67

VIII