Anti-GPIbα mediated platelet desialylation and activation: a

novel Fc-independent platelet clearance mechanism and

potential therapeutic and diagnostic target in ITP

by June Li

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Laboratory medicine and Pathobiology

University of Toronto

© Copyright by June Li 2014

ii

Anti-GPIbα mediated platelet desialylation and activation: a novel

Fc-independent platelet clearance mechanism and potential

therapeutic and diagnostic target in ITP

June Li

Master of Science

Department of Laboratory Medicine and Pathobiology

University of Toronto

2014

Abstract

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by

autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests

antibody-mediated platelet destruction occurs in the spleen via Fcγ receptors (FcγR). However, it

has been demonstrated that anti-GPIb-mediated ITP is often refractory to therapies targeting

FcγR pathways. Utilizing a panel of murine monoclonal antibodies (mAbs) against murine and

human GPIIbIIIa and GPIbα, it was found that anti-GPIbα induces not only platelet activation to

a much greater extent than anti-GPIIbIIIa antibodies, but also significant surface expression of

neuraminidase 1 and platelet desialylation. Utilizing inhibitors of platelet activation and

desialylation, it was found that these two processes are not mutually exclusive, but rather exist in

a positive feedback loop, leading to FcγR-independent platelet clearance in the liver likely via

Ashwell-Morell receptors. Furthermore, in a murine model of ITP, sialidase inhibitor treatment

rescued platelet counts in predominantly anti-GPIbα -mediated thrombocytopenia.

.

iii

Acknowledgements

”Science is Mother Nature’s way of communicating with us, it should be respected”

Dr. Heyu Ni

I would like to thank first and foremost Dr. Heyu Ni for giving me the opportunity, the tools, and

skills which has made it possible for me to have had the most enriching and exciting dialogue

with “Mother Nature” for the past 2 years. You have been tough on me, but it has driven me to

excel and in the end made me even more passionate about research and equipped me with the

faculties to flourish in this field. For this I am forever grateful.

I would also like to thank my committee members Dr. Alan Lazarus and Dr. Gerald Prud’homme

for your tough questions, encouragement and great advice, which have shaped my work to be

something I can be proud of. And Dr. John Semple, thank you for your feedback on my thesis,

and during my defense, your time and attention is invaluable and greatly appreciated.

To the lab members of the Ni Lab both past and present most notably Oliver and Guangheng.

Oliver, thank you for listening to my endless hypothesis and answering my many obscure

technical questions, you are a wealth of knowledge that I was privileged to have access to.

Guangheng, thank you for your endless skepticism about my data, it pushed me to produce solid

work. And thank you for your experimental suggestions, the most interesting revelations usually

resulted from those experiments, many of which I still don’t have the answer to. Thanks to

Dianne van der Waal for initiating this project, you gave me the backbone on which this

fascinating story was built. Yiming and Adili thanks for your help in understanding the

complicated being that is the platelet. Chris Spring, thank you for all your help, I am sorry I can’t

be more specific as you were an integral part of everything. I am not exaggerating when I say I

would have been lost without your help. I owe you a foie gras.

iv

Elisa Simpson, you were my first friend and supporter in this lab, I would not be here today if it

wasn’t for your initial belief in me. To all of my other colleagues and now also friends, Sean

Lang, Conglei Li, Alison Cameron-Vendrig, Miao Xu and Brian Vadasz thanks for the laughs

and the sometimes very needed break from research.

Thank you to the Vivarium staff, especially Alex and Donna, for taking such good care of my

sometimes very sick mice, and for your patience in teaching me how to be humane yet efficient

in my in vivo work. And of course I can’t forget the mice, thank you for the sacrifice of your

lives, they have not been in vain. And a million apologies for poking so many holes in you.

Lastly, thanks to those I love most in this world, both my birth family and the family I chose.

Virginia, Marc, Anne and Gregory, thanks for your love and support. I am very lucky to have

you all in my life.

This is only the beginning of a very long and rewarding conversation with “Mother Nature” and I

cannot wait to hear what she has to say next.

v

Table of Contents

Abstract ........................................................................................................................................... ii

Acknowledgements ........................................................................................................................ iii

Table of Contents ............................................................................................................................ v

List of Tables ................................................................................................................................. ix

List of Figures ................................................................................................................................. x

Abbreviations ................................................................................................................................ xii

Chapter 1: Introduction and Literature Review .............................................................................. 1

1.1 Evolutionary origin of platelets .................................................................................... 1

1.2 Formation of platelets ................................................................................................... 1

1.3 Platelet hemostatsis ....................................................................................................... 2

1.4 Immune Thrombocytopenia .......................................................................................... 4

1.4.1 Clinical perspectives ............................................................................................... 4

1.4.2 Pathogenesis of ITP ................................................................................................ 5

1.4.3 Antibodies in ITP .................................................................................................... 8

1.4.3.1 Clonality of Antibodies ............................................................................... 8

1.4.4 Antigenic targets in ITP .......................................................................................... 9

1.4.4.1 GPIb-IX-V complex.................................................................................... 9

1.4.4.2 GPIIbIIIa ................................................................................................... 13

1.4.5 Platelet clearance in ITP ....................................................................................... 14

1.4.5.1 Fc-dependent platelet clearance ................................................................ 14

1.4.5.2 Fc-independent platelet clearance ............................................................. 14

1.5 Treatments in ITP ....................................................................................................... 17

vi

1.5.1 Corticoidsteroids ................................................................................................... 17

1.5.2 Anti-D ................................................................................................................... 18

1.5.3 Intravenous Immunoglobulin ................................................................................ 18

1.5.4 Splenectomy .......................................................................................................... 19

Chapter 2: Rationale, hypothesis and aims ................................................................................... 20

2.1 Rationale ..................................................................................................................... 20

2.2 Hypothesis................................................................................................................... 21

2.3 Specific Aims .............................................................................................................. 22

2.3.1 Aim 1: Comparison of anti-GPIbα and anti-GPIIbIIIa antibody induced platelet

effects in vitro ....................................................................................................... 22

2.3.2 Aim 2: To further characterize anti-GPIbα mAb mediated desialylation including

identifying the primary platelet surface desialylation target and identification of

putative sialidase ................................................................................................... 22

2.3.3 Aim 3: Characterize the link between platelet desialylation and platelet activation

............................................................................................................................... 22

2.3.4 Aim 4: Assess the utilization of the Fc-independent pathway by anti-GPIbα

antibodies both in vitro and in vivo ...................................................................... 23

2.3.5 Aim 5: Assess the therapeutic potential of a sialidase inhibitor ameliorating anti-

GPIbα antibody mediated thrombocytopenia in mice .......................................... 24

Chapter 3: Materials and Methods ................................................................................................ 26

3.1 Patients ........................................................................................................................ 26

3.2 Reagents ...................................................................................................................... 26

3.3 Blood collection and platelet isolation ........................................................................ 26

vii

3.4 Platelet incubations ..................................................................................................... 27

3.5 Flow cytometry ........................................................................................................... 28

3.6 Western Blotting ......................................................................................................... 28

3.7 Antibody Fab generation............................................................................................. 29

3.8 In vitro phagocytosis ................................................................................................... 29

3.9 Immunocytochemistry ................................................................................................ 30

3.10 In vivo platelet activation and desialylation ............................................................... 30

3.11 In vivo platelet clearance ............................................................................................ 31

3.12 Immunohistochemistry ............................................................................................... 31

3.13 Statistical analysis ....................................................................................................... 32

Chapter 4: Results ......................................................................................................................... 33

4.1 Aim 1: Comparison of anti-GPIbα and anti-GPIIbIIIa antibody mediated platelet

effects .......................................................................................................................... 33

4.1.1 Anti-GPIbα antibodies induces significantly higher platelet activation than anti-

GPIIbIIIa in murine and human platelets .............................................................. 33

4.1.2 Anti-GPIbα antibodies induces significantly higher platelet desialylation than

anti-GPIIbIIIa in murine and human platelets ...................................................... 34

4.1.3 The overall more desialylated state of GPIbα mAb does not contribute to the

detected platelet desialylation ............................................................................... 35

4.2 Aim 2: Characterization of anti-GPIbα antibody mediated desialylation ................... 36

4.2.1 Increased surface expression of NEU1 following anti-GPIbα incubation ............ 36

4.2.2 Platelet desialylation is predominantly localized to the GPIbα subunit ............... 37

viii

4.3 Aim 3: Characterization of link between anti-GPIbα mediated platelet activation and

desialylation ................................................................................................................ 38

4.3.1 Anti-GPIbα mediated platelet effects requires F(Ab)2 ......................................... 38

4.3.2 Platelet activation and desialylation are linked in a positive feed-back loop ....... 39

4.3.3 Human FcγRIIa may not contribute to anti-GPIbα mediated platelet desialylation

............................................................................................................................... 40

4.4 Aim 4: Assess the utilization of the Fc-independent pathway by anti-GPIbα opsonized

platelets both in vitro and in vivo ............................................................................... 40

4.4.1 Anti-GPIbα opsonized platelets can be uptaken by macrophages via non-FcγR

routes ..................................................................................................................... 40

4.4.2 Anti-GPIbα mAb causes platelet activation and desialylation in vivo ................. 41

4.4.3 ASPGR contributes to anti-GPIbα opsonized platelet clearance .......................... 42

4.4.4 Anti-GPIbα opsonized platelets are predominantly cleared by ASPGR in the

absence of macrophages ....................................................................................... 42

4.4.5 More anti-GPIbα opsonized platelets are localized to the liver in macrophage

depleted mice ........................................................................................................ 43

4.5 Sialidase inhibitor DANA ameliorates thrombocytopenia caused predominantly by

anti-GPIbα mAb .......................................................................................................... 44

Chapter 5: Discussion ................................................................................................................... 46

Chapter 6: Figures ......................................................................................................................... 57

Chapter 7: Future directions.......................................................................................................... 85

Chapter 8: References ................................................................................................................... 86

ix

List of Tables

Table 1 Novel mouse anti-mouse anti-GPIb and anti-GPIIbIIIa monoclonal antibodies .. 57

x

List of Figures

Figure 1 Immunopathogenesis of ITP ................................................................................... 7

Figure 2 Schematic of structure of GPIb-IX-V complex .................................................... 12

Figure 3 Fc-independent platelet clearance mediated by anti-GPIb antibodies .................. 25

Figure 4 Anti-GPIbα mAb causes murine platelet activation and apoptosis ...................... 58

Figure 5 Anti-GPIbα mAb causes human platelet activation and apoptosis ....................... 59

Figure 6 Anti-GPIbα mAb causes human platelet aggregation........................................... 60

Figure 7 Polyclonal anti-GPIbα sera but not purified IgG from GPIb-/- mice causes platelet

activation ............................................................................................................... 61

Figure 8 Anti-GPIbα mAb causes murine platelet desialylation......................................... 62

Figure 9 Anti-GPIbα mAb causes human platelet desialylation ......................................... 63

Figure 10 Polyclonal anti-GPIbα sera and purified IgG from GPIb-/- mice causes platelet

desialylation .......................................................................................................... 64

Figure 11 Anti-GPIbα mAb are more desialylated than anti-GPIIbIIIa................................ 65

Figure 12 Sialidase inhibitor DANA can inhibit anti-GPIbα mediated platelet desialylation

............................................................................................................................... 66

Figure 13 Anti-GPIbα mAb binding to fixed platelets does not increase platelet desialylation

............................................................................................................................... 67

Figure 14 Anti-GPIbα antibody induces murine platelet surface expression of Neu1 .......... 68

Figure 15 Human ITP anti-GPIbα sera induces human platelet NEU1 surface expression .. 69

Figure 16 Anti-GPIbα mAb causes murine platelet surface expression of Neu1 as detected

by flow cytometry ................................................................................................. 70

xi

Figure 17 Anti-GPIbα mAb induces human platelet surface expression of NEU1 as detected

by flow cytometry ................................................................................................. 71

Figure 18 Anti-GPIbα mAb mediated desialylation is localized to the GPIbα subunit ........ 72

Figure 19 Removal of GPIbα subunit by OSGE decreases desialylation state of platelets to

baseline ................................................................................................................. 73

Figure 20 NIT B Fab binds murine and human platelets ...................................................... 74

Figure 21 NIT Fab does not cause murine or human platelet activation or desialylation ..... 75

Figure 22 NIT B Fab induces significant platelet clearance when injected in vivo .............. 76

Figure 23 Platelet activation and desialylation exists in a positive feedback loop ............... 77

Figure 24 Anti-GPIbα opsonized platelets are phagocytosed by Fc-independent pathways 78

Figure 25 Anti-GPIbα mAb causes platelet activation and desialyation in vivo .................. 79

Figure 26 The ASPGR contributes to the clearance of anti-GPIbα mAb but not anti-

GPIIbIIIa mAb opsonized platelets ...................................................................... 80

Figure 27 Anti-GPIbα opsonized platelets are cleared predominantly by ASPGR in the

absence of macrophages ....................................................................................... 81

Figure 28 Very low localization to the spleen of anti-GPIIbIIIa or anti-GPIbα opsonized

platelets in the absence of macrophages ............................................................... 82

Figure 29 Significant amounts of anti-GPIbα opsonized platelets but not anti-GPIIbIIIa are

localized to the liver and Ashwell-Morell receptors in the absence of macrophages

............................................................................................................................... 83

Figure 30 Sialidase inhibitor DANA ameliorates predominantly anti-GPIbα mediated

thrombocytopenia ................................................................................................. 84

xii

Abbreviations

ADP Adenosine-5'-diphosphate

APC Antigen presenting cell

ASPGR Asialogylcoprtein receptor

BAFF B-cell activating factor

Ca2+ Calcium

CD Cluster of differentiation

DANA N-acetyl-2,3 deoxy neuramic acid

DNA Deoxyribonucleic acid

ECL Chemiluminescence

EDTA Ethylenediaminetetraacetic acid

Fab Fragment, antigen binding

Fc Fragment, crystallizable

FITC Fluorescein isothiocyanate

FSC Forward-scatter

GP Glycoprotein

GM-CSF Granulocyte-Macrophage colony stimulating factor

Ig Immunoglobulin

IL Interleukin

xiii

INF Interferon

ITAMs Intracellular tyrosine activation motif

IVIG Intravenous immunoglobulin

LRR Leucine-rich repeat

Mac-1 Macrophage 1 antigen

MAIPA Monoclonal antibody immobilization of platelet antigens

mAb Monoclonal antibody

NEU1 Neuraminidase 1

OSGE O-sialo-glycoprotein endopepsidase

P38MAPK p38 mitogen activated protein kinase

PBS Phosphate buffered solution

PFA Paraformaldehyde

PGI2 Prostaglandin-2

PI3K Phosphoinositide 3-kinase

PPP Platelet-poor plasma

PRP Platelet-rich plasma

PSGL-1 P-selectin glycoprotein ligand-1

PVDF Polyvinylidene fluoride

RCA-1 Ricinus communis agglutinin-1

SDS Sodium dodecyl sulfate

xiv

SEM Standard error of the mean

SSC Side-scatter

TM Transmembrane

TNF Tumor necrosis factor

TPO Thrombombopoetin

TxA2 Thromboxane A2

VWF von Willebrand factor

1

Chapter 1: Introduction and Literature Review

1.1 Evolutionary origin of platelets

Platelets are small anucleated hematopoietic cells, shed from the cytoplasm of the

megakaryocyte, which are fundamentally required for the maintenance of normal hemostasis.

The anucleate platelet is found only in mammals. In all other vertebrates, the analogous cell to

the platelet which are primarily involved in hemostasis are termed thrombocytes and are

nucleated. Thrombocytes and platelets are thought to have evolved from cells found in lower-

invertebrates termed haemocytes which circulates in the haemolymph and are involved in not

only hemostatic functions, but are also in multiple defense mechanisms of the animal.

Interestingly, unlike the platelet or the thrombocyte, the haemocyte is capable of aggregating and

sealing wounds without any other exogenous factors. The factors which promote cell aggregation

and coagulation are released from the haemocyte, and do not require factors in the circulating

fluid. Despite the similarities between haemocytes and mammalian platelets, there is no

definitive proof that they are the evolutionary progenitor of platelets. In addition, the biological

advantage of having anucleated platelets produced from the cytoplasm of a larger cell is to this

day, unclear (1)

1.2 Formation of platelets

Platelets are derived from megakaryocytes, a rare polyploidal precursor cell descended from

pluripotent stem cells (2). Megakaryocytes reside primarily in the bone marrow (and to a lesser

extent in the blood and lungs) and their primary function is to produce platelets. Thrombopoietin

(TPO) which binds to the c-MPL receptor is the primary cytokine responsible for promoting

growth and development of megakaryocyte precursors. During platelet biogenesis,

2

megakaryocytes undergo a series of remodeling events including specialized DNA amplification

(endomitosis) during which DNA undergoes replication without cellular division. Subsequently,

the megakaryocyte matures and rapidly fills with a large quantities of platelet-specific proteins

and organelles, and membrane systems that will be subdivide and packaged into platelets.

Studies into the regulation of allocation of platelet granules and proteins is of great interest.

Megakaryocytes then undergo extension events which include the development of a demarcation

membrane system (an extensive tubular system) which is thought to function as a membrane

reservoir to be passed on to formed platelets. In addition, a dense tubular network and open

canalicular system and platelet granules are also formed. The process of platelet biogenesis

involves the formation of proplatelets along pseudopodial extensions from the megakaryocyte.

The necessary platelet proteins and organelles are then shuttled to the proplatelets, which are

then extended into the bone marrow sinusoids and released into circulation (3).

1.3 Platelet hemostatsis

The primary function of platelets is hemostatic. In humans, they circulate at 150-400 x 109

platelets/L, and have a life-span of 7-10 days. Normally in the absence of vessel injury platelets

circulate the body in a quiescent state which is maintained by the intact endothelium that releases

nitrous oxide and prostaglandin-2 (PGI2) and expresses CD39 (1). The hemostatic response to

injury is dictated by the extent of damage, the specific matrix proteins exposed and flow

conditions. Typically, platelets hemostatic function is particularly relevant at sites of high shear

stress, such as micro arteriolar circulation. In pathological conditions, such as arterial thrombosis

of which platelets are a major contributor, the narrowed vessel lumen from plaque formation

artificially forms a high-shear environment. At sites of endothelial damage, the underlying sub

endothelium is exposed. Plasma VWF binds Type VI collagen in the extracellular matrix which

3

exposes the A1 domain, which tethers the platelet via GPIbα. The short on-off rate is sufficient

to promote platelet rolling along the surface allowing for multiple platelet surface protein

interactions with underlying extracellular matrix proteins, including GPVI to collagen. This then

initiates platelet signaling through GPIb resulting in inside-out signaling activation GPIIbIIIa,

and stable adhesion of VWF. After the initial adhesion of platelets, granule secretion of ADP, α-

thrombin, and thromboxane A2 which acting as further agonists on G-coupled protein coupled

receptors is required for full activation of GPIIbIIIa and platelet shape change, required for

platelet aggregation and fibrin generation (1).

4

1.4 Immune Thrombocytopenia

1.4.1 Clinical perspectives

Immune thrombocytopenia (ITP) describes a heterogeneous group of disorders characterized by

immune-mediated platelet destruction. It is estimated the incidence of ITP range from 1.6 to 2.68

per 100 000 persons per year, with prevalence estimates ranging from 9.5 to 23.6 per 100 000

persons (4). Primary ITP (previously known as idiopathic ITP) is characterized by isolated

thrombocytopenia without any co-existing disorders. Secondary ITP encompasses all forms of

ITP that is not primary, this includes posttransfusion purpura, drug induced purpura and all

disease associated ITP (5). Epidemiological studies suggests the rate of incidence in adults is

approximately equal for both sexes except between the ages of 30-60 where more women are

affected. A patient is diagnosed with ITP when they have a platelet count of <100x109/L (6). In

children, ITP usually acute, appearing within 1-2 weeks after viral infection or 2-6 weeks after

standard immunizations. And resolves spontaneously in a few weeks. In adults, the onset is

usually insidious and tend to persist to chronic (7). Unfortunately the diagnosis of primary ITP is

based on a criterion of exclusion, and currently there is no reliable gold-standard in clinical or

laboratory parameters which can predict diagnosis with absolute accuracy (8). The

unpredictability of disease course is further reflected with the required additional criteria of

characterizing of ITP as newly diagnosed (within 3 months of diagnosis), persistent (3-12

months following diagnosis) and chronic (longer than 12 months (5). Increased bleeding is the

primary morbidity associated with ITP, and symptoms can vary from asymptomatic and mild

such as petechiae, purpura, epistaxis to severe hemorrahage. Although severity of

thrombocytopenia does not necessarily correlate to bleeding, patients with persistently lower

than 30x109 platelets/L are at much higher risk for severe bleeding and mortality (9). Severe

5

bleeding has been observed in the gastrointestinal tract and in the central nervous system.

Intracranial hemorrhage is the primary cause for mortality in ITP, with mortality estimated to be

around or less than 5% (9).

1.4.2 Pathogenesis of ITP

The seminal experiment in 1951 by Dr. William Harrington involving self-infusion of the plasma

from an ITP patient resulted in himself becoming severely thrombocytopenic. This led to the

discovery that a factor in the plasma was the culprit responsible for mediating decrease in

platelet counts (10). Subsequent studies by Shulman et al. established that this factor could be

adsorbed by platelets and was localized to the IgG-rich plasma fraction, thus identifying it as

autoantibodies (11). Indeed, autoantibodies are generally considered to be the primary factor

responsible for decreased platelet counts in ITP. However, as an autoimmune disease, the

dysregulation of the immune system is multifactorial and a myriad of immune cells including

antigen presenting cells (APC) and T-cells are intimately involved in perpetuating the anti-

platelet response (Figure 1) (12, 13). In addition, platelet destruction is no longer considered the

only mechanism responsible for low platelet counts, over the years, increasing evidence has

shown megakaryocytic abnormalities in ITP, thus also impeding the production of platelets (14,

15). The central dogma surrounding ITP as an autoimmune disease is the loss of self-tolerance.

Regulatory cells, particularly CD4+CD25+FOXP3+ T-cells (Treg) which are key factors in

maintaining peripheral tolerance, have been consistently shown to be dysfunctional or decreased

in ITP patients (16) . Recent studies which further emphasize the magnitude of the impact of

regulatory cells in ITP suggest that successful therapies which raise platelet counts appear to do

so through normalization of the Tregs (17, 18). The decrease of Tregs results in an imbalance

and subsequent increase in pathogenic T-cells, which, through cytokines, mediate interactions

6

with APCs and B-cells. The repertoire of CD4+ T-cells in the anti-platelet response are generally

considered Th1 biased (19, 20). This means their cytokine profile is predominant in INF-γ, IL-2,

TNFα and granulocyte-macrophage colony-stimulating factor (GM-CSF), which generally

promote pro-inflammatory and cell-mediated immune responses. Recent studies are now also

shedding light on the importance of T-helper (Th)17 cells in ITP. IL-17, a pro-inflammatory

cytokine and IL-18, which stimulates the development of Th1 cells and IFN-γ production are

shown to be increased in patients with active ITP (21, 22). Collectively the pro-inflammatory

response is self-perpetuating, inflammatory cytokines and chemokines acting on APCs

perpetuate platelet antigen uptake and presentation via MHC II molecules to further drive T-cell

responses (23, 24). B-cells in ITP also exhibit abnormalities. It has been shown that there is

increased circulating B-cell activating factor (BAFF) in ITP patients. BAFF is a regulator of B-

cell development and pro-survival factor, thus maintaining pathogenic antibody production (25,

26)

7

Figure 1 Immunopathogenesis of ITP

Stasi et al. Thromb Haemost 2008; 99: 4–13

8

1.4.3 Antibodies in ITP

Although still considered to be the primary cause for low platelet counts in ITP, antibodies can

only be detected in ~50% of ITP patients (27), further there are patients with detectable anti-

platelet antibodies but no decrease in platelet number (28). Thus an important determinant of the

role of antibodies in the pathogenesis of ITP is their antigenic targets.

Measurement of autoantibodies has proven to be challenging in the past. Early attempts to detect

autoantibodies using agglutination, complement activation or platelet lysis proved unsuccessful

due to low sensitivity (29). In 1987, two assays were developed, the immunobead assay (30) and

the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay (31). These

two assays have been proven to be the most clinically useful with resulting sensitivities ranging

from 49%-66% and specificities ranging from 78%-93% (32). Utilizing these assays, it was

found that the predominant antigenic targets in chronic ITP were GPIIbIIIa and GPIb/IX, with

70-80% of antibody positive patients with antibodies against GPIIbIIIa and 20-40% of patients

with antibodies against GPIb/IX or both (33, 34).

1.4.3.1 Clonality of Antibodies

Although antibodies in ITP are generally considered to be polyclonal (35), there is mounting

evidence which suggest the repertoire of antigenic targets is limited and likely derived from the

expansion of a limited number of B-cell clones. For example studies of both serum and platelet-

associated antibodies from ITP patients have been shown to be light chain-restricted, suggesting

antibody clonality (36, 37). And more recently Roark et al generated a Fab/phage display library

of all the autoantibodies from 2 ITP patients (38). Analysis of genetics of platelet reactive Fab

revealed from both patients almost exclusive rearrangements of the VH3-30 IgG heavy chain

9

variable gene. Thus they concluded that the anti-platelet antibodies appeared to be produced by a

limited number of B-cells clones as a result of antigen-driven somatic mutation in which

reactivity was highly dependent on specific heavy- and- light chain pairings.

1.4.4 Antigenic targets in ITP

1.4.4.1 GPIb-IX-V complex

1.4.4.1.1 Structure

The GPIb-IX complex is expressed exclusively on platelets and megakaryocytes. The complex is

composed of 3 subunits GPIbα, GPIbβ, GPIX, which all possess similar structures comprising of

long N-terminal extracellular domain containing a leucine-rich-repeat (LRR) motif, single-pass

transmembrane domain, and a relatively short C-terminus intracellular domain which lacks any

associated G-coupled proteins or tyrosine kinases (39). GPIbα is the largest subunit and

possesses all the known ligand binding sites of the complex. As the second most abundant

platelet surface protein, it is expressed at about 25,000 copies/platelet (40). GPIbα is linked via

disulfide bond to GPIbβ, and non-covalently associated with GPIX in a 1:2:1 stoichiometry (41,

42). GPIbα has 610 residues and represents the largest subunit of the complex. The N-terminal

domain (residues 1-282) is composed of 7 LRRs and flanking or capping sequences, and ends in

a negatively charged sulfated tyrosine C-tail (43). Crystal structure reveals a single di-sulfide

bond in the N-capping region, and 2 disulfide bonds in the C-capping region. The LRR region

adopts a concave shape comprised of β-strands. The binding sites for VWF has been localized in

the C-flanking and sulfated tyrosine segments, and binding sites for VWF A1 in the leucine-rich

N- and C-flanking regions (44, 45). The binding site for thrombin are located to the acidic

10

residue-rich sequence containing sulfated tyrosines (46). Following the N-terminal there is a long

highly glycosylated mucin-like macroglycopeptide domain (residues 303-485), containing O-

linked, sialylated hex saccharide on average every 3-4 amino acids (47). This region extends the

N-terminal globular domain and VWF binding site approximately 45 nm from the platelet

surface (48). The length of which is highly polymorphic between any individual (49).

Amazingly, macroglycopeptide region is approximately 60% carbohydrate by weight and

contributes up to 64% of total sialic acid content of the entire platelet. (50) A stalk region of

approximately 40-50 residues follows the macroglycopeptide domain. Covalent-binding to the

GPIbβ occurs at the junction of the extracellular and TM domains. It had been previously

thought one GPIbα subunit can form one disulfide bond with one GPIbβ, but recently it has been

demonstrated that one GPIbα can form two di-sulfide bonds with two GPIbβ via adjacent

cysteines (42). The cytoplasmic tail of GPIbα can bind to filamin A, 14-3-3γ, and

phosphoinositide 3-kinase (PI3K) (51).

Expression of all three GPIbα, GPIbβ, and GPIX are required for proper expression of the

complex. This is strongly evidenced in Bernard Soulier patients for whom mutations have been

mapped to GPIbα, GPIbβ and GPIX genes resulting in complete absence of expression the GPIb-

IX complex (52).

GPIbα is weakly associated with GPV, and has little impact on the expression of GPI-IX.

However, GPI-IX is required for efficient expression of GPV on the platelet surface. Traditional

stoichiometry postulated GPV to be associated in the transmembrane domain (TM) between two

GPIbα, however, recent quantitative analysis indicate similar number of GPIX and GPV

11

molecules (53), and further the putative binding interface in the GPV TM region is too small to

support simultaneous association with two GPIbα (54)

12

Figure 2 Schematic of structure of GPIb-IX-V complex

( ) N-linked glycosylation ( ) O-linked-glycosylation

Li. et al Cardiovasc Hematol Disord Drug Targets.2013 Mar 1;13 (1):50-8.

1.4.4.1.2 GPIb-Signaling

Interaction of GPIb-V-IX with VWF on the subendothelial matrix following endothelial damage

under high flow conditions is crucial for primary hemostasis and initiation of thrombus

formation. However, because GPIb-V-IX is not associated with any G-coupled proteins nor does

possess any intrinsic tyrosine kinase activity, the previous thought was that GPIb-IX-VWF

interactions were for solely for the purpose of tethering. However, it is now widely accepted that

GPIbα do in fact transduce intracellular signaling, similar to the collagen receptor GPVI, which

leads to GPIIbIIIa integrin activation and cytoskeleton re-arrangements (55). Published data to

date strongly support the Src-family tyrosine kinases and tyrosine phosphorylation as an essential

13

component and early regulator of GPIbα signaling (56). Cross-linking of GPIbα by multimeric

VWF initiates lateral clustering of GPIb-IX (57), is followed by translocation to lipid rafts (58)

where associations with immunoreceptor tyrosine activation motif (ITAM) bearing FcγR occurs.

FcγR association with GPIbα is positively contributes but is not essential for signal transduction

(59), during which tyrosine phosophorylation by GPIbα-associated-Src (60). leads to further

signaling and GPIIbIIIa activation.

1.4.4.1.3 Target epitopes of GPIb-IX

Epitope mapping of autoantibodies to GPIb/IX revealed that binding sites at both the cytosolic

and extracellular regions. Further studies from the same group utilizing recombinant fragments

of the extracellular domain of GPIb and sera from 16 ITP patients positive for anti-GPIb/IX

revealed 38% of the ITP sera reacted with extracellular portion of GPIbα subunit. Presumably,

the remaining patients had autoantibodies against GPIbβ, GPIX, a complex-dependent epitope,

or intracellular regions of the complex. Further analysis revealed 6 of the 16 patients shared a

limited epitope 9 amino acids long (333-341) within the macroglycopeptide region. Only one

patient had autoantibodies against the globular N-terminus region.

1.4.4.2 GPIIbIIIa

GPIIbIIIa is the most abundant platelet surface protein with copy number ~80,000 (61). It is an

integrin which requires inside-out signaling which triggers a conformational change in GPIIIa

subunit permissive for binding. Ligands which binds to activated GPIIbIIIa include VWF,

fibrinogen, fibronectin and vitronectin. Further there may be other yet to be identified ligands

which may positively contribute to platelet activation through binding of GPIIbIIIa independent

of the known ligands as has been observed by our lab(62, 63). Following binding, GPIIbIIIa

14

initiates outside-in signaling, involving activation of Src and Syk protein tyrosine kinases, and

signaling to the actin cytoskeleton, resulting in platelet spreading (64). Epitope determination

studies have shown the dominant target of autoantibodies is on the GPIIb subunit of GPIIbIIIa,

however, binding is cation dependent suggesting it requires the GPIIIa subunit to be present as

an intact complex (65). Less common are autoantibodies directed to the GPIIIa subunit (66).

1.4.5 Platelet clearance in ITP

1.4.5.1 Fc-dependent platelet clearance

Following antibody binding, the Fc-region may mediate various ways of platelet clearance. The

most common mechanism is platelet phagocytosis in the spleen via FcγRIIa and FcγRIII. The

high affinity receptor FcγRI does not seem to play a relevant role in ITP (67) Increased

complement deposition has also been demonstrated, whereby normal platelets incubated with

selected ITP serum autoantibodies resulted in increased C4 and C3 binding followed by platelet

lysis (68). In addition, increased platelet associated C3, C4 and C9 have been found on ITP

platelets (69).

1.4.5.2 Fc-independent platelet clearance

Fc-independent phagocytosis is an ancient mechanism of the innate immune system in clearing

invading organisms prior to the development of the adaptive immune system (70). Today, in

complex mammals, Fc-independent phagocytosis play essential roles in the clearance of

apoptotic/senescent cells and aiding in maintaining peripheral tolerance, while at the same time

acting as the first line of defense and maintaining their native roles in the clearance of foreign

pathogens (70). Fc-independent phagocytosis of platelets in normal physiological situations may

15

occur as a natural process in the clearance of senescent platelets (71), or clearance of activated

platelets.

1.4.5.2.1 Fc-independent platelet clearance of cold stored platelets

Cold-storage of platelets prior to transfusion for the purpose of bleeding prophylaxis is not

practiced despite the benefits of refrigeration in prolonging platelet storage. The underlying

reason being cold-stored platelets are rapidly cleared from circulation once transfused, thus

negating any potential benefits in prevention of bleeding (72). The cause for platelet clearance

remained a mystery until the Hoffmeister group in 2003 published dual papers in Cell and

Science demonstrating cooling of platelets to 40C causes GPIb-receptor clustering leading to

rapid clearance by the Mac-1 receptor expressing Kupffer cells in the liver, through recognition

of clustered β-glycans on GPIbα (73, 74). Unfortunately a Phase I clinical trial administering

autologous platelets modified with addition of sugar to cover the exposed β-glycan failed at

extending the circulation time of platelets (75). This setback prompted further investigation

which resulted in the discovery of the role of the Ashwell-Morell receptor in the clearance of

long-term cold stored platelets. It was found following long-term refrigeration platelet sialidase

Neuraminidase-1 (NEU1) is translocated to the platelet surface where it mediates removal of

terminal sialic residues on GPIbα, leading to clearance in the liver via the AMR and not Mac-1

on macrophages (71, 76).

1.4.5.2.2 Mammalian neuraminidases

Sialidases or neuraminidases are enzymes which catalyze the hydrolytic cleavage of non-

reducing sialic acid (N-acetylneuramic acid) residues linked to saccharide chains of

glycoproteins and gangliosides(77). They are ubiquitous in all the animal kingdoms including

16

bacteria and viruses, but are absent in plants, insects and yeast (78). In veterbrates, four

sialidases have been identified to date. They are encoded on different genes and differ in their

subcellular localizations and substrate preference. NEU1 is typically considered lysosomal with a

substrate preference of glycoproteins with α2,3 and α2,6 linkages . NEU2 is cytosolic with a

preference for α2,3 linkages present on gangliosides and glycoproteins (79). NEU3 is

constitutively expressed on the plasma membrane and is highly specific for gangliosides (80).

NEU4 has been localized to the mitochondrial membrane and lysosomal lumen (81). It has a

wide substrate specificity from glycoproteins to gangliosides and oligosaccharides, and has been

shown to complement the function of defective NEU1 (82).

1.4.5.2.3 Ashwell-Morell receptor

The Ashwell Morell receptor is a C-type lectin comprised of 2 transmembrane glycoproteins

(ASPGR-1 and ASGPR-2). It is part of the asialoglycoprotein receptor family which comprises

of other lectin receptors including the Kupffer cell receptor, the macrophage galactose receptor

and galectins The AMR is expressed almost exclusively on hepatocytes. It is the predominant

receptor involved in the removal of desialylated glycoproteins (asialoglycoproteins) from the

blood through binding of exposed penultimate galactose residues (83) Endogenous ligands for

the AMR have been difficult to identify as AMR-/- mice do not accumulate asialoglycoproteins

in their blood (84). However, the AMR has been identified to cause rapid hemostatic modulation

in response to reduced sialylation state of platelets and VWF in pathological situations such as

infections with microbes expressing sialidase activity. Furthermore it was also found that in these

situations the AMR is protective by delaying onset of disseminated intravascular coagulopathy of

sepsis by rapidly clearing desialylated platelets (85).

17

1.5 Treatments in ITP

Treatment is initiated once platelet counts fall below 25-30 x 109 platelets/L (high-risk bleeding)

or when signs of bleeding appear. Purpose of treatment in ITP is to raise platelet counts to a low-

level risk of bleeding range. But other factors such as patient’s age, lifestyle and possible side

effects are also considered (6, 86). First-line treatments most commonly used are corticosteroids,

IVIG and less common is Anti-D. Response rates vary considerably between the aforementioned

treatments studies, which makes direct comparisons of effectiveness between treatments

difficult. Newer treatment options including TPO mimetics (eg. romiplosim, eltrombopag) are

generally considered a maintenance therapy and long-term study data are just now emerging.

There are no significant differences between the responses to the variations of mimetics used

(87).

1.5.1 Corticoidsteroids

Corticosteroids are the standard initial treatment (6). It is administered either as low-dose

(prednisolone) over several weeks, or high-dose (dexamethasone, 40mg/day for 4 days) pulse

(given every 14 days). The high-dose “pulse” dexamethasone is preferred as an initial treatment

due to lower-risk of steroid related side effects with an 86% initial response rate and a 50% of

sustained response in newly diagnosed adults with ITP (88). However, a recent study comparing

prednisolone and dexamethasone treatment courses in 25 patients showed longer sustained

responses in the prednisolone group (89).

Corticosteroids is a general immunosuppressive agent acting at the transcription level. The exact

mechanisms of action have not been fully elucidated but generally it promotes transcription of

anti-inflammatory factors such as IL-10, while down regulating transcription of pro-

18

inflammatory factors such as IL-1β, TNFα, IL-6, IL-8, IL-12 and IL-18 (90). Corticosteroids

have also been reported to modulate the expression profile of FcγR macrophages, by decreasing

expression of activating FcγR and increasing expression of inhibitory FcγRIIb. Corticosteroids

are also associated with severe side-effects including changes in facial features, osteoporosis,

menstrual problems, excessive appetite, brittle skin, sleeping difficulties, and increased

susceptibility to infections (91). For these reasons, it is an extremely difficult regimen for patient

compliance and relapses are not uncommon.

1.5.2 Anti-D

Anti-D is less commonly administered than IVIG and corticosteroids, as patients must be Rh (D)

positive, be non-splenectomised and not have autoimmune hemolytic anemia. Its efficacy has

been demonstrated to be just as, or more effective than IVIG at 75ug/kg. Mild-anemia may be a

common side effect, more severe side effects such as intravascular hemolysis, disseminated

intravascular coagulation and renal failure are possible but very rare (6).

1.5.3 Intravenous Immunoglobulin

Intravenous Immunoglobulin (IVIG) recipients are more likely to attain a platelet increase within

a much shorter time (24 hours) than corticosteroid treatment. And in many patients it is used

concomitantly with steroids. Although the modes of action are still unclear, several mechanisms

have been suggested including mononuclear phagocytic system blockade, competitive inhibition

of FcRn resulting in rapid clearance of pathogenic antibodies, and many others (92)

19

1.5.4 Splenectomy

Splenectomy is a second-line treatment option for those who fail or develop intolerance to initial

therapies or who develop chronic ITP with persistent bleeding. Around 80% of patients respond

to splenectomy, and for around 66% the response is sustained with no additional therapy for at

least 5 years. There is no standard test which can accurately predict response to splenectomy.

Interestingly, indium-labeled autologous platelet scanning show for patients with splenic platelet

destruction the response is higher than if it shows hepatic sequestration.

20

Chapter 2: Rationale, hypothesis and aims

2.1 Rationale

Autoantibodies in ITP are mainly directed against the two most abundant platelet surface

receptors: GPIb-XI and GPIIbIIIa. These antigens belong to two distinct protein families. They

are of distinct protein structures, contribute differently to platelet adhesion and aggregation, and

bind different ligands which mediate diverse signaling cascades. Despite considerable

investigation, the pathogenesis of ITP remains incompletely understood, and we currently lack

consensus for effective therapy. ITP has long been thought of as a single, homogenous disease.

However, variability in natural history, response to therapy, clinical manifestation, target

receptors of autoantibodies and distinct antibody effects on platelets, suggest that ITP is best

thought of as an autoimmune syndrome.

This theory has been supported by several lines of evidence. Interestingly, Nieswandt’s group in

2000 discovered certain anti-GPIbα monoclonal antibodies (mAb) were able to induce

thrombocytopenia in mice with their F(ab)2 fragments alone, independent of the Fc-portion,

which was not observed with anti-GPIIbIIIa mAb F(ab)2. These findings were also observed in

our lab in FcγR -/- mice and further, we found most of anti-GPIα mediated thrombocytopenia

was refractory to IVIG treatment. Recently we published findings in a murine model of fetal-

neonatal alloimmune thrombocytopenia (FNAIT) where it was shown that anti-GPIbα polysera

caused platelet activation and apoptosis. These previous lessons in murine models of ITP suggest

antibodies targeting anti-GPIbα may lead to anti-platelet effects, resulting in additional Fc-

independent clearance pathways.

21

Other lines of evidence in support of anti-GPIbα antibodies causing Fc-independent platelet

clearance include F(ab)2 fragments of various anti-GPIbα antibodies inducing platelet

agglutination and GPIIbIIIa-dependent aggregation in vitro (93, 94) and causing

thrombocytopenia in vivo when tested as an anti-thrombotic agent (95, 96). In addition,

retrospective patient data have shown patients with anti-GPIbα antibodies are associated with

poorer responses to IVIG and steroid treatments. Recently a link between platelet activation and

platelet desialylation was demonstrated in cold-stored platelets. Whether anti-GPIbα induced

platelet activation can also lead to desialylation leading to a novel mechanism of platelet

clearance is unknown (Figure 3). Currently, given that lack of standard criteria to determine

success of ITP diagnosis and therapy, this study is crucial in characterizing novel platelet

clearance mechanisms as determined by antibody specificity and identifying new therapeutic and

biomarkers for more effective treatment and diagnosis of ITP.

2.2 Hypothesis

Antibody specificity dictates differential responses to therapy, whereby anti-GPIb antibodies

induces platelet activation, leading to platelet desialylation resulting in Fc-independent

clearance mechanisms

22

2.3 Specific Aims

2.3.1 Aim 1: Comparison of anti-GPIbα and anti-GPIIbIIIa antibody

induced platelet effects in vitro

Human and mouse platelets will be incubated with a new panel of mouse anti-mouse

GPIb and GPIIbIIIa monoclonal antibodies (mAb) generated in knock-out mice. Flow

cytometry will be used to measure platelet activation through P-selectin expression,

platelet apoptosis through Annexin-V binding and platelet desialylation through binding

of fluorescein conjugated Ricinus Communis Agglutinin-I (RCA-1)

2.3.2 Aim 2: To further characterize anti-GPIbα mAb mediated

desialylation including identifying the primary platelet

surface desialylation target and identification of putative

sialidase

Western blot assay on entire platelet lysate following anti-GPIbα mAb

incubation and probed with RCA-1 will be used to determine the dominant

desialylated residue Incubation of platelets in vitro

Cleavage of GPIbα residue via O-sialo-glycoendopepsidase (OSGE) and assess

changes in platelet desialylation

2.3.3 Aim 3: Characterize the link between platelet desialylation

and platelet activation

Various inhibitors of platelet activation will be utilized to assess impact on antibody

mediated platelet desialylation

Inhibitors of platelet desialylation will be utilized to assess impact on platelet activation

23

Generation of monovalent Fab fragments and assessment of its ability to mediate

antiplatelet effects both in vivo and in vitro

Elucidate the contribution FcγRIIa on human platelets to the magnitude of antibody

response compared to murine platelets

2.3.4 Aim 4: Assess the utilization of the Fc-independent

pathway by anti-GPIbα antibodies both in vitro and in

vivo

A macrophage cell-line (RAW 264.7) in the presence of FcγR blockers will be used to

determine FcγR-independent phagocytic potential of anti-GPIbα mAb treated platelets

Injection of anti-GPIbα and anti-GPIIbIIIa antibodies in wild-type (WT) Balb/c mice to

determine if the antibodies causes platelet activation and desialylation in vivo

To assess the role of the hepatocyte, more specifically, it’s ASPGR, the Ashwell-Morell

receptor in clearance of anti-GPIbα bound platelets, in both macrophage depleted and

non-depleted mice we will utilize an AMR blocker and assess its ability to increase

circulation of anti-GPIbα bound platelets

To assess tissue localization of anti-GPIbα or anti-GPIIbIIIa bound platelets spleen and

livers will be harvested following injection of fluorescently labeled antibody opsonize

platelets and visualized via immunofluorescence

24

2.3.5 Aim 5: Assess the therapeutic potential of a sialidase

inhibitor ameliorating anti-GPIbα antibody mediated

thrombocytopenia in mice

Co-injection of the sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid

sodium salt (DANA) with anti-GPIb or anti-GPIIbIIIa antibodies and assess differences

in platelet number the following day

25

Figure 3 Fc-independent platelet clearance mediated by anti-GPIb

antibodies

Source: Li. et al Cardiovasc Hematol Disord Drug Targets.2013 Mar 1;13(1):50-8.

26

Chapter 3: Materials and Methods

3.1 Patients

Patient 17 sera was part of patient samples collected from Anhui Medical University Hospital,

Hefei, China as previously reported (97). Previous MAIPA tests confirmed presence of anti-

GPIbα antibodies. Sunnybrook ITP patient was obtained from Sunnybrook Health Science

Center following death. Permission was obtained from spouse. Anti-GPIbα antibodies, with no

anti-GPIIbIIIa antibodies were detected by MAIPA. The research was approved by a local

research ethics committee.

3.2 Reagents

Inhibitors were used for the following: intracellular Ca2+ increases: BAPTA-AM (Calbiochem),

P38-MAPK: SB203580 (Alexis Biochemicals,), neuraminidase: N-acetyl-2,3-dehydro-2-deoxy

neuraminic acid sodium salt (DANA; EMD Biosciences), pan-caspase inhibitor: Q-VD-OPh

(Sigma) cAMP: prostacyclin (PGI2; Cayman Chemical) and Ashwell-Morell receptor:

asialofetuin and its control fetuin (Sigma). O-sialoglycoprotein endopeptidase (OSGE; Cederlane

Laboratories) was used to cleave GPIb. Sialidase neuraminidase originated from Clostridium

perfringens (Roche Applied Sciences).

3.3 Blood collection and platelet isolation

Procedures were approved by the Research Ethics Board of St. Michael’s Hospital (Toronto, ON,

Canada) and conducted as previously described (98). Venous blood was obtained from from

healthy volunteers by venipuncture into 3.2% trisodium citrate. Platelet-rich plasma (PRP) was

prepared by centrifugation (10 minutes, 300 g, no brake, 22°C). Platelets were isolated from PRP

27

and washed (15 minutes, 1050 g, no brake, 22°C, with 10 ng/mL PGI2) and resuspended in

HEPES Tyrode’s solution (145 mM NaCl, 5 mM KCl, 0.5 mM Na2HPO4, 1 mM MgSO4, 5 mM

HEPES, 5 mM glucose, pH 7.2). Platelets were counted by a Z2 Series Coulter Counter

(Beckman Coulter), adjusted to 200x106 platelets/mL, and stored at 22°C for 30 minutes to

regain responsiveness. Sera was isolated from blood of healthy donors by high-speed

centrifugation of the platelet-poor plasma and red blood cell fraction (5 minutes, 2000 g, 22°C).

For isolation of murine platelets, blood was collected from anesthetized mice in 100 μL of 130

mM trisodium citrate by retro-orbital eye bleed. and washed platelets were prepared as

previously described (99, 100).

3.4 Platelet incubations

Platelets were treated with various mAbs (5μg/mL) or with sera from ITP patients (1/50 v/v) in

HEPES Tyrode’s solution, pH 6.5, for 60 minutes at 22°C. Unbound antibody was removed by

centrifugation (15 minutes, 1050 g, no brake, 22°C, with 10 ng/mL PGI2). Platelets were

resuspended in Tyrode’s solution (pH 7.2) and incubated (30 minutes, 22°C) to restore

responsiveness before measurements. In some experiments, various inhibitors were used, prior to

addition of mAbs (30 minutes, 22°C), p38-MAPK inhibitor SB203580 (10 μM), Ca2+ quencher

BAPTA-AM (10 μM), neuraminidase inhibitor DANA (1mM), O-sialoglycoprotein

endopeptidase (OSGE, 80 μg/mL) to remove the extracellular part of GPIbα, as previously

described (101).

28

3.5 Flow cytometry

P-selectin expression was detected with FITC-labelled anti–P-selectin antibody (BD

Biosciences) for mouse platelets, or with PE-labelled anti-P-selectin antibody (eBioscience) for

human platelets. To quantify desialylation of GPIbα, platelets were incubated with fluorescein-

labeled RCA-1 (Vector Laboratories,) (0.5 μg/mL, 15 minutes, 22°C) to measure exposure of

galactose residues. Surface-exposed PS was deduced from the binding of FITC-conjugated

Annexin V (BD biosciences) and measured in the presence of 2.5 mM CaCl2. Surface Neu1

expression was measured with rabbit anti-NEU1 IgG antibody (Santa Cruz, 1:50 dilution) and

detected with Alexa Fluor 488 secondary antibody (Molecular Probes/Invitrogen). Murine IgG

binding was detected with Alexa fluor 647 goat anti-mouse IgG (H+L)(Invitrogen) Fold-increase

is represented as percentage increase from gated control platelets set at 1% positive. 10,000

platelet events were acquired and analyzed on a FACS Calibur (BD Biosciences) or

MACSQuant (Miltenyi Biotech).

3.6 Western Blotting

Human platelets were incubated with mAb (NIT F) and DANA as described above. Platelet

lysate was separated by SDS-PAGE, transferred on to PVDF membrane (GE Healthcare), and

probed with biotin conjugated RCA-1 (Vector Labs) according to manufacturer instructions and

with mouse anti-human CD41b antibody (clone EPR6995, Abcam) at 4°C overnight. Avidin

conjugation to RCA-1 was performed with Vectastain ABC kit (Vector Labs) according to

manufacturer instructions. HRP-conjugated secondary anti-mouse antibody (1:5000, Santa Cruz)

was then utilized. Blots were developed by ECL (Thermo Scientific) and visualized on the

VersaDoc MP 4000 imaging system (BioRad).

29

3.7 Antibody Fab generation

NIT B and PSI CI Fab fragments were generated with Thermoscientific IgG1 F(Ab)2 and Fab

preparation kits according to manufactures’ protocol. Briefly, 1mg/mL of antibody was desalted

and digested with immobilized Ficin (beaded agarose resin) in the presence of 25mM cysteine.

Digest products were purified with Protein G beads and dialyzed and concentrated with Amicon

Ultra-4 Centrifugal Filter Units (Millipore). Purified Fab was run on 10% sodium dodecyl sulfate

(SDS)-polyacrylimide gel for visualization.

3.8 In vitro phagocytosis

Macrophage cell-line RAW 264.7 (ATCC) was seeded at 4 x 105 cells/well in 24-well plates,

maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin, and

incubated at 37°C, 5% CO2 for 24 hours until they were approximately 80% confluent. Platelets

were treated with 9D2 or NIT G as described above and labelled with CFMDA (2.5 μM,

Invitrogen). An FcγR-blocker cocktail consisting of commercial FcγR blocker (BD Biosciences)

and naïve WT BALB/c sera in a 1:4 ratio was added at a 25x dilution to RAW 263.7 cells and

incubated for 30 minutes before addition of 107 CFMDA-labeled and antibody-treated platelets.

Platelets and macrophages were incubated together for 30 minutes at 37°C. To stop the assay,

RAW 264.7 cells were washed 3 times with PBS. RAW 264.7 were dissociated from plates and

adherent platelets were removed with 0.05% trypsin-EDTA (Invitrogen) treatment. RAW 264.7

cells containing ingested CFMDA platelets were identified by fluorescence and quantified by

flow cytometry. Unbound platelets were separated from macrophages by their forward and side

scatter characteristics. A total of 10,000 events were counted for each sample.

30

3.9 Immunocytochemistry

Platelets treated with anti-GPIbα or anti-GPIIbIIIa antibodies as described above were fixed in

4% PFA at 22°C for 20 minutes and spun down onto poly-L-lysine coated coverslips (BD

Biosciences, 500g for 5 minutes). Cells were blocked overnight in PBS with 1% BSA at 4°C.

Cells were incubated with primary antibodies rabbit anti-NEU1 IgG (Santa Cruz) and our anti-

GPIIbIIIa mAb M1 for 4 hours at 4°C. The platelets were washed in triplicate with PBS before

addition of species appropriate secondary antibodies Alexa Fluor 488 and 647 (Invitrogen).

Images were taken with a Zeiss LSM 700 Confocal laser scanning microscope with a 63X

objective. Images were analyzed with ZEN 2010 software (Zeiss) and Adobe Photoshop 7

3.10 In vivo platelet activation and desialylation

The passive murine ITP model was used. Various anti-platelet mAbs were injected

intraperitoneally. The amount of mAbs used was determined following titration to induce similar

levels of thrombocytopenia. After titration, 0.75 μg of anti-GPIbα mAb (0.03 μg/g total body

weight) was found to be comparable to injection of 4 μg of anti-GPIIbIIIa mAb (0.16 μg/g total

body weight). In some experiments, neuraminidase inhibitor DANA (2 mg or 0.08 mg/g total

body weight) was injected intraperitoneally 2 minutes prior to injection of mAbs. 24 hours later,

mice were bled via the saphenous vein, platelet count was measured with Coulter Z counter, and

whole blood platelets were analysed for activation and apoptosis markers via flow cytometry as

described above.

31

3.11 In vivo platelet clearance

108 washed murine platelets were prepared as described above and labeled with 5 μM of

CMFDA and incubated with anti-platelet mAbs (5 μg/mL, 1 hour, 22°C) and injected into WT

BALB/c mice as described in the immunohistochemistry section. Platelet circulation was

measured after transfusion of CMFDA-labelled platelets via enumeration of fluorescently-

labelled platelets in whole blood samples after 1 (baseline), 10, and 30 minutes. To assess the

contribution of Ashwell-Morell receptors on hepatocytes, a bolus injection of inhibitor

asialofetuin (0.2 mg/g total body weight) was administered via the tail vein and fetuin was used

as control, as previously described (71). Ten minutes post-transfusion, a booster injection

asialofetuin (0.1 mg/g total body weight) was administered intraperitoneally. Some mice were

depleted of their macrophages via intravenous (IV) injection of Clondrate encapsulated

liposomes (0.01mL/g body weight) 48 hours prior.

3.12 Immunohistochemistry

Following in vivo platelet clearance studies as described above,, mice were anesthetized as

described above and drained of their blood via retro-orbital bleeding. Liver and spleen were harvest

and snap-frozen in liquid nitrogen. Frozen tissue was sectioned (5µM) with Leica Cryostat, and

fixed onto slides in ice-cold methanol. Slides were washed in 2% BSA and then incubated with

primary rat anti-mouse F4/80 (clone BM8, ebioscience) or rabbit anti-ASPGR1/1 (Santa Cruz)

overnight at 40 C. Slides were then stained or anti-rat Cy3 or anti-rabbit Alexa Fluor 647 secondary

antibody for 2 hours. Sections were mounted with Vectashield mounting medium containing

DAPI. Images were captured using Olympus upright fluorescence microscope and analysed with

Adobe photoshop 7.

32

3.13 Statistical analysis

Data are presented as mean ± SEM. Statistically significant differences among groups was

assessed by either a Student’s unpaired t test (2 tailed) or by a 1-way ANOVA with Bonferroni

post-hoc analysis, as appropriate. Statistical analyses were performed using Prism software

(GraphPad). A P value of 0.05 or less was considered significant.

33

Chapter 4: Results

4.1 Aim 1: Comparison of anti-GPIbα and anti-GPIIbIIIa antibody

mediated platelet effects

4.1.1 Anti-GPIbα antibodies induces significantly higher platelet activation

than anti-GPIIbIIIa in murine and human platelets

To determine if anti-GPIbα antibodies causes platelet activation and desialylation, a panel of

murine anti-GPIbα and anti-GPIIbIIIa mAbs generated in GPIb -/- and GPIIIa -/- mice were used.

These antibodies possess cross-reactivity against other species, including human, rabbit, rat and/or

pig antigens (Table 1). These are the first reported syngeneic anti-GPIbα and anti-GPIIbIIIa

antibodies utilized in the study of ITP thus circumventing xenogenic antibody complications.

It was found that when 5µg/mL of anti-GPIbα mAbs was incubated with murine washed platelets,

3 out of the 6 (NIT E, NIT F, NIT H) induced significant platelet activation (P-selectin). The

remaining anti-GPIbα antibodies (NIT A, NIT B, NIT G) induced visibly increased, although not

significant P-selectin expression (Figure 4A). Platelet apoptosis was also assessed, and it was

found that only NIT E induced significant PS-exposure of murine platelets (Figure 4B). Thus NIT

E was found to induce the highest murine platelet activation and apoptosis, which was also

indicated in the most marked elongated forward and side scatter characteristic of activated platelets

(Figure 4C).

Assays with human platelets utilized only NIT A, NIT B, and NIT F as they were the only ones

reactive to human GPIbα. It was found that incubations of 10µg/mL of anti-GPIbα mAb with

washed human platelets, 2 out of the 3 (NIT B and NIT F) induced significant platelet activation

(P-selectin) (Figure 5A) and all 3 induced significant platelet apoptosis (PS-exposure) (Figure 5B).

34

Based on these results, it appears that the response of human platelets to anti-GPIbα mAb is much

stronger than murine platelets. This may be attributable to the human platelet FcγRIIa, which is

not expressed on murine platelets. Further, only the anti-GPIbα mAb induced platelet aggregation

in human platelets (Figure 6), whereas there was no aggregation observed with murine platelets in

the presence of the tested anti-GPIbα mAb.

To better represent a more physiological situation, polyclonal sera generated from 4x immunized

GPIb-/- and GPIIIa -/- was also tested. It was found that, as expected, anti-GPIb antibodies induced

significant platelet activation dose-dependently (Figure 7A), whereas anti-GPIIIa antibodies did

not. Protein G seraphose purified IgG from both GPIb-/- and GPIIIa -/- 4x immunized sera was

also tested, and interestingly it was found that anti-GPIb antibodies no longer induced significant

platelet activation (Figure 7B). This suggests that there may be factors in the sera of the immunized

GPIb -/- mice which are contributing to antibody induced platelet activation.

4.1.2 Anti-GPIbα antibodies induces significantly higher platelet desialylation

than anti-GPIIbIIIa in murine and human platelets

The most surprising and significant discovery was that anti-GPIbα antibodies induced platelet

desialyation in both murine and human platelets. (Figure 8, Figure 9) Desialylation was detected

via flow with fluorescein conjugated Ricinus Comminus Agglutinin-1 (RCA-1), a lectin derived

from the castor oil plant, which binds to underlying desialylated galactose residues. Overall, the

level of desialylation was much more marked than that of the observed platelet activation in both

human and murine platelets. In murine platelets, NIT A and NIT G which did not induce significant

platelet P-selectin expression, does induce significant platelet desialylation. This antibody

mediated desialylation was also shown to occur in a dose dependent manner when tested with

increasing titrations of NIT E, and NIT A (Figure 8B). Similarly, polyclonal sera from 4x

35

immunized GPIb -/- mice also induced dose-dependent platelet desialylation (Figure 10A).

Interestingly, the purified IgG from the GPIb -/- sera was able to induce significant desialylation

even though it did not induce any detectable platelet activation (Figure 10B).

4.1.3 The overall more desialylated state of GPIbα mAb does not contribute to

the detected platelet desialylation

As previously mentioned, antibodies themselves are significantly sialylated. Thus both the anti-

GPIbα and anti-GPIIIa mAb were tested for levels of desialylation via western blot. It was found

that all of the anti-GPIbα mAb were significantly desialylated (Figure 11) whereas none of the

anti-GPIIIa mAb were. To confirm the detection of platelet desialylation in the presence of anti-

GPIbα mAb was due to a platelet derived process rather than passive binding of the mAb, two

methods were adopted. In the first, the sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuramic

acid (DANA) was used in the co-incubation of human and murine platelets with anti-GPIbα mAb.

It was found that DANA did have an inhibitory effect on anti-GPIbα mAb mediated platelet

desialylation, indicating that initiation of desialylation was occurring following binding of anti-

GPIbα mAb (Figure 12). Second, anti-GPIbα mAb mediated desialylation was tested on fixed

human platelets, whereby platelets were first fixed in 2% PFA before incubation with mAb. It was

found that contrary to live-platelets, there was no increased in desialylation following anti-GPIbα

mAb binding of fixed platelets (Figure 13). These tests confirm that anti-GPIbα antibody binding

to platelets induces platelet desialylation.

36

4.2 Aim 2: Characterization of anti-GPIbα antibody mediated

desialylation

4.2.1 Increased surface expression of NEU1 following anti-

GPIbα incubation

To identify the candidate enzyme mediating anti-GPIbα antibody mediated desialylation, the

mammalian neuraminidase-1 (NEU1) was considered. Although NEU1 is typically located within

platelet granules, it has been previously shown to translocate to the platelet surface following long-

term cold storage (102). Further, NEU1 has a narrow substrate specificity with preference for

terminal glycans primarily in α2,3 and α2,6 linkages (103). All this makes NEU1 a likely

candidate for mediating anti-GPIbα antibody induced platelet desialylation.

Surface expression of NEU1 was detected via flow and immunofluorescence with an antibody

which binds the C-terminus of human and murine NEU1 (clone H-300). Immunofluorescence

studies revealed increased expression of NEU1 in the presence of anti-GPIbα antibodies either

with the polyclonal sera from 4x immunized GPIb -/- mice or anti-GPIbα mAb (NIT G) (Figure

14). No detectable surface NEU1 expression was seen with anti-GPIIbIIIa antibodies (polyclonal

sera from 4x immunized GPIIIa -/- mice and 9D2). For human platelets, sera from ITP patients

were used. The first was from an ITP patient at Sunnybrook who passed away from ITP related-

bleeding complications. The sera obtained was found to contain only anti-GPIbα antibodies with

no detectable anti-GPIIbIIIa antibodies (case report submitted to Haematologica). The second

was from a patient at Anhui Medical Hospital in China as part of a cohort in a previously

published report. This patient is also positive for only anti-GPIbα antibodies. Both sera from

these anti-GPIbα positive patients induced surface NEU1 expression as detected by

immunofluorescence (Figure 15). It should be mentioned that both the sera samples also induces

37

platelet desialylation (data not shown). Surface expression of NEU1 was also detectable by flow

cytometry in both murine and human platelets (Figure 16). Further, surface NEU1 expression

increases dose-dependently in murine platelets in the presence of anti-GPIbα antibodies,

congruent with the dose-dependent increases in desialylation (Figure 17).

4.2.2 Platelet desialylation is predominantly localized to the GPIbα subunit

The GPIbα subunit is the most heavily glycosylated platelet surface receptor, which suggests that

if it were a substrate of anti-GPIbα antibody mediated desialylation, it will likely be the primary

contributor to the overall desialylation state of the platelet. Thus whether GPIbα was a substrate

of antibody mediated desialylation was investigated.

First a western blot was run on the entire human platelet lysate following incubations with anti-

GPIbα mAb NIT A, NIT B and NIT F. Biotinlylated RCA-1 was used to probe for all proteins

which were heavily desialylated. Co-currently the same sample was also probed with anti-GPIbα

antibody. The resulting membrane revealed the strongest band corresponded to ~150kD which is

the same molecular weight of GIbα. In addition, the anti-GPIbα antibody revealed a band of the

same size. Co-incubations with the salidase inhibitor DANA was able to inhibit desialylation of

the GPIbα subunit. No other protein in the platelet lysate above 68kD was significantly

desialylated as indicated in the western blot.(Figure 18)

Second, flow cytometry was used to measure differences in levels of anti-GPIbα mAb (NIT F)

mediated human platelet desialylation following cleavage of the GPIbα subunit with the enzyme

O-sialyloglycoendopepsidase (OSGE). OSGE has been previously reported to selectively remove

the N-terminus of GPIbα subunit by irreversibly cleaving O-linked glycoproteins in the stalk

region (5). It was found that NIT F induced platelet desialylation returned to baseline following

38

removal of the extracellular domain of GPIbα subunit (Figure 19A). To confirm that the

extracellular region of GPIbα was removed, the platelet was also checked by flow for NIT F

binding, and it was found to no longer bind NIT F (Figure 19B).

These results strongly implicate GIbα as the dominant substrate for anti-GPIbα antibody

mediated platelet desialylation.

4.3 Aim 3: Characterization of link between anti-GPIbα mediated

platelet activation and desialylation

4.3.1 Anti-GPIbα mediated platelet effects requires F(Ab)2

Our group have demonstrated before anti-GPIbα antibodies induce platelet activation, thus the

next step was to investigate the link between anti-GPIbα mediated platelet activation and

desialylation. It is hypothesized that anti-GPIbα mediates platelet activation by cross-linking

adjacent GPIbα-subunits causing receptor clustering. Clustering of the receptor mimics

multimeric VWF binding causing intracellular signaling events leading to platelet activation and

desialylation. To test this hypothesis, monovalent Fab fragments of anti-GPIbα mAb NIT B were

generated (Figure 20) and tested in its ability to induce in vitro platelet activation, desialylation

and thrombocytopenia in vivo. It was found that contrary to the intact NIT B antibody, the Fab

fragment did not induce murine platelet activation or desialylation (Figure 21). Injection of the

NIT B Fab via the IP route at 3ug (3x higher than the dose used for intact antibody) was unable

to induce thrombocytopenia at 24 hours but was able to induce a significant platelet decrease

(Figure 22). Interestingly, in human platelets it was found that NIT B Fab fragment did cause

desialylation in one of the three normal donor platelet populations, but overall the increase was

not significant (Figure 21). As a control, Fab fragments of an anti-GPIIbIIIa antibody (PSI CI)

39

was also generated, and as expected it did not induce any changes in the platelet either in vivo or

in vitro (data not shown).

4.3.2 Platelet activation and desialylation are linked in a positive feed-back

loop

Preliminary data from our lab have suggested that anti-GPIbα mediated platelet desialylation in

murine platelets is downstream of platelet activation, but interestingly platelet desialylation

further potentiates platelet activation in a positive feedback loop. This positive link between

platelet desialylation and activation hadn’t been tested in human platelets. Utilizing general

inhibitors of platelet activation including BAPTA-AM (inhibits intracellular Ca2+ flux) and

SB203580 (inhibits P38MAPK enzymatic function) it was found that like mouse platelets,

inhibition of platelet activation had profound effects in suppressing antibody mediated platelet

desialylation. BAPTA-AM had the most significant inhibitory effect, inhibiting desialylation

almost to baseline levels. Suggesting that perhaps Ca2+ influx is the most upstream signal

leading to desialylation. An inhibitor of apoptosis, Q-VD-OPh (a pan caspase inhibitor) was also

tested, and it had minimal and non-significant effect on platelet desialylation (Figure 23A).

DANA was also tested in its ability to inhibit platelet activation. It was found that there was a

slight but significant decrease in anti-GPIbα mAb mediated platelet activation, supporting the

data from murine platelets whereby desialylation contributes to platelet activation (Figure 23B).

In addition, it was found treatment of murine platelets with neuraminidase induced platelet

activation, suggesting that desialylation of platelet surface proteins on its own is sufficient to

induce platelet activation

40

4.3.3 Human FcγRIIa may not contribute to anti-GPIbα mediated platelet

desialylation

As previously shown, anti-GPIbα mAb mediated platelet activation and desialylation is stronger

in human platelets than murine. One possibility is the presence of FcγRIIa on the human platelets

but not on murine which could potentially enhance platelet activation. The human FcγRIIa

blocker IV.3 was employed to investigate the contribution of human FcγRIIa on antibody

mediated platelet activation and desialylation. Surprisingly, FcγRIIa blocker had no significant

effect on anti-GPIbα mAb mediated platelet desialylation (Figure 23C).

4.4 Aim 4: Assess the utilization of the Fc-independent pathway by

anti-GPIbα opsonized platelets both in vitro and in vivo

4.4.1 Anti-GPIbα opsonized platelets can be uptaken by macrophages via non-

FcγR routes

Previous published reports from our lab have shown that anti-GPIbα antibodies can cause

thrombocytopenia in FcγR -/- mice (104), however direct Fc-independent phagocytosis by

macrophages of anti-GPIbα antibody opsonized platelets have never been investigated. Here a

murine macrophage cell-line (RAW 267.4) was incubated with CFMDA-fluorescently labeled

platelets pre-treated and opsonized with anti-GPIbα or anti-GPIIbIIIa mAb. An FcγR blocker

cocktail consisting of commercial FcγRIIa/III blocker and naïve WT sera was used to inhibit

FcγR phagocytosis. The difference between phagocytosed platelets in the absence and presence

of FcγR blockers was measured by flow cytometry. It was found that when platelets were

opsonized with anti-GPIIbIIIa mAb, in the presence of FcγR blockers, phagocytosis was

inhibited ~85% while FcγR blockers only inhibited ~20% of phagocytosis of anti-GPIbα

opsonized platelets (Figure 24). This indicates whilst anti-GPIIbIIIa mediated platelet clearance

41

is dependent on FcγR, anti-GPIbα bound platelets in addition to the FcγR pathway, also utilize

FcγR independent pathway

4.4.2 Anti-GPIbα mAb causes platelet activation and desialylation in vivo

To assess whether anti-GPIbα antibodies can cause platelet activation and desialylation in vivo, a

passive model of murine thrombocytopenia was adopted. Here 1µg of randomly selected anti-

GPIbα mAb (NIT B, NIT F, NIT G) or 4µg of anti-GPIIIa mAb (9D2, PSI BI, PSI C1) was I.P

.injected into WT Balb/C mice. The dose of antibody used was based on the amount required to

induce comparable levels of thrombocytopenia. It was found that much higher doses of anti-

GPIIIa mAb than anti-GPIbα mAb were required to induce thrombocytopenia (< 400 x 106

platelets/mL). Control mice were injected with PBS. Following antibody injection platelet counts

were acquired at selected time points via saphenous vein bleed and enumeration was done on the

Beckman© Z2 coulter counter. It was found that platelet numbers began to fall at around the 6

hour time point post injection. At 24 hours post-injection, the platelets were also analyzed via

flow cytometry for P-selectin expression and RCA-1 binding. It was found that all of the injected

anti-GPIbα mAb (NIT B, NIT F and NIT G) induced in-vivo platelet desialylation, but no

significant P-selectin expression individually. In addition, it was observed that the anti-GPIIIa

mAb induced similar levels of P-selectin, but no significant desialylation (Figure 25A). Overall,

when the results from individual mAb were pooled and analyzed it was found that only anti-

GPIbα antibodies caused significant platelet P-selectin expression and desialylation in vivo

(Figure 25B)

42

4.4.3 ASPGR contributes to anti-GPIbα opsonized platelet clearance

Anti-GPIbα antibody mediated Fc-independent clearance routes has never been investigated in

vivo. Previously, the clearance of desialylated cold-stored platelets have been implicated in the

liver via Ashwell-Morell receptors. Here, it was tested whether anti-GPIbα opsonized platelets

may be cleared via the same route. CFMDA-fluorescently labelled platelets were incubated with

anti-GPIbα antibodies ex-vivo before I.V. re-injection into WT Balb/c mice. Mice were bled at 0,

15, and 30 minutes post inject via saphenous vein, and the amount of CFMDA-labeled, anti-

GPIbα antibody opsonized platelets remaining in circulation were enumerated via flow

cytometry. It was found that both anti-GPIbα and anti-GPIIIa antibody opsonized platelets were

cleared relatively fast from circulation, with circulating levels returning close to 0% at around

the 30 minute mark (Figure 26). To assess the contribution of the ASGPR in antibody opsonized

platelet clearance, a competitive inhibitor of the ASPGR receptor was used. Asialofetuin is a

fetal-calf serum protein with high affinity for ASPGR, in particular the Ashwell-Morell receptor

in the liver. A sialylated form (fetuin) was injected as a negative control. It was found co-

injection of 2mg of asialofetuin with anti-GPIbα mAb (NIT F and NIT G) opsonized platelets

was able to maintain antibody opsonized platelets in circulation by ~20% compared to injection

with fetuin. Injection of asialofetuin with anti-GPIIbIIIa opsonized platelets had no effect on

platelet circulation. These results indicate that ASPGRs, most likely the Ashwell-Morell receptor

is utilized in the clearance of anti-GPIbα but not anti-GPIIbIIIa mediated platelet clearance

4.4.4 Anti-GPIbα opsonized platelets are predominantly cleared by ASPGR in

the absence of macrophages

Common first-line ITP therapies target FcR-mediated clearance of platelets. Given that there is a

subset of patients who are refractory to first-line therapies, and whom are linked to having anti-

43

GPIbα antibodies, it was worth investigating the contribution of the ASPGR in the clearance of

anti-GIbα mediated platelet clearance in the context of absence of macrophages. Thus

macrophages were depleted from WT Balb/c mice with clondrate encapsulated-liposomes.

Macrophages were found to ~90% depleted from the spleen and liver at 48 hours following I.V.

injection of clondrate liposomes. The above described circulation study was then preformed on

these macrophage-depleted mice. In addition, to better depict a physiological situation, instead of

anti-GPIbα mAb, anti-GPIbα sera from GPIb-/- mice or anti-GPIIbIIIa sera from GPIIIa-/- mice

were incubated with platelets at a 50x dilution prior to injection into mice. It was observed that in

the macrophage depleted mice, anti-GPIIbIIIa opsonized platelets remained in circulation at

close to 100% of initial injected platelets even at the 30 min time point (Figure 27). This

indicates, as expected, anti-GPIIbIIIa antibody mediated platelet clearance is largely dependent

on FcγR on macrophages. In contrast, platelets pre-incubated with anti-GPIbα antibodies still

experienced clearance from the circulation in macrophage depleted mice by around 40%. Most

notably, co-injections with asialofetuin was able to almost completely rescue platelet circulation.

This data clearly illustrates the dominant role of ASPGR, most likely the Ashwell-Morell

receptor in mediating platelet clearance in the absence of macrophages. It is also of interest to

note that anti-GPIbα opsonized platelets were not cleared from circulation in the normal, non-

macrophage depleted mice with the same efficacy as anti-GPIIbIIIa opsonized platelets, which

warrants further investigation.

4.4.5 More anti-GPIbα opsonized platelets are localized to the liver in

macrophage depleted mice

Livers and spleens from mice were harvested following circulation studies with anti-GPIbα or

anti-GPIIbIIIa opsonized fluorescent platelets. They were flash-frozen and sectioned and stained

44

with anti-F4/80 (macrophage marker) and anti-ASPGR1/2 (Ashwell-Morell receptor).

Fluorescent platelets are labelled with CFMDA. It was found on average there were significantly

more anti-GPIbα opsonized platelets localized to the liver, particularly in macrophage depleted

mice. Further, the platelets can be seen co-localizing with the Ashwell-Morell receptor (Figure

29). There was significantly less anti-GPIbα and anti-GPIIbIIIa opsonized platelets in the spleen

of the macrophage depleted mice (Figure 28). There were also some anti-GPIIbIIIa opsonized

platelets seen in the liver of macrophage depleted mice, but not nearly as much with anti-GPIbα

bound platelets. This data may be representative of patients who are refractory to splenectomy,

or those who may have platelet clearance localized to the liver.

4.5 Sialidase inhibitor DANA ameliorates thrombocytopenia caused

predominantly by anti-GPIbα mAb

The use of sialidase inhibitor DANA was effective in vitro at attenuating anti-GPIbα mediated

platelet desialylation. The next step was to assess its therapeutic potential in vivo. Thus we

randomly selected a panel of anti-GPIbα mAb (NIT B, NIT E, NIT F, and NIT G) and anti-

GPIIbIIIa mAb (9D2, PSI C1, PSI E1 and M1) and injected into WT Balb/c mice via the IP

route. Co-injections of 2mg of DANA was assessed for its impact on thrombocytopenia (Figure

28). Platelets were enumerated 24 hours later. It was found that most of the anti-GPIbα mAb co-

injected with DANA could increase platelet counts and ameliorate thrombocytopenia. NIT E was

the only one which did not respond to DANA, this may be due to the small n- number tested, as

there is an observable increase in platelet numbers in these DANA treated mice. Unexpectedly,

PSI E1 was also responsive to DANA, which is in stark contrast to the other anti-GPIIbIIIa mAb

45

tested. These results suggest a strong potential for sialidase inhibitors such as DANA to be used

therapeutically in ITP.

\

46

Chapter 5: Discussion

The objective of Aim 1 was to assess the anti-platelet effects of anti-GPIb and anti-GPIIbIIIa

antibodies in both mouse and human platelets. Although generally it was found that anti-GPIbα

mAbs induced more platelet activation, apoptosis and desialylation, individually, not every

antibody induced the same degree of anti-platelet effects. This may be a consequence of different

epitope targets on GPIbα. Those antibodies which induces the greatest amount of platelet

activation and desialylation (eg. NIT E) may be binding at a site on GPIbα similar to the

endogenous VWF-binding site most conducive to GPIb-cross linking and signal transduction.

Those which has minimal effect may be binding at a site, perhaps in the stalk region, which

sterically hinders conformational changes following GPIb-IX activation. The exact epitopes of

the GPIbα mAb have not yet been determined, however preliminary aggregation data revealed

pre-incubation with murine and human platelets can inhibit ristocetin/botrocetin/VWF induced

platelet aggregation, suggesting they are binding at sites similar to VWF. The use of polyclonal

sera from immunized GPIb-/- mice mitigates the variation seen with the mAb and is a better

physiological representation. However, the differences seen between purified IgG and sera raise

further questions, and suggest that in ITP patients, factors in the blood may promote or

antagonize anti-platelet effects mediated by binding of antibodies.

A surprising result was the overall desialylated state of all the anti-GPIbα mAb (Figure 11).

Whether this is a consequence of generation of the mAb itself through hybridoma formation, or a

side-effect of GPIb -/- genotype, or the general anti-GPIbα immune response deserves further

investigation. As well, ITP patient antibody samples should also be tested for this bias. Antibody

glycosylation states in ITP has been recently noted to play a significant role in platelet

phagocytosis and complement deposition, and overall platelet clearance (105).

47

Aim 2 focused on characterizing anti-GPIbα mediated desialylation. Immunofluorescence

microscopy and flow cytometry identified increased platelet surface expression of NEU1

exclusively in the presence of anti-GPIbα antibodies. However, there is no direct evidence that

NEU1 is the sialidase mediating desialylation. NEU3 is constitutively expressed on the platelet

surface (92), however its preferred substrate are gangliosides which makes it an unlikely

candidate in mediating desialylation of glycoproteins. NEU1 has previously been localized to

granules of the platelet (92), whether these granules are released in a regulated manner, or under

which specific platelet signals would be of interest to investigate.

The second part of Aim 2 focused on determining the primary substrate of anti-GPIbα mediated

desialylation. Given that GPIbα subunit is the most heavily sialylated structure on the platelet

surface, it was the most likely candidate. Western blots of entire platelet lysate confirmed GPIbα

as the dominant substrate of desialylation. Assays with the compound OSGE further supports the

GPIbα desialylation. However, although OSGE is effective at removing almost all GPIbα from

the platelet surface, it cannot be excluded that it is also removing other O-linked glycoprotein

surface proteins. It has been reported that P-selectin and VWF are also targets of OSGE. It is

interesting to speculate how surface NEU1 specifically targets GPIbα. It is known that GPIb

complex localize to lipid rafts upon engagement of VWF and platelet activation (95). It may be

possible that anti-GPIbα mAb causes similar mobilization of GPIb to lipid rafts, and if

translocation of NEU1 is also targeted to lipid rafts it could explain specific targeting of GPIbα.

It is interesting that in the immunofluorescence images of human platelets treated with anti-GPIb

human ITP plasma, surface NEU1 expression is not diffuse and does not co-localize with anti-

CD41 (Figure 3.2.1B). This suggests that it is possible that there is specific localization of NEU1

on the plasma membrane. Further investigations utilizing fluorescence energy transfer (FET) and

48

flow cytometry or immunofluorescence microscopy with an anti-GPIbα platelet marker may be

useful in determining co-localization of NEU-1 and GPIbα.

The purpose of Aim 3 was to further characterize the link between anti-GPIbα mediated platelet

activation and platelet desialylation. Our hypothesis is that cross-linking of the GPIbα subunit by

the Fab arms of the antibody mimics the clustering of GPIbα induced by VWF, which is

conducive platelet activation (57). As expected the NIT B Fab fragments lost the ability to

mediate platelet activation and desialylation in murine and human platelets. However,

interestingly in vivo, it was observed that while NIT B Fab did not induce thrombocytopenia in

mice (<400 x106 /mL) it did result in significant platelet clearance. This may be explained

through shear stress induced conformational changes of GPIbα as a consequence of Fab binding

to the N-terminus, resulting in minor platelet activation and clearance. Unpublished data from

Li’s group at Emory University has demonstrated conformational changes of GPIbα subunit

following VWF binding coupled with a retraction force is sufficient to induce Ca2+ flux

indicating signal transduction. Further tests with different GPIb Fab is required to confirm this

data.

VWF induced GPIb activation initiates signaling cascades shown to involve intracellular Ca2+

and P38MAPK. Thus inhibitors of both Ca2+ (BAPTA-AM ) and P38MAPK (SB203580) was

utilized to determine if anti-GPIbα mediated platelet activation was through the similar signaling

cascades. Overall, platelet activation was significantly inhibited in the presence of both these

inhibitors indicating induction of similar signaling pathways as physiological VWF mediated

GPIb activation. In addition, Q-VD-OPh, a broad spectrum caspase inhibitor was the least

effective at inhibiting anti-GPIbα mediated platelet activation (Figure 3.2.3A). This may be

49

explained by the distinct pathways of PS-exposure in platelets. One of which is an activation

induced PS-exposure dependent on Ca2+ flux and the other is the slow bona-fide apoptotic

processes, involving mitochondrial depolarization. In the presence of GPIbα mAb, it is observed

platelet PS-exposure is a consequence of platelet activation signals, thus not dependent on

caspase activation and mitochondrial depolarization. It was also found that BAPTA-AM almost

completely inhibited platelet desialylation, this suggests that desialylation requires and is

downstream of platelet activation. Interestingly, it was found that sialidase inhibitor DANA also

decreased anti-GPIbα mediated platelet activation. This suggests that desialylation itself

potentiates further anti-GPIbα mediated platelet activation, resulting in a positive feed-back loop.

Logically, this is a likely scenario as removal of bulky-terminal sialic residues could facilitate

GPIb clustering and anti-GPIbα mediated platelet activation. It is currently unknown whether

GPIIbIIIa activation and subsequent outside-in signaling is also a significant contributing factor

for anti-GPIbα mediated platelet activation and desialylation. Given that there are a significant

portion of patients positive for both autoantibodies, it would be interesting to investigate.

FcγRIIa signaling, through its tyrosine-based activation motif, has been shown to positively

contribute to platelet activation and has been correlated with antibody-mediated platelet

activation (96-98), particularly when associated with the GPIb complex (59, 95, 96, 98). This

may also be the mechanism behind higher anti-GPIbα antibody induced platelet activation and

desialylation in human platelets compared to murine. This may also be a target mechanism for

FcγR-dependent therapies such as IVIG in anti-GPIbα positive ITP patients who are responsive

to the therapy (99). Thus it would have been expected that FcγRIIa blocker IV.3 would

significantly decrease anti-GPIbα antibody mediated desialylation, however that was not the

observed result. This may be explained by the dose of IV.3 used, whereby higher doses of IV.3

50

may be causing cross-linking of FcγRIIa contributing to platelet activation. Thus lower doses of

IV.3 may be utilized in the future to test the contribution of FcγRIIa on human platelets.

Aim 4 examined FcγR independent phagocytosis as a consequence of anti-GPIbα mediated

platelet activation and desialylation. Here a murine macrophage cell-line (RAW 267.4) was

incubated with fluorescently labeled platelets pre-treated and opsonized with anti-GPIbα or anti-

GPIIbIIIa mAb. FcγR blocker cocktail consisting of commercial FcγRIIa/III blocker and naïve

WT sera was used to inhibit FcγR phagocytosis. It was found that blocking of the FcγR pathway

was effective in blocking anti-GPIIbIIIa antibody mediated platelet phagocytosis but not anti-

GPIbα. This indicates whilst anti-GPIIbIIIa mediated platelet clearance is dependent on FcγR,

anti-GPIbα bound platelets in addition to the FcγR pathway, also utilize FcγR independent

pathways. Further studies into inhibition of platelet desialylation or activation caused by anti-

GPIbα mAb prior to phagocytosis by FcγR blocked macrophages will better answer which Fc-

independent ligands are utilized by macrophages in the uptake of anti-GPIbα opsonized platelets.

In addition, since many first-line therapies target the innate immune system phagocytic

pathways, it would be interesting to investigate their efficacy directly on the inhibition of

macrophage uptake of anti-GPIbα or anti-GPIIbIIIa opsonized antibodies.

Subsequently, began the investigation of anti-GPIbα mediated platelet effects and Fcγ-

independent platelet clearance in vivo. It was observed that contrary to in vitro assays, none of

the anti-GPIbα mAb individually induced significant platelet activation, although overall,

platelet activation was significant. This could be due to sequestration or removal of the activated

platelet aggregates which prevented them from being isolated for analysis, or anti-GPIbα

mediated platelet activation may be causing shedding of P-selectin which decreases its detection,

51

or anti-GPIbα mediated platelet activation is generally lower in vivo. However, every mAb

induced significant desialylation. These results suggest firstly, that in vivo, minor anti-GPIbα

mediated platelet activation can cause significant desialylation, and secondly, platelet activation

as mediated by anti-GPIIbIIIa is not conducive to platelet desialylation. This may be due to a

requirement of anti-GPIb activation, and as mentioned before, co-localization with NEU1 on the

plasma membrane to undergo desialylation. How anti-GPIIbIIIa mAb mediates platelet

activation and apoptosis is unclear, but it may involve the FcγRs on platelet surface.

As mentioned before, the Ashwell-Morell receptor on hepatocytes are a key component in

removing desialylated glycoproteins within the blood. And given that it has been demonstrated

anti-GPIbα mAb mediates platelet desialylation both in vitro and in vivo, Aim 4.2 assessed its

direct contribution in clearing anti-GPIbα bound platelets. Asialofetuin, a well-recognized

inhibitor of ASPGR particularly the AMR, was injected in conjunction with fluorescently-

labelled anti-GPIbα mAb opsonized platelets. At various time points, mice were bled to measure

the number of anti-GPIbα mAb opsonized platelets remaining in circulation. It was found that

asialofetuin was effective in increasing the number of anti-GPIbα mAb opsonized platelets

compared with injection of fetuin, a non-specific control. In addition, asialofetuin had no effect

in the presence of anti-GPIIbIIIa opsonized platelets (Figure 3.3.2B). These results indicate that

ASPGR play a significant role in clearance of anti-GPIbα opsonized desialylated platelets.

However, it is unclear currently if it is indeed the Ashwell-Morell receptor which mediates

phagocytosis of anti-GPIbα antibody opsonized platelets. In addition, even though it is

demonstrated that the GPIbα subunit is the predominant subunit which undergoes desialylation,

it is unknown whether it is the ligand which mediates binding to the ASGPR/Ashwell-Morell

receptor. Given that the Ashwell-Morell receptor preferentially binds penultimate galactose

52

residues (106) which is only present on N-linked glycosylation. Since mice lack N-linked

glycosylation on their GPIbα subunit doubts regarding whether GPIbα may be the ligand for

Ashwell-Morell is raised. Further investigations utilizing a hepatic cell line to directly assess

uptake of anti-GPIbα bound platelets would elucidate the ASPGR dependent phagocytosis of

anti-GPIbα opsonized platelets.

Aim 5 assessed the therapeutic potential of DANA in ameliorating anti-GPIbα antibody

mediated thrombocytopenia. Overall, it appeared that in general anti-GPIbα mediated

thrombocytopenia was responsive to DANA. However, the platelet rescue was far from

complete. Most mice which responded saw only a slight increase in platelet number, but were

still thrombocytopenic. In addition there is a lot of variation within the groups. Unexpectedly,

PSI E (an anti-GPIIbIIIa mAb) injection was also responsive to DANA treatment, this is contrary

to the in vitro observations in which PSI E did not induce any significant desialylation. Perhaps,

in vivo other factors in the blood may affect the antibody mediated platelet effects which could

not be seen in vitro with washed platelets. Although the therapeutic effects of DANA was not

total, it has the potential to play a therapeutic role. Further studies comparing responsiveness to

IVIG in this model may further support this claim. Although IVIG has been shown to be

predominantly ineffective with previous anti-GPIb mAb mediated thrombocytopenia in mice, it

has never been tested with this new panel of mouse anti-mouse mAb. In addition, it would

extremely interesting to test DANA as treatment in the active murine model of ITP. The active

model involves engrafting splenocytes from GPIb -/- mice immunized against GPIb into wild

type mice (100). This model not only encompasses the innate component of ITP, but also the

adaptive components involving T and B- cells, and thus is more representative of the human

disease. It has been previously shown anti-GPIb mediate ITP in this model is less responsive to

53

steroid treatments, whether sialidase inhibitors may play a role attenuating the anti-platelet

adaptive immune response, and antibody generation deserves investigation.

54

Conclusions

Overall this study has demonstrated a novel FcR-independent mechanism of platelet clearance

in ITP mediated by anti-GPIbα autoantibodies, which is distinct from anti-GPIIbIIIa-mediated

ITP in both mechanism and therapeutic management. It was demonstrated both in vitro and in

vivo that binding of mAbs to GPIbα induced significant platelet activation, apoptosis, NEU1

translocation, and platelet desialylation.

Anti-GPIbα antibody-driven desialylation of platelet glycoproteins was found to have significant

impact on the development of thrombocytopenia. The sialylation state of platelets has been

previously reported to directly affect platelet clearance in various infectious diseases. For

example, enhanced removal of desialylated platelets was observed in sepsis and was caused by

neuraminidase activity of Streptococcus pneumonia (85). As well, infection by the parasite

Trypansoma cruzi, which causes Chagas’ disease, caused release of high levels of

neuraminidase, leading to desialylation of platelets and enhanced platelet clearance (107). It is

unknown whether ITP patients infected with influenza undergo a period of more severe

thrombocytopenia due to increased viral-derived sialidase activity, but, notably, a recent case

report documented that the sialidase inhibitor oseltamivir (Tamiflu) elevated platelet counts in a

pediatric ITP patient suffering from Influenza A (108), thus highlighting the clinical relevance of

these findings..

Traditional approaches to ITP pathophysiology hold that platelet clearance in ITP is primarily

mediated by FcγRs of macrophages in the RES, primarily in the spleen (109). However, it is now

55

clear that FcγR-independent mechanisms exist (70). Hepatic sequestration of platelets in patients

with ITP is an important criterion in determining responsiveness to splenectomy (110, 111). The

discovery that hepatocytes play a significant role in the clearance of anti-GPIbα-opsonized

platelets through interaction between desialylated residues on the platelet surface and the AMR

provides a potential explanation for refractoriness seen in splenectomized ITP patients. Given

the lack of treatment options for patients refractory to current ITP therapies, these findings are

important not only in revealing a new mechanism of ITP, but also for the introduction of a new

diagnostic tool (e.g. RCA-1 staining) and a potential novel therapeutic method (e.g. sialidase

inhibition) for treatment of these patients.

In summary, this study unveils the FcR-independent mechanism of platelet clearance. This

finding may not only enhance our understanding of anti-GPIbα-mediated ITP, but may also

provide insights into the platelet activation-desialylation dialogue and its positive feedback loop,

which may contribute to thrombosis and cardiovascular diseases. In addition, this novel FcR-

independent platelet clearance pathway may also be involved in embolus clearance. Currently, it

is largely unknown why subsets of ITP patients are refractory to first-line treatments, including

IVIG and steroids. Given that IVIG is extremely costly and of limited supply and that steroids

are associated with severe side effects (112), these treatments should be implemented with

maximum efficacy and not for patients who will not benefit. This evidence that anti-GPIbα

mAbs cause platelet desialylation and FcγR-independent clearance via the liver offers valuable

implications for the diagnosis, prognosis, and successful treatment of ITP. Patients with ITP that

test positive for anti-GPIbα antibodies and present with significant platelet desialylation may be

identified as likely non-responders to conventional first-line treatments or splenectomy. Clinical

trials are required to test the diagnostic value of platelet desialylation and the therapeutic

56

potential of sialidase inhibitors, either alone or in conjunction with standard treatments in these

ITP patients.

57

Chapter 6: Figures

Table 1 Novel mouse anti-mouse anti-GPIb and anti-GPIIbIIIa

monoclonal antibodies

58

Figure 4 Anti-GPIbα mAb causes murine platelet activation and

apoptosis

2.5 x 107 washed murine platelets were incubated in HEPES tyrode buffer with 5µg/mL of the

indicated anti-GPIbα or anti-GPIIbIIIa mAb for 1 hour at room temperature. Platelets were

washed in the presence of PGI2 and incubated with (A) anti-P-selectin antibody or (B) Annexin

V in the presence of 2.5mM CaCl2 (C) SSC-A vs FSC-A plot of NIT E treated platelets. Control

platelets were incubated with naïve WT sera. Data is represented as fold-change from control.

Mean ± SEM (n>3). P<0.05(*), P<0.01 (**),P<0.0001 (****).

Ctr

l

NIT

A

NIT

B

NIT

E

NIT

F

NIT

G

NIT

H9D

2M

1

PS

I B

1

PS

I C

1

PS

I E

1

HU

TA

B

0

5

1 0

1 5

An

ne

xin

V (

fold

-ch

an

ge

)

a n ti-G P Ib a n ti-G P IIb IIIa

****

P-s

ele

cti

n (

fold

-ch

an

ge

)

Ctr

l

NIT

A

NIT

B

NIT

E

NIT

F

NIT

G

NIT

H9D

2M

1

PS

I B

1

PS

I C

1

PS

I E

1

HU

TA

B

0

2

4

6

8

a n ti-G P Ib a n ti-G P IIb IIIa

****

***

AA B

C

59

Figure 5 Anti-GPIbα mAb causes human platelet activation and

apoptosis

2.5 x 107 washed human platelets were incubated in HEPES tyrode buffer with 5µg/mL of the

indicated anti-GPIbα or anti-GPIIbIIIa mAb for 1 hour at room temperature. Platelets were

washed in the presence of PGI2 and incubated with (A) anti-P-selectin antibody or (B) Annexin

V in the presence of 2.5mM CaCl2 Control platelets were incubated with IVIG. Data is

represented as fold-change from control. Mean ± SEM (n>3). P<0.05(*), P<0.01 (**),P<0.001

(***).

P-s

ele

cti

n (

fold

-ch

an

ge

)

Ctr

l

NIT

A

NIT

B

NIT

F9D

2M

1

PS

I B

1

PS

I C

1

PS

I E

1

HU

TA

A

HU

TA

B

0

2

4

6

8

a n ti-G P Ib a n ti-G P IIb IIIa

****

A

An

ne

xin

V (

fold

-ch

an

ge

)C

trl

NIT

A

NIT

B

NIT

F9D

2M

1

PS

I B

1

PS

I C

1

PS

I E

1

HU

TA

A

HU

TA

B

0

5

1 0

1 5

*

a n ti-G P Ib a n ti-G P IIb IIIa

***

**

B

60

Figure 6 Anti-GPIbα mAb causes human platelet aggregation

3 x 108 human PRP was used for aggregation. 5µg of indicated mAb was added at the arrow.

Time is represented on the X-axis. Platelets were tested from 2 different platelet donors.

61

Figure 7 Polyclonal anti-GPIbα sera but not purified IgG from

GPIb-/- mice causes platelet activation

(A) Polyclonal sera from 4x WT platelet immunized GPIb-/- and GPIIIa-/- mice were used in the

indicated dilutions. Sera is representative of pooled sera from 3 mice from each group.(B) IgG

from sera was protein G purified and incubated with washed murine platelets at 5µg/mL in the

same conditions as previously described. Control platelets were incubated with naïve GPIb-/- or

GPIIIa-/- sera/purified IgG. Platelets were analyzed via flow cytometry for P-selectin. Data is

represented as fold-change from control. Mean ± SEM (n=3). P<0.05(*)

A

P-s

ele

cti

n (

fold

-ch

an

ge

)

C tr l 5 0 0 x 1 0 0 x 5 0 x 5 0 0 x 1 0 0 x 5 0 x

0

2

4

6

8

a n ti-G P Ib s e ra a n ti-G P IIIa s e ra

*

P-s

ele

cti

n (

fold

-ch

an

ge

)

C tr l

0

2

4

6

8

a n ti-G P Ib

IgG

a n ti-G P IIb IIIa

IgG

B

62

Figure 8 Anti-GPIbα mAb causes murine platelet desialylation

(A) Washed murine platelets were incubated with anti-GPIbα or anti-GPIIbIIIa mAb as

previously described. Desialylation was detected via flow cytometry with fluorescein conjugated

RCA-1.Data is represented as fold-change from control. Mean ± SEM (n>3).

P<0.05(*),P<0.001(***), P<0.0001(****) (B) Murine platelets were incubated with 1µg/mL,

2.5µg/ml and 5µg/mL of NIT E and NIT A. Histogram is representative of dose-dependent

increase in RCA-1 binding with NIT E. Inset graph is representative of the Mean ± SEM of NIT

E and NIT A dose-dependent response

RC

A-1

bin

din

g (

fold

-ch

an

ge

)

Ctr

l

NIT

A

NIT

B

NIT

E

NIT

F

NIT

G

NIT

H9D

2M

1

PS

I B

1

PS

I C

1

PS

I E

1

HU

TA

B

0

2

4

6

8

a n ti-G P Ib a n ti-G P IIb IIIa

****

******

*

A n ti-G P Ib (g /m L )

RC

A-1

bin

din

g (

fold

-ch

an

ge

)

0

2

4

6

8

1 0

2 .5 51

A

B

63

Figure 9 Anti-GPIbα mAb causes human platelet desialylation

(A) Human platelets were incubated mAb as previously described. RCA-1 binding was detected

via flow cytometry. Data is represented as fold-change from control. Mean ± SEM (n>3).

P<0.05(*),P<0.01(**).

RC

A-1

bin

din

g (

fold

-ch

an

ge

)

Ctr

l

NIT

A

NIT

B

NIT

F9D

2M

1

PS

I B

1

PS

I C

1

PS

I E

1

HU

TA

A

HU

TA

B

0

5

1 0

1 5

a n ti-G P Ib a n ti-G P IIb IIIa

**

**

*

A

B

64

Figure 10 Polyclonal anti-GPIbα sera and purified IgG from GPIb-/-

mice causes platelet desialylation

(A) Polyclonal sera from 4x WT platelet immunized GPIb-/- and GPIIIa-/- mice were used in the

indicated dilutions. Sera is representative of pooled sera from 3 mice from each group.(B) IgG

from sera was protein G purified and incubated with washed murine platelets at 5µg/mL in the

same conditions as previously described. Control platelets were incubated with naïve GPIb-/- or

GPIIIa-/- sera/purified IgG. Platelets were analyzed via flow cytometry for RCA-1 binding. Data

is represented as fold-change from control. Mean ± SEM (n=3). P<0.05(*),P<0.0001(****).

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l 5 0 0 x 1 0 0 x 5 0 x 5 0 0 x 1 0 0 x 5 0 x

0

2

4

6

8

a n ti-G P Ib s e ra a n ti-G P IIIa s e ra

*

*

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l

0

2

4

6

8

a n ti-G P Ib

Ig G

a n t i-G P IIb II Ia

Ig G

****

A B

65

Figure 11 Anti-GPIbα mAb are more desialylated than anti-GPIIbIIIa

All anti-GPIbα and anti-GPIIbIIIa mAb indicated in Table 1 were analyzed for desialylation via

western blot. The membrane was probed with biotinylated-RCA-1.

NIT

A

NIT

B

NIT

E

NIT

F

NIT

G

NIT

H

A n ti-G P Ib m A b A n ti-G P IIb IIIa

66

Figure 12 Sialidase inhibitor DANA can inhibit anti-GPIbα mediated

platelet desialylation

Murine and human platelets were pre-incubated with 1mM of DANA for 20 minutes at room

temperature before addition of anti-GPIbα mAb as previously described. (A) NIT A, NIT B, NIT

F, and NIT G were used (n=3) (B) NIT A, NIT B and NIT F were used (n=4). Data is

represented as fold-change from control and mean ±SEM of individual mAb.

P<0.05(*),P<0.01(**),P<0.0001(****).

H u m a n p la te le ts

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l D A N A

0

2

4

6

8

a n ti-G P Ib

**

****

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l D A N A

0

2

4

6

8

**

*

a n ti-G P Ib

M u rin e p la te le tsA B

67

Figure 13 Anti-GPIbα mAb binding to fixed platelets does not increase

platelet desialylation

Washed human platelets were fixed in 2% PFA overnight then incubated with 5µg/mL of NIT A,

NIT B or NIT F and then washed and analyzed via flow cytometry for (A) Antibody binding via

secondary anti-mouse IgG (B) Platelet activation via P-selectin expression. 0.125U of Thrombin

was added to ensure platelets were nonreactive. (C) Desialylation via RCA-1 binding

A

B

C

68

Figure 14 Anti-GPIbα antibody induces murine platelet surface

expression of Neu1

Murine platelets treated with (A) NIT G or 9D2 or (B) 100x dilution of anti-GPIbα or anti-

GPIIbIIIa sera were fixed onto poly-L-lysine coated coverslips and incubated with anti-NEU1

and anti-CD41 antibodies. They were then visualized via immunofluorescence microscopy Zeiss

LSM 700 Confocal laser scanning microscope at (A) 63X oil-immersion and (B) 63x oil-

immersion 3.2x magnification

A1

00

x d

ilu

tio

n

an

ti-G

PIb

p

oly

clo

na

l

se

ra

10

0x

dil

uti

on

an

ti-G

PII

bII

Ia

po

lyc

lon

al

se

ra

A n ti-C D 4 1 A n ti-N E U 1 M e r g e d

B

69

Figure 15 Human ITP anti-GPIbα sera induces human platelet NEU1

surface expression

Human platelets treated with (A) 50x ITP sera from China (B) 100x dilution of ITP sera from

Sunnybrook were fixed onto poly-L-lysine coated coverslips and incubated with anti-NEU1 and

anti-CD41 antibodies. They were then visualized via immunofluorescence microscopy on a Zeiss

LSM 700 Confocal laser scanning microscope at 63x oil-immersion and 3.2x magnification

Pa

tie

nt

17

se

ra

Su

nn

yb

ro

ok

IT

P

pa

tie

nt

se

ra

A

B

70

Figure 16 Anti-GPIbα mAb causes murine platelet surface expression

of Neu1 as detected by flow cytometry

(A)Washed murine platelets were incubated with anti-GPIbα or anti-GPIIbIIIa mAb as

previously described. Neu1 expression was detected via flow. .Data is represented as fold-change

from control. Mean ± SEM (n>3). P<0.05(*),P<0.001(***), P<0.0001(****) (B) Murine

platelets were incubated with 1µg/mL, 2.5µg/ml and 5µg/mL of NIT E and NIT A. Histogram is

representative of dose-dependent increase in Neu1 following binding with NIT E. Inset graph is

representative of the Mean ± SEM of NIT E and NIT A dose-dependent response.

Ne

u1

ex

pre

ss

ion

(fo

ld-c

ha

ng

e)

C tr l a n t i-G P Ib a n ti-G P IIb II Ia

0

2

4

6

8

*

a n ti-G P Ib (g /m L )

Ne

u-1

ex

pre

ss

ion

(fo

ld-c

ha

ng

e)

0 .0

0

1

2

3

1 2 .5 5

M u r in e p la te le ts

A

B

71

Figure 17 Anti-GPIbα mAb induces human platelet surface

expression of NEU1 as detected by flow cytometry

(A) Human platelets were incubated mAb as previously described. NEU1 binding was detected

via flow cytometry. Data is represented as fold-change from control. Mean ± SEM (n>3).

P<0.01(**), P<0.001(***)...

Ne

u1

ex

pre

ss

ion

(fo

ld-c

ha

ng

e)

C tr l a n t i-G P Ib a n ti-G P IIb II Ia

0

2

4

6

8

**

***

H u m a n p la te le ts

72

Figure 18 Anti-GPIbα mAb mediated desialylation is localized to the

GPIbα subunit

Human platelets were treated with NIT B and NIT F +/- 1mM of DANA as previously described.

Western blot was performed on platelet lysate to determine desialylation of platelet proteins,

with biotinylated-RCA-1, anti-GPIbα antibody was used to confirm the strongest band detected

by RCA-1 was GPIα.

P la te le ts N IT B N IT F

+ 1

mM

DA

NA

+ 1

mM

DA

NA

P la te le ts N IT B N IT F

+ 1

mM

DA

NA

+ 1

mM

DA

NA

P ro b e d w ithR C A -1 a n ti-G P Ib

-a c tin

73

Figure 19 Removal of GPIbα subunit by OSGE decreases

desialylation state of platelets to baseline

(A) Human platelets treated with NIT F was analyzed for desialylation via flow cytometry.

Following which the same sample was treated with OSGE to remove GPIbα, and reanalyzed

again for desialylation. It was found platelet desialylation returned to almost baseline following

GPIbα removal Graph is represented as fold-change from control and mean ±SEM of individual

mAb. n=3 P<0.01(**) (B) NIT F bound GPIbα was checked before and after OSGE treatment to

ensure GPIbα removal

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l O S G E

0

2

4

6

8

1 0

*

an ti-G P Ib

**

**

R C A -1 b in d in g

M u rin e Ig G b in d in g

A

B

74

Figure 20 NIT B Fab binds murine and human platelets

(A) NIT B Fab was generated and confirmed by SDS-gel. (B) NIT B binding to murine and

human platelets was confirmed via flow cytometry

4 8 k D

H u m a n p la te le ts

M o u s e p la te le ts

A

B

75

Figure 21 NIT Fab does not cause murine or human platelet

activation or desialylation

10µg/mL NIT B Fab binding to murine platelets does not cause murine or human platelet (A)

activation or (B) desialylation compared to intact NIT B as detected by flow cytometry. Data is

represented as fold-change from control and mean ±SEM. n =3 P<0.05(*)

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l N IT B N IT B F a b

0

2

4

6

8

*

A B

P-s

ele

cti

n (

fold

-ch

an

ge

)

C tr l N IT B N IT B F a b

0

2

4

6

8

*

P-s

ele

cti

n (

fold

-ch

an

ge

)

C tr l N IT B N IT B F a b

0

2

4

6

8

**

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l N IT B N IT B F a b

0

2

4

6

8

*

H u m a n p la te le ts

M u r in e p la te le ts

C D

76

Figure 22 NIT B Fab induces significant platelet clearance when

injected in vivo

(A) IP injection of 3µg of NIT B Fab causes significant platelet clearance but not

thrombocytopenia at 24 post injection (B) circulating platelets at 24 hours and 48 hours post

injection have bound NIT B Fab as detected by flow cytometry

H o u rs

Pla

tele

t c

ou

nt

(x1

06/m

L)

2 4 4 8

0

2 0 0

4 0 0

6 0 0

8 0 0P B S

N IT B F a b

**

H o u rs

% o

f F

ab

bo

un

d p

late

lets

2 4 4 8

0

2 0

4 0

6 0

8 0

1 0 0

2 4 h o u rs 4 8 h o u rs

A

B

77

Figure 23 Platelet activation and desialylation exists in a positive

feedback loop

Anti-GPIbα mAb-mediated platelet desialylation is downstream of platelet activation yet also

potentiates activation in a positive feedback loop. (A) RCA-1 binding was measured after

platelets were pretreated with vehicle (DMSO) or platelet activation inhibitors prior to incubation

with anti-GPIbα mAbs (NIT A, NIT B, and NIT F). Inhibitor SB203580 inhibits P38MAPK;

BAPTA-AM inhibits [Ca2+]i increases, Q-VD-OPh is a pan-caspase inhibitor. All inhibitors

except Q-VD-OPh significantly inhibited platelet desialylation (B) Platelets were incubated with

DANA prior to addition of anti-GPIbα mAbs and was found to significantly decrease P-selectin

expression (C) 2µg of human FcγRII/III blocker IV.3 was added 20 minutes prior to addition of

anti-GPIbα mAbs no significant decrease in RCA-1 binding was detected. All data are

represented as mean ± SEM (n=3) of individual mAb. *P<0.05, **P<0.01, and ***P<0.001

versus control unless otherwise indicated.

P-s

ele

cti

n (

fold

-ch

an

ge

)

C tr l D M S O D A N A

0

2

4

6

8

**

*

a n ti-G P Ib

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l IV .3

0

2

4

6

8

a n ti-G P Ib

**

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

C tr l D M S O B A P T A S B Q -V D -O P h

0

2

4

6

8

a n ti-G P Ib

***

*

**

*

A B

C

78

Figure 24 Anti-GPIbα opsonized platelets are phagocytosed by Fc-

independent pathways

Anti-GPIbα-opsonized platelets are cleared by FcγR-independent mechanisms. RAW 264.7

macrophage ingestion of fluorescently-labeled (CFMDA) platelets pre-incubated with anti-

GPIbα mAbs (NIT A, E, and G) or anti-GPIIbIIIa mAbs (9D2 and PSIC 1) and in the presence

of FcγR blockers. Percentage decrease is represented as percent difference between platelets

ingested without vs. with FcγR blocker treatment. Percentage of macrophages with ingested

platelets was normalized to those with ingested untreated platelets. Mean ± SEM (n=3).

**P<0.005.

De

cre

as

e o

f p

ha

go

cy

tos

ed

pla

tele

ts %

a n ti-G P Ib a n ti-G P IIb II Ia

-1 0 0

-8 0

-6 0

-4 0

-2 0

0

* *

79

Figure 25 Anti-GPIbα mAb causes platelet activation and

desialyation in vivo

Anti-GPIbα mAb causes platelet activation and desialylation in vivo. (A) 1µg of anti-GPIbα

mAb (NIT B, NIT F or NIT G) and 4µg of anti-GPIIbIIIa (9D2, PSI B1 and PSI C1) were

injected via IP into WT Balb/c mice. Platelets from anti-GPIbα mAb treated mice had significant

platelet desialylation, and increased P-selectin expression as determined by flow cytometry. (B)

Overall anti GPIbα mAb induces significant P-selectin expression and (C) desialylation. Data are

represented as mean ± SEM (C and D) of individual mAb. (n=3) *P<0.05,

Fo

ld-c

ha

ng

e

Ctr

l

NIT

B

NIT

F

NIT

G9D

2

PS

I B

1

PS

I C

1

0

5

1 0

1 5

2 0

P -s e le c t in

R C A -1

*

**

*

C tr l a n t i-G P Ib a n ti-G P IIb II Ia

0

5

1 0

P-s

ele

cti

n (

fold

-ch

an

ge

)

**

C tr l a n t i-G P Ib a n ti-G P IIb II Ia

0 .0

2 .5

5 .0

7 .5

1 0 .0*

RC

A-1

bin

din

g

(fo

ld-c

ha

ng

e)

A

B C

80

Figure 26 The ASPGR contributes to the clearance of anti-GPIbα

mAb but not anti-GPIIbIIIa mAb opsonized platelets

The ASPGR receptor clears anti-GPIbα mAbs bound platelets. NIT F or NIT G

opsonized CFMDA-platelets were intravenously injected into WT BALB/c mice with

ASPGR inhibitor (asialofetuin) or control (fetuin). Blood was collected at 0, 15, and 30

minutes and percentage of labeled platelets remaining in the circulation was assessed

by flow cytometry. Asialofetuin was able to rescue circulation of mAb-treated labeled

platelets, notably at 15 minutes. Fetuin co-injections did not affect circulating platelet

numbers. Results are representative of 3 different experiments. Mean ± SEM

(n=4).*P<0.05 versus fetuin platelets at the same time point.

T im e (m in )

CF

MD

A-l

ab

ell

ed

cir

cu

lati

ng

pla

tele

ts (

% o

f b

as

e l

ine

)

0 5 1 0 1 5 2 0 2 5 3 0

0

2 0

4 0

6 0

8 0

1 0 0

N IT G a n d fe tu in

N IT G a n d a s ia lo fe tu in*

N IT F

0 5 1 0 1 5 2 0 2 5 3 0

0

5 0

1 0 0 N IT F + fe tu in

N IT F + a s ia lo fe tu in

T im e (m in )

CF

MD

A-l

ab

ell

ed

cir

cu

lati

ng

pla

tele

ts (

% o

f b

as

e l

ine

)

*

N IT G

81

Figure 27 Anti-GPIbα opsonized platelets are cleared predominantly

by ASPGR in the absence of macrophages

The ASPGR is the predominant clearance pathway of anti-GPIbα mAbs bound platelets in the

absence of macrophages. Splenic and hepatic macrophages were depleted via clondrate

liposomes (MΦ dep.) 48 hours prior to study. 50x dilution polyclonal anti-GPIb-/- or anti-

GPIIIa-/- opsonized CFMDA-platelets were intravenously injected into WT BALB/c mice with

ASPGR inhibitor (asialofetuin) or control (fetuin). Blood was collected at 0, 15, and 30 minutes

and percentage of labeled platelets remaining in the circulation was assessed by flow cytometry.

Asialofetuin was able to completely rescue anti-GPIbα opsonized platelet clearance in

macrophage depleted mice. (n=3)

T im e (m in )

% o

f C

FM

DA

-la

be

lle

d c

irc

ula

tin

g

pla

tele

ts (

% o

f b

as

e l

ine

)

0 1 0 2 0 3 0

0

2 0

4 0

6 0

8 0

1 0 0

M d e p . a n ti-G P Ib + A S

M d e p . a n ti-G P I

M d e p . a n ti-G P IIb IIIa

a n t i-G P IIb IIIa

a n ti-G P Ib

82

Figure 28 Very low localization to the spleen of anti-GPIIbIIIa or

anti-GPIbα opsonized platelets in the absence of

macrophages

Immunofluorescence studies of splenic tissue of macrophage depleted (MΦ dep.) reveal little

platelet localization to the spleen compared to non-macrophage depleted tissue. Spleens were

harvested following circulation studies (Figure 27) and sectioned and stained for anti-F4/80.

Platelet localization is visualized as green fluorescence. DAPI is blue

M

de

p a

nti

-GP

Ib

M

de

p a

nti

-GP

IIb

IIIa

an

ti-G

PII

bII

Ia

C F M D A -p lts a n ti-F 4 /8 0 M e rg e d

83

Figure 29 Significant amounts of anti-GPIbα opsonized platelets but

not anti-GPIIbIIIa are localized to the liver and Ashwell-

Morell receptors in the absence of macrophages

Immunofluorescence studies of hepatic tissue of macrophage depleted (MΦ dep.) mice reveal

significant anti-GPIbα opsonized platelet localization to the liver and to the Ashwell-Morell

receptor not seen with anti-GPIIbIIIa opsonized platelets. Livers were harvested following

circulation studies (Figure 27 ) and sectioned and stained for anti-ASPGR1/2. Platelet

localization is visualized as green fluorescence. DAPI is blue.

M

de

p a

nti

-GP

Ib

M

de

p a

nti

-GP

IIb

IIIa

C F M D A -p lts a n ti-A S P G R 1 /2 M e rg e d

84

Figure 30 Sialidase inhibitor DANA ameliorates predominantly anti-

GPIbα mediated thrombocytopenia

WT Balb/c mice were injected via IP route with either 1µg of anti-GPIbα mAb (NIT B, NIT E,

NIT F and NIT G) or 4µg of anti-GPIIbIIIa mAb (9D2, PSI C1, PSI E1, M1). 2mg of DANA

was injected in treatment groups. Platelets counts were enumerated 24 hours post injection.

DANA was able to rescue platelet numbers in anti-GPIbα mAb injected mice but not anti-

GPIIbIIIa mAb injected mice with the exception of PSI E1. Significance was determined via

Whitney-Mann unpaired two-tailed student t-test P<0.05 (*), P<0.01 (**)

D A N A

0

5 0 0

1 0 0 0

pla

tele

t c

ou

nt

(x 1

06/m

L)

*

N IT B

pla

tele

t c

ou

nt

(x 1

06/m

L)

D A N A

0

5 0 0

1 0 0 0

N IT E

D A N A

0

5 0 0

1 0 0 0

pla

tele

t c

ou

nt

(x 1

06/m

l)

*

N IT G

D A N A

0

5 0 0

1 0 0 0

pla

tele

t c

ou

nt

(x1

06/m

l)

P S I C 1

D A N A

0

5 0 0

1 0 0 0

pla

tele

t c

ou

nt

(x1

06/m

l)

9 D 2

D A N A

0

5 0 0

1 0 0 0

P S I E

pla

tele

t c

ou

nt

(x 1

06/m

l)

**

D A N A

0

5 0 0

1 0 0 0M 1

pla

tele

t c

ou

nt

(x1

06/m

l)

D A N A

0

5 0 0

1 0 0 0 *

pla

tele

t c

ou

nt

(x1

06/m

l)

N IT F

85

Chapter 7: Future directions

1. Assess the effect of anti-GPIbα mediated platelet responses in the

active murine model of ITP

2. How desialylation of GPIbα affects antigen processing and

maintenance of the anti-platelet response. Given that sugar

residues mask antigenic epitopes, the removal of them may

enhance the response: treat platelets with NEU1 before

immunization into GPIb-/- and see if immune response is increased

3. Investigate whether desialylation of platelets also can mediate

desialylation of the bound antibody to GPIbα. A recent paper has

shown desialylated antibodies are more pathogenic (113)

4. Further elucidate the exact downstream signaling mechanisms of

GPIbα which results in NEU1 expression, i.e. utilize Src inhibitors

5. Look for co-localization of NEU1 on platelet surface with GPIbα

6. Determine if there is increased GPIbα shedding following NEU1

treatment

7. Determine if it N-linked or O-linked sialic residues which are

removed from GPIbα

86

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