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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