Pathogenesis of Integrin αIIb-mediated Fetal and Neonatal

Alloimmune Thrombocytopenia: The Effect of Maternal

Antibodies on Hematopoietic Stem Cells

by

Brigitta Elaine Oswald

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 Brigitta Elaine Oswald 2018

ii

Pathogenesis of Integrin αIIb-mediated Fetal and Neonatal Alloimmune

Thrombocytopenia: The Effect of Maternal Antibodies on

Hematopoietic Stem Cells

Brigitta Elaine Oswald

Master of Science

Department of Laboratory Medicine and Pathobiology

University of Toronto

2018

Abstract

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disorder caused

by maternal immune responses to fetal platelet antigens, particularly αIIbβ3 integrin. Although

αIIb and β3 have similar genetic polymorphic frequencies, the reported incidence of αIIb-

mediated FNAIT is at least 20 times less than β3-mediated FNAIT, the mechanism of which is

unknown. Since hematopoietic stem cells (HSCs) express αIIb, I tested whether HSCs are

affected in αIIb-mediated FNAIT. A mouse model was established in which fetal death was more

frequently observed compared to β3-mediated FNAIT. Maternal anti-αIIb antibodies bound fetal

HSCs in vivo, and impaired HSC migration was observed in embryonic day (E) 14.5 fetuses and

post natal day one (PND1) neonates. I found that the E14.5 liver may be a site of macrophage-

mediated clearance, prior to complete splenic development. PND1 HSC distribution was also

irregular: significantly decreased in bone marrow and blood and significantly increased in lung

and spleen.

iii

Acknowledgments

First and foremost, I'd like to thank my supervisor, Dr. Heyu Ni, for his continued support

throughout my undergraduate and graduate career. He's taught me that you need to be a little

crazy to pursue science and the inner workings of Mother Nature and her creations. He always

expects the best and pushes all of his students to be excellent outstanding.

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

their guidance, support and insight. Your tough questions and shrewd feedback which helped

move my project forward has been greatly appreciated. Another thank you Dr. Don Branch for

chairing my Master’s thesis defence.

To the many colleagues at St. Michael's Hospital: thank you. To Oliver, for being a wealth of

knowledge on the entire FNAIT project and for developing the animal model in the first place.

To Brian, for getting the αIIb project going. To Issaka, for being my teacher when I knew

absolutely nothing and staying patient with all the questions I could come up with. To Jade, for

sharing in my misery. To Miguel, for keeping me sane. To Reid, June, Tyler, Miao, Sahil,

Tiffany, Juan and Si-Yang, for always keeping the student room a lively and supportive place.

To Guangheng, for your astute questions causing cacophonous discussions (arguments) during

lab meetings. To Xi, I know we haven't had the opportunity to work together much but I respect

your steady hands. To Ruby, for your optimistic attitude and your knack for tailoring important

documents to their readers. To all past Ni lab members who have helped shape it into the

community it is today. To the Research Core Facilities Specialists, for teaching me how to use

and care for our shared equipment, especially Chris Spring because we all know that the Fortessa

really is a diva. And finally, to the Research Vivarium staff who care for our mice 24/7/365, and

to the mice themselves for their sacrifice in the name of science.

iv

I would like to acknowledge the funding I've received: The Queen Elizabeth II Graduate

Scholarship in Science and Technology. And further, the Canadian Institutes of Health Research

and the Canadian Blood Services for supporting Dr. Heyu Ni and by extension, me.

I would like to thank my family and friends (Juliana) for all their interest in what it really is that I

do, even if it was feigned or I started to sound like the teacher from Charlie Brown at some

points. Being surrounded by your positivity and support has meant the world to me.

And finally, a big thank you to my main man Zack for listening to me, continually supporting

me, calming me down when I'm overwhelmed, and your unwavering belief in me.

v

Table of Contents

Acknowledgments.......................................................................................................................... iii

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

List of Figures ................................................................................................................................ ix

Abbreviations ................................................................................................................................. xi

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

1.1 Platelets ................................................................................................................................1

1.1.1 Platelet Formation ....................................................................................................1

1.1.2 Platelet Structure ......................................................................................................3

1.1.3 Platelet Function ......................................................................................................4

1.1.4 Platelet Alloimmunization .......................................................................................6

1.2 Fetal and Neonatal Alloimmune Thrombocytopenia ...........................................................7

1.3 Pathogenesis of FNAIT........................................................................................................8

1.4 Human Platelet Antigens ...................................................................................................10

1.4.1 GPIII/Integrin β3 subunit .......................................................................................12

1.4.2 GPIIb/Integrin αIIb subunit ...................................................................................18

1.4.3 GPIbα and GPIbβ ...................................................................................................20

1.4.4 GPIα/Integrin α2 subunit .......................................................................................22

1.4.5 CD109 ....................................................................................................................23

1.4.6 CD36 ......................................................................................................................24

1.5 Diagnosis............................................................................................................................25

vi

1.6 Treatment ...........................................................................................................................27

1.6.1 Postnatal Treatment ...............................................................................................28

1.6.2 Antenatal Treatment...............................................................................................29

1.6.3 Future Treatments ..................................................................................................34

1.7 Hematopoietic stem cells ...................................................................................................37

1.7.1 Murine hematopoietic development ......................................................................37

1.7.2 HSC markers ..........................................................................................................38

Chapter 2: Rationale, Hypothesis, and Aims ................................................................................39

2.1 Rationale ............................................................................................................................39

2.2 Hypothesis..........................................................................................................................39

2.3 Specific Aims .....................................................................................................................40

2.3.1 Aim 1: To assess whether our anti-αIIb-mediated model of

FNAIT is more severe than our β3-mediated model and

determine its characteristics. ..............................................................................40

2.3.2 Aim 2: To determine whether maternal anti-αIIb antibodies

can target HSCs and determine their effects on HSC

populations at E14.5 in the yolk sac, fetal liver, and fetal blood. ....................41

2.3.3 Aim 3: At PND1, study the effects of maternal anti-αIIb

antibodies on HSC populations in the neonatal liver, blood,

bone marrow, lung, and spleen. ..........................................................................41

Chapter 3: Chapter 3: Materials and Methods ..............................................................................42

3.1 Mice ...................................................................................................................................42

3.2 Reagents .............................................................................................................................43

3.3 Blood collection and platelet preparation ..........................................................................44

3.4 Animal model of αIIb-mediated FNAIT ............................................................................45

3.5 Antibody Titration .............................................................................................................47

vii

3.6 Preparation of fetal tissue ...................................................................................................48

3.7 Preparation of neonatal tissues...........................................................................................49

3.8 Flow cytometry gating strategy .........................................................................................50

3.9 Statistical analysis ..............................................................................................................52

Chapter 4: Results .........................................................................................................................53

4.1 Aim 1: Characterization of αIIb-mediated FNAIT and comparison

with β3-mediated FNAIT ...................................................................................................53

4.1.1 Immunized mice generated a sufficient antibody titer for

the active αIIb-mediated FNAIT model .................................................................53

4.1.2 Platelet count was significantly decreased in FNAIT PND1

neonates compared to naïve controls .....................................................................55

4.1.3 Fetal death rate in αIIb-mediated FNAIT is significantly higher

than β3-mediated FNAIT .......................................................................................57

4.1.4 FNAIT fetuses experience bleeding and death ......................................................59

4.1.5 E14.5 FNAIT fetuses had significantly decreased body, placenta,

and liver masses ....................................................................................................61

4.2 Aim 2: Targeting of fetal HSCs by maternal antibodies and their effects

on HSC populations ...........................................................................................................63

4.2.1 Maternal antibodies bound to fetal liver tissue in vivo ..........................................63

4.2.2 No significant difference in non-blood cell populations in the

liver or yolk sac of E14.5 FNAIT fetuses compared to controls ...........................65

4.2.3 E14.5 yolk sac total and αIIb+ HSC populations were

significantly increased in FNAIT fetuses compared to controls ............................67

4.2.4 Total and αIIb+ HSC populations in the liver of E14.5 FNAIT

fetuses remained similar to controls ......................................................................69

4.2.5 The blood of E14.5 FNAIT fetuses had significantly increased

total and αIIb+ HSC populations compared to controls .........................................71

4.3 Aim 3: The effect of maternal antibodies on neonatal HSC populations ..........................73

viii

4.3.1 Lin- populations in the liver and lung of PND1 neonates were

preserved between FNAIT and controls ................................................................73

4.3.2 PND1 FNAIT neonates had an increased liver αIIb+ HSC

population compared to controls ............................................................................75

4.3.3 PND1 FNAIT neonates had significantly decreased total HSCs

in the blood compared to controls ..........................................................................77

4.3.4 HSC populations of PND1 FNAIT neonates were significantly

decreased in the bone marrow compared to controls .............................................79

4.3.5 Significant increases of total and αIIb+ HSC populations in the

lung of PND1 FNAIT neonates compared to controls...........................................81

4.3.6 Total and αIIb+ HSC populations in the spleen of PND1 FNAIT

neonates were significantly increased compared to controls .................................83

Chapter 5: Discussion ...................................................................................................................85

Chapter 6: Future Directions.........................................................................................................92

Chapter 7: References ...................................................................................................................94

ix

List of Figures

Figure 1: Hematopoietic Development ........................................................................................... 2

Figure 2: Waves of Hemostasis ...................................................................................................... 5

Figure 3: Platelet surface proteins................................................................................................. 11

Figure 4: Schematic of αIIb-mediated active FNAIT mouse model ............................................ 46

Figure 5: Gating strategy for flow cytometric analyses ................................................................ 51

Figure 6: Antibody generation in naïve and twice immunized αIIb-/- mice .................................. 54

Figure 7: Significantly decreased platelet count in FNAIT PND1 neonates ................................ 56

Figure 8: Significantly increased fetal death rates in αIIb-mediated versus

β3-mediated FNAIT .................................................................................................... 58

Figure 9: Bleeding and fetal death of FNAIT fetuses ................................................................... 60

Figure 10: Significantly decreased body, placenta, and liver mass of E14.5

FNAIT fetuses ............................................................................................................. 62

Figure 11: Maternal antibodies were capable of binding to fetal liver tissue ............................... 64

Figure 12: Lin- population remained stable in the liver and yolk sac of E14.5

FNAIT and control fetuses.......................................................................................... 66

Figure 13: Total and αIIb+ HSC populations were increased in the yolk sac of

E14.5 FNAIT and control fetuses ............................................................................... 68

Figure 14: No significant difference in total and αIIb+ HSC populations in the

liver of E14.5 FNAIT fetuses compared to controls ................................................... 70

Figure 15: Total and αIIb+ HSC populations were significantly increased in the

blood of E14.5 FNAIT fetuses compared to controls ................................................. 72

x

Figure 16: No significant difference in Lin- populations in the liver or lung of

PND1 FNAIT neonates compared to controls ............................................................ 74

Figure 17: αIIb+ HSC population was significantly increased in the liver of

PND1 FNAIT neonates compared to controls ............................................................ 76

Figure 18: Total HSCs in the blood of PND1 FNAIT neonates were significantly

decreased compared to controls .................................................................................. 78

Figure 19: Total and αIIb+ HSC populations were significantly decreased in the

bone marrow of PND1 FNAIT neonates compared to controls ................................. 80

Figure 20: Total and αIIb+ HSC populations were significantly increased in the

lung of PND1 FNAIT neonates compared to controls................................................ 82

Figure 21: Total and αIIb+ HSC populations were significantly increased in the

spleen of PND1 FNAIT neonates compared to controls ............................................ 84

xi

Abbreviations

-/- Deficient

AMIS Antibody mediated immune suppression

APC Antigen presenting cell

AUC Area under the curve

CEACAM Carcinoembryonic antigen-related cell adhesion molecule

E Embryonic day

FNAIT Fetal and neonatal alloimmune thrombocytopenia

GP Glycoprotein

HDFN Hemolytic disease of the fetus and newborn

HLA Human leukocyte antigen

HPA Human platelet antigen

HSC Hematopoietic stem cell

ICH Intracranial hemorrhage

ISBT International Society of Blood Transfusion

ISTH International Society of Thrombosis and Hemostasis

ITP Immune thrombocytopenia

IUGR Intrauterine growth restriction

IVIG Intravenous immunoglobulin

LPS Lipopolysaccharide

MAIPA Monoclonal antibody-specific immobilization of platelet antigens

MHC Major histocompatibility complex

NK Natural killer

xii

PECAM Platelet endothelial cell adhesion molecule

PNC Platelet Nomenclature Committee

PND Post natal day

Poly I:C Polyinosinic:polycytidylic acid

TGF-β Transforming growth factor β

TH2 CD4+ T helper cell

WT Wild type

VWF von Willebrand Factor

1

Chapter 1: Introduction and Literature Review

1.1 Platelets

1.1.1 Platelet Formation

During hematopoietic development hematopoietic stem cells (HSCs) differentiate into a common

myeloid progenitor which can then form a rare polyploidal platelet precursor cell known as a

megakaryocyte1 (Figure 1). Megakaryocytes normally reside in the bone marrow and, as recently

demonstrated, in the lung2, but can also be found in the blood. The primary function of these

cells is platelet production3. Maturing megakaryocytes undergo endomitosis, DNA replication

without cellular division, followed by rapid manufacture of platelet-specific proteins and

organelles. The plasma membrane then extends and develops a demarcation membrane system

(an extensive tubular system), a dense tubular network, the platelet open cannalicular system,

and forms platelet granules. Pseudopodia with proplatelets extend from the megakaryocyte, and

platelet proteins and organelles are transported to the proplatelets. Platelet biogenesis concludes

with the release of platelets into circulation. Approximately 100 billion platelets are formed daily

to support a concentration of 150-400 x109 platelets/L in the plasma with a lifespan of

7-10 days4.

2

Figure 1: Hematopoietic Development

Hematopoietic stem cells can self-renew or differentiate into common myeloid or lymphoid

progenitors. Common myeloid progenitors can then go on to differentiate into megakaryocytes

which produce platelets. This figure is adapted from OpenStax (2016), Anatomy and Physiology,

Chapter 3: The Cellular Level of Organization, OpenStax CNX5.

3

1.1.2 Platelet Structure

Platelets are the smallest of the blood cells with a diameter of 1.5-3 µm and thickness of

0.5 µm4,6. Despite being anucleate they are versatile and dynamic cells7–9 capable of rapid

changes to their membrane10–12 and structure13–15. Resting platelets are discoid in shape, but upon

recognition of vascular damage, they quickly adhere to and spread over the injured vessel wall,

made possible by cytoskeletal remodeling and de novo synthesized actin filament

polymerization13–15. The resting platelet membrane is asymmetrical; the outer membrane is made

up of neutral phospholipids and the inner membrane contains negatively charged phospholipids,

such as phosphatidylserine, which are transferred to the outer membrane by flippases during

activation16,17. The platelets’ plasma membrane is also folded into a system of open channels

known as the open cannalicular system18. Platelets can vastly increase their surface area upon

activation through the membrane made available by this system. Plasma can also be endocytosed

and platelet granules can be released into this space which allows for diffusion of granule

contents into the plasma19.

In addition to lysosomes, platelets also contain α granules and dense granules. The most

numerous are α granules (~50-80 per platelet) which contain many proteins contributing to the

hemostatic and thrombotic functions of platelets4,20. These granules also contain

immune-modulatory and pro-inflammatory factors21–23, and proteins that can have both pro- and

anti-angiogenic effects24,25. Dense granules (~3-8 per platelet) also contain hemostatically-active

components which aid in blood coagulation. Platelet granules can be exocytosed into the open

cannalicular system as mentioned above, or directly into the plasma.

4

1.1.3 Platelet Function

Platelets are best known for their critical roles in hemostasis and thrombosis4,6. Due to a

fluid-mediated interaction between platelets and larger cells in the vessel called margination,

platelets are found in higher concentrations near the vessel wall26,27. This position allows them to

constantly monitor the vessel for injury or damage. Upon vascular damage, the underlying sub

endothelium is exposed. At sites of injury under high shear stress, such as micro arteriolar

circulation, platelet hemostatic function is of particular importance. There are three waves of

hemostasis: the protein wave, the first wave, and the second wave (Figure 2). The "protein wave

of hemostasis" is constituted by the deposition of plasma fibronectin on the vessel wall. Platelet

adhesion to the site of injury, the beginning of the classical "first wave of hemostasis," is

initiated by the GPIb-IX-V complex-von Willebrand Factor (VWF) interaction. Proteins released

from platelet granules can also contribute to the protein wave. Platelet surface proteins (Figure 3)

such as the glycoprotein (GP)Ib-IX-V complex, GPVI, and integrins αIIbβ3, α2β1, α5β1, α6β1

are responsible for the detection of subendothelial matrix proteins and platelet adhesion to the

vessel wall4,28–32. Platelet activation through these and other interactions such as granule

secretion of ADP, αthrombin, and thromboxane A2, and platelet membrane surface proteins:

integrin αIIbβ3, thrombin receptors (protease activated receptors), ADP receptors (P2Y1, P2Y12

and P2YX), and thromboxane receptors4,6,30–34 results in platelet shape change and αIIbβ3

integrin transitioning to a high-affinity ligand binding state to mediate platelet aggregation.

Platelets also contribute to the "second wave of hemostasis" which is also known as blood

coagulation. Platelet activation involves phosphatidylserine exposure which provides a

negatively charged surface to harbour coagulation factors, contributing to the pro-coagulant

activity of platelets11,12.

5

Figure 2: Waves of Hemostasis

At a site of injury, hemostasis begins with the "protein wave of hemostasis" where plasma

fibronectin deposits on the vessel wall. This is followed by platelet accumulation at the site of

injury, the beginning of the classical "first wave of hemostasis," initiated by the GPIb-IX-V

complex-VWF interaction. Proteins released from platelet granules can also contribute to the

protein wave. Interactions between several other platelet surface receptors and their ligands

mediate stable platelet adhesion to the vessel wall. Platelet activation through these and other

interactions results in platelet shape change and αIIbβ3 integrin transitioning to a high-affinity

ligand binding state to mediate platelet aggregation. Platelets also contribute to the "second wave

of hemostasis" which is also known as blood coagulation. Platelet phosphatidylserine exposure

provides a negatively charged surface to harbour coagulation factors and promote cell-based

thrombin generation and fibrin formation. Adapted from Xu X R et al. Crit Rev Clin Lab Sci

2016; 53(6):409-4304.

6

1.1.4 Platelet Alloimmunization

When an individual has an immune response against a polymorphic protein after exposure to

cells or tissues from a member of the same species with a distinct polymorphism, this is known

as alloimmunization35. Alloimmunization against platelets can occur following platelet

transfusion, pregnancy, or transplantation; in these cases, immunization against foreign platelet

proteins can occur. Unlike infectious agents which cause an immune response in almost 100% of

non-immune-compromised individuals, only about 8% of platelet transfusion recipients develop

a detectable immune response against human platelet antigens (HPAs)36. Although transfusion of

foreign antigen-bearing or incompatible platelets (expressing antigens to which a patient can/has

developed antibodies) is not considered dangerous, if these platelets are transfused they will be

rapidly cleared from circulation. This is known as platelet refractoriness and the clinical benefit

of the transfusion will be precluded. For patients requiring multiple platelet transfusions, platelet

alloimmunization can be devastating and potentially result in death if a compatible blood donor

cannot be located. Post-transfusion purpura is an acute episode of severe immune

thrombocytopenia. It can also occur in rare cases, usually within the first week after transfusion

of incompatible platelets37–41. Platelet alloimmunization in the context of pregnancy is known as

fetal and neonatal alloimmune thrombocytopenia (FNAIT) and will be the focus of this thesis.

7

1.2 Fetal and Neonatal Alloimmune Thrombocytopenia

FNAIT is described as a maternal alloantibody-mediated decrease in fetal and neonatal platelet

count. An estimated 0.5-1.5/1000 new cases in live born neonates occur each year42–46 and unlike

hemolytic disease of the fetus and newborn (HDFN), FNAIT can occur in primigravida as well

as subsequent antigen-positive pregnancies42,47–50. Currently, maternal and paternal HPA

mismatching is not routinely pre-screened45,51; consequently, FNAIT is normally only diagnosed

after the birth of a neonate with petechial hemorrhages, ecchymoses, or other bleeding

symptoms49,52. Confirmation of diagnosis requires serological testing, however initial diagnosis

is often dependent on platelet count and other clinical findings since FNAIT is the leading cause

of severe neonatal thrombocytopenia (<50x109/L)48,49. Symptoms of FNAIT include low platelet

count, severe bleeding such as intracranial hemorrhage (ICH), intrauterine growth restriction

(IUGR), and miscarriage42,52–56.

The most common cause ICH in full term neonates is FNAIT57. It occurs in 8-28% of FNAIT

cases58–60, results in death in approximately 5% of cases, but can also lead to neurological

impairment55–57,61–65. Diagnosis of ICH in FNAIT can occur as early as 14 weeks gestation and

up to 80% of ICH can be detected in utero; most diagnoses are made at ≥20 weeks66–71. The early

onset of bleeding and the severity of outcomes highlight the importance of detection and

preventive treatments to attempt to alleviate harmful neurological consequences and mortality.

8

1.3 Pathogenesis of FNAIT

FNAIT occurs when fetal platelets or platelet-antigen bearing cells enter maternal circulation and

are recognized by the maternal immune system as foreign antigens72. The mechanism by which

these fetal platelet antigens (Figure 3) enter maternal circulation or are recognized as foreign is

currently unclear. In immune thrombocytopenia (ITP - considered the adult/autoimmune

counterpart of FNAIT), CD4+ and CD8+ T regulatory cells are highly involved in immune

suppression and maintaining immune tolerance towards platelet antigens73–76. The roles these

cells may play during pregnancy and in the maternal immune response against fetal platelet

antigens has yet to be elucidated. It has been shown that paternal spermatozoa can express some

platelet antigens which may cause pre-fertilization immunization against these antigens77–79. As

early as 7 weeks gestation, fetal material, such as microparticles and cell-free DNA, is detectable

within maternal circulation80–84 which may allow for a break in the immunoprivileged site

(placenta), potentiating the maternal immune response. Placental trophoblast cells are also

known to express some platelet antigens85–89. These examples of exposure of fetal platelet

antigens to the maternal immune system may initiate the maternal immune response, beginning

with the engulfment of fetal tissue by maternal antigen presenting cells (APCs)72. APCs then

present the paternally inherited antigen via their MHC-Class II receptor. T-cell receptors

recognize the antigen in the context of the MHC-Class II receptor and cause stimulation of CD4+

helper T-cells (TH2). These activated TH2 cells go on to interact with B cells presenting the

antigen in their MCH-Class II receptor and activate B cells through CD40-CD40L interaction.

B cells subsequently proliferate and generate alloantibodies against the foreign platelet antigen.

Maternal IgG can then be transported across the placenta from maternal to fetal circulation via

the neonatal Fc receptor (FcRn) expressed by syncytiotrophoblast cells of the chorionic villi90,91.

9

Fetal FcRn, but not maternal is required for this translocation of IgG as was elegantly

demonstrated in a mouse model using FcRn+/+ (wild-type, WT) and FcRn-/- (deficient) mice91.

Detection of maternal antibodies in fetal circulation can be done at approximately 13 weeks

gestation in humans92,93 and embryonic day 10.5 (E10.5) in mice.

The mechanisms of platelet clearance in FNAIT are unknown but thought to be similar to its

adult/autoimmune counterpart, ITP. In ITP, the Fab portion of antibodies binds to platelets

leaving the Fc portion free to bind Fcγ receptors on macrophages and other phagocytic cells94.

Opsonized platelets and platelet-antigen bearing cells are phagocytosed and cleared to the spleen;

this is known to as Fc-dependent platelet clearance95,96. There is evidence supporting other

platelet clearance mechanisms in ITP which are referred to as Fc-independent platelet clearance

pathways97,98, however the importance of these mechanisms in FNAIT has not been addressed.

10

1.4 Human Platelet Antigens

An antigen present in part of a population but absent in another due to genetic polymorphisms is

known as an alloantigen99. In 1990, the platelet working party of the International Society of

Blood Transfusion (ISBT) developed a nomenclature for HPAs100. It was then revised in 2003 by

the Platelet Nomenclature Committee (PNC) formed by collaboration between the ISBT platelet

working party and the scientific subcommittee on platelet immunology of the International

Society of Thrombosis and Hemostasis (ISTH)101. HPA nomenclature begins first with a number

where HPAs are numbered by date of discovery. Alleles of each HPA are then organized

alphabetically by their frequency from high to low. An up-to-date list of PNC approved HPAs is

included on the Immuno Polymorphism Database website at https://www.ebi.ac.uk/ipd/hpa/101.

Any polymorphic and antigenic platelet protein, except for major histocompatibility complex

(MHC) genes which have their own HLA designations, may be designated as an HPA. However,

there is strict criteria which must be met for an HPA to be approved by the PNC. There are

currently 37 known HPAs (Figure 3); 12 have been grouped into six bi-allelic systems (HPA-1,

-2, -3, -4, -5, -15) for which alloantibodies to both alleles have been observed, for the remaining

sites alloantibodies have only been reported against the rare allele. 27 sites are caused by

bi-allelic polymorphisms and two are caused by tri-allelic polymorphisms102. All but one of these

sites are caused by single nucleotide substitutions resulting in a single amino acid substitution;

HPA-14bw is caused by a tri-nucleotide in-frame deletion resulting in a single amino acid

deletion103,104.

11

Figure 3: Platelet surface proteins

There are currently 37 human platelet antigens (HPAs) located on six platelet proteins, GPIIIa,

GPIIb, GPIbα, GPIbβ, GPIa, and CD109. HPA-1 through -21 are displayed with their location on

their respective protein, HPA-22 to -29 have been added below. Adapted from Murphy F and

Pamphilon DH, Practical Transfusion Medicine, 2013, 3rd ed. Wiley-Blackwell105.

12

1.4.1 GPIII/Integrin β3 subunit

The integrin β3 subunit is also known as CD61 or GPIIIa. It has the ability to pair with either the

αIIb or αV integrin subunits, and must be paired to be expressed on the surface of a cell. 106. It is

widely expressed, not only on platelets and their progenitors, but also several other cell types

such as trophoblast cells85–87, endothelial cells and endothelial progenitors107,108, spermatozia77,78,

microglia109, and astrocytes110. On the surface of platelets β3 is expressed with either the αV or

the αIIb subunit. αVβ3 has a low copy number on the platelet surface111. It is known as the

vitronectin receptor; however, it can bind other proteins with an RGD motif. It can also bind

osteopontin which is not normally found in healthy vasculature, but interestingly is expressed by

atherosclerotic plaques29. It is currently unknown whether this receptor is involved in the

pathogenesis of atherosclerosis. αIIbβ3 is the major receptor for fibrinogen. It is quite

promiscuous, however, and can bind several other proteins such as VWF, fibronectin,

vitronectin, and other ligands which may be able to mediate platelet aggregation in the absence

of both fibrinogen and VWF.

β3 integrin is responsible for the vast majority of reported cases of FNAIT. More than half of

HPA sites, 15 to be exact, are located on the integrin β3 subunit (HPA-1, -4, -6, -7, -8, -10, -11,

-14, -16, -17, -19, -21, -23, -26, and -29)103. An estimated 75-85% of FNAIT cases in the

Caucasian population are a result of HPA-1a (leucine to proline substitution at residue 33)

incompatibility alone42,46,112. Although the prevalence of HPA-1a-mediated FNAIT is lower in

populations with different ethnic backgrounds, β3 HPAs are responsible for a far greater amount

of FNAIT cases than all other HPAs combined, regardless of race56. Variation in ethnic

background corresponds with variation in HPA allele genetic frequency; the allele frequencies of

HPAs -1 to -6 and -15 can be found on the Immuno Polymorphism Database website (above).

13

1.4.1.1 Fetal genotype association with alloimmunization

HPA-1a-mediated FNAIT, as the most common cause of FNAIT in the Caucasian population has

been studied more intensively than any other HPA. For example, a study by Kjeldsen-Kragh

et al. investigated in part the HLA DRB3 (MHC-Class II) genotype of immunized HPA-1a

negative women113. The DRB3*01:01 allele of this MHC-Class II gene has previously been

reported to be strongly associated with alloimmunization against HPA-1a; women possessing

this allele had an approximately 25 times higher risk of HPA-1a-immunization when compared

to women who lack this allele114,115. In this study, 90% of the immunized HPA-1a negative

women were found to be DRB3*01:01 positive113; these women were also found to have

significantly increased alloantibody titers116. Since the pathogenesis of FNAIT depends on

presentation of the paternally inherited antigen via the MHC-Class II receptor on the surface of

maternal APCs and B cells, the efficacy of this presentation makes a difference. The

DRB3*01:01 allele has been shown to produce a version of the MCH-Class II receptor which is

highly effective at presenting the antigenic peptide containing the HPA-1a epitope (residue 33)

to TH2 cells117. In the absence of this allele, presentation of the antigenic peptide is less efficient,

therefore its presentation, T cell activation, and subsequent B cell activation is less effective.

Interestingly, in a study of 165 HPA-1a-mediated FNAIT pregnancies screened from 100 448

women, significantly lower birth weight was observed in male neonates, and after adjusting for

other factors, this decreased in birth weight was linearly correlated to maternal anti-HPA-1a

alloantibody titer118. While female birth weight also displayed a similar trend toward decrease,

this was not significant118. These findings can be put to use as predictors of clinical outcomes,

however they also raise questions about other sex differences which may be occurring.

14

1.4.1.2 Anti-HPA-1a Prophylaxis

The prevalence of HPA-1a-mediated FNAIT has prompted the investigation of potential benefits

of screening women for HPA-1a deficiency and the presence of HPA-1a alloantibodies by

several groups44,45,51,113,114,119. In the populations studied, the rate of HPA-1a deficiency was

1.6-2.1% and between 3-12% of those deficient individuals developed alloantibodies against

HPA-1a. This, however, does not mean that that FNAIT is guaranteed to occur in HPA-1a

positive pregnancies44,45,51,113,114,119. Through treatment of identified at-risk pregnancies, one

group was able to decrease the number of FNAIT-related complications to approximately 25% of

a historic value113. FNAIT can be considered the platelet analogue of HDFN, another

alloantibody mediated pregnancy complication. HDFN is treated using anti-D prophylaxis which

is known as antibody mediated immune suppression (AMIS). Prophylactic administration of

anti-D antibodies prevents a maternal immune response. It was worthwhile to investigate the

potential clinical benefit of anti-HPA-1a prophylaxis due to 1) success in mitigating

complications of FNAIT by antenatal screening and treatment and 2) a finding that anti-HPA-1a

alloantibodies were developed in relation to delivery in about 75% of women113,120,. This was

executed using the β3-/- mouse model described below121. The proof-of-concept study of this

preventative AMIS strategy showed that AMIS could also be an effective prophylactic treatment

in a β3-mediated model of FNAIT121. In order to manufacture an anti-HPA-1a IgG product for

human testing, human plasma is currently being collected in the US and Europe. This product

will be used to test the efficacy of AMIS prophylactic treatment in human FNAIT122.

15

1.4.1.3 Animal models of β3-mediated FNAIT

Due to ethical constraints it is difficult to perform basic research on human FNAIT patients,

creating a demand for animal models of the disease. There are several passive models of

thrombocytopenia which involve injecting anti-β3 antibodies into animals such as mice123,124,

rats125, dogs126, or baboons127. Genetically or chemically modified anti-HPA-1a antibodies

(B2G1 and SZ21) which no longer had the Fc portion of the antibody have been used in murine

models123,124. Injection of anti-HPA-1a antibodies into a chimeric mouse model usually results in

clearance of human HPA-1a+ platelets, however concurrent injection with these modified

antibodies prevented that clearance123,124. It is now known, however, that FcRn is the receptor

responsible for translocation of maternal antibodies across the placenta, and FcRn requires an

intact Fc region for this to occur91. Unfortunately, this limits the therapeutic potential of these

modified antibodies, as they could only be useful with direct administration to fetal circulation.

Using a passive model also negates the effects of the maternal immune response and its effects

on fetal tissues.

The Ni laboratory was the first to develop an active mouse model using β3-/- BALB/c

background mice79. To generate an immune response, β3-/- females are immunized with WT

platelets. These females are then bred with WT males to generate heterozygous pups which

present with FNAIT. The pups exhibit symptoms such as thrombocytopenia, severe bleeding,

ICH, and fetal death, similar to the human disease. Consistently, this model has been put to use

to reveal the pathophysiology of FNAIT41. First, it was demonstrated that both intravenous

immunoglobulin (IVIG) and anti-FcRn antibodies that blocked the function of FcRn were able to

ameliorate FNAIT79,91. During pregnancy it is possible and probable for mothers to be exposed

to infectious agents. In an effort to imitate this scenario, mice were intraperitoneally injected

16

with either lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) during the

immunization phase of the FNAIT model. Acting as stimulatory adjuvants, both compounds

were able to enhance the anti-β3 immune response. This result demonstrates that

alloimmunization against β3 HPAs may be enhanced by infection, viral or bacterial, during

pregnancy.

The Ni lab active β3-mediated murine FNAIT model has also been used to investigate

off-target/non-platelet effects of maternal antibodies. Other cells, particularly endothelial cells

which highly express αVβ3, may also be affected by these antibodies90. Interestingly, an

anti-angiogenic effect of maternal anti-β3 antibodies was found through antibody interaction

with αVβ390,128. When comparing a similar GPIbα-mediated FNAIT model with the β3-mediated

FNAIT model, ICH was not observed in the GPIbα-mediated model despite a similar reduction

in platelet count observed in both models. Upon examining the brains and retinas of β3 FNAIT

pups, it was observed that they had reduced vessel density compared to naïve controls. These

pups also experienced endothelial cell apoptosis and inhibited angiogenic signalling. αIIb-/-

neonates also developed ICH when injected with anti-β3 sera, even though this treatment did not

result in a decreased platelet count. This data seems to indicate that hemostasis in the fetus is not

dependent on platelets. This is further supported by studies with fibrinogen-deficient mice,

NF-E2-deficient mice which lack circulating platelets, and double deficient NF-E2-/-/fibrinogen-/-

mice. None of these mouse strains exhibit in utero bleeding129,130, which indicates that fibrin clot

formation is also not required for fetal hemostasis. If this is the case, it is likely that the anti-

angiogenic effect of maternal antibodies, not thrombocytopenia, is actually the main mechanism

in FNAIT causing severe bleeding. Although it cannot be excluded that there may by a

synergistic effect between anti-angiogenesis and thrombocytopenia, the widely-held view of fetal

bleeding and its mechanism in FNAIT is shifted by this evidence.

17

Since placental trophoblast cells also express αVβ3 and αIIbβ385–87, investigation into the effect

of maternal anti-β3 alloantibodies on these cells was warranted. This investigation brought about

the discovery of uterine natural killer (NK) cells which normally contribute to

decidualization90,131–135. Under FNAIT conditions however, these cells were altered to an

activated cytotoxic state from their quiescent non-cytotoxic phenotype. Once in this activated

state, NK cells became capable of antibody-mediated cell cytotoxicity. This change was likely

mediated by the maternal immune response inflammation88. The NK cell-mediated pathology

was ameliorated with the use of anti-NK cell antibodies; meaning that NK cells may be a

potential therapeutic target. Within the placenta there are several other types of immune cells

along with uterine NK cells, and so targeting those cells, especially those responsible for the

inflammation, may also have therapeutic potential. These findings may also be applicable to

FNAIT mediated by other HPAs or other types of immune-mediated pregnancy loss.

18

1.4.2 GPIIb/Integrin αIIb subunit

The integrin αIIb subunit also goes by the names GPIIb or CD41, only pairs with the β3 subunit

and just like the β3 subunit, both subunits must be expressed and paired for proper cell surface

expression106. It was originally thought that αIIb was only expressed by platelets and their

progenitors, megakaryocytes, but it is now known to be expressed by certain populations of

HSCs and is one of the earliest known markers of hematopoietic commitment136–143. Although

the αIIb and β3 subunits share a similar frequency of genetic polymorphisms144–146, suggesting

the two subunits might have a similar FNAIT incidence rates, current data on reported cases

shows that the incidence of αIIb-mediated FNAIT is actually more than 20 times less than that of

β3-mediated FNAIT. There are 7 HPA sites within the αIIb subunit: HPA-3, -9, -20, -22, -24,

-27, and -28, which overall account for only 3-5% of all FNAIT cases43,53,63,103,147. These cases

are reportedly difficult to detect, particularly clinically severe, and may often result in

miscarriage which likely causes underreporting52,63,148. The most common αIIb antigens are

HPA-3a and -9bw, both of which are found on the αIIb calf-2 domain and are only 19 base pairs

apart. These two HPAs are in a linkage disequilibrium102. There are two other HPAs in the calf-2

domain: HPA-27bw and -28bw103. HPA-22bw, located in the β-propeller domain which is close

to the ligand binding domain, is notable because it is currently unknown whether maternal

alloantibodies against this HPA have the ability to block or partially block receptor function149.

The Ni laboratory has also been experimenting with an αIIb-mediated murine FNAIT model

similar to the previously described β3 model. Both αIIb-/- and human αIIb transgenic mice have

been employed to recapitulate the disease. While testing these models, variation in the

immunization phase was investigated by either immunizing twice or four times. All mothers

immunized four times failed to deliver pups, and fetal death was also prevalent among the twice

19

immunized mothers150. This data suggests that αIIb-mediated FNAIT, with this high incidence of

fetal death, is more severe than β3-mediated FNAIT. The elevated rate of fetal death in this

model may explain the paucity of reported cases and the disparity of incidences between

αIIb- and β3-mediated FNAIT.

Since αIIb is also expressed by HSCs and hematopoietic progenitors136–143, there is a possibility

that these cells could be targeted by maternal alloantibodies. Hematopoietic progenitors can be

found as early as E7.5 expressing αIIb.

20

1.4.3 GPIbα and GPIbβ

The reported incidence of FNAIT due to either GPIbα or GPIbβ is rare56,112,151–153. These two

proteins together only each have one HPA site, HPA-2 and -12 respectively103. Cases of FNAIT

mediated by HPA-12 on GPIbβ are exceedingly uncommon154,155. GPIbα or GPIbβ are also

known as CD42b and CD42c. These two proteins are components of the GPIb-IX-V complex

which is one of the most abundant platelet surface receptors. The GPIb-IX-V complex is known

as the major receptor for VWF that plays a role in platelet adhesion, and is especially important

under high shear conditions156. Other ligands of this receptor include thrombin,

P-selectin, integrin αMβ2, factor XI, and factor XII157–162. Unlike αIIbβ3 integrin, expression of

the GPIb-IX-V complex is restricted to platelets and megakaryocytes156. The GPIbα subunit has

recently been implicated as an essential player indispensable for platelet-mediated hepatic

thrombopoietin generation163. Whether fetal thrombopoietin generation is affected by maternal

anti-GPIbα alloantibodies is currently unknown. Interestingly, 20-40% of autoantibodies in the

adult analogue of FNAIT, ITP, are directed against GPIbα41,164–166, a staggering difference

compared to the infrequency of GPIb-mediated FNAIT. GPIbα has been shown to be less

immunogenic than β3 integrin which may account in part for this discrepancy. However, the

immune response against GPIbα was enhanced by the addition of LPS or Poly I:C in the

immunization phase of a GPIbα-mediated murine FNAIT model167. This murine model,

developed by the Ni laboratory, involves the use of BALB/c background GPIbα-/- mice. With

approximately 80% of GPIbα-/- FNAIT pregnancies resulting in fetal death, this model

demonstrated an extremely high rate of fetal death once a maternal immune response had been

achieved168. FNAIT mediated by GPIbα is unlike most other forms of FNAIT in that there is a

lack of bleeding symptoms, and so it is described as "non-classical." Maternal anti-GPIbα

21

antibodies were observed to cause platelet activation, resulting in placental fibrin deposition that

was not observed in the β3 FNAIT model. GPIbα-mediated FNAIT was also abrogated through

the use of IVIG or anti-FcRn therapies in this model168, however the efficacy of this treatment

has yet to be studied in human patients with either HPA-2 or HPA-12 mediated FNAIT as they

are both rare forms of the disease. A decreased or refractory response to steroid therapies or

IVIG treatment is often observed in patients with anti-GPIb-IX antibody specificities in

ITP95,169–171. This is likely due to different, Fc-independent mechanisms of platelet clearance in

GPIb-mediated ITP97,98. Whether a similar phenomenon of Fc-independent platelet clearance

occurs in anti-GPIb-mediated FNAIT or whether maternal alloantibodies abrogate

thrombopoietin, and therefore platelet production has yet to be explored.

Recent evidence may indicate that there is a new platelet alloantigen within the GPIb-IX-V

complex, present on the GPIX subunit172. Although the PNC has not yet approved this platelet

alloantigen as an HPA, Jallu et al. found a GPIX variation (NM_000174.4:c.368C>T) paternally

present in an FNAIT case where other known HPA fetomaternal incompatibilities were absent.

This group transiently transfected the GPIX variant into HEK293 cells where it was

co-expressed with GPIb. Maternal sera reacted with the variant expressed on these cells. This

may represent the finding of a novel HPA.

22

1.4.4 GPIα/Integrin α2 subunit

The integrin α2 subunit, also referred to as Cd49b, GPIa, or VLA-2, is estimated to be the second

leading protein responsible for cases of FNAIT56,112. Four HPA sites: HPA-5, -13, -18, and -25,

are found on α2; HPA-5 is the most prevalent58,173,174. Surprisingly, the most common

platelet-specific alloantibody occurring during pregnancy is anti-HPA-5b175–177. The antibody

titer against HPA-5b, however, does not correlate with FNAIT taking place in those pregnancies.

The α2 subunit is expressed by vascular endothelial cells, dendritic cells, and other

hematopoietic lineage cells such as NK cells and leukocytes178,179. It may also be expressed on

other cell types. The only partner of the α2 subunit is the integrin β1 subunit. α2β1 integrin is a

collagen receptor. When FNAIT is mediated by α2, patients in these cases can present with ICH.

The mechanism(s) of ICH in this version of the disease has not been studied, however they may

be similar to β3-mediated FNAIT where there was an anti-angiogenic effect of maternal

alloantibodies. Investigation of this possibility is warranted.

23

1.4.5 CD109

Only one HPA site can currently be found on CD109: HPA-15103. CD109 can be expressed by

several cell types, including HSCs, T helper cells, activated platelets, and some endothelial

cells180–182. It is a glycosylphosphatidylinositol-linked glycoprotein which, upon proteolytic

cleavage, becomes activated181,182. This cleaved, activated portion is capable of

thioester-mediated covalent binding. It may be able to bind with other molecules on the cell

surface, or those on nearby cells182. It has also been found to play a role in negatively regulating

the TGF-β pathway in keratinocytes180. Its specific function on platelets is not currently known,

however both of these known functions may play a role in platelet activation, adhesion or

aggregation. FNAIT mediated by HPA-15 has been reported to cause profound

thrombocytopenia (3x109/L), severe ICH resulting in death, and amegakaryocytosis, which has

not been reported in FNAIT mediated by any other HPA181–183. The severity of

HPA-15-mediated FNAIT may explain the rarity of reported cases183–185.

24

1.4.6 CD36

CD36 does not have an HPA designation because when an individual becomes immunized

against CD36, it is due to a deficiency of the protein in the immunized individual rather than a

specific polymorphic difference. Some scientists in the field of platelet immunology consider

immunization against CD36 to be an isoimmune response, a specialized type of alloimmune

response, which occurs in individuals lacking the antigenic protein. In 1989, immunization

against this protein was first reported186. It was then identified as CD36, also known as GPIV,

one year later by the same group187. Deficiency in CD36 is more common in individuals of Asian

or African ethnic backgrounds, occurring in an estimated 2-5% of people of either

descent186,188–190. Pregnant women deficient in CD36 who are exposed to CD36 by an antigen

positive fetus may develop an immune response causing fetal symptoms similar to fetal and

neonatal alloimmune thrombocytopenia187,191.

25

1.5 Diagnosis

Pre-screening for maternal and paternal/fetal HPA incompatibilities is not currently practiced,

thus FNAIT is usually diagnosed after birth49,59,192,193. Neonates who are diagnosed with FNAIT

usually present with petechia, purpura, ecchymoses, or other bleeding, along with unexplained

thrombocytopenia (platelet count <150x109/L59,192,194), but are otherwise healthy. Another

symptom, particularly in males, is low birth weight118. During normal human gestation, the

platelet count of a healthy fetus reaches 150x109/L by the end of the first trimester195,196 and

increases to 300x109/L by 30-35 weeks197. FNAIT is the leading cause of severe neonatal

thrombocytopenia (platelet count <50x109/L)48,49,194, so when thrombocytopenia is present, this

may be a sign of FNAIT.

Serological testing is required to confirm the diagnosis of FNAIT, this tests for the presence of

alloantibodies in maternal sera48,49,53,192. The most common method used for serological testing is

the monoclonal antibody immobilization of platelet antigens (MAIPA) assay 198–201. MAIPA is

considered the gold standard for the detection of platelet antibodies53. The assay is quite versatile

and can be used for the detection of rare HPA alloantibodies202: complexes are formed by a

platelet antigen sandwiched between a murine monoclonal antibody and the human anti-platelet

alloantibody from maternal sera. Rarely, however, alloantibodies with low avidity may not be

detectable at childbirth, in this case follow-up testing is highly recommended, especially in the

event of subsequent pregnancies202. There are several washing steps in the protocol of the

MAIPA assay, so some low avidity maternal alloantibodies are unable to remain bound to their

targets, resulting in a false negative. It may be required to detect low avidity alloantibodies with

other assays such as surface plasmon resonance203.

26

A new detection strategy which is currently in development utilizes a self-assembling monolayer

(SAM) coated on a glass or silica surface and covalently linked to platelet proteins163,204,205.

Dr. Miguel Neves of the Ni laboratory has made use of silica magnetic beads coated with a SAM

designed specifically to link with any –OH terminated surface to allow for variability of coated

surfaces and provide effective resistance to non-specific binding. Purified αIIbβ3 integrin or

GPIbα recombinant ectodomains have been covalently linked to the surface. The chemical

properties of the SAM open the possibility to covalently link almost any protein to the surface.

The assay also does not require any blocking or washing steps due to the properties of the SAM

and its effective resistance to fouling. The lack of washing steps may overcome problems with

avidity experienced in the MAIPA assay while remaining highly sensitive.

In an ideal situation the infant and both parents should be genotyped for at least the most

common HPAs. Recurrence rates in subsequent antigen-positive pregnancies is nearly 100%.

Antigen positivity of subsequent pregnancies can be determined by the paternal genotype: if the

father is homozygous, the recurrence rate will be nearly 100%, if the father is heterozygous this

rate will be approximately 50%202. A history of FNAIT represents a significant risk to

multigravidous women and subsequent fetuses should undergo genetic testing. Genetic testing

can be done non-invasively and is relatively low risk when done utilizing fetal cell-free DNA

collected from maternal plasma. HPA-1a fetal genetic testing can be done routinely48,206 and this

process is in development for several other HPAs (HPA-3, -5, and -15) 207. Using fetal cell-free

DNA from maternal plasma has proven highly effective and accurate in HDFN208, therefore will

likely be useful for FNAIT. These tests, both serological and genetic, are time-consuming, so

they are only well suited to confirming diagnosis; treatment should be commenced as soon as

FNAIT is suspected.

27

1.6 Treatment

Treatment of women and infants affected by FNAIT can prove to be challenging. Since there is

no routine pre-screening for FNAIT and it can occur in a first pregnancy, antenatal treatment is

not possible for all cases49,59,192,193. Treatments for FNAIT have not been standardized192,209, and

so regimens are often based on the severity of thrombocytopenia and bleeding in the

infant59,192–194. Current treatment options include IVIG, corticosteroids, and neonatal platelet

transfusions59,192,194.

28

1.6.1 Postnatal Treatment

Since it takes time to confirm a diagnosis of FNAIT, it often begins only as a suspicion for

infants with bleeding and unexpected thrombocytopenia. This suspected diagnosis comes from

first excluding other common causes of thrombocytopenia such as infection, pre-eclampsia or

eclampsia, maternal ITP, neonatal limb abnormalities, or cutaneous hemangiomas59,192. If FNAIT

is suspected, treatment must be initiated promptly. Although there is no standardized treatment,

some guidelines call for IVIG at a dose of either 400mg/kg/d given over 2-4h for 5 days or

1g/kg/d for 2 days to which approximately 75% of babies respond49,192,194,210. In cases of severe

thrombocytopenia, a platelet transfusion can also be beneficial – a threshold of 30x109/L is

normally used. Below this value infants are immediately transfused with random donor platelets

and a post-transfusion count must be obtained to exclude refractoriness to platelet transfusion.

Those infants with counts remaining below the threshold should then be transfused with

compatible antigen-negative donor platelets or plasma-depleted washed maternal platelets. If the

platelet count is above the threshold, it is more economical to await results of initial serological

studies in order to transfuse compatible platelets, either from an antigen-negative donor platelets

or plasma-depleted washed maternal platelets49,59,193,211. Platelet counts in affected infants should

be monitored for one week or more following transfusion to ensure a positive response to

treatment192.

29

1.6.2 Antenatal Treatment

For women with a history of FNAIT, or for those in whom anti-platelet alloantibodies were

detected, antenatal treatment becomes possible. The recurrence rate is nearly 100% in subsequent

antigen-positive pregnancies and FNAIT is often more severe if left untreated49.

1.6.2.1 Intravenous Immunoglobulin

IgG is pooled from >1000 donors to produce a blood product called IVIG. It is routinely used in

the treatment of several immune-mediated diseases such as ITP212,213, and it is used by many

centres as an off-label antenatal treatment for FNAIT53,214. Although IVIG is generally thought to

be safe, some patients do experience some mildly inclement side effects, such as

headache, nausea, fatigue, and dizziness, which can deter some of those patients from its

use215–217. IVIG has been used since positive outcomes were first reported by Bussel et al. in

1988. However, the efficacy of IVIG is controversial218–223 despite several studies whose

outcomes displayed a fetal benefit71,220. Treatment with IVIG can vary from 0.5-2g/kg/week,

dosing can be consistent throughout pregnancy or by stratified based on clinical monitoring of

the fetus or severity of previous pregnancies224.

30

1.6.2.2 Steroids – Prednisone

Prednisone is often given in conjunction with IVIG. A study comparing IVIG and prednisone

showed that a favourable treatment response occurred in the IVIG treatment group more than

twice as often than the prednisone group219. When comparing IVIG alone and IVIG with

prednisone, both treatments resulted in comparable favourable outcomes, however, women on

prednisone were reported to have higher rates of gestational diabetes, insomnia, jitteriness and a

tendency to greater fluid retention225. Now, it is generally accepted that patients should be treated

based on relative risk determined by maternal history. In this way, patients are stratified into

different risk groups where higher risk indicates that treatment should be delivered earlier and

more aggressively. Treatment options start at simply monitoring for HPA-specific alloantibodies,

and range to high dose IVIG with prednisone, with therapy escalated as needed226.

31

1.6.2.3 Caesarean Section

Regardless of risk, elective Caesarean is frequently recommended in cases of FNAIT. It seems as

though this is mainly done to avoid the trauma of birth which may cause ICH. However, up to an

estimated 80% of ICH can already by detected in utero66–71. The majority of in utero ICH is

detected at 20 weeks, however it can be detected as early as 14 weeks66–71, suggesting that the

choice to deliver by Caesarean due to the risk of ICH may not be as desirable. A method of

assigning risk has been suggested by Bertrand et al. through the measurement of antibody titer

over time227. Bertrand et al. measured antibody concentration using MAIPA and analyzed the

data by calculating the area under the curve (AUC) weighted by weeks between quantifications.

Interestingly, they found that AUC negatively correlated with neonatal platelet count where high

AUC correlated with severe thrombocytopenia but low AUC correlated with a safe neonatal

platelet count. This allowed a threshold to be set for when the AUC is 23 IU/mL to determine

whether to recommend vaginal or Caesarean delivery.

32

1.6.2.4 Fetal platelet transfusion and Fetal blood sampling

In 1984 Daffos et al. executed and reported the first successful fetal platelet transfusion.

Approximately 6 hours prior to an elective Caesarean section, maternal platelets were transfused

into a 37-week-old fetus228. This group also reported on their performance of fetal blood

sampling from 606 pregnant women for many different contraindications. Only approximately

2% of these procedures resulted in fetal death, excluding those which were chosen not to be

carried to term229. This study is often used to show the safety of the procedure; the reported rate

of this serious complication may not truthfully reflect the risks associated with fetal blood

transfusions or fetal blood sampling for patients with FNAIT or severe thrombocytopenia in

which the risk may be elevated. When used in FNAIT, fetal blood sampling is sometimes used to

measure fetal platelet count and determine the efficacy of therapy. Fetal platelet count, however,

may not be as important a determinant of fetal and treatment outcomes as was once believed.

It has recently been shown in a murine model of FNAIT that maternal anti-β3 antibodies had an

anti-angiogenic effect which was the major cause of ICH. These antibodies caused vascular

endothelial cell apoptosis in vivo, inhibited proliferation and network formation of these cells in

vitro, and inhibited angiogenic signalling both in vivo and in vitro90. It was also indicated that

fetal hemostasis is not dependent on platelets or fibrin clot formation129,130. If the effect of

maternal anti-β3 alloantibodies on human angiogenesis is similar to that observed in mouse

models, fetal blood sampling may be useful for in utero diagnosis, but neither it nor fetal platelet

transfusion may be necessary for antenatal treatment. Although it has yet to be confirmed in

human patients, in the mean time it would likely be beneficial to reduce the use of these invasive

techniques. Perhaps the best use of fetal blood sampling is for confirmation of diagnosis, and

33

fetal blood transfusion should mainly be used to increase fetal platelet count prior to elective

Caesarean like the first demonstrated use by Daffos et al.228.

34

1.6.3 Future Treatments

1.6.3.1 Anti-FcRn therapy

Monoclonal antibodies targeting FcRn have been studied in several FNAIT mouse models, such

as the β3-mediated model, with promising results91,168. Anti-FcRn is thought to block fetal FcRn

and prevent the transport of maternal (pathogenic) IgG into fetal circulation, although this

mechanism has not been confirmed. FcRn is an Fc receptor which has several functions

including: extending the life of IgG in circulation, transporting maternal IgG from milk across

the gut epithelium to fetal circulation, and transporting maternal IgG across the placenta from

maternal to fetal circulation. For the first two functions, the microenvironment has a low pH

which facilitates the binding of IgG to FcRn. IgG is then released at physiological pH. It is

currently unclear how FcRn transports IgG across the placenta and further study to elucidate the

mechanism is required. Monoclonal antibodies are frequently used as therapeutics, however the

safety and efficacy of anti-FcRn as a therapeutic must be tested before its use in a clinical setting.

35

1.6.3.2 Anti-NK cell therapy

Since trophoblast cells express αIIbβ3 integrin, a recent study using the β3-mediated FNAIT

model investigated the effect of maternal antibodies on the placenta. Surprisingly, it was found

that uterine resident NK cells which normally have a quiescent non-cytotoxic phenotype were

switch to an activated cytotoxic phenotype during the pathogenesis of the disease88. In an attempt

to remedy the cytotoxic effects of these cells on the placenta, monoclonal antibodies were used

to either block NK cell activating receptors (anti-NKp46 or anti-FcγRIIIa) or deplete NK cells,

both of which ameliorated the fetal death and bleeding associated with β3-mediated FNAIT to a

degree. This was the first time that NK cells were identified as players in the pathogenesis of

FNAIT. Depletion or blocking the activation of NK cells may not be an ideal therapy for clinical

use, however these observations indicate that there may be other targets on NK cells which could

block their transition from a quiescent to cytotoxic state, or potentially there are other placental

resident immune cell therapeutic targets89.

36

1.6.3.3 Prophylaxis against HPA-1a-mediated FNAIT

It has recently been shown that anti-HPA-1a alloantibodies were developed in about 75% of

women in relation to delivery120, meaning that FNAIT may be even more similar to HDFN than

currently believed. Screening for HDFN is done routinely, however this in not the case for

FNAIT. HDFN pre-screening is required in order to administer anti-D, a prophylactic treatment

which prevents a maternal immune response, i.e. AMIS. Similarly, administration of

anti-HPA-1a antibodies as a prophylactic treatment was successful in preventing a maternal

immune response and FNAIT in a murine model121. With this promising result in mind, HPA-1a

antibodies are currently being collected and pooled from donors in the USA and Europe to make

an anti-HPA-1a transfusion product. This product will be used to test the efficacy of prophylactic

treatment in human patients. Provided the results are positive, such a study will likely have an

impact on future detection and treatment of FNAIT122.

37

1.7 Hematopoietic stem cells

HSCs are the pluripotent progenitors of all myeloid and lymphoid blood cells. Although they are

found in adult bone marrow, their location in the body during development varies greatly.

1.7.1 Murine hematopoietic development

Beginning at E7.5 in murine development, the generation of hematopoietic progenitor cells can

first be detected in what are known as the yolk sac blood islands230–232. These first hematopoietic

cells are produced in the yolk sac from hemangioblasts, the common mesodermal stem cell for

endothelial cells18,233,234. Erythroid–myeloid–lymphoid progenitors can also begin to be detected

at 8.5 in the Para-aortic Splanchnopleura which later develops into what is known as the

aorta-gonad-mesonephros region (AGM; formed by the dorsal aorta, genital ridge and

mesonephros)136,235. Colony-forming HSCs are first found in the AGM at E10.5136,235. These

early hematopoietic cells have been found to express known HSC markers CD117236 and

CD34237 along with αIIb. Starting at E10.5 HSCs begin to migrate to the placenta. Their

migration continues to the fetal liver at E11.5 where they develop until beginning to migrate to

the bone marrow at E17.5232. Once HSCs reach the liver, αIIb expression is down-regulated so

only a portion remains αIIb+. Due to their expression of αIIb, these important progenitor cells

may also be targeted by maternal alloantibodies in αIIb-mediated FNAIT, potentially

contributing to the severity.

38

1.7.2 HSC markers

Interestingly, αIIb integrin has been found to be associated with the onset of hematopoiesis136–143.

That is, αIIb is one of the earliest markers of hematopoietic commitment. At early stages in the

yolk sac and AGM, hematopoietic cells were found to co-express αIIb with known HSC markers

CD117236 and CD34237. By the time they reach the liver however, a portion of the cells have

started to down-regulate αIIb, so only a portion remain αIIb+ 137. Due to their expression of αIIb,

these important progenitor cells may also be targeted by maternal alloantibodies in

αIIb-mediated FNAIT.

39

Chapter 2: Rationale, Hypothesis, and Aims

2.1 Rationale

Integrins αIIb and β3 are known to have a similar number of polymorphisms in humans144–146

which might indicate that the likelihood of maternal-paternal mismatch could be comparable for

the two subunits. As previously mentioned, the reported incidence of αIIb-mediated FNAIT is

only 3-5% of cases43,53,147, compared to β3-mediated FNAIT (HPA-1a) which accounts for

75-85% in Caucasians53,144. There is difficulty in the detection of antibodies against αIIb

HPAs53,238, and so the actual incidence of αIIb-mediated FNAIT may be much greater than

currently reported. Some of our lab’s previous work found that severe bleeding seen in

β3-mediated FNAIT was due to an anti-angiogenic (anti-αVβ3) effect, rather than

thrombocytopenia88,90. This work showed that fetal development was impaired by antibody

opsonization of cell types other than platelets, such as endothelial cells and trophoblasts, which

lead to ICH, IUGR, and fetal death88,168. Although HSCs have been shown to express αIIb at

very early stages of hematopoietic commitment136–143, the possibility that maternal anti-αIIb

alloantibodies in FNAIT opsonise these cells and affect their development has not been

investigated.

2.2 Hypothesis

Maternal anti-αIIb antibodies in αIIb-mediated-FNAIT target and bind to fetal

hematopoietic stem cells causing decreased HSC populations and impaired HSC migration,

resulting in a more severe form of the disease than β3-mediated FNAIT, with a higher rate

of fetal death.

40

2.3 Specific Aims

2.3.1 Aim 1: To assess whether our anti-αIIb-mediated model of FNAIT is

more severe than our β3-mediated model and determine its

characteristics.

• After a female mouse is found to be pregnant by plug detection, designated E0.5, a loss

of >1g/day by a pregnant mouse will be considered fetal death, as will the birth of dead

pups.

• Some pregnant mice will be sacrificed at E14.5. These fetuses, their placenta and liver

will be weighed. Detection of dead pups at this time point will be recorded as fetal death.

• Some pregnant mice will be allowed to go to term and neonates sacrificed at post-natal

day one (PND1). Blood will be collected from these neonates to measure platelet count.

41

2.3.2 Aim 2: To determine whether maternal anti-αIIb antibodies can target

HSCs and determine their effects on HSC populations at E14.5 in the

yolk sac, fetal liver, and fetal blood.

• Fetal liver tissue collected from fetuses of pregnant mice sacrificed at E14.5 will be

homogenized into single cell suspensions and stained with fluorescent antibodies to

detect HSCs and murine cell-bound IgG. These samples will be analyzed by flow

cytometry to determine the degree of binding of maternal antibodies to fetal HSCs in

vivo.

• Blood, fetal liver tissue, and the yolk sac will be collected from fetuses of pregnant mice

sacrificed at E14.5. These tissues will be homogenized into single cell suspensions (if

required, i.e. not for blood) and stained with fluorescent antibodies marking HSCs. Flow

cytometric analysis will be employed to detect HSC populations in these tissues.

2.3.3 Aim 3: At PND1, study the effects of maternal anti-αIIb antibodies on

HSC populations in the neonatal liver, blood, bone marrow, lung, and

spleen.

• Liver, blood, bone marrow, lung, and spleen will be collected from neonates of pregnant

mice allowed to survive past parturition. These tissues will be homogenized into single

cell suspensions (if required, i.e. not for blood) and stained with fluorescent antibodies

marking HSCs. Flow cytometric analysis will be employed to detect HSC populations in

these tissues.

42

Chapter 3: Materials and Methods

3.1 Mice

αIIb deficient (αIIb-/-) mice were provided by Jonathan Frampton (Birmingham Medical School,

Birmingham, United Kingdom)27 and backcrossed onto a C57BL/6 background 10 times.

Genotypes of experimental animals were confirmed by polymerase chain reaction analysis.

Wild-type (WT) C57BL/6 mice (6-10 weeks old) were purchased from Charles River

Laboratories (Montreal, QC, Canada). All mice were housed in the Research Vivarium at St.

Michael’s Hospital and the Animal Care Committee approved the experimental protocols.

43

3.2 Reagents

Isoton II Diluent was purchased from Beckman Coulter Canada (Mississauga, ON, Canada).

Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, was obtained from

Sigma-Aldrich Canada (Oakville, ON, Canada). Phycoerythrin (PE)-conjugated rat anti-mouse

αIIb monoclonal antibody and purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block™)

were obtained from BD Biosciences (Mississauga, ON, Canada). Brilliant Violet 421™

(BV421)-conjugated anti-mouse CD34 antibody and a FITC-conjugated anti-mouse Lineage

(Lin) Cocktail (Components include anti-mouse CD3e, clone 145-2C11; anti-mouse Ly-6G/

Ly-6C, clone RB6-8C5; anti-mouse CD11b, clone M1/70; anti-mouse CD45R/B220, clone

RA3-6B2; and anti-mouse TER-119/Erythroid cells, clone Ter-119) were obtained from

BioLegend (San Diego, CA, USA). Allophycocyanin (APC)-conjugated rat anti-mouse CD117

(c-Kit) antibody was obtained from Thermo Fisher Scientific (Walthan, MA, USA).

44

3.3 Blood collection and platelet preparation

6-10-week-old WT mice were anesthetized by injecting 20 µL/g of 10% avertin

(2, 2, 2- tribromoethyl and tertiary amyl alcohol) and bled via the retro-orbital plexus using

heparin-coated glass capillary tubes. Blood was collected into a tube containing 100μL of the

anticoagulant 3% acid citrate dextrose (ACD; 1/9, v/v). Platelets were obtained by centrifugation

at 300x g for 5 min, collection of the buffy coat and re-addition of 400μL phosphate buffered

saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4),

followed by 300x g for 5 min and collection of the remaining buffy coat. Platelets were washed

with 10 mL PBS and pelleted at 1050x g for 12 min and re-suspended in PBS. 10μL of the

platelet solution was diluted in 240μL Isoton II Diluent, and 50μL of this solution was added to

10ml of Isoton II Diluent in an accuvette to count platelets with a Z2 Series Coulter Counter

(Beckman Coulter, Brea, CA, USA). A high concentration of platelets (108/immunization) was

used to immunize αIIb-/- female mice.

45

3.4 Animal model of αIIb-mediated FNAIT

Washed WT platelets, prepared as described above, were used to immunize αIIb-/- female mice

twice at weekly intervals. Blood was then collected from the saphenous vein for the purpose of

measuring antibody titer, described below. Immunized αIIb-/- females were then bred with WT

males to produce heterozygous pups presenting with FNAIT (Figure 4). Naïve αIIb-/- females

bred with WT males were used as controls. The day a vaginal plug was found was designated

E0.5, and body weight of pregnant females was monitored throughout pregnancy. Fetal death

was detected by weight loss of >1g per day or discovery of dead fetuses/pups.

46

Figure 4: Schematic of αIIb-mediated active FNAIT mouse model

αIIb-/- female mice were immunized with WT platelets, antibody titer measured, and bred with

WT males to generate heterozygous pups with FNAIT.

47

3.5 Antibody Titration

The hind leg of immunized αIIb-/- mice was shaved to reveal the saphenous vein. The vein was

punctured using a 23G needle and whole blood collected. After centrifugation at 10,000x g for

5 min, the sera (supernatant) was separated. Detection of total IgG was performed by serially

diluting sera from 1:50 to 1:3200. Diluted sera were incubated with 106 WT washed platelets for

one hour. Platelet-IgG samples were washed with 10mL PBS and centrifugation at 1050x g for

10 min, then labelled with FITC-conjugated goat anti-mouse IgG for one hour. After a second

wash, samples were analyzed on a Fortessa X20 (BD Biosciences). Titer was determined as the

highest dilution which still produced a noticeable increased in mean fluorescence intensity

(MFI). Sera from naïve mice was used as a control.

48

3.6 Preparation of fetal tissue

Pregnant female mice were anesthetized by injecting 20 µL/g of 10% avertin and bled via the

retro-orbital plexus using heparin-coated glass capillary tubes. Whole blood was collected to

isolate sera. The uterus was then harvested into PBS and kept on ice. Individual conceptuses

were then separated, uterus removed and yolk sac collected, and warmed with 37oC PBS to

restart the heartbeat. The umbilical cord was cut and fetal blood was collected in 30 μL

PBS/Ethylenediaminetetraacetic acid (EDTA), pH 7.4. Placenta and body mass were measured

and the fetal liver was then harvested for weighing. Using the frosted ends of two microscope

slides, the yolk sac and liver were homogenized and filtered through a 40μm cell strainer with

ice-cold PBS/EDTA. After centrifugation at 300x g for 5 min and removal of the supernatant,

yolk sac, liver, and blood samples were incubated with purified rat anti-mouse CD16/CD32

(Mouse BD Fc Block™, 1:100 dilution) in PBS/EDTA at room temperature for 20 min. Samples

were washed with 3ml PBS/EDTA by centrifugation at 300x g for 5 min, and removal of the

supernatant. This was followed by incubation with PE-conjugated rat anti-mouse αIIb

monoclonal antibody (1:100 dilution), BV421-conjugated anti-mouse CD34 antibody (1:100

dilution), and APC-conjugated rat anti-mouse CD117 (c-Kit) antibody (1:100 dilution), and

either FITC-conjugated goat anti-mouse IgG (1:100 dilution) for determination of in vivo

maternal antibody binding, or FITC-conjugated anti-mouse Lin Cocktail (1:50 dilution) to

investigate HSC populations for 45 min at room temperature. Samples were washed,

reconstituted with 400μL PBS with 5% bovine serum albumin (BSA), and filtered a second time

through a 40μm cell strainer before 20,000-100,000 events were acquired and analyzed on a

Fortessa X20 (BD Biosciences).

49

3.7 Preparation of neonatal tissues

Neonates at age PND1 were sacrificed by decapitation. 10μL whole blood was collected into

240μL PBS/EDTA for platelet enumeration as described above. Remaining blood was collected

into 10μL PBS/EDTA. Tissues (liver, lung, femur for bone marrow, and spleen) were harvested

and homogenized as described above. After centrifugation at 300x g for 5 min and removal of

the supernatant, tissue and blood samples were incubated with purified rat anti-mouse

CD16/CD32 (Mouse BD Fc Block™, 1:100 dilution) in PBS/EDTA at room temperature for

20 min. Samples were washed with 3ml PBS/EDTA by centrifugation at 300x g for 5 min, and

removal of the supernatant. This was followed by incubation with PE-conjugated rat anti-mouse

αIIb monoclonal antibody (1:100 dilution), BV421-conjugated anti-mouse CD34 antibody

(1:100 dilution), and APC-conjugated rat anti-mouse CD117 (c-Kit) antibody (1:100 dilution),

and FITC-conjugated anti-mouse Lin Cocktail (1:50 dilution) for 45 min at room temperature.

Samples were washed, reconstituted with 400μL PBS with 5% BSA, and filtered a second time

through a 40μm cell strainer before 20,000-100,000 events were acquired and analyzed on a

Fortessa X20 (BD Biosciences).

50

3.8 Flow cytometry gating strategy

Fetal liver samples obtained as described above were prepared by homogenizing, filtering,

washing, blocking with purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block™, 1:100

dilution) in PBS/EDTA at room temperature for 20 min, and washing a second time. Samples

were then either left unstained; stained with only one of PE-conjugated rat anti-mouse αIIb

monoclonal antibody (1:100 dilution), BV421-conjugated anti-mouse CD34 antibody (1:100

dilution), and APC-conjugated rat anti-mouse CD117 (c-Kit) antibody (1:100 dilution), and

FITC-conjugated anti-mouse Lin Cocktail (1:50 dilution); or all but one of the described

fluorescent antibodies for fluorescence-minus-one (FMO) controls. These samples were washed,

reconstituted with 400μL PBS with 5% BSA, and filtered a second time through a 40μm cell

strainer before 10,000 events were acquired and analyzed on a Fortessa X20 (BD Biosciences).

The samples in Figure 5 were used to create the gates for further analysis.

51

Figure 5: Gating strategy for flow cytometric analyses

Tissue samples were prepared by homogenizing, filtering, washing, blocking with Fc Block™,

washing, incubating with Mouse IgG-FITC, Lineage (Lin)-FITC, CD34-BV421, CD117-APC,

and/or αIIb-PE, washing, and filtering before samples were acquired and analyzed by flow

cytometry. Single stained and fluorescence-minus-one (FMO) stained tissues were employed to

create the gates to denote FITC+, FITC-, BV421+, APC+, and PE+ cells populations which were

used for further analysis of maternal antibody binding and hematopoietic stem cell (HSC)

populations. Lineage (differentiated blood cell) markers included anti-CD3e, anti-Ly-6G/Ly-6C,

anti-CD11b, anti-CD45R/B220, and anti-TER-119.

52

3.9 Statistical analysis

Data are presented as mean ± SD or SEM as stated. Statistical significance was assessed by a

Student’s unpaired t test (2 tailed) using Excel software (Microsoft Office). A P value of 0.05 or

less was considered significant.

53

Chapter 4: Results

4.1 Aim 1: Characterization of αIIb-mediated FNAIT and comparison

with β3-mediated FNAIT

4.1.1 Immunized mice generated a sufficient antibody titer for the active αIIb-

mediated FNAIT model

Female αIIb-/- mice were immunized twice, once weekly, with WT platelets to initiate an

immune response. Sera was collected one week following the second immunization, diluted, and

incubated with WT platelets and then FITC-conjugated anti-mouse antibodies for detection of

antibody titer by flow cytometry. Antibodies were generated and detected (Figure 6). The

antibody titer generated was deemed sufficient to cause FNAIT in heterozygous pups from

pairings with WT male mice due to a significant decrease in platelet counts in these offspring

(Figure 7).

54

Figure 6: Antibody generation in naïve and immunized αIIb-/- mice

Antibody titers were measured in either naïve or immunized αIIb-/- mice by serial dilution of sera

from 1:50 to 1:3200, incubation with wild type platelets, and staining with FITC-conjugated goat

anti-mouse IgG. Mean fluorescence intensity (MFI) in the FITC channel was used to determine

the highest dilution at which there was still a noticeable increase, and this was designated as the

antibody titer. Two rounds of immunization generated an immune response capable of causing

FNAIT in heterozygous pups (active FNAIT model). Control (Naïve) n = 9, Immunized n = 13;

Error bars are ±SEM.

55

4.1.2 Platelet count was significantly decreased in FNAIT PND1 neonates

compared to naïve controls

Immunized αIIb-/- female mice were bred with WT males to produce heterozygous FNAIT pups.

Naïve αIIb-/- female mice bred with WT males were used as controls. Pups were sacrificed at

PND1 and whole blood was collected. Whole blood was used to determine platelet counts.

FNAIT pups born to immunized αIIb-/- mothers were significantly decreased compared to those

from pups delivered by naïve αIIb-/- mothers (Figure 7).

56

Figure 7: Significantly decreased platelet count in FNAIT PND1 neonates

Whole blood was collected from post-natal day 1 (PND1) neonates and platelet count measured

using a Z2 Series Coulter Counter. As expected, FNAIT neonates present with significantly

lower platelet counts than their naïve control counterparts. Control (Naïve) n = 16 neonates,

3 pregnancies; FNAIT (Immunized) n = 35 neonates, 5 pregnancies; **** = P < 0.001.

Error bars are ±SD.

57

4.1.3 Fetal death rate in αIIb-mediated FNAIT is significantly higher than β3-

mediated FNAIT

Fetal death was detected by either weight loss of pregnant mothers of >1 g/day, or observation of

dead fetuses when pregnant mice were sacrificed at E14.5. Delivery of stillborn pups was also

counted as fetal death when it was observed. Fetal death rates were recorded in four different

scenarios: immunized αIIb-/- female mice crossed with WT male mice (αIIb-mediated FNAIT),

naïve αIIb-/- female mice crossed with WT male mice (αIIb control), immunized β3-/- female

mice crossed with WT male mice (β3-mediated FNAIT), and naïve β3-/- female mice crossed

with WT male mice (β3 control). The rate of fetal death observed in αIIb-mediated FNAIT (n =

8/13 = 61.5%) was much greater than the rate observed in β3-mediated FNAIT

(n = 12/22 = 35%). When taking the genetic background of these mice into account, the αIIb-/-

mice were C57BL/6 background and the β3-/- mice were BALB/c background, the relative rates

of fetal death between these two forms of the disease to their controls were significantly different

with αIIb-mediated FNAIT resulting in more fetal death (Figure 8; n = 13-34 pregnancies/group;

X2 relative rates of fetal death = 4.47; P < 0.05).

58

Figure 8: Significantly increased fetal death rates in αIIb-mediated versus

β3-mediated FNAIT

Fetal death was determined either by loss of >1g/day in a pregnant mouse, delivery of dead pups,

or discovery dead fetuses or reabsorbed conceptuses at E14.5. αIIb-mediated FNAIT data was

compared with our lab’s previously published β3-mediated FNAIT data52,53. X2 relative rates of

fetal death = 4.47; P < 0.05; n = 13-34 pregnancies/group.

59

4.1.4 FNAIT fetuses experience bleeding and death

Some control and immunized pregnant female mice were sacrificed at E14.5 to investigate

phenomena occurring at this time point. Uteri were collected from these mice and imaged before

separating and imaging each fetus. Representative images are shown below in Figure 9. Control

fetuses appeared light pink with a dark red liver and distinct eyeballs (Figure 9A). Live FNAIT

fetuses often presented with bleeding which is lighter red in colour (Figure 9C, yellow arrows).

Dead FNAIT pups either appeared as small red pockets within the uterus (Figure 9B, red arrows)

which indicated early fetal death, or as discoloured fetuses which have just begun to be

reabsorbed (Figure 9D). Both live and recently deceased FNAIT fetuses were observed to have

placentas which were lighter in colour than those of the relatively healthier control fetuses.

60

Figure 9: Bleeding and fetal death of FNAIT fetuses

Fetuses and placenta were harvested from naïve (control) or immunized (FNAIT) pregnant

female mice at E14.5 and imaged, these images are representative. (A) Control live E14.5 fetuses

and placenta, (B) Early fetal death (FNAIT uterus, red arrows) (C) FNAIT live fetuses with

bleeding (yellow arrows) and placenta (D) FNAIT deceased fetuses and placenta.

61

4.1.5 E14.5 FNAIT fetuses had significantly decreased body, placenta, and liver

masses

Live E14.5 FNAIT and control fetuses and placentas collected from naïve and immunized αIIb-/-

pregnant female mice were weighed, fetuses were dissected to collect and mass the liver. FNAIT

fetuses had decreased body (P < 0.001), placenta (P < 0.005), and liver (P < 0.001) masses when

compared to naïve controls (Figure 10). These significant decreases are indicative of IUGR.

62

Figure 10: Significantly decreased body, placenta, and liver mass of E14.5 FNAIT

fetuses

αIIb-/- pregnant female mice were sacrificed at E14.5 to harvest fetal tissues. Fetal (A) body,

(B) placenta, and (C) liver masses were recorded. Miscarried fetuses were not included. Control

(Naïve) n = 27 fetuses, 4 pregnancies; FNAIT (Immunized) n = 20 fetuses, 3 pregnancies;

*** = P < 0.005, **** = P < 0.001. Error bars are ±SD.

63

4.2 Aim 2: Targeting of fetal HSCs by maternal antibodies and their

effects on HSC populations

4.2.1 Maternal antibodies bound to fetal liver tissue in vivo

Livers of live E14.5 FNAIT and control fetuses collected from naïve and immunized αIIb-/-

pregnant female mice were homogenized to be prepared as single cell suspensions. These

preparations were then stained with fluorescent antibodies for CD34-BV421, CD117-APC, αIIb-

PE, and mouse IgG-FITC for flow cytometric analysis, where HSCs were considered

CD34+CD117+ cells. This was to test for in vivo binding of maternal antibodies to fetal liver

tissues. There was a significant increase (P < 0.001) in the percentage of HSCs bound by

maternal antibodies from the control (n = 7 fetuses, 1 pregnancy) to FNAIT liver tissue (n = 16

fetuses, 2 pregnancies) (Figure 11A). Figure 11B displays a representative overlaid histogram of

HSC populations of control and FNAIT samples.

64

Figure 11: Maternal antibodies were capable of binding to fetal liver tissue

Livers were collected from E14.5 fetuses and were prepared by homogenizing, filtering,

washing, blocking with Fc Block™, washing, incubating with CD34-BV421, CD117-APC, αIIb-

PE, and mouse IgG-FITC, washing, filtering to be acquired and analyzed by flow cytometry.

HSCs were considered CD34+CD117+ cells. (A) Significant increase in percentage of αIIb+

HSCs bound with maternal IgG, averaged from control (n = 7 fetuses, 1 pregnancy) and FNAIT

(n = 16 fetuses, 2 pregnancies) fetuses; **** = P < 0.001. Error bars are ±SEM.

(B) Representative overlaid histogram of HSC populations of control (Naïve) and FNAIT

(Immunized) samples.

65

4.2.2 No significant difference in non-blood cell populations in the liver or yolk

sac of E14.5 FNAIT fetuses compared to controls

Fetal liver and yolk sac samples collected from E14.5 FNAIT and control fetuses were prepared

as single cells suspensions and stained with fluorescent antibodies for Lin-FITC, CD34-BV421,

CD117-APC, and αIIb-PE for analysis by flow cytometry. These samples were analyzed to

investigate populations of HSCs; the same cytometric data (.fcs files) were analyzed to exclude

the possibility of confounding effects on a separate, unrelated, αIIb- cell population. The Lin-

population was chosen as it excludes only blood cells, so its percentage of total cells should be

high and relatively stable. As expected, no significant difference was observed in the Lin- cell

population as a percentage of total cells in the liver or lung between E14.5 FNAIT and control

fetuses (Figure 12).

66

Figure 12: Non-blood cell populations remained stable in the liver and yolk sac of

E14.5 FNAIT and control fetuses

Fetal liver and yolk sac samples obtained as described above were prepared by homogenizing,

filtering, washing, blocking with Fc Block™, washing, incubating with Lineage-FITC,

CD34-BV421, CD117-APC, and αIIb-PE, washing, filtering to be acquired and analyzed by flow

cytometry. (A) No significant difference in Lineage- cells as a percentage of total cells in the

liver of E14.5 fetuses from control to FNAIT. Control (Naïve) n = 52 fetuses, 9 pregnancies;

FNAIT (Immunized) n = 97 fetuses, 13 pregnancies. (B) No significant difference in Lineage-

cells as a percentage of total cells in the yolk sac of E14.5 fetuses from control to FNAIT.

Control (Naïve) n = 42 fetuses, 6 pregnancies; FNAIT (Immunized) n = 84 fetuses,

12 pregnancies. ns = not significant. Error bars are ±SEM. Lineage (differentiated blood cell)

markers included anti-CD3e, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and

anti-TER-119.

67

4.2.3 E14.5 yolk sac total and αIIb+ HSC populations were significantly increased

in FNAIT fetuses compared to controls

The yolk sac is the first location in the embryo where HSCs can be found, as early as E7.5 in

murine gestation. Yolk sac samples collected from E14.5 FNAIT and control fetuses were

prepared as single cells suspensions and stained with fluorescent antibodies for Lin-FITC,

CD34-BV421, CD117-APC, and αIIb-PE to be analyzed by flow cytometry. HSCs were

identified as Lin-CD34+CD117+ cells, with a subset of αIIb+ HSCs. Surprisingly, both total (P <

0.005) and αIIb+ (P < 0.05) HSC populations were significantly increased in FNAIT pups when

compared with controls (Figure 13A). The proportion of αIIb+ HSCs, however, remained

unchanged (Figure 13B).

68

Figure 13: Total and αIIb+ HSC populations were increased in the yolk sac of

E14.5 FNAIT and control fetuses

Fetal yolk sac samples obtained as described above were prepared by homogenizing, filtering,

washing, blocking with Fc Block™, washing, incubating with Lineage-FITC, CD34-BV421,

CD117-APC, and αIIb-PE, washing, and filtering for flow cytometric analysis. HSCs were

considered Lineage-CD34+CD117+ cells. (A) Significant increase in Total and αIIb+ HSC

populations as a percentage of total cells in the yolk sac of E14.5 fetuses from control to FNAIT.

(B) No significant difference in the fraction of αIIb+ HSCs as a percentage of total HSCs in the

yolk sac of E14.5 fetuses from control to FNAIT. Control (Naïve) n = 42 fetuses, 6 pregnancies;

FNAIT (Immunized) n = 84 fetuses, 12 pregnancies; * = P < 0.05, *** = P < 0.005, ns = not

significant. Error bars are ±SEM. Lineage (differentiated blood cell) markers included anti-

CD3e, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

69

4.2.4 Total and αIIb+ HSC populations in the liver of E14.5 FNAIT fetuses

remained similar to controls

HSCs begin migration to the murine fetal liver at E11.5 and only start migrating to the bone

marrow at E17.5. Fetal liver tissue samples were collected from E14.5 FNAIT and control

fetuses, prepared as single cells suspensions and stained with fluorescent antibodies for

Lin-FITC, CD34-BV421, CD117-APC, and αIIb-PE to be analyzed by flow cytometry. HSCs

were identified as Lin-CD34+CD117+ cells, with a subset of αIIb+ HSCs. The total HSC

population (not significant, ns) and αIIb+ HSC population (ns) as a percentage of total cells were

comparable between FNAIT and control fetuses (Figure 14A). The proportion of total HSCs

which were αIIb+ also remained unchanged (Figure 14B).

70

Figure 14: No significant difference in total and αIIb+ HSC populations in the liver

of E14.5 FNAIT fetuses compared to controls

Fetal liver samples obtained as described above were prepared by homogenizing, filtering,

washing, blocking with Fc Block™, washing, incubating with Lineage-FITC, CD34-BV421,

CD117-APC, and αIIb-PE, washing, filtering to be acquired and analyzed by flow cytometry.

HSCs were considered Lineage-CD34+CD117+ cells. (A) No significant difference in Total and

αIIb+ HSC populations as a percentage of total cells in the liver of E14.5 fetuses from control to

FNAIT. (B) No significant difference in the fraction of αIIb+ HSCs as a percentage of total HSCs

in the liver of E14.5 fetuses from control to FNAIT. Control (Naïve) n = 52 fetuses,

9 pregnancies; FNAIT (Immunized) n = 97 fetuses, 13 pregnancies; ns = not significant.

Error bars are ±SEM. Lineage (differentiated blood cell) markers included anti-CD3e,

anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

71

4.2.5 The blood of E14.5 FNAIT fetuses had significantly increased total and

αIIb+ HSC populations compared to controls

HSCs migrate several times throughout gestation and they are carried between their niches by the

blood. Blood samples collected from E14.5 FNAIT and control fetuses were treated with PBS-

EDTA in order to maintain them as single cells suspensions and stained with fluorescent

antibodies for Lin-FITC, CD34-BV421, CD117-APC, and αIIb-PE to be analyzed by flow

cytometry. HSCs were identified as Lin-CD34+CD117+ cells, with a subset of αIIb+ HSCs. The

total (P < 0.05) and αIIb+ (P < 0.005) HSC populations as a percentage of total cells were

significantly increased compared to naïve controls (Figure 15A). The αIIb+ subset of total HSCs

was also significantly increased (P < 0.05, Figure 15B).

72

Figure 15: Total and αIIb+ HSC populations were significantly increased in the

blood of E14.5 FNAIT fetuses compared to controls

Fetal blood samples obtained as described above were prepared by blocking with Fc Block™,

washing, incubating with Lineage-FITC, CD34-BV421, CD117-APC, and αIIb-PE, washing,

filtering to be acquired and analyzed by flow cytometry. HSCs were considered Lineage-

CD34+CD117+ cells. (A) Significant increase in Total and αIIb+ HSC populations as a

percentage of total cells in the blood of E14.5 fetuses from control to FNAIT. (B) Significant

increase in the fraction of αIIb+ HSCs as a percentage of total HSCs in the blood of E14.5 fetuses

from control to FNAIT. Control (Naïve) n = 6 fetuses, 1 pregnancy; FNAIT (Immunized)

n = 46 fetuses, 6 pregnancies; * = P < 0.05, *** = P < 0.005. Error bars are ±SEM.

Lineage (differentiated blood cell) markers included anti-CD3e, anti-Ly-6G/Ly-6C, anti-CD11b,

anti-CD45R/B220, and anti-TER-119.

73

4.3 Aim 3: The effect of maternal antibodies on neonatal HSC

populations

4.3.1 Lin- populations in the liver and lung of PND1 neonates were preserved

between FNAIT and controls

Fetal liver and lung samples collected from PND1 FNAIT and control neonates were prepared as

single cells suspensions and stained with fluorescent antibodies for Lin-FITC, CD34-BV421,

CD117-APC, and αIIb-PE to be analyzed by flow cytometry. These samples were analyzed to

investigate populations of HSCs; the same cytometric data (.fcs files) were analyzed in order to

exclude the possibility of confounding effects on a separate, unrelated, αIIb- cell population. The

Lin- population was chosen as it excludes only blood cells, so its percentage of total cells should

be high and relatively stable. As expected, no significant difference was observed in the Lin- cell

population as a percentage of total cells in the liver or lung between PND1 FNAIT and control

neonates (Figure 16).

74

Figure 16: No significant difference in non-blood cell populations in the liver or

lung of PND1 FNAIT neonates compared to controls

Neonatal liver and lung samples obtained as described above were prepared by homogenizing,

filtering, washing, blocking with Fc Block™, washing, incubating with Lineage-FITC, CD34-

BV421, CD117-APC, and αIIb-PE, washing, filtering to be acquired and analyzed by flow

cytometry. (A) No significant difference in Lineage- cells as a percentage of total cells in the

liver of PND1 neonates from control to FNAIT. Control (Naïve) n = 27 neonates, 5 pregnancies;

FNAIT (Immunized) n = 46 neonates, 6 pregnancies. (B) No significant difference in Lineage-

cells as a percentage of total cells in the lung of PND1 neonates from control to FNAIT. Control

(Naïve) n = 21 neonates, 4 pregnancies; FNAIT (Immunized) n = 46 neonates, 6 pregnancies.

ns = not significant. Error bars are ±SEM. Lineage (differentiated blood cell) markers included

anti-CD3e, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

75

4.3.2 PND1 FNAIT neonates had an increased liver αIIb+ HSC population

compared to controls

By PND1, the liver is no longer the main niche of HSCs, however some HSCs do still remain

here. Liver samples collected from PND1 FNAIT and control neonates were prepared as single

cells suspensions and stained with fluorescent antibodies for Lin-FITC, CD34-BV421,

CD117-APC, and αIIb-PE to be analyzed by flow cytometry. HSCs were identified as

Lin-CD34+ CD117+ cells, with a subset of αIIb+ HSCs. Although there was no significant

difference in the total HSC population between the FNAIT and control neonates (ns), the

percentage of αIIb+ HSCs of total cells was significantly increased (P < 0.001, Figure 17A). The

proportion of αIIb+ HSCs within the total HSC population was correspondingly significantly

increased (P < 0.001, Figure 17B).

76

Figure 17: αIIb+ HSC population was significantly increased in the liver of PND1

FNAIT neonates compared to controls

Neonatal liver samples obtained as described above were prepared by homogenizing, filtering,

washing, blocking with Fc Block™, washing, incubating with Lineage-FITC, CD34-BV421,

CD117-APC, and αIIb-PE, washing, filtering to be acquired and analyzed by flow cytometry.

HSCs were considered Lineage-CD34+CD117+ cells. (A) Significant increase in αIIb+ HSC

population as a percentage of total cells in the liver of PND1 neonates from control to FNAIT.

(B) Significant increase in the fraction of αIIb+ HSCs as a percentage of total HSCs in the liver

of PND1 neonates from control to FNAIT. Control (Naïve) n = 27 neonates, 5 pregnancies;

FNAIT (Immunized) n = 46 neonates, 6 pregnancies; **** = P < 0.001, ns = not significant.

Error bars are ±SEM. Lineage (differentiated blood cell) markers included anti-CD3e,

anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

77

4.3.3 PND1 FNAIT neonates had significantly decreased total HSCs in the blood

compared to controls

HSCs, when migrating, can be found in the blood; they are not often found here under other

circumstances. Blood samples collected from PND1 FNAIT and control neonates were treated

with PBS-EDTA in order to maintain them as single cells suspensions, and then stained with

fluorescent antibodies for Lin-FITC, CD34-BV421, CD117-APC, and αIIb-PE to be analyzed by

flow cytometry. HSCs were identified as Lin-CD34+CD117+ cells, with a subset of αIIb+ HSCs.

While total HSCs as a percentage of total cells was significantly decreased (P < 0.01), the αIIb+

HSC populations within total cells was similar between the FNAIT and control neonates

(ns, Figure 18A). The proportion of αIIb+ HSCs as a percentage of total HSCs was significantly

increased (P < 0.005, Figure 18B).

78

Figure 18: Total HSCs in the blood of PND1 FNAIT neonates were significantly

decreased compared to controls

Neonatal blood samples obtained as described above were prepared by blocking with Fc

Block™, washing, incubating with Lineage-FITC, CD34-BV421, CD117-APC, and αIIb-PE,

washing, filtering to be acquired and analyzed by flow cytometry. HSCs were considered

Lineage-CD34+CD117+ cells. (A) Significant decrease in total HSC population as a percentage of

total cells in the blood of PND1 neonates from control to FNAIT. (B) Significant increase in the

fraction of αIIb+ HSCs as a percentage of total HSCs in the blood of PND1 neonates from

control to FNAIT. Control (Naïve) n = 16 neonates, 3 pregnancies; FNAIT (Immunized)

n = 35 neonates, 5 pregnancies; ** = P < 0.01, *** = P < 0.005, ns = not significant. Error bars

are ±SEM. Lineage (differentiated blood cell) markers included anti-CD3e, anti-Ly-6G/Ly-6C,

anti-CD11b, anti-CD45R/B220, and anti-TER-119.

79

4.3.4 HSC populations of PND1 FNAIT neonates were significantly decreased in

the bone marrow compared to controls

The bone marrow is the major adult HSC niche. During murine development, HSCs begin to

migrate here at E17.5. By PND1, the vast majority of HSCs should be found in the bone marrow

stem cell niche. Bone marrow samples collected from PND1 FNAIT and control neonates were

prepared as single cells suspensions and stained with fluorescent antibodies for Lin-FITC,

CD34-BV421, CD117-APC, and αIIb-PE to be analyzed by flow cytometry. HSCs were

identified as Lin-CD34+ CD117+ cells, with a subset of αIIb+ HSCs. All populations of HSCs,

total (P < 0.05) and αIIb+ (P < 0.05) as percentages of total cells (Figure 19A), and αIIb+

HSCs as a percentage of total HSCs (P < 0.05, Figure 19B) were significantly decreased between

FNAIT and control neonates.

80

Figure 19: Total and αIIb+ HSC populations were significantly decreased in the

bone marrow of PND1 FNAIT neonates compared to controls

Neonatal bone marrow samples obtained as described above were prepared by homogenizing,

filtering, washing, blocking with Fc Block™, washing, incubating with Lineage-FITC,

CD34-BV421, CD117-APC, and αIIb-PE washing, filtering to be acquired and analyzed by flow

cytometry. HSCs were considered Lineage-CD34+CD117+ cells. (A) Significant decrease in

Total and αIIb+ HSC population as a percentage of total cells in the bone marrow of PND1

neonates from control to FNAIT. (B) Significant decrease in the fraction of αIIb+ HSCs as a

percentage of total HSCs in the bone marrow of PND1 neonates from control to FNAIT. Control

(Naïve) n = 28 neonates, 5 pregnancies; FNAIT (Immunized) n = 34 neonates, 5 pregnancies;

* = P < 0.05. Error bars are ±SEM. Lineage (differentiated blood cell) markers included

anti-CD3e, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

81

4.3.5 Significant increases of total and αIIb+ HSC populations in the lung of

PND1 FNAIT neonates compared to controls

The lung, although not considered a hematopoietic organ, has recently been shown to be a site of

significant platelet biogenesis and a reservoir for HSCs2. Thus, it was an interesting organ to

investigate during the pathogenesis of αIIb-mediated FNAIT in the context of the hypothesis of

this project. Lung tissue samples collected from PND1 FNAIT and control neonates were

prepared as single cells suspensions and stained with fluorescent antibodies for Lin-FITC,

CD34-BV421, CD117-APC, and αIIb-PE to be analyzed by flow cytometry. HSCs were

identified as Lin-CD34+ CD117+ cells, with a subset of αIIb+ HSCs. Surprisingly, all populations

of HSCs; total (P < 0.01) and αIIb+ (P < 0.01) as percentages of total cells (Figure 20A), and

αIIb+ HSCs as a percentage of total HSCs (P < 0.001, Figure 20B); were significantly increased

between FNAIT and control neonates.

82

Figure 20: Total and αIIb+ HSC populations were significantly increased in the

lung of PND1 FNAIT neonates compared to controls

Neonatal lung samples obtained as described above were prepared by homogenizing, filtering,

washing, blocking with Fc Block™, washing, incubating with Lineage-FITC, CD34-BV421,

CD117-APC, and αIIb-PE, washing, filtering to be acquired and analyzed by flow cytometry.

HSCs were considered Lineage-CD34+CD117+ cells. (A) Significant increase in the Total and

αIIb+ HSC populations as a percentage of total cells in the lung of PND1 neonates from control

to FNAIT. (B) Significant increase in the fraction of αIIb+ HSCs as a percentage of total HSCs in

the lung of PND1 neonates from control to FNAIT. Control (Naïve) n = 21 neonates,

4 pregnancies; FNAIT (Immunized) n = 46 neonates, 6 pregnancies; ** = P < 0.01,

**** = P < 0.001. Error bars are ±SEM. Lineage (differentiated blood cell) markers included

anti-CD3e, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

83

4.3.6 Total and αIIb+ HSC populations in the spleen of PND1 FNAIT neonates

were significantly increased compared to controls

As a site of clearance of opsonized cells and particles, the spleen is highly involved in

immunosurveillance. Along with its role in the immune system, it does normally house a small

number of hematopoietic progenitors which mainly produce B lymphoid cells. Splenic tissue

samples collected from PND1 FNAIT and control neonates were prepared as single cells

suspensions and stained with fluorescent antibodies for Lin-FITC, CD34-BV421, CD117-APC,

and αIIb-PE to be analyzed by flow cytometry. HSCs were identified as Lin-CD34+ CD117+

cells, with a subset of αIIb+ HSCs. Unexpectedly, both total (P < 0.05) and αIIb+ (P < 0.05) HSC

populations as percentages of total cells were significantly increased in FNAIT neonates

(Figure 21A). However, the proportion of αIIb+ HSC population of total HSCs (ns, Figure 21B)

remained unchanged between FNAIT and control neonates.

84

Figure 21: Total and αIIb+ HSC populations were significantly increased in the

spleen of PND1 FNAIT neonates compared to controls

Neonatal spleen samples obtained as described above were prepared by homogenizing, filtering,

washing, blocking with Fc Block™, washing, incubating with Lineage-FITC, CD34-BV421,

CD117-APC, and αIIb-PE, washing, filtering to be acquired and analyzed by flow cytometry.

HSCs were considered Lineage-CD34+CD117+ cells. (A) Significant increase in total and αIIb+

HSC populations as a percentage of total cells in the spleen of PND1 neonates from control to

FNAIT. (B) No significant difference in the fraction of αIIb+ HSCs as a percentage of total HSCs

in the spleen of PND1 neonates from control to FNAIT. Control (Naïve) n = 17 neonates,

3 pregnancies; FNAIT (Immunized) n = 42 neonates, 6 pregnancies; * = P < 0.05, ns = not

significant. Error bars are ±SEM. Lineage (differentiated blood cell) markers included

anti-CD3e, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220, and anti-TER-119.

85

Chapter 5: Discussion

The objective of Aim 1 was to assess whether our anti-αIIb-mediated model of FNAIT is more

severe than our β3-mediated model and determine its characteristics. The model utilizes WT

platelet transfusion to facilitate a maternal immune response. It was found that two rounds of

weekly platelet transfusion generated a sufficient antibody titer for the purpose of causing

FNAIT in αIIb heterozygous pups (Figure 6). As confirmation, platelet enumeration was

performed on the PND1 neonates from the FNAIT model and control pregnancies (Figure 7) to

ensure the FNAIT neonates indeed had a significantly decreased platelet count.

Miscarriage is a devastating outcome of severe FNAIT. Unfortunately, αIIb-/- female mice lose

pregnancies even under normal, non-diseased conditions. To take this into account, rates of fetal

death were measured in the αIIb-mediated FNAIT model and a naïve αIIb control group to be

compared to previous data52,53 for the β3-mediated FNAIT model and a naïve β3 control group.

Naïve β3 mice did not appear to have difficulty carrying a pregnancy to term. Even when taking

into account the baseline fetal death rate for these two strains of mice, the αIIb-mediated FNAIT

model had a higher rate of fetal death than that of the β3-mediated model (Figure 8). This shows

that αIIb-mediated FNAIT is a more severe form of the disease and opens the potential that

miscarriage may account for a significant portion of non-reported cases. Hence, due to

underreporting, αIIb-mediated FNAIT may be more prevalent than current data suggests.

αIIb-mediated FNAIT, besides causing a significantly decreased platelet count, also caused other

noticeable symptoms. Severe bleeding and early, otherwise undetectable fetal death was

observed when pregnant females were sacrificed at E14.5 (Figure 9). Fetuses still alive at this

time point had significantly decreased body, placenta, and liver masses than their naïve control

86

counterparts (Figure 10). These results indicate that αIIb-mediated FNAIT fetuses experience

IUGR, severe bleeding, and a high risk of miscarriage.

Aim 2 focused on the targeting of fetal HSCs by maternal antibodies and their effects on fetal

HSC populations. It was imperative for this project to determine whether maternal anti-αIIb

antibodies were capable of binding to fetal HSCs. E14.5 fetal liver samples were prepared from

both naïve and FNAIT group fetuses. Antibody binding to HSCs was determined through

staining for αIIb+ HSCs and mouse IgG followed by analysing cells with flow cytometry. There

was a significant increase in the binding of mouse IgG to fetal HSCs from the FNAIT group

(Figure 11), meaning that maternal anti-αIIb antibodies are able to bind to fetal HSCs in vivo.

The second part of Aim 2 examined the effects of maternal anti-αIIb antibodies on HSC

populations in the E14.5 fetal yolk sac, liver and blood. First, to exclude the possibility of

confounding effects on αIIb- cell populations, the non-blood cell Lin- population was

investigated (Figure 12). There was no significant difference between FNAIT and control fetuses

in this population, reducing the risk that the following observations are false. It was found that

there were significant increases in total and αIIb+ HSCs as a percentage of total cells in the yolk

sac (Figure 13) and significant increases in these populations and the αIIb+ HSC population as a

percentage of HSCs in the fetal blood (Figure 15). In the fetal liver, however, there was no

significant change in any HSC populations (Figure 14).

Since the HSC populations in the liver were not significantly different, but those in both the yolk

sac and blood were significantly increased, this may indicate an overall increase in the total HSC

population in FNAIT pups; a surprising result. One possible explanation is that the maternal anti-

αIIb antibodies caused increased proliferation of these cells. It has been previously demonstrated

by our lab that certain monoclonal anti-GPIbα antibodies can activate platelets56,57. Therefore it

87

may be possible for these anti-αIIb antibodies to be activating HSC proliferation. Another

possibility is that the anti-αIIb antibodies are impairing HSC migration to their niche in the liver.

The stem cell niche is an important factor in the growth, development, and control of HSC

populations, in that the niche provides local signals and gradients to help guide HSCs in the

process of differentiation and self-renewal58-60. In the absence of signals from the niche, stem

cells have been known to proliferate until exhaustion where the lack of cytokine and chemokine

signals results in impaired self-renewal61. In this way, maternal anti-αIIb antibodies may be

indirectly causing increased HSC proliferation by impairing their ability to migrate to the fetal

liver.

The apparent increase in the HSC population could also be due to decreased differentiation or

increased self-renewal of the stem cells. Only 3 organs of the fetus were examined for HSCs,

thus the overall effect on HSCs is not clear. Despite this, it is odd that the HSC populations were

only significantly increased at E14.5 in the yolk sac and blood, but not the liver. The increase in

fetal blood alone is an indication of impaired migration since HSCs are usually only in the blood

while transitioning to a new location. Increased HSC populations in the yolk sac and blood may

mean that these cells are not appropriately transiting their new niche in the liver.

Another argument for impaired migration comes from the function of αIIbβ3 integrin. On

platelets, it is responsible for platelet aggregation through its interaction with fibrinogen and its

other ligands4,6,30,33. The function of αIIbβ3 integrin on HSCs is unknown but based on its role

on the surface of platelets, it may aid in HSC adhesion to the cells of their niche. Maternal

anti-αIIb antibodies may be able to block this interaction, resulting in impaired migration. The

degree to which αIIbβ3 integrin may contribute to cell-to-cell adhesion would then influence the

extent of impairment. With impaired migration, a decrease in the HSC population of the fetal

88

liver would be expected. It remained unchanged, however, and in the face of increased

populations elsewhere, this may indicate that αIIbβ3 integrin plays a small role in migration.

Another possible explanation for the lack of decreased HSC population in the liver is that they

may be chaperoned here by immature macrophages. Since fetal HSCs are opsonized by maternal

anti-αIIb antibodies, this means that they can be targeted by mononuclear phagocytes for

phagocytosis and clearance. In an adult, mononuclear phagocytes transit to the spleen in order to

act as professional APCs for lymphocytes62. This is part of a complex system known as the

mononuclear phagocyte system (MPS)62. During embryonic development, however, the spleen is

not fully formed until E16.563. Little is known about embryonic development of the MPS, and so

the possibility exists that clearance of these opsonized cells may be occurring in the liver, which

at E14.5 is a hotbed for hematopoietic activity. Therefore, clearance of opsonized HSCs in the

liver may be masking the expected decrease in liver HSC populations from impaired migration.

The purpose of Aim 3 was to determine the effects of maternal anti-αIIb antibodies on neonatal

HSC populations. Again, to exclude confounding effects of these antibodies on αIIb- cell

populations, the Lin- population was examined (Figure 16) and no significant differences were

observed between the FNAIT and control neonates. In the neonatal liver, there was no significant

difference in the total HSC populations between the FNAIT and control groups, however the

αIIb+ population as a percentage of total cells and as a percentage of HSCs was increased

(Figure 17). This may be an indication of impaired migration of the αIIb+ HSCs. Further

indication of impaired migration of αIIb+ HSCs comes from the neonatal blood where despite a

significant decrease in the total HSC population (Figure 18A), the αIIb+ portion of that

population was significantly increased (Figure 18B) in addition to significant decreases in all

measured HSC populations in the neonatal bone marrow (Figure 19). The significant decreases

89

in the bone marrow populations may also be evidence of HSC exhaustion from earlier

(potentially observed at E14.5) uncontrolled proliferation. Surprisingly, there was a significant

increase in all HSC populations in the neonatal lung (Figure 20). The lung has recently been

identified as a major contributor to platelet production and a reservoir of hematopoietic

progenitors64. Migration of hematopoietic cells to the lung may be mediated by a unique set of

proteins (i.e. not involving αIIbβ3 integrin), enabling migration to this newly uncovered

hematopoietic organ. Another unexpected result was the increased total and αIIb+ populations in

the spleen (Figure 21A). This may be similar to the masking of the expected decrease in fetal

liver HSC populations since the spleen, at this point in development, is now the main organ of

clearance for the MPS63. There was no change in the proportion of αIIb+ HSCs relative to total

HSCs (Figure 21B), however, and so this could be a sign that HSCs are migrating to the spleen

in the same or a similar way to the lung rather than as a mechanism of clearance. Further

investigation is required to robustly determine the effect of maternal anti-αIIb antibodies on

HSCs and the mechanisms by which changes in HSC populations are executed.

90

Conclusions

The results of this investigation indicate that αIIb-mediated FNAIT is a more serious version of

this disease when compared with β3-mediated FNAIT due to a higher rate of fetal death. It is

also characterized by severe bleeding and IUGR in addition to the decreased platelet count. This

higher rate of fetal death means that >50% of αIIb-mediated FNAIT cases may go unreported

and it is likely significantly more prevalent than current data suggest.

This report also reveals an intriguing new pathology of the disease. Maternal anti-αIIb antibodies

that cross the placenta can bind to fetal HSCs in vivo and likely cause impaired migration of

these cells. Due to the significantly increased circulating and yolk sac-residing HSC populations

at E14.5 it appears that their migration to the liver is impaired. The HSC population data at this

time point suggests an overall increase in HSCs. This change in the population may be due to

increased proliferation caused either directly or indirectly by the maternal anti-αIIb antibodies, or

decreased differentiation/increased self-renewal of these cells. The function of αIIbβ3 integrin on

HSCs is currently unknown, but it is likely similar to its function on platelets, where it is mainly

responsible for platelet aggregation. On HSCs it may play a role in cell-to-cell adhesion to the

niche and maternal anti-αIIb antibodies could have the potential to block this interaction,

impairing migration to the same degree to which αIIbβ3 contributes. There is also a distinct

possibility that the E14.5 fetal liver is a site of clearance for the fetal, immature MPS. This data

exposes a plethora of possibilities and research questions to be answered.

The data from neonates seems to support the hypothesis of impaired migration, since αIIb+ HSCs

remain in the liver but are decreased in the blood and bone marrow. HSCs were found in the

neonatal lung, which has recently been identified as having notable haematopoietic potential,

providing a niche for hematopoietic progenitors and megakaryocytes64. This could be

91

considered a hallmark of impaired migration as HSCs do not normally flock to the lung in

such magnitude but were given the opportunity since they remained in circulation. The spleen

also had increased HSC populations, however it is unclear whether this is due to clearance of

opsonized cells by the MPS or if the spleen is an additional "safe harbour" for HSCs like the

lung appears to be.

Finally, αIIb-mediated FNAIT has been revealed to progress through a previously undefined

pathway which results in a higher risk of miscarriage and increased disease severity.

92

Chapter 6: Future Directions

1. Genotype FNAIT and control fetuses and neonates for gender to determine whether there

is gender difference in body, placenta or liver masses.

2. Examine earlier and later time points, such as E12.5, E16.5, E18.5, and PND2-5, during

fetal and postpartum development in order to investigate HSC migration patterns

throughout gestation and early neonatal growth while maternal antibodies are still intact

in neonatal circulation.

3. Investigate HSCs in the entire fetus, including placenta to elucidate the overall effect on

HSCs in FNAIT fetuses, whether it is proliferation, no change, or degradation of these

cells.

4. Investigate the conditions of HSCs using apoptotic markers either with flow cytometry

using a similar procedure as outlined above or using immunohistochemical staining of

fetal and neonatal tissues.

5. Use a passive model where αIIb-specific Fab fragments are directly injected to fetal

circulation. The modified antibodies would still bind to HSCs, but any Fc-dependent

interaction would be removed.

6. Backcross the αIIb-/- mice which are currently C57BL/6 background onto a BALB/c

background and repeat the investigations to determine if the strain of the mice affects the

disease.

93

7. Obtain an HSC line for cell culture cells, and a macrophage cell line. Culture HSCs under

different conditions to determine the effect of maternal antibodies on these cells: in

medium, with maternal anti-αIIb sera, with maternal anti-αIIb sera and macrophages,

with purified polyclonal maternal anti-αIIb antibodies, with purified polyclonal maternal

anti-αIIb antibodies and macrophages, with maternal anti-β3 sera, with maternal anti-β3

sera and macrophages, with purified polyclonal maternal anti-β3 antibodies, with purified

polyclonal maternal anti-β3 antibodies and macrophages, and as a control since HSCs do

not express GPIbα: with maternal anti-GPIbα sera, with maternal anti-GPIbα sera and

macrophages, with purified polyclonal maternal anti-GPIbα antibodies, with purified

polyclonal maternal anti-GPIb antibodies and macrophages. The last four could also be

accomplished with naïve mouse sera/purified polyclonal IgG.

94

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