i

Analysis of platelets during malaria infection, and

their interaction with Plasmodium-infected

erythrocytes.

Nazneen Adenwalla

A thesis submitted for the degree of Master of Philosophy of The Australian

National University

May 2017

ii

Declaration

I declare that the experimental work presented in this thesis is the original work by myself.

Intravenous and intraperitoneal injections were carried out by either Lora Starrs or Hong

Ming Huang.

This thesis has not been submitted before for any examination in this or any other

universities, and conforms to the Australian National University guidelines and regulations.

iii

Acknowledgements

I would like to thank my supervisors Associate Professor Brendan McMorran and Professor

Simon Foote for giving me the opportunity to be a part of their lab and for all their help with

the project over the last few years.

A big thank you to Lora Starrs and Ming Huang. I could not have asked for kinder, friendlier

and more helpful lab colleagues turned friends to have made my coming into work daily

enjoyable. I cannot thank you enough for all the help you both have given me over the past

few years and will forever be grateful not only for the help, but for the advice and

encouragement over the last few years which has no doubt led to me submitting this thesis. I

will miss our conversations, words of wisdom, general hilarity and hope the book of “wise

quotes” happens one day!

Thank you to other members of the lab including Dr Gaetan Burgio, Anna Ehmann, Hao

Yang, Minette Salmon, Nay Chi Khin, Jing Gao and Ceri Flowers and past members Elinor

Hortle, Bernadette Pederson, Juliana Anokye and Edison Johar for making the lab a fun

working environment.

To Mick Devoy and Dr Harpreet Vohra – thank you for your endless help whenever I visited

the FACS laboratory. No matter how silly the question may have been (or times asked!) you

were both always so friendly and willing to help which I appreciate a lot.

iv

To other members of JCSMR especially Sarita Dhounchak, Melanie Johnson-Saliba, Fui Jiun

Choong, Thilaga Velusamy and James O’Connor for your friendship, chats, tea breaks and

encouragement. I will miss you all.

Thank you to Melanie, Lora, Stacey D’Mello and my mum for your help proofreading and

editing my chapters.

Thank you to the Australian Government Research Training Program for providing financial

support.

Finally, to my family and friends back home in New Zealand – for the support from the time

I left NZ and throughout my time in Australia. Always appreciated.

v

Abstract

Malaria is an infectious disease caused by Plasmodium parasites, transmitted by the female

Anopheles mosquito. Malaria can cause mild symptoms as well as more severe complications

which may lead to death. Annually, there are half a million deaths worldwide with millions

more newly infected with malaria. Although antimalarials exist, due to the prevalence of drug

resistance, novel, more effective treatments are needed to combat malaria by aiding the host

immune response. One of the earliest signs of a malarial infection is a decrease in the

concentration of platelets in the blood of infected individuals - clinically referred to as

thrombocytopenia. Platelets have been shown to play a protective role during a malaria

infection by targeting the parasite vacuole via platelet factor 4 (PF4). PF4 is only released

from activated platelets upon contact with infected red blood cells (iRBCs), and not

uninfected red blood cells (uRBCs). The molecules responsible for platelet activation and

subsequent release of PF4 from iRBCs were to be determined in this thesis. Platelet activation

via infected lysate from trophozoite stage parasites was originally observed but not

consistent. Purified trophozoites were unable to activate platelets hence the molecule of

interest was unable to be determined.

The second part of this thesis was to measure the contribution of platelets binding to RBCs

in thrombocytopenia. It has been previously shown that platelets bind preferentially to iRBCs

compared with uRBCs. No definite mechanism of thrombocytopenia has been elucidated to

date, although a number, such as splenic pooling and endothelial activation, have been

thought to play a role. In this thesis, it was hypothesised that platelet binding and subsequent

clearance may be responsible for malaria-induced thrombocytopenia. Before the onset of

thrombocytopenia, preferential binding of platelets to iRBCs was observed and although

vi

platelets were cleared more quickly in infected mice, the contribution of platelet bound RBCs

was unable to be definitely identified.

vii

Table of Contents

Declaration ........................................................................................................................................... ii

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

Abstract ................................................................................................................................................ v

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

List of Tables ..................................................................................................................................... xii

List of Abbreviations ........................................................................................................................ xiii

Chapter 1 Introduction .........................................................................................................................1

1.1 Malaria ..........................................................................................................1

1.2 History of Malaria ..........................................................................................................2

1.3 Life cycle of Malaria ..........................................................................................................3

1.4 Consequences of a Malaria Infection .........................................................................................4

1.5 Malaria Treatment ..........................................................................................................6

1.5.1 Antimalarials .......................................................................................................................6

1.5.2 Vaccine Protection ..............................................................................................................7

1.6 Host Response to Malaria 8

1.6.1 Resistance to Malaria: Genetic Polymorphisms .................................................................8

1.6.2 Innate Immune System .................................................................................................... 10

1.6.3 Adaptive Immune System ................................................................................................ 11

1.7 Platelets ....................................................................................................... 11

1.7.1 Traditional Role of Platelets ............................................................................................. 12

1.7.2 Platelets as Regulators of the Immune Cell ..................................................................... 13

1.8 Molecular Mechanisms of P. falciparum Adhesion ................................................................ 14

1.8.1 CD36 ................................................................................................................................. 15

1.8.2 ICAM1 ............................................................................................................................... 15

1.8.3 CD62P ............................................................................................................................... 16

1.8.4 Thrombospondin .............................................................................................................. 16

1.9 Platelets During a Malaria Infection ....................................................................................... 16

1.10 Platelet Factor 4 ....................................................................................................... 19

1.11 Thrombocytopenia ....................................................................................................... 20

1.12.1 The role of the spleen in malaria induced thrombocytopenia ...................................... 23

1.12.2 The role of the liver in malaria induced thrombocytopenia .......................................... 24

1.12.3 Platelet Consumption and Endothelial Activation ......................................................... 25

1.12.4 Platelet Binding to Red Blood Cells ................................................................................ 26

Hypothesis ........................................................................................................................................ 28

viii

Project aims ....................................................................................................... 28

Chapter 2 Materials and Methods ..................................................................................................... 29

2.1 Materials for Human Platelet Activation Studies.................................................................... 29

2.1.1 10x Platelet Wash Buffer ................................................................................................. 29

2.1.2 1x Platelet Wash Working buffer (PWB) .......................................................................... 29

2.1.3 10x Tyrode Solution ......................................................................................................... 29

2.1.4 Tyrode working buffer ..................................................................................................... 30

2.1.5 10x Human Tonicity Buffer (HTB) .................................................................................... 30

2.1.6 FACS Buffer ...................................................................................................................... 30

2.1.7 Hypotonic Lysis Buffer...................................................................................................... 30

2.1.8 10x RPMI .......................................................................................................................... 30

2.1.9 90% Percoll ....................................................................................................................... 31

2.1.10 70% Percoll ..................................................................................................................... 31

2.1.11 Phosphate Buffered Saline (PBS) ................................................................................... 31

2.1.12 Red Cell Wash/Sorbitol .................................................................................................. 31

2.1.13 PF4 ELISA Wash Buffer ................................................................................................... 31

2.1.14 Reagent Diluent ............................................................................................................. 31

2.1.15 Stop Solution .................................................................................................................. 32

2.2 Materials for Mouse Models of Thrombocytopenia Studies ................................................... 32

2.2.1 Citrate Buffer.................................................................................................................... 32

2.3 Methods for Human Platelet Activation Studies ..................................................................... 32

2.3.1 Human Platelet Purification ............................................................................................. 32

2.3.2 Plasmodium falciparum Culture ...................................................................................... 33

2.3.3 Giemsa smears: Human Blood ......................................................................................... 34

2.3.4 Percoll Gradient ............................................................................................................... 34

2.3.5 Whole Protein Purification ............................................................................................... 34

2.3.6 Platelet Activation Assay .................................................................................................. 35

2.3.7 Platelet Factor 4 ELISA ..................................................................................................... 35

2.3.8 Flow Cytometry Staining .................................................................................................. 36

2.3.9 Platelet Rich Plasma Assay ............................................................................................... 36

2.3.10 Gating strategy for positive events ................................................................................ 37

2.4 Methods for Mouse Models of Thrombocytopenia ................................................................ 39

2.4.1 Blood collection from tail ................................................................................................. 39

2.4.2 Giemsa smears: Mouse Blood .......................................................................................... 39

ix

2.4.3 Biotinylation of Cells ........................................................................................................ 40

2.4.4 Gating Strategy for Platelets, Red Blood Cells, iRBCs and biotinylated platelets ............ 40

2.4.5 Gating Strategy for Platelet Bound Red Blood Cells ........................................................ 42

2.4.6 Statistical Analysis ............................................................................................................ 44

Chapter 3 Platelet Activation in Response to Plasmodium falciparum ............................................ 46

3.1 Platelet Activation Using Infected Lysate to Activate Platelets ............................................. 47

3.1.1 Platelet Activation Using Infected Lysate: anti-CD62P .................................................... 48

3.1.2 Platelet Activation Using Infected Lysate: anti-PAC1 ...................................................... 50

3.1.3 Platelet Factor 4 Release Using Infected Lysate .............................................................. 52

3.1.4 Platelet Activation using Infected and Uninfected Lysate ............................................... 54

3.2 Platelet activation using purified trophozoite stage red blood cells ........................................ 56

3.2.1 Platelet Activation Using Purified Trophozoite stage: anti-CD62P and anti-PAC1 .......... 56

3.2.2 Platelet Factor 4 Release Using purified trophozoite stage red blood cells: anti-PAC1 and

anti-CD62P ................................................................................................................................ 57

Discussion ....................................................................................................... 59

Chapter 4 Thrombocytopenia in Mouse Models of Plasmodium chabaudi ...................................... 63

4.1.1 Preferential Binding of Platelets to Red Blood Cells During a Malaria Infection ............. 69

4.2 Challenge 2: Platelet Clearance in Male Mice ........................................................................ 71

4.2.1 Platelet and Red Blood Cell Concentration during a Plasmodium chabaudi infection in

male mice .................................................................................................................................. 71

4.2.2 Platelet Clearance During a Plasmodium chabaudi Infection in Male Mice .................... 74

4.2.3 Platelet Consumption or Inhibition of Production ........................................................... 76

4.2.4 Preferential Binding of Platelets to Red Blood Cells ........................................................ 78

4.2.5 Rate of Loss of Free Platelets vs Platelet Bound Red Blood Cells .................................... 80

4.3 Challenge #3: Platelet Clearance in Male Mice ...................................................................... 82

4.3.1 Platelet and Red Blood Cell Concentration During a Plasmodium chabaudi Infection in

Male Mice ................................................................................................................................. 82

4.3.2 Platelet Clearance During a Plasmodium chabaudi Infection in Male Mice .................... 85

4.3.3 Platelet Consumption or Inhibition of Production ........................................................... 87

4.3.4 Preferential Binding of Platelets ...................................................................................... 89

4.3.5 Rate of Loss of Free Platelets vs Platelet Bound Red Blood Cells .................................... 91

Discussion ....................................................................................................... 93

Chapter 5 General Discussion ........................................................................................................... 98

References ....................................................................................................................................... 102

x

List of Figures

Figure 1.1: Worldwide distribution of malaria.

Figure 1.2: Life cycle of Plasmodium falciparum.

Figure 1.3: Role of platelets in clotting.

Figure 1.4: Survival curve of mice infected with Plasmodium chabaudi.

Figure 1.5: Percentage growth inhibition with purified human platelets.

Figure 1.6: Intraerythrocytic parasites are killed by platelets.

Figure 1.7: Thrombocytopenia in mice infected with Plasmodium chabaudi.

Figure 1.8: Thrombocytopenia in healthy human volunteers infected with P. falciparum.

Figure 1.9: Platelet recruitment to the liver post- P. berghei infection.

Figure 1.10: Platelet binding to infected and uninfected red blood cells.

Figure 2.1: Gating strategy for positive events.

Figure 2.2: Gating strategy for platelets/biotinylated platelets, RBCs and iRBCs.

Figure 2.3: Gating strategy for platelet bound RBCs.

Figure 2.4: An example of a linear regression

Figure 3.1: Activation of platelets using infected lysate: anti-CD62P.

Figure 3.2: Activation of platelets using infected lysate: anti-PAC1.

Figure 3.3: Platelet Factor 4 ELISA with infected lysate.

Figure 3.4: anti-CD62P and anti-PAC1 Positive events and mean fluorescent intensity.

Figure 3.5: Platelet activation by RBCs at trophozoite stage

Figure 3.6: Platelet Factor 4 ELISA with trophozoite stage RBCs

Figure 4.1: Platelet and RBC concentration compared to parasitaemia in male C57BL/6 mice.

Figure 4.2: Relationship between platelet binding to iRBC and uRBCs in male C57BL/6

mice.

xi

Figure 4.3: Platelet and RBC concentration compared to parasitaemia in male C57BL/6 mice.

Figure 4.4: Clearance of biotinylated platelets.

Figure 4.5 Biotinylated and nonbiotinylated platelets in infected and uninfected mice over

the course of infection

Figure 4.6: Relationship between platelet binding to iRBC and uRBCs in male C57BL/6

mice.

Figure 4.7: Preferential binding of platelets to infected and uninfected cells vs free platelets

Figure 4.8: Platelet and RBC concentration compared to parasitaemia in male C57BL/6 mice.

Figure 4.9: Clearance of biotinylated platelets.

Figure 4.10: Biotinylated and nonbiotinylated platelets in infected and uninfected mice over

the course of infection

Figure 4.11: Relationship between platelet binding to iRBC and uRBCs in male C57BL/6

mice.

Figure 4.12: Preferential binding of platelets to infected and uninfected cells vs free platelets

xii

List of Tables

Table 2-1 Cell markers used to distinguish human and mouse populations of interest on flow

cytometry

xiii

List of Abbreviations

ACT Artemisinin-based combination therapy

ATP Adenosine triphosphate

CCM Complete Culture Media

DIC Disseminated intravascular coagulation

DV Digestive Vacuole

ICAM Intercellular Adhesion Molecule 1

iRBCs Infected Red Blood Cells

HTB Human Tonicity Buffer

IP Intraperitoneal

IV Intravenous

mins Minutes

mL MilliLiter

PF4 Platelet Factor 4

PfCRT Plasmodium falciparum chloroquine resistance

transporter

PfEMP1 Erythrocyte Membrane Protein 1 Family

P. chabaudi Plasmodium chabaudi

P. falciparum Plasmodium falciparum

PRP Platelet Rich Plasma

PWB Platelet Wash Buffer

RBCs Red Blood Cells

rpm Revolutions per minute

xiv

RT Room Temperature

secs Seconds

SM Severe malaria

TLRs Toll-like receptors

TWB Tyrode Wash Buffer

µL Microliter

uRBC Uninfected Red Blood Cells

Vwf Von Willebrand factor

1

Chapter 1 Introduction

1.1 Malaria

Malaria is caused by protozoan parasites belonging to the genus Plasmodium and is one of

the deadliest diseases affecting the human population [1]. Of the five species of human

malarial parasites (Plasmodium falciparum, P. vivax, P. malariae, P. knowlesi and P. ovale),

[2], P. falciparum is the predominant species threatening the human population in endemic

areas. The 2016 World Health Organization (WHO) report stated 212 million new cases and

429,000 deaths due to malaria [3] occurred in 2015, with the majority of the cases and deaths

recorded in the Sub-Saharan African region. This was followed by South-East Asia and the

Eastern Mediterranean region [3]. To reduce and prevent malaria transmission in endemic

areas, vector control has been implemented either by implementation of insecticide-treated

mosquito nets, or indoor residual spraying [4]. In Sub-Saharan Africa, insecticide-treated

mosquito nets were heavily used with an estimated 53% of the population at risk of malaria

sleeping under a net in 2015 compared to 30% in 2010 [3]. Furthermore, 106 million people

world-wide were protected from malaria transmission by indoor residual spraying including

49 million people in Africa, with the proportion of the population at risk declining from a

peak of 5.7% globally in 2010 to 3.1% in 2015 [3].

Figure 1.1 shows the current global malaria landscape by country where more than half the

world’s countries have eliminated malaria from their borders [5].

2

Figure 1.1: Worldwide distribution of malaria. Current landscape of malaria showing

more than half the world’s countries have eliminated malaria. Regions including Sub-

Saharan Africa and Asia are clearly still at the lowest end of the spectrum, though highlighted

in pink as "controlling malaria". Figure taken from [5, 6].

1.2 History of Malaria

Malaria infections have been recorded for thousands of years, with texts from ancient

civilizations of India, China and Greece alluding to fevers which identify as malaria. The

writings of Hippocrates described the reoccurrence of malaria and the association between

an enlarged spleen and a patient’s residence in a low-lying, marshy area [7]. Malaria was

coined from the medieval Italian words ‘mala’ and ‘aria’ meaning ‘bad air’ [8]. It was not

until 1880 where Alphonse Laveran performed necropsies on malaria victims and found, in

the majority of cases, pigmented bodies which were moveable were found in the blood. He

concluded the rapid movement must be parasites and that these must be the cause of malaria

which he called Oscilliaria malariae which was later reclassified to Plasmodium [9].

3

1.3 Life cycle of Malaria

The life-cycle of the malaria parasite begins when the female Anopheles mosquito harboring

the parasite in its salivary gland, takes a blood meal from a human host. Subsequent to the

mosquito bite, the parasite, in the form of sporozoites, travels to the liver via the bloodstream

where they invade hepatocytes and proliferate asexually. From here they develop into

schizonts, and release from hepatocytes as merozoites [10]. Merozoites re-enter the blood

stream and invade red blood cells (RBCs), after which the intra-erythrocytic parasite mature

and asexually reproduces into schizonts, eventually rupturing the RBC and releasing newly

formed merozoites to invade new RBCs. The sexual stage occurs when some merozoites

mature into gametocytes. Following ingestion by mosquitoes during a blood meal, an

individual gametocyte may form one female macrogamete or up to eight male microgametes

[11]. In the mosquito midgut, gamete fusion produces a zygote that develops into a motile

ookinete. At this stage, there is an opportunity for recombination between genetically distinct

parasite isolates, or outcrossing to create genetic diversity [12]. The ookinetes subsequently

penetrate the midgut of the wall and form oocysts [11]. Over time, the oocysts enlarge and

burst to release sporozoites that migrate to the salivary glands and can infect humans during

the next blood meal. The lifecycle of the malaria parasite is described in Figure 1.2. It is

during the RBC stage of the malarial cycle that clinical manifestations of malaria occur and

continue until either the host immune response eliminates infection, it is cleared via

antimalarial treatment [13] or the host dies.

4

Figure 1.2: Life cycle of Plasmodium falciparum. The life cycle begins when 1) the female

Anopheles mosquito takes a blood meal 2) the parasite in the form of sporozoites travel via

the bloodstream to the liver where they 3) invade hepatocytes and proliferate asexually into

merozoites 4) upon release of merozoites they reenter the bloodstream and invade RBCs

where they replicate asexually into merozoites and burst from RBCs to invade new RBCs 5)

some merozoites form gametocytes and a mosquito may ingest them during their next blood

meal thus continuing the cycle. Figure adapted from [14] by Farzin Adenwalla.

1.4 Consequences of a Malaria Infection

Malaria has many symptoms similar to the common cold and, with the hallmark pathological

feature being fever, it often resembles viral infections. Symptoms common to fever such as

nausea, chills, headaches and vomiting are present and it is therefore difficult to pinpoint a

malarial diagnosis [15]. The clinical symptoms manifest during the asexual blood stage of

the life cycle [16] (Figure 1.2 Step 4). After a period of symptoms where severity can vary,

parasite load is controlled by the host’s immune response, although symptoms recur at

intervals over weeks and months, associated with rises in parasitaemia [17]. The successive

1

1

5

5

3

2

4

3

2

2

5

waves of parasitaemia are lower and symptoms are less pronounced until eventually the

infection is cleared [17].

In the case where malaria infection is not controlled, infection can progress to severe malaria

(SM) which may result in death. P. falciparum is the predominant species causing SM in

humans and accounts for over half a million deaths each year mainly in children in Sub-

Saharan Africa [18]. The main forms of SM are different in children and adults in various

epidemiological settings: 1) severe anaemia occurs in infants in areas of stable, intense

transmission; 2) cerebral malaria and respiratory distress (as a result of metabolic acidosis)

occur in young children in areas of moderate transmission and 3) cerebral malaria, organ

dysfunction (e.g., renal failure, severe jaundice, and pulmonary oedema) and acidosis occur

in individuals of all ages in areas of low and unstable transmission [19].

Various factors play a role in the pathophysiology of SM including exponential parasite

growth and microvascular obstruction from adherence of mature parasites to blood vessels

[20]. If the exponential phase increases by 10-fold every 48 hours, then total body

parasitaemia is reached more rapidly [17]. This high parasite load triggers an acute

inflammatory response and therefore SM arises due to excessive or poorly controlled

responses that have evolved primarily to control acute infection [18]. Another key feature of

SM is microvascular obstruction caused by P. falciparum sequestration of parasites within

microvascular beds. This occurs via modifications on the RBC surface by the insertion and

display of variant proteins such as P. falciparum erythrocyte membrane protein (PfEMP1)

[21] which are able to bind to receptors on other cells including CD36 which is one of the

well characterized receptors [22]. Infected cells can also bind to uninfected RBCs (uRBCs)

(a process known as resetting) [23] and activated platelets, which lead to clumps. Both of

these can lead to microvascular obstruction and SM.

6

1.5 Malaria Treatment

Many drugs were developed to treat malaria in the 20th century, with chloroquine and

artemisinin being the most widely-implemented and effective. However, with the rise in

antimalarial resistance now threatening their efficacy, the need to discover novel antimalarial

drug candidates are required [24].

1.5.1 Antimalarials

The first line of treatment for uncomplicated P. falciparum malaria in endemic areas is

artemisinin combination treatment (ACT) [25]. ACT consists of an artemisinin derivative

and a partner drug, which is another structurally unrelated antimalarial compound such as

lumefantrine or mefloquine [26]. The mechanism behind artemisinin is based on the presence

of the endoperoxide bond present in artemisinins, while their derivatives (as molecules

without the endoperoxide bond) have no antimalarial activity [27]. This bond, in the presence

of haem (from the degradation of host haemoglobin from the parasite) decomposes into free

radicals [28]. These free radicals may then damage specific intracellular targets, possibly via

alkylation [29]. The artemisinin derivative clears the majority of the parasites but it has a

short half-life whilst the partner drug, has a longer half-life and targets parasites that remain

in the system [30].

Another well-known antimalarial is chloroquine. At a physiological pH of ~7.4, chloroquine

is a diprotic weak base and in its un-protonated state, is able to enter the digestive vacuole

(DV) of the parasite within a RBC [31]. Within the DV, chloroquine molecules become

protonated and accumulate inside the acidic DV as the membrane is not permeable to charged

species [32, 33]. The protonated chloroquine subsequently binds to haematin, which is a toxic

byproduct of haemoglobin proteolysis, thereby preventing haematin differentiating to the

7

haemozoin crystal [34]. Due to its inability to crystalize, free haematin interferes with the

parasite detoxification processes thereby damaging the plasmodium membrane [35].

Due to antimalarial drug resistance (including chloroquine resistance), novel effective

treatments are needed to combat malaria. [36]. The mechanisms behind drug resistance are

spontaneous, and thought to be independent to the drug used; rather, it is thought to be due

to mutations in genes encoding the drug’s parasite target or influx/efflux pumps that affect

the intraparasitic concentrations of the drug [37]. One of these pumps involved in

chloroquine transport is the P. falciparum chloroquine-resistance transporter (PfCRT). In the

presence of a mutation, resistance occurs via a decrease in the concentration of chloroquine

within the DV [38] due to the transport of drug away from the DV via PfCRT [39] and hence

the site of action. The vital mutation seems to be the replacement of lysine with threonine at

position 76 [40]. However, PfCRT is not the sole determinant of chloroquine resistance, as

it has been shown that mutations in the homolog of the major multidrug-transporter P.

falciparum multidrug gene also modulates the extent of resistance [38].

1.5.2 Vaccine Protection

In terms of vaccines, RTS,S/AS01 is the most advanced P. falciparum vaccine candidate

developed globally [41]. The vaccine consists of hepatitis B surface antigen virus-like

particles and incorporates a portion of the P. falciparum-derived circumsporozoite protein

and a liposome-based adjuvant [41]. The vaccine targets the circumsporozoite protein of

P. falciparum to induce specific CD4-positive T cells that are associated with protection to

P. falciparum infection and episodes of malaria [42, 43]. In a 2009 study of 15,000 infants

and young children from Sub-Saharan Africa, in infants aged 6-12 weeks and young children

5-15 months, the efficacy of the vaccine decreased rapidly [44]. A booster shot 20 months

8

after the initial dose increased protection slightly. Individuals in the 5-17 month group

contracted meningitis compared to children who received control vaccines [45].

Another vaccine that seemed promising and underwent early phase I/IIa clinical trials was

using the radiation-attenuated, whole-cell sporozoite vaccine which delivered sterile

protection against injection with sporozoites of the same parasite strain [46]. However, this

vaccine had drawbacks including lack of cross-strain protection, high numbers of parasites

required, route of delivery and the logistical requirements for a liquid nitrogen cold chain to

maintain viability of vaccine [46]. Despite these drawbacks, the ultimate goal for a successful

vaccine is to induce strain-transcendent protection [17].

1.6 Host Response to Malaria

Malaria is the strongest known selective pressure on the human genome in recent history

[47]. Genetic polymorphisms such as sickle cell anaemia [47] and Duffy negativity [48] in

their asymptomatic heterozygous form, confer resistance to the malaria parasite providing an

evolutionary advantage for these polymorphisms and, along with the host response, play an

important role in controlling infection. Along with these genetic polymorphisms, two lines

of host immune defence play a role in combating malaria infection; innate immune system

and the adaptive immune system.

1.6.1 Resistance to Malaria: Genetic Polymorphisms

Genetic polymorphisms have been shown to confer resistance to malaria and have been

associated as an evolutionary force for genetic traits such as sickle cell disease [49] and Duffy

negativity [48]. Sickle cell disease results when there is a substitution of valine for glutamic

acid at its sixth amino acid of the β-globin chain [50]. Individuals homozygous for the

9

mutation develop symptomatic, and potentially deadly sickle disease whereby the

polymerized haemoglobin causes RBCs to become sickle shaped and occlude blood vessels,

whilst heterozygous individuals have sickle cell trait and are generally asymptomatic [51].

Sickle cell trait confers resistance to malaria [52] and the frequency of carriers of this trait is

high in malaria endemic regions, furthering evidencing the evolutionary advantage and

history of sickle cell disease with malaria prevalence [53]. Resistance is thought to be due

to parasitised mutant polymorphic RBCs subjected to enhanced phagocytosis by monocytes

which suggests P. falciparum is cleared by the immune system more rapidly [54].

Progressive dehydration of RBCs and increased cellular density have also been associated

with decreased invasion by P. falciparum suggesting that structural features of the host cell

play a role in resistance [55].

The Duffy-negative red cell phenotype is another well-known polymorphism that can cause

malarial resistance. The Duffy antigen encodes a chemokine receptor (DARC, also known

as Fy) and is expressed on the RBC surface. A polymorphism at a GATA-1 binding site in

the promotor of the DARC gene alters receptor expression, leading to no expression on the

RBC surface [56]. Almost all Central and West African people are Duffy-negative and as

such these individuals are resistant to Plasmodium vivax infection which requires Duffy to

invade the RBC [57].

Understanding the variability of genes in human populations, and how they may provide

resistance against malaria, may provide greater insight into developing new interventions,

therapies and working towards better management of malaria.

10

1.6.2 Innate Immune System

The innate immune system comprises of cells such as natural killer cells and dendritc cells.

This is the first line of defence in regard to bacteria [58], virus [59] and parasite [60] invasion.

Studies in mice and humans show the important role of macrophages in phagocytosing

iRBCs in the absence of cytophilic or opsonizing malaria-specific antibody [61]. This

interaction of iRBCs is possibly due to the presence of CD36 on the surface of various cell

types which subsequently results in sequestration of parasites in the microvascular endothelia

[62].

Furthermore, studies in mice have found that cytokines, such as interferon-γ, are released

within the first few hours of a malaria infection, and subsequently predict the course of

infection and its final outcome [63, 64]. Interferon-γ is an important pro-inflammatory

cytokine [65] and it plays a protective role against infection by protozoan parasites [66, 67].

The production of interferon-γ in naïve animals may result from either pre-existing, cross-

reactively primed effector memory T-cells or from cells of the innate immune system, e.g.

phagocytic granulocytes, macrophages, Natural Killer cells or γδ T-cells [68].

Platelets constiture an important part of the innate immune system defense against malaria.

Although the traditional role of platelets is in haemostasis and thrombosis, platelets have

been recently defined as an immune cell and have been shown to play a role in malaria by

killing the parasite which resides within the RBC [69, 70]. The role of platelets in malaria

infection is discussed further in Section 1.9.

11

1.6.3 Adaptive Immune System

The adaptive immune system consists of cells such as B and T lymphocytes. Mechanisms of

the adaptive immune system that play a role in parasite defense include antibodies blocking

hepatocyte invasion, antibodies binding to adhesion molecules on the vascular endothelium

thus preventing sequestration of iRBCs, and antibodies blocking merozoite invasion into

RBCs [71].

When immune adults leave malaria-endemic regions, their immunity to malaria wanes

quickly, which suggests that continued exposure to malarial antigens is needed not only for

the generation of memory cells and effector cells but also for their persistence [72]. Rapid

boosting of antibody responses to various antigens after reinfection does take place which

indicates the presence of memory B cells [72].

During the erythrocytic stage of infection, CD4+ T-cells can play a protective role via

interferon-γ production. CD4+ T-cell help is also required for the B-cell response which is

involved in control and elimination of infected red blood cells [73]. CD4+ T-cells are also

important for controlling pre-erythrocytic stages of Plasmodium infection through the

activation of parasite-specific CD8+ T-cells [73] which are a subpopulation of MHC class-I

restricted T cells and mediators of adaptive immunity.

1.7 Platelets

Platelets have been well described for their role in haemostasis and thrombosis [74],

however, in recent times, the versatility of platelets has recently been discovered due to their

ability to interact with invading pathogens [75]. Platelets interact with pathogens using

receptors that are present on classical immune cells such as Toll-Like Receptors (TLRs),

CD40 and ICAM-2 [76].

12

The following section discusses not only their traditional role as initiators of clotting but also

how platelets play a role in the immune system, their role in a malaria infection and how a

loss of platelets during a malarial infection is commonly observed (known as

thrombocytopenia).

1.7.1 Traditional Role of Platelets

The “father of the platelet” was Giulio Bizzozero, who in 1862, described a novel

morphological element in the blood that played a crucial role in haemorrhage and thrombosis

[77], and was later termed 'platelet'. Platelets are anuclear cells and the smallest cells within

the blood, ranging from ~2-5 µm in diameter and 0.5 µm in thickness [78]. They are produced

from megakaryocytes in the bone marrow and after being released into circulation, have a

life span of around 7-10 days in humans [79].

In healthy vasculature, platelets are kept in an inactive state via nitric oxide and prostacyclin

which are released by endothelial cells that line the blood vessels [80]. However, when a

vascular injury occurs, collagen and basement membrane proteins are exposed that allow

platelets to adhere to the substratum [74]. Platelets then adhere and aggregate and release

platelet activation factors such as adenosine diphosphate and thromboxane A2 and, following

activation, platelets produce thrombin catalyzing the coagulation cascade, eventually

generating a mesh like structure that stops bleeding [74]. The role of platelets in the formation

of a blood clot is described in Figure 1.3.

13

Figure 1.3: Role of platelets in clotting. Vascular injury occurs exposing collagen and

extracellular matrix proteins 1) activated platelets adhere to site of injury due to the exposure

of collagen and 2) aggregate and release other factors such as adenosine diphosphate,

thromboxane A2 and thrombin which in turn activates more platelets 3) thrombin turns

fibrinogen into fibrin which forms a meshlike structure and stops bleeding. Adapted from

[74] by Farzin Adenwalla

1.7.2 Platelets as Regulators of the Immune Cell

Besides their role in haemostasis and thrombosis, platelets exert immunological functions

and participate in the interaction between pathogens and host defense due to their binding

ability since they have a broad repertoire of receptor molecules [75]. For example, CD154 is

a transmembrane protein that is expressed by CD4+ T cells, however in 1998, Henn and

colleagues found that CD154 was also expressed by activated platelets [81] and binds to

CD40. CD40 was first described on human bladder carcinoma cells [82] but is also expressed

on B lymphocytes, monocytes, dendritic cells and endothelial cells [83]. Interaction of

CD154 and CD40 on endothelial cells induces various inflammatory responses including the

expression of inflammatory adhesion receptors such as intercellular adhesion molecule 1

(ICAM1) and vascular cell adhesion molecule 1 [84].

1 2 3

14

Platelets also express TLRs which are a class of pattern recognition receptors able to detect

distinct evolutionarily conserved structures on pathogens (termed PAMPs – pathogen

associated molecular patterns) [85]. TLRs are also expressed by neutrophils, macrophages

and dendritic cells which highlight the role of various TLRs in both innate and adaptive

immune responses. Human platelets for example, have been shown to express TLR1, TLR2,

TLR4, TLR6, TLR8, and TLR9 and TLR3 [86, 87] and platelet expressed TLRs act as a

bridge between platelets and inflammatory responses further illustrating the intricate

interactions between innate and adaptive immunity [87, 88]. TLR4 is one of the most

extensively studied TLRs on human platelets [89]. An example of the role of TL4 between

platelets and infection occurs by interacting with lipopolysaccharides from gram-negative

bacteria which are able to activate platelets and induce platelet-neutrophil interactions

leading to neutrophil degranulation and release of extracellular traps which in turn can kill

bacteria [90].

1.8 Molecular Mechanisms of P. falciparum Adhesion

Due to parasite-derived adhesins expressed on the surface of mature iRBCs, they are able to

adhere to various receptors and be sequestered [91]. Three types of adhesions have been

described; cytoadherence to endothelial cells [92], rosetting with uRBCs [93] and

interactions with platelets that can lead to clumping of iRBCs in vitro [94]. The cloning of

the var genes encoding the variant surface antigen family PfEMP1 in 1995 provided the

groundwork for the molecular basis of adhesion in P. falciparum malaria [95-97]. The

variants are expressed on the surface of the iRBC and are responsible for some of the

adhesive properties of iRBCs. Some of the receptors that promote adhesion are discussed

below.

15

1.8.1 CD36

iRBCS are able to bind to the scavenger receptor CD36 which is expressed on macrophages,

epithelial cells, monocytes, RBC precursors and platelets [98]. PfEMP1 variants are the

parasite-induced iRBC ligands for CD36, and CD36 binding is a characteristic of almost all

P. falciparum isolates derived from malaria patients. CD36 is a key element in innate defense

due to the diverse range of cells CD36 is expressed by [61]. In dermal microvasculature,

iRBC adhesion to CD36 may help to create a microenvironment that enhances parasite

replication, at least in murine parasites such as P. berghei [99].

It has been observed that platelet activation can occur when iRBCs bind to CD36 on platelets.

When platelets were incubated with control RBC or P. falciparum, iRBCs pre-treated with

Fab fragments of non-specific IgG or a CD36 blocking antibody, there was a significant

increase in platelet activation as determined by increased binding of anti-PAC1 [100] which

recognizes the fibrinogen binding sites in the glycoprotein IIb/IIIa (also known as CD41)

complex of activated platelets. The binding of iRBC to platelets has been shown to release

platelet factor 4 which has been implicated in parasite killing [69, 70].

1.8.2 ICAM1

ICAM1 belongs to the immunoglobulin superfamily expressed on endothelial cells and

leukocytes [91]. Research using isolates from patients found that ICAM1 mediated rolling

adhesion, while CD36 mediated stationary adhesion, suggesting that ICAM1 capture and

CD36 stationary adhesion [101] may work together to promote efficient sequestration. In a

malaria infection, there are elevated serum levels of the soluble form of ICAM1 [102, 103]

and post-mortem tissue samples have demonstrated widespread systemic upregulation of

ICAM1 expression at microvascular endothelial sites [104, 105].

16

1.8.3 CD62P

CD62P is a glycoprotein that is expressed on activated platelets and endothelial cells, and is

important for leukocyte trafficking [91]. From field isolates in Thailand, CD62P has been

shown to mediate rolling of iRBCs on endothelial cells, and facilitates adhesion [106, 107].

The parasite ligand for CD62P is thought to be PfEMP1 since purified PfEMP1 can bind to

CD62P in vitro [108].

1.8.4 Thrombospondin

Thrombospondin is an adhesive glycoprotein that is released into plasma in response to

platelet activation by thrombin [91]. It was the first molecule associated with P. falciparum

cytoadherence. It has previously been shown that iRBCs bind to purified thrombospondin

in static assays [109], and bind to endothelial cells via thrombospondin under flow conditions

[110].

1.9 Platelets During a Malaria Infection

During malaria infections, it has been found that platelets played a protective role by killing

the parasite [70, 111]. Evidence for the protective effect of platelets resulted from a study

using mice that were deficient in platelets [70]. Mice that display a homozygous disruption

of Mpl which encodes for the megakaryocyte growth and differentiation factor C-mpl (the

receptor for thrombopoietin), ended up with only 1/10th as many platelets as their wild-type

counterparts [112]. When these mice were infected with the mouse malaria strain P.

chabaudi, Mpl-/- mice had a lower survival rate than wild-type mice [70] (Figure 1.4a and

1.4b).

17

Figure 1.4: Survival curve of mice infected with Plasmodium chabaudi. Mice were

infected with P. chabaudi and survival rates monitored in a) male mice b) female mice [70].

In vitro studies were also carried out to demonstrate platelet protection using a P. falciparum

culture. When human platelets at increasing concentrations were cultured with purified with

P. falciparum at trophozoite stage, a concentration-dependent increase in the inhibition of

parasite growth was observed (Figure 1.5a). Aspirin inhibits platelet cyclooxygenases which

results in a decrease in thromboxane A2 and therefore decreased platelet activation [113].

When platelets were incubated with aspirin, the killing effects of platelets on parasites were

no longer present. (Figure 1.5b), further confirming platelet-mediated parasite killing [114].

18

Figure 1.5: Percentage of growth inhibition with purified human platelets. a) Parasite

growth inhibition with increasing concentrations of purified platelets. Chloroquine was used

as a positive control. b) Parasite growth in the presence of platelets preincubated with aspirin

before being added to parasite culture (in vitro aspirin) or untreated platelets (control)

compared with platelets taken from the same donor on separate occasions after taking aspirin

twice daily for one week before platelet collection (in vivo aspirin) [70].

More evidence in the above study of platelet protection playing a role in the protection

against P. falciparum was via the use of terminal deoxynucleotidyl transferase deoxyuridine

triphosphate nick end labeling (TUNEL) which stains for degraded DNA. Twice as many

dead intraerythrocytic parasites were seen in wild-type mice compared to the mutant mice

(Figure 1.6a) which suggested platelets play a role in the killing of the parasite. TUNEL-

stained intracellular parasites were also observed in the P. falciparum cultures where the

frequency of stained parasites was more than three times as high in platelet-treated cultures

(Figure 1.6b).

a

a

b

a

19

Figure 1.6: Intraerythrocytic parasites are killed by platelets. a) Percentage of all P.

chabaudi parasites staining positive with TUNEL in mice wild-type and mutant mice. b)

Percentage of TUNEL-positive parasites in P. falciparum cultures incubated with platelets

and without platelets for 24 hours [70].

1.10 Platelet Factor 4

The molecule associated with the killing of the parasite via platelets was identified as

platelet factor 4 (PF4) [69, 111]. PF4 is a platelet derived CXC-type chemokine which is

released from the α-granules upon platelet activation [115]. PF4 is a human defense peptide

that has antimicrobial activity to pathogens including bacteria [116] and fungi [117].

The mechanism of action of PF4 was shown to be via lysis of the DV [69]. The DV is the

site where the parasite obtains essential amino acids and energy in order to survive, and it

appears that PF4 works solely on the DV, as the parasite plasma membrane is undisturbed,

as is mitochondrial function [69].

PF4 does not work alone but needs the Duffy antigen to initiate the killing mechanism. The

Duffy protein has been identified as a receptor for chemokines and hence has been named

Duffy antigen receptor for chemokines (DARC) [118]. It is expressed on the RBC surface

and, when parasites where cultured in Duffy negative blood, parasite growth was unaffected

by platelets or PF4 treatment [111]. Likewise, when chemokines were used with a higher

a b

20

affinity to Duffy than PF4, or when monoclonal antibodies that block PF4 binding were used,

it blocked the ability of PF4 to kill parasites suggesting PF4 was the parasite killing molecule.

1.11 Thrombocytopenia

In 1924, it was reported that platelet numbers in malaria-infected individuals were reduced

in the peripheral blood of patients with acute malaria [119]. The decrease in platelet number

is known as thrombocytopenia, characterised by a platelet count less than <150,000/mm3

[120].

In a malaria infected individual, thrombocytopenia is commonly observed and can be a result

of platelet activation, splenic pooling, or a decreased platelet life-span due to antibody and

cellular immune responses [120]. On rare occasions, thrombocytopenia may result from

antibodies produced against antimalarials, interacting with platelets [121]. In most cases,

bleeding is not associated with thrombocytopenia and requires no treatment, with platelet

count returning to normal after successful treatment of the malarial episode [122].

Along with anaemia, thrombocytopenia is the most frequent malaria-associated

haematological complication observed [123] as concentration of platelets have been shown

to be inversely correlated with the level of parasitaemia. Thrombocytopenia has been shown

to occur early in infection in both humans and mice before severe forms of the disease

develop [70, 124], therefore thrombocytopenia is an excellent diagnostic tool for those

returning from an endemic region.

It has been previously shown in human [124], mouse [70] and monkey [125] models of

malaria that a loss of platelets accompanies an increase in parasitaemia. In mice infected

with P. chabaudi, the relationship between platelet concentration and parasitaemia is inverse

and is shown in Figure 1.7. A decrease in platelet concentration is observed as concentration

21

of parasites increase, however RBC concentration remains constant; thus, thrombocytopenia

is noted before the symptoms such as anaemia is observed. Platelet numbers also reached the

lowest point before the onset of severe disease symptoms and anaemia.

Figure 1.7: Thrombocytopenia in mice infected with P. chabaudi. An inverse relationship

between platelet concentration (green) and parasitaemia (blue). Red blood cell levels stay the

same during infection (red) [70].

Another study, conducted in 1983 by DeGraves and colleagues [126] observed rats infected

with P. chabaudi. This study showed there was an association with infection and decreased

numbers of RBCs and platelets with increasing percentage of parasitised cells. When parasite

numbers were reduced to a level not detectable, anaemia seemed to persist for several weeks

whereas thrombocytopenia was no longer observed.

Another malaria model investigating thrombocytopenia was conducted on monkeys. It has

been observed that severe thrombocytopenia developed in Aotus monkeys [125] infected

with P. vivax. From 26 monkeys, 77% developed severe thrombocytopenia and showed an

inverse relation to parasitaemia.

a

22

In human studies, healthy individuals were infected with P. falciparum, and showed a

significant decrease in platelets between five-six days after the onset of blood stage infection

(Figure 1.8). With this decrease in platelet number, levels of von Willebrand factor (vWF –

a marker of chronic endothelial activation) and vWF propeptide (a marker of acute

endothelial activation) significantly increased at a very early stage in infection which suggest

acute endothelial cell activation early in malaria [124]. The authors hypothesised that

endothelial cell activation with subsequent release of activated vWF is related to the

development of early thrombocytopenia. Increased amounts of activated vWF which expose

the glycoproteinIba-binding site of vWF for platelets were also observed. Platelet numbers

and levels of both vWF and activated vWF showed a strong inverse correlation in the

volunteers. Activated vWF may therefore be an important inducer of thrombocytopenia

during early malaria and may contribute to the pathogenesis of malaria.

Figure 1.8: Thrombocytopenia in healthy human volunteers infected with

P. falciparum. Percentage of platelet baseline during infection from healthy individuals

infected with P. falciparum. A significant difference was seen on days five and six. [124].

23

Altogether, the above models suggest that thrombocytopenia does indeed occur during a

malaria infection, the exact mechanism of malaria-induced thrombocytopenia has not been

elucidated. A number of proposed mechanisms have been investigated and some of these are

discussed in the following section.

1.12 Mechanisms of Thrombocytopenia

A number of possible mechanisms of malaria-induced thrombocytopenia have been

postulated. Splenic pooling, liver sequestration, endothelial activation, platelet consumption

and platelet binding to RBCs have been proposed, although the exact mechanisms are still

under investigation.

1.12.1 The role of the spleen in malaria induced thrombocytopenia

In 1973, Skudowitz and colleagues suggested that platelets were sequestered in the spleen

during an acute infection. Within the hematopoietic system, the spleen is an important

structure as it stores 1/3 of the platelets that are produced by the bone marrow whilst the

remaining are in circulation [127].

In a malaria infected individual, an increase in spleen size is commonly noted, as well as in

subjects who are often exposed to malaria. The spleen is known to clear away infected RBCs,

and splenectomised patients usually show an increase of parasitaemia during a P. falciparum

infection regardless of the antimalarial agent used, showing the crucial importance of the

spleen in parasite clearance [121].

In a P. chabaudi infection, where mice were splenectomised, thrombocytopenia was absent

which reinforced the idea that the spleen was important in thrombocytopenia [128].

However, in 2004, Karanikas et al [129] evaluated the platelet kinetics and sequestration site

24

by isotopic studies in uncomplicated malaria-induced thrombocytopenia. Although they

showed that platelet lifespan was reduced, recovery was normal, and the platelet turnover

rate was enhanced, the sequestration site of uncomplicated malaria-induced

thrombocytopenia appeared to be non-splenic. This, therefore, refuted the original hypothesis

that malaria-induced thrombocytopenia was splenic. Another piece of evidence that supports

the idea that thrombocytopenia is non-splenic is that when five Rhesus monkeys were

inoculated with sporozoites of P. cynomolgi, two of which had their spleens removed, all five

monkeys showed a significant fall in platelets correlating with maximum parasitaemia [130].

There was no difference between those with and without their spleens, with platelet levels

returning to normal levels two to six days later.

1.12.2 The role of the liver in malaria induced thrombocytopenia

Another possible organ responsible for thrombocytopenia is the liver. The relationship

between the liver and platelets is strong, as the liver is the source of thrombopoietin (the

glycoprotein made in the liver responsible for platelet production in the bone marrow) [131].

The earliest stage of the innate immune response to infection is the acute-phase response

resulting in the production of acute-phase protein by the liver [132]. Stimulators of acute-

phase proteins production include IL-1β and TGF-β which are platelet-derived immune

mediators. Activated platelets express adhesion molecules such as P-selectin which interacts

with lectin receptors on hepatic sinusoids to induce the production of acute-phase proteins,

indicating that platelets may play a role in the induction of the acute-phase response [132].

In a P. berghei model it was found that platelets are activated early during blood stage

infection and platelets initiate the acute-phase response [132]. In this experiment, mice were

infected with P. berghei and 24 h post-infection, mice were injected intravenously with

25

fluorescently labelled anti-platelet antibodies. Mice were then euthanized, livers thinly sliced

and imaged on a fluorescent microscope (Figure 1.9a). Infected mice had ∼3-fold more

platelet foci in liver sections compared with uninfected controls, indicating an increase in

platelet trafficking to the liver during acute-phase response induction (Figure 1.9b).

Figure 1.9: Platelet recruitment to the liver post- P. berghei infection. a) Platelets (white

arrows) are recruited to the liver post infection compared to control. b) Platelet foci were

quantified from 10 different fields [132].

1.12.3 Platelet Consumption and Endothelial Activation

Platelet consumption (as a result of platelet activation) is hypothesised to be one mechanism

of malaria-induced thrombocytopenia in humans. Adenosine diphosphate can cause

activation and aggregation [133] and it has been suggested that when adenosine diphosphate

is released during erythrocytes haemolysis in a malaria infection, platelets are activated and

in turn are cleared away by the spleen [134].

Another proposed mechanism of malaria-induced thrombocytopenia regarding platelet

consumption can be a result of disseminated intravascular coagulation (DIC). DIC is a

condition in which small blood clots develop throughout the bloodstream, blocking small

blood vessels which in turn can deplete platelets due to increased clotting [135]. In a study

of 21 patients [136] with falciparum malaria, six developed DIC. The authors observed that

26

the patients with more severe thrombocytopenia also had DIC and that there was a correlation

between platelet count and C3 protein levels, with the reduction in C3 proportional to that of

parasitaemia. This suggested that thrombocytopenia was not independently associated with

C3 (a protein of the immune system that plays a role in the complement system and

contributes to innate immunity) [137]. In 2004, a study with P. falciparum and P. vivax

patients demonstrated a negative correlation between platelet counts, thrombin-anti-

thrombin complex and D-dimers which are DIC markers, suggesting that the activation of

coagulation could be partially responsible for thrombocytopenia [138].

1.12.4 Platelet Binding to Red Blood Cells

Previous studies have shown that platelets can bind to uRBCs and iRBCs [70, 139]. In mice

that were infected with P. chabaudi, when parasitaemia was ~10-15%, the proportion of

platelets bound to iRBC was greater than that of platelets bound to uRBCs. CD41 (a platelet

surface marker) on the surface of RBC was used as a marker for detecting platelet-bound

RBCs. There were three times as many infected CD41 positive RBCs compared to uRBCs,

indicating that platelets preferentially bound to iRBCs (Figure 1.10a). Similar results were

seen with cultured P. falciparum with twice the number of infected RBCs bound by human

platelets compared to uRBCs (Figure 1.10b). Therefore, the aim of Chapter 4 was to measure

the contribution platelet binding played towards thrombocytopenia in a mouse model of

malaria.

27

Figure 1.10: Platelet binding to infected and uninfected red blood cells. a) Platelets bind

three times more to infected RBCs than uninfected RBCs in mice (P <0.05) b) Platelets bind

two times more to infected RBCs than uninfected RBCs in in vitro human-model assay (P <

0.01) [70].

a b

28

Hypothesis

The first hypothesis of this thesis was that iRBCs express or secrete molecules(s) unique to

the iRBC and not to uRBCs. The molecule(s) that attract platelets and induce subsequent

activation and release of PF4 onto the surface of the infected RBC was to be determined.

The second hypothesis was that platelet binding to iRBCs contributed to thrombocytopenia

in a mouse model of malaria.

The following aims were carried out to test the above hypotheses.

Project aims

• To determine the protein(s) involved in activating platelets when platelets are

incubated with iRBCs at the trophozoite stage (chapter three).

• To investigate thrombocytopenia in a mouse model of malaria using flow cytometry

by measuring platelet, RBC and platelet bound RBC concentrations (chapter four).

• To determine platelet clearance during an infection (chapter four).

• To determine if platelet complexes contribute to the loss of free circulating platelets

and ultimately contribute to thrombocytopenia (chapter four).

29

Chapter 2 Materials and Methods

2.1 Materials for Human Platelet Activation Studies

2.1.1 10x Platelet Wash Buffer

43 mM K2HPO4

43 mM Na2HPO4

243 mM NaH2PO4

1.13 M NaCl

55 mM Glucose

pH should be ~6.8, filter sterilised and stored at -20oC.

2.1.2 1x Platelet Wash Working buffer (PWB) – made fresh on the day

1x platelet wash buffer, 10mM theophylline (Simga-Aldrich), 0.35% (w/v) human serum

albumin (Sigma-Aldrich).

2.1.3 10x Tyrode Solution

1.34 M NaCl

120 mM NaHCO3

29 mM KCl

3.4 mM NaH2PO4.2H2O (monobasic)

10 mM MgCl2

50 mM HEPES

50 mM Glucose

30

pH should be 7.35, filter sterilized and stored at -20oC.

2.1.4 Tyrode working buffer - made fresh on day of prep

0.35% (w/v) human serum albumin, 1x Tyrode solution.

2.1.5 10x Human Tonicity Buffer (HTB)

4 mM NaH2PO4.H2O

16 mM Na2HPO4.2H2O

120 Mm NaCl

2.1.6 FACS Buffer

1x HTB containing 1% Bovine Serum Albumin (Sigma-Aldrich).

2.1.7 Hypotonic Lysis Buffer

50 mM Hepes pH 7.5

0.5 M KCl

5 mM Dithiothreitol

50 mM Lysine

3 mM MgCl2

2.1.8 10x RPMI

Add one sachet of RPMI 1640 Medium (Thermo Fisher Scientific) to 80 mL milliQ water

and top up to 100 mL when dissolved.

31

2.1.9 90% Percoll

Make up 90% Percoll (v/v) from 100% Percoll (Sigma-Aldrich) in 10x RPMI.

2.1.10 70% Percoll

Make up 70% Percoll v/v solution from 90% Percoll solution in RCW/Sorbitol.

2.1.11 Phosphate Buffered Saline (PBS)

137mM NaCl

2.7mM KCl

8.1mM Na2HPO4

pH 7.2-7.4, 0.2µm filter sterilized.

2.1.12 Red Cell Wash/Sorbitol

13.3% (w/v) Sorbitol

10mM Sodium phosphate,

160mM NaCl

pH 7.4 and filter sterilized.

2.1.13 PF4 ELISA Wash Buffer

0.05% Tween™ 20 (Sigma-Aldrich) in PBS

2.1.14 Reagent Diluent

1% Bovine Serum Albumin (Sigma-Aldrich) in PBS pH 7.2-7.4, 0.2µm filter sterilised.

32

2.1.15 Stop Solution

2 N H2SO4

2.2 Materials for Mouse Models of Thrombocytopenia Studies

2.2.1 Citrate Buffer

97 mM Na3C6H5O7

43 mM C6H8O7

243 mM C6H12O6

Citrate buffer was stored at 4ºC.

2.3 Methods for Human Platelet Activation Studies

2.3.1 Human Platelet Purification

Human platelets were obtained from freshly drawn blood from volunteers on the day of the

experiment by an experienced phlebotomist and according to the ANU ethics protocol

2014/732 and Macquarie University ethics protocol 5201200714. Collection of blood was

into BD Vacutainer™ Plus Plastic Citrate tubes (BD Biosciences) and subsequently

centrifuged for 13 mins at room temperature (RT) (23ºC) with brakes set at zero. After

centrifugation, platelet rich plasma (PRP) was carefully pipetted into pre-warmed 15 mL

falcon tubes and left to rest at 37ºC for 30 mins. PRP was subsequently centrifuged for a

further seven mins at 1700g to pellet platelets. After the centrifugation, the supernatant was

carefully removed to ensure minimal disruption to the platelet pellet. The platelet pellet was

then washed in an equal volume of platelet wash buffer and left to rest at 37ºC for a further

30 mins. One final centrifugation step at 1500g for seven mins was carried out. The

33

supernatant was removed and the platelet pellet was resuspended in 500µL Tyrode’s Buffer

containing a final concentration of ADPase at 0.02U/ml. A diluted sample of platelets was

run on the ADVIA 120 Hematology System (Siemens) to measure platelet concentration and

baseline platelet activation levels using mean platelet volume and mean platelet component

measurement.

2.3.2 Plasmodium falciparum Culture

The human malarial species P. falciparum 3D7 laboratory strain was used for human platelet

activation studies. 3D7 parasites were cultured with blood donated by the Australian Red

Cross, to a maximum of 10% parasitaemia and 2% haematocrit in Complete Culture Medium

(CCM) (Sigma-Adrich). Cultures were fed every second day to remove waste products,

retain correct pH and provide fresh nutrients to the parasites. This was done by transferring

the culture to sterile centrifuge tubes and centrifuged at 500g for five mins (brakes up 5,

down 3) at RT. The supernatant was removed and replaced with fresh media. To split the

cells to a desired level of parasitaemia, an appropriate volume of parasitised cells were mixed

with fresh uninfected blood to a final haematocrit of 2% using the below calculation. Cultures

were maintained in sealed flasks filled with a gas mixture (1% O2/5 % CO2 in N2) and

incubated at 37 oC, shaking at 50rpm.

Amount of infected of blood required = (% parasitaemia required/current % parasitaemia)*

total volume of blood

Amount of fresh blood required = total volume of blood – amount of infected blood

34

2.3.3 Giemsa smears: Human Blood

In order to determine parasitaemia, 100µL of P. falciparum culture was centrifuged to pellet

infected blood. 80µL of supernatant was removed and the remaining media was used to

resuspend the pellet. A smear was made using the blood and was then subsequently fixed in

methanol for 45 secs. The slide was left to air dry and stained in Giemsa for 20 mins, before

air-drying again, and visualizing under a light microscope.

2.3.4 Percoll Gradient

When parasitaemia reached 5-10% trophozoite stage (as determined via Giemsa stain), a

Percoll gradient was used to purify trophozoite stage RBCs to use in subsequent experiments

(section 2.3.6). Culture was decanted into a 50mL falcon tube and centrifuged for five mins

at 500g (brakes up 5 down 3). The supernatant was removed and the infected RBC pellet was

gently layered on top of 70% percoll and centrifuged for 10 mins at 3500rpm (brakes up 3

down 0). Three layers were seen after the centrifugation; ring stage and uRBCs at the bottom,

middle layer of percoll and only the top layer was the trophozoites. The top layer was

carefully pipetted and used for platelet activation assays.

2.3.5 Whole Protein Purification

When parasitaemia reached 5-10% trophozoite stage, tropohozoite stage RBCs were purified

as per section 2.3.4 and resuspended in 20 vols of ice-cold hypotonic lysis buffer containing

cOmplete, Mini, EDTA-free tablets (Roche). Three cycles of freeze thawing in dry ice was

carried out and the samples were centrifuged at 10,000g for 15 mins. Supernatants were

removed and pellets were washed in buffer and, after the final wash, stored in aliquots at -80

oC.

35

2.3.6 Platelet Activation Assay

Purified human platelets (300µL) collected as described in section 2.3.1 were incubated at

37ºC with either trophozoite stage RBCs (Section 2.3.4) or infected lysate from hypotonically

lysed iRBCs collected as described Section 2.3.5. 100µL was fixed with 1% cytofix (BD

Biosciences; diluted 1/4 in PBS to achieve 1%) to stain with anti-CD62P-FITC (AK4,

BioLegend) prior to flow cytometry (Section 2.3.8), 100µL was stained with 10µg/mL

anti-PAC1-FITC (PAC1, BioLegend) for 45 mins at 37ºC and fixed with 1% cytofix and the

remaining 100µL centrifuged at 1000g for two mins supernatant was collected and kept at -

80ºC for PF4 detection.

2.3.7 Platelet Factor 4 ELISA

PF4 detection was carried out using the commercially available Human CXCL/PF4 DuoSet

ELISA (R & D systems). Briefly, 96-well plates were coated with capture antibody by

incubating overnight at RT. Wells were aspirated and then washed with PF4 ELISA wash

buffer (3x 400 µL) and blocked with 300µL of reagent diluent. A subsequent washing step

was carried out. 100µL of sample was added to each well before being incubated for two

hours at RT and then washed. 100µL of detection antibody was added, incubated for two

hours at RT and washed. 100µL of Streptavidin-HRP was added to each well and left covered

in the dark for 20 mins at RT and subsequently washed. 100 µL of substrate solution

(1-Step™ Turbo TMB-ELISA) (BioRad) was added to each well, left in the dark for 20 mins

and washed. In order to stop the reaction, 50µL of stop solution was added to each well. The

SpectraMax 190 Microplate Reader (Molecular Devices) was immediately used to measure

the optical density at 450 nm.

36

2.3.8 Flow Cytometry Staining

Samples fixed for CD62P (Section 2.3.6) staining were centrifuged for two mins and fixation

buffer was removed carefully to not disrupt the platelet pellet. The pellet was resuspended

with 500µL FACS buffer and centrifuged for two mins at 1000g. FACS buffer was removed

and the pellet resuspended in 50µL FACS buffer containing 20µg/mL

anti-CD62P-FITC on ice for ~30 mins. 1mL FACS buffer was added and centrifuged for two

mins at 1000g. Supernatant was removed and the pellet was resuspended in 500µL FACS

buffer containing 1/1000 Hoechst and left at RT for ~20 mins before running on flow

cytometry. A total of 50,000 events were collected.

The PAC1 sample (described in 2.3.6) was centrifuged for two mins at 1000g and supernatant

removed. The pellet was resuspended in 500µL FACS buffer and run on flow cytometry with

a total of 50,000 events collected.

2.3.9 Platelet Rich Plasma Assay

The concentration of ATP released from platelet rich plasma (PRP) was determined using

the Chrono-Log Model 700 Whole Blood /Optical Lume Aggregometer (Chrono-Log

Corporation). The Chrono-Log measures ATP using a luminescent assay using firefly

luciferin-luciferase (CHRONO-LUME reagent) as the ATP binds to the luciferin-luciferase

and generates light. Briefly, 900µL (450µL PRP and 450µL saline) was transferred to

cuvettes containing magnetic stir bars and allowed to pre-warm to 37ºC in the aggregometer

wells for three minutes. 100µL CHRONO-LUME was added to each cuvette and incubated

for two minutes before the addition of purified trophozoites (Section 2.3.4) or infected lysate

(Section 2.3.5) and monitored for ATP release.

37

2.3.10 Gating strategy for positive events

In order to measure percentage of positive events, flow cytometry was carried out using

samples prepared as described in Section 2.3.6 with anti-PAC1 or anti-CD62P both

conjugated to FITC (but different samples) and the following gating strategy was

implemented (Figure 2.1).

38

Figure 2.1: Gating strategy for positive events. a) singlets gated using FSC-H vs FSC-A

b) SSC-A vs FSC-A was used to gate for population of interest (POI) from a c) unstained

sample d) CD41 positive sample

b

b

c

c

d

d

CD41

Hoescht

CD41

Hoescht

CD41 positive

events

CD41 positive

events

a

b

39

2.4 Methods for Mouse Models of Thrombocytopenia

All animal work was carried out in accordance with protocol A2014/55 at The Australian

National University and housed under a 12 hour:12 hour light:dark cycle with food and water

provided ad libitum.

2.4.1 Blood collection from tail

In order to measure platelet and RBC concentration, 5uL of blood was collected via pipette

from each mouse through a small tail snip. Blood was added to 45uL of ACD buffer

containing 0.01mg/mL anti-CD41-FITC (MWReg30, BD Biosciences) and 0.004mg/mL

anti-TER-119-PeCy7 (TER-119, eBioscience). The blood/ACD mixture was incubated for

30 mins at RT and 10µL was then added to 980µL PBS to subsequently run on flow

cytometry. Before running the sample on flow cytometry, 10µL of 123ecounting beads

(eBioscience) were added to the sample. 1000 bead events were collected to ensure

statistically significant results. Using counting beads the concentration of platelets and RBCs

were able to be back calculated using the following formula.

Concentration of Platelets or RBCs/mL = (Events Collected/Bead volume (uL)*Dilution

Factor*1000

2.4.2 Giemsa smears: Mouse Blood

In order to determine parasitaemia in mice, a drop of blood was collected from a tail snip and

smeared. The slide was fixed in methanol for 45 secs and left to air dry and subsequently

stained in Giemsa for 20 mins, before air-drying again, and visualizing under a light

microscope.

40

2.4.3 Biotinylation of Cells

When parasites were detected in the bloodstream via Giemsa staining (Section 2.4.2),

200µL of PBS containing 1.5mg of EZ-Link Sulfo-NHS-LC-Biotin (ThermoFisher) was

injected into the lateral vein of the mouse via intravenous (IV) injection. This labels all the

cells of the blood, however for this assay biotinylated platelets were of interest. Four hours

after IV injection, 5uL of blood was collected via a small tail snip. Blood was added to 45uL

of ACD buffer containing. 5µL of blood/ACD mixture was subsequently added to 45µL 1x

PBS containing 0.01mg/mL anti-CD41-FITC and 0.006mg/mL anti-TER-119-PeCy7 and

0.01mg/mL streptavidin APC-Cy7 (eBioscience). The blood/ACD mixture was incubated

for 30 mins at RT and added to 940µL PBS to subsequently run on flow cytometry. Before

running the sample on flow cytometry, 10µL of counting beads was added to the sample, and

1000 beads were collected to ensure statistically significant results.

2.4.4 Gating Strategy for Platelets, Red Blood Cells, iRBCs and biotinylated platelets

Using cell-specific markers, anti-CD41 for platelets, anti-TER-119 for RBCs, Hoechst for

iRBCs and streptavidin for biotinylated platelets (Table 2.1), the following gating strategy

was implemented (Figure 2.2)

41

Figure 2.2: Gating strategy for platelets/biotinylated platelets, RBCs and iRBCs. Gating

strategy for platelets/biotinylated platelets, RBCs and iRBCs. a) beads were gated using a

bead only control b) singlets gated using FSC-H vs FSC-A c) SSC-A vs FSC-A was used to

gate for population excluding beads d) CD41-FITC vs TER-119-PeCy7 to distinguish

platelet and RBC populations respectively e) platelet gate from d was used to measure

populations double positive for biotin and CD41 and termed biotinylated platelets f) RBC

gate from d was used to measure populations double positive for Hoechst and TER-119 and

determined as an iRBCs.

Bio

tin

Ho

esc

ht

b

bb

b

e

e

f

f

TER-119

Ter119

TER-119

Ter119

CD

41

CD41

Hoescht

a

e

b

e

c

e

d

e

42

2.4.5 Gating Strategy for Platelet Bound Red Blood Cells

Using cell-specific markers, platelets bound to iRBCs and uRBCs were distinguished using

the gating strategy described in Figure 2.3.

43

Figure 2.3: Gating strategy for platelet bound RBCs. a) beads were gated using a bead

only control b) singlets and doublets gated using FSC-H vs FSC-A c) SSC-A vs FSC-A was

used to gate for population excluding beads d) CD41 vs TER-119 to gate for all populations

positive for TER-119 e) RBC gate from d was used to measure populations for TER-119+

and TER-119+ Hoechst + to gate uRBC and iRBCs respectively f) uRBC gate from e to gate

for CD41+TER-119+ population representing platelet bound uRBCs g) iRBC gate from e to

gate for CD41+TER-119+ population representing platelet bound iRBCs.

TER-119

Ter119

Ter119

Ter119

TER-119

Ter119

TER-119

Ter119

CD

41

Ter119

Ter119g

f

d

d

f

f

a

a

b

d

c

d

CD

41

CD

41

g

ge

f

e

f

TER-119

Ter119

Ter119

Ter119

g

f

44

2.4.6 Statistical Analysis

Statistical analysis was performed with GraphPad Prism 5 software using two-way-ANOVA

or t-tests. P-values are listed in the legends of figures. Statistical significance was determined

as P<0.05.

Platelet consumption was examined using the biotinylated population, classified as the

''original'' population at day of biotinylation. 50% (where y=50) of this biotinylated original

platelet population was calculated using linear regression (Figure 2.4 shows an example) for

each individual mouse. The mean from each group of mice (infected or uninfected) was used

to determine the time taken for 50% clearance of biotinylated platelets.

Figure 2.4 An example of a linear regression. Linear regression for one mouse, to find the

time it took post-biotinylation for 50% of the original biotinylated platelets to remain in

circulation.

0 1 2 3 4 50

20

40

60

80

100

y= -24.74x + 88.6

Day post biotinlation

% b

ioti

nyla

ted

pla

tele

ts

45

Table of cell markers used for flow cytometry

Table 2-1 Cell markers used to distinguish human and mouse populations of interest

on flow cytometry

.

Parameter Measured Cell marker and fluorophore used

Platelets (human) Anti-human PAC1-FITC (PAC1+)

(PAC1, Biolegend)

Anti-human CD62P-FITC (CD62P+)

(AK4, BioLegend)

Platelets (mouse) Anti-mouse CD41-FITC (CD41+)

(MWReg30 BD Biosciences)

Red Blood Cells (mouse) Anti-mouse TER-119-PeCy7 (TER-119+)

(Ter-119 eBioscience)

Infected Red Blood Cells (mouse) Anti-mouse TER-119-PeCy7 and Hoechst

( TER-119+/ Hoechst +)

Biotinylated Platelets (mouse) Anti-mouse CD41-FITC and Streptavadin-

APC-Cy7 (eBioscience) (CD41+/Biotin+)

46

Chapter 3 Platelet Activation in Response

to Plasmodium falciparum

Platelets have been found to play a protective role against infection by the malaria parasite

P. falciparum by targeting its’ DV. PF4 has been implicated as the molecule responsible for

the destruction of the parasite [69, 111], however the exact mechanism of how infected red

blood cells (iRBCs) attract platelets and release PF4 is yet to be elucidated.

It has been demonstrated that platelets can bind to iRBCs and uRBCs [70, 111] where, upon

contact of platelets to iRBCs, PF4 is released by the platelet, and deposited onto the surface

of the iRBC but not the uRBC [111]. After deposition onto the iRBC surface, PF4 is

translocated to the DV and kills the parasite [69]. Platelets can interact with iRBCs via the

platelet cell surface molecule CD36 binding to PfEMP1 variants translocated to the iRBC

cell surface by the parasite [91]. However, the molecule(s) that parasites express resulting in

PF4 release from platelets and subsequent deposition onto iRBCs, but not uRBCs, has yet to

be determined. The aim of this chapter was to identify what molecule(s) the iRBC expresses

to stimulate platelet secretion of PF4. Flow cytometry was used to measure platelet activation

using the platelet markers anti-CD62P and anti-PAC1 (table 2.1). Purified trophozoite stage

RBCsand lysed purified trophozoite stage RBCs (infected lysate) were used as agonists to

investigate platelet activation.

47

Aims

The aims of this chapter were:

1. Confirm platelet activation using:

1) Infected lysates from cultured cells at trophozoite stage by flow cytometry using

anti-PAC1 and anti-CD62P as markers of activation. PF4 detection was carried out

via ELISA.

2) Intact RBCs at trophozoite stage using anti-PAC1 and anti-CD62P as platelet

activation markers. PF4 detection was carried out via ELISA.

2. Test for platelet activation using platelet:iRBC ratios to find the optimal

concentration of molecule of interest to activate platelets.

The above is described in more detail in Materials and Methods (Chapter 2).

3.1 Platelet Activation Using Infected Lysate to Activate Platelets

In order to identify the molecule(s) that iRBCs may express which activate platelets, infected

lysates from cultured parasites were incubated with platelets and activation was quantified

by flow cytometry. Briefly, RBCs infected with P. falciparum at ~5-10 % parasitaemia were

purified using a Percoll gradient to obtain purified trophozoites (~80-90 %) (Section 2.3.4).

Trophozoites were subsequently lysed using the hypotonic lysis method, as described in

Section 2.3.5. Platelets at a concentration of 4.2x107platelets/mL were incubated with

infected lysate (0.53mg/mL) for 45 mins at 37ºC. Activated platelets were quantified by flow

cytometry using anti-PAC1 and anti-CD62P as markers of platelet activation. The assay was

carried out three independent times in either duplicate or triplicate.

48

3.1.1 Platelet Activation Using Infected Lysate: anti-CD62P

Results from three independent experiments carried out in either duplicate or triplicate using

infected lysate from purified trophozoites are presented in Figure 3.1. Platelet activation was

measured using anti-CD62P. Anti-CD62P is secreted from α-granules upon activation of

platelets and is cell surface expressed. Platelets only and platelets that had been treated with

hypotonic lysis buffer served as the negative control.

Figure 3.1a shows that there was baseline activation of control, untreated platelets being

positive for anti-CD62P (7% +/- 0.4%). When platelets were treated with thrombin which

served as a positive control as it is a platelet agonist, there was almost 100% activation of

platelets (96% +/- 1.0%) compared to buffer treated platelets (5.4% +/- 0.7%). When platelets

were treated with infected lysate (6.7% +/- 0.6%), there was no significant difference

between platelets that were treated with infected lysate, untreated platelets and buffer treated

platelets. Mean fluorescent intensity (MFI) was also measured but no difference was seen in

treatment groups except with thrombin treated platelets (23.1 +/- 0.4) (Figure 3.1b).

49

Figure 3.1 Activation of platelets using infected lysate: anti-CD62P. Positive events and

mean fluorescent intensity was measured using flow cytometry using anti-CD62P as a

marker of platelet activation. a) Percentage of positive events b) Mean fluorescent intensity.

Thrombin was used as a positive control. Bars of graphs represent the mean of three

independent experiments. Error bars indicate SEM ***P <0.05. 1way ANOVA using

Tukey’s Multiple Compariston Test was used.

0

50

100

150Untreated

Thrombin

Whole Protein

Buffer

***

a

Treatment

% p

osti

ve e

ven

ts

0

5

10

15

20

25Untreated

Thrombin

Whole Protein

Buffer

***b

Treatment

Mean

Flu

ore

scen

t In

ten

sit

y

50

3.1.2 Platelet Activation Using Infected Lysate: anti-PAC1

Another marker of platelet activation that was used was anti-PAC1. Anti-PAC1 recognizes

an epitope on the glycoprotein IIb/IIIa (also known as CD41) complex of activated platelets

located with, or near, the platelet fibrinogen-binding site [140]. Results from three

independent experiments carried out in either duplicate or triplicate using infected lysate

from purified trophozoites are presented in Figure 3.2.

Figure 3.2a shows that compared to untreated platelets, treatment with thrombin resulted in

increased expression of the anti-PAC1 marker, distinguished by both the number of

positively stained platelets (96.5% +/- 0.7%), and the population MFI (93.0 +/- 12.1).

Platelets treated with infected lysate also showed a significant increase in the percentage of

positive events (79.0% +/- 2.8%) when compared to untreated platelets (18.0% +/- 1.7%),

and buffer treated platelets (37.5% +/- 7.0%). This indicated that the infected lysate activated

platelets and in turn exposed the integrin which resulted in increased levels of anti-PAC1

being detected by flow cytometry. Mean MFI was also measured but no difference was seen

in treatment groups except with thrombin treated platelets (Figure 3.2b). Taken together,

these experiments indicated platelets were unable to be activated via the lysate of iRBCs as

measured by anti-CD62P but possibly via anti-PAC1 as it may have been that the molecule

acted on the glycoprotein IIb/IIIa causing activation and hence bind anti-PAC1 but not

activate the alpha granule.

51

0

50

100

150Untreated

Thrombin

Whole Protein

Buffer***

***

Treatment

% p

osti

ve e

ven

ts

0

50

100

150Untreated

Thrombin

Whole Protein

Buffer

***

b

Treatment

Mean

Flu

ore

scen

t In

ten

sit

y

Figure 3.2 Activation of platelets using infected lysate: anti-PAC1. Positive events and

mean fluorescent intensity was measured using flow cytometry using anti-PAC1 as a marker

of platelet activation. a) Percentage of positive events b) Mean fluorescent intensity.

Thrombin was used as a positive control. Bars of graphs represent mean of three experiments

independent experiments. Error bars indicate SEM ***P<0.05. 1way ANOVA using Tukey’s

Multiple Compariston Test was used.

a

a

52

3.1.3 Platelet Factor 4 Release Using Infected Lysate

Another marker of platelet activation is PF4. PF4 is a chemokine secreted from the platelet

alpha granule upon platelet activation. PF4 secretion by platelets in response to treatment

with iRBC lysates was monitored using ELISA (Section 2.3.7) on the supernatant collected

from the treated platelets. As seen in Figure 3.3, the supernatant from untreated platelets

contained some PF4 (baseline level, approx. 1000 pg/mL). A significant increase in PF4

concentration was seen when platelets were incubated with thrombin, consistent with the

thrombin-mediated activation of platelets. This indicated that the platelets were able to be

activated upon stimulation with an appropriate agonist. Platelets treated with hypotonic lysis

buffer had no effect on platelet activation similar to the untreated control. There was no

difference observed between negative controls and infected lysate treated platelets.

53

0

1000

2000

3000Untreated

Thrombin

Infected lysate

Buffer

***

Treatment

PF

4 c

on

cen

trati

on

(p

g/m

L) ***

Figure 3.3 Platelet Factor 4 ELISA with infected lysate. PF4 levels were measured using

ELISA. Thrombin was used as a positive control. Bars of graphs represent mean of three

independent experiments. Error bars indicate SEM. ***P <0.05. 1way ANOVA using

Tukey’s Multiple Compariston Test was used.

54

3.1.4 Platelet Activation using Infected and Uninfected Lysate

As shown in Figure 3.2a, infected lysate activated platelets. However, whether this was due

to a parasite protein or an uRBC protein, was determined next. Figure 3.4a shows platelet

activation using uninfected lysate. When uninfected lysate from control uRBCs was

incubated with platelets to check activation levels, no platelet activation was observed

between platelets treated with infected lysate or uninfected lysate (Figure 3.4a and Figure

3.b). Thrombin did however activate platelets as shown by anti-PAC1 (88.1% +/- 0.6%)

(Figure 3.4a) and anti-CD62P (56.5% +/- 3.3%) (Figure 3.4b), suggesting that this was not

due to a lack of activation ability by the platelets. MFI was again measured but no difference

was seen, except with thrombin treated platelets via anti-PAC1 (27.8 +/- 0.8) (Figure 3.4c)

and anti-CD62P (12.0 +/- 0.3) (Figure 3.4d). Even though no activation was observed with

either infected or uninfected lysate, it is possible that the different batch of infected lysate

used may have affected the lack of activation. This experiment was carried out once

compared to three times previously (Figures 3.1 and 3.2) with a different preparation of lysate

hence no definite conclusion can be made regarding activation of platelets using infected

lysate. Hence, the next step was to determine if intact trophozoites activated platelets.

55

0

20

40

60

80

100Untreated

Thrombin

Infected Lysate

Uninfected Lysate

Buffer

***

Treatment

po

sit

ive e

ven

ts (

%)

0

5

10

15Untreated

Thrombin

Infected Lysate

Uninfected Lysate

Buffer

***

TreatmentM

ean

Flu

ore

scen

t In

ten

sit

y

Figure 3.4 anti-CD62P and anti-PAC1 Positive events and mean fluorescent intensity.

Positive events and mean fluorescent intensity was measured using flow cytometry using

anti-PAC1 and anti-CD62P as a marker of platelet activation. a) anti-PAC1 positive events

b) anti-CD62P positive events c) anti-PAC1 MFI D) anti-CD62P MFI. Thrombin was used

as a positive control. Bars of graphs represent mean of one independent experiment carried

out in triplicate. Error bars indicate SEM, ***P<0.05 1way ANOVA using Tukey’s Multiple

Compariston Test was used.

0

20

40

60

80Untreated

Thrombin

Infected Lysate

Uninfected Lysate

Buffer

***

Treatment

po

sti

ve

ev

en

ts (

%)

0

10

20

30

40Untreated

Thrombin

Buffer

Infected Lysate

Uninfected Lysate

***

Treatment

Mean

Flu

ore

scen

t In

ten

sit

y

a

a

c

a

a

b

a

a

d

a

a

56

3.2 Platelet activation using purified trophozoite stage red blood cells

To further investigate the inconsistent effects of parasitised cell lysates on platelet activation,

additional studies were conducted examining the ability of RBCs at trophozoite stageto

activate platelets using the same three markers of platelet activation. It was hypothesized that

intact trophozoites may express a protein(s), which when expressed appropriately on the cell

surface, are responsible for platelet:iRBC contact and subsequent platelet activation. In

addition, it has previously been shown, using PRP incubated with purified iRBC membranes,

that platelets were able to be activated using anti-PAC1 as a marker [100].

3.2.1 Platelet Activation Using Purified Trophozoite stage: anti-CD62P and anti-PAC1

Infected RBCs at trophozoite stage were purified via a Percoll gradient which yielded

approximately 80-90% trophozoites (Section 2.3.4). Initial trophozoite-platelet incubations

were conducted using 1:10 platelet to trophozoite ratio. Using anti-CD62P and anti-PAC1,

the percentage of positive events were measured using flow cytometry. Infected RBCs at

trophozoite stage RBCs did not activate platelets when anti-CD62P was used as a marker as

there was no difference between trophozoite treated platelets (6.1% +/- 0.6%) and uRBC

treated platelets (5.3% +/- 1.5%). There was a significant difference in thrombin treated

platelets (58.8% +/- 4.7%) compared to untreated platelets (11.1% +/- 3.7%) indicating that

platelets were able to be activated upon stimulation with an appropriate agonist (Figure 3.5a).

MFI was measured but no difference was observed except with thrombin treated platelets

(Figure 3.5c) (22.2 +/- 3.0). Using anti-PAC1 as a marker for platelet activation, no platelet

activation was seen using trophozoite stage parasites (3.1% +/- 1.3%) compared to uRBC

treated platelets (3.4% +/- 1.5%) (Figure 3.5b). MFI was unable to be analyzed due to data

not saved in the correct format for CD62P.

57

0

20

40

60

80Untreated

Thrombin

Trophozoites

Red Blood Cells (uninfected)

Media

***

Treatment

po

sti

ve e

ven

ts (

%)

0

20

40

60

80

Untreated

Thrombin

Trophozoites

Red Blood Cells (uninfected)

Buffer

***

Treatment

po

sit

ive e

ven

ts (

%)

0

10

20

30Untreated

Thrombin

Trophozoites

Red Blood Cells (uninfected)

Media

***

Treatment

Mean

Flu

ore

scen

t In

ten

sit

y

Figure 3.5 Platelet activation by RBCs at trophozoite stage. Positive events and mean

fluorescent intensity was measured using flow cytometry using anti-PAC1 and anti-CD62P

as a marker of platelet activation. a) anti-PAC1 positive events b) anti-CD62P positive events

c) anti-PAC1 MFI. Thrombin was used as a positive control. Bars of graphs represent mean

of two independent experiments. Error bars represent SEM. ***P <0.05. 1way ANOVA

using Tukey’s Multiple Compariston Test was used.

3.2.2 Platelet Factor 4 Release Using purified trophozoite stage red blood cells: anti-

PAC1 and anti-CD62P

Another set of trophozoite stage RBC-platelet incubation experiments were performed using

various cell ratios starting from a 1:1 platelet to trophozoite ratio. This experiment included

additional controls of platelets incubated with uRBCs at the same ratios, as well as thrombin

as a positive control. All of platelet:trophozoite ratio supernatants were assayed for PF4

concentrations. Figure 3.6 shows, there was baseline activation as seen by PF4 release in

untreated and media treated platelets. Thrombin was able to cause PF4 secretion, albeit at a

a

a

c

a

a

b

a

a

58

lower level to that which was previously observed, which may suggest the platelets were not

functioning normally; however the experiment was carried out two independent times in

triplicate each time with different platelet donors. Neither trophozoite stage RBCs nor

uninfected RBCs caused PF4 secretion. At the ratio of 1:0.0625, although significant (P

0.008) there was no significant difference between the platelet:trophozoite ratio and media

(p 0.1066) which acted as a control.

Figure 3.6 Platelet Factor 4 ELISA with trophozoite stage RBCs a) a significant

difference was seen in PF4 when thrombin was incubated with platelets. PF4 was measured

in trophozoite treated platelets at various ratios. Bars of graphs represent mean of two

independent experiments *** p <0.001.

Statistics was carried out on GenStat using two-way ANOVA.

1:11:0

.5

1:0.2

5

1:0.1

25

1:0.0

625

0

100

200

300

400

Trophozoites

Red Blood Cells (uninfected)

Untreated

Thrombin

Media

Platelet:Trophozoite

PF

4 c

on

cen

trati

on

(p

g/m

L)

59

Discussion

Platelets have been shown to play a role in the killing of the malaria parasite by targeting the

(DV [69]. It has been shown that PF4, in conjunction with the Duffy receptor on RBCs,

targets the DV, lysing it and killing the parasite [111]. Platelets can bind to iRBCs, however

it is only when a platelet adheres to an iRBC that PF4 is secreted [111]. The molecule(s) an

iRBC expresses or secretes that ensures only a platelet bound to an iRBC secretes PF4 has

yet to be determined and was the main aim of this chapter.

This study used three markers of platelet activation; anti-CD62P, PF4, and anti-PAC1.

Anti-CD62P [141] and PF4 [142] are released from α-granules upon platelet activation,

whilst anti-PAC1 recognizes an epitope on the glycoprotein IIb/IIIa (also known as CD41)

complex of activated platelets located with, or near, the platelet fibrinogen-binding site

[140].

In order to measure platelet activation, flow cytometry was used with the use of two

parameters; percent of positive events and mean fluorescent intensity (MFI). MFI is

the mean of the fluorescent intensity in the fluorescence channel in use, whilst percent of

positive events is the number of cells that have bound antibodies of interest to it. However,

sometimes using positive events is a more precise way of interpreting FACS data. This is

due to MFI being a mean of intensity – such that it depends on the amount of antibody

attached to a certain population of cells and assuming that the antibody is evenly distributed

across the cell population of interest. In the above experiments, the percent of positive events

and MFI were used using the platelet activation markers anti-PAC1 and anti-CD62P. PF4

detection was carried out via a commercially available PF4 ELISA kit.

60

The first aim of the project was to observe platelet activation via the addition of infected

lysate. RBCs at trophozoite stage were lysed hypotonically which was subsequently termed

“infected lysate” and incubated with purified human platelets. This was conducted to narrow

down the potential parasite expressed molecule(s) responsible for platelets solely binding to

iRBCs and subsequent deposition of PF4 on to the surface (i.e if a cytoplasmic protein was

responsible). Using one batch of infected lysate, a consistent activation of platelet

glycoprotein IIb/IIIa receptor (PAC1) was observed, but not alpha granule activation.

However, using second lysate preparation no activation through either pathway was

observed.

It must be noted that two batches of purified lysate were used. The first batch was used for

the three experiments (Figure 3.2a) which showed that whole protein caused activation. The

second batch of protein however showed that no activation occurred (Figure 3.4a). After

purification for both lysates, Giemsa smears indicated ~80-90% trophozoite stage parasites

were present. Although unlikely, it may be possible that the first batch of infected lysate that

was used expressed a parasite protein in a given point in time in which the second batch of

parasites did not, thus contributing to the differences in platelet activation observed between

experiments.

Due to the lack of reproducibility no conclusive statement was able to be made regarding the

activation of platelets via parasites.

It has been previously shown using flow cytometry, with anti-PAC1 as markers of platelet

activation, that when PRP was incubated with Percoll purified intact trophozoite stage

iRBCs, there was a significant increase in MFI for anti-PAC1 [100]. PF4 levels were also

61

measured and showed a significant difference in PRP treated with iRBCs compared to PRP

control uRBCSs. Another study also showed PF4 release in the presence of human purified

platelets [111]. In order to determine what molecule(s) the iRBC expressed was responsible

for activating platelets, Srivastava and colleagues [100] incubated PRP with saponin purified

P. falciparum or hemozoin. Pure trophozoites did not stimulate platelets, nor did purified

hemozoin. Purified membranes of iRBC did however activate platelets whilst uRBC

membranes did not which demonstrated that Plasmodium iRBCs express membrane proteins

that are pro-thrombotic [100].

In this thesis, Percoll purified intact trophozoite stage RBCs were incubated with human

purified platelets using the same markers as the previously published data; anti-CD62P and

anti-PAC1. However, platelet activation was not observed. PF4 detection was also measured

via ELISAs after human purified platelets were incubated with Percoll purified intact

trophozoite stage RBCs but no PF4 was detected.

A difference in the designs of the studies must be noted. In the current study, activation of

platelets was carried out using purified platelets and not PRP as the study conducted by

Srivastava and colleagues [100]. Although PRP has a high concentration of platelets it

contains other components such as chemokines, cytokines, growth factors, adhesive proteins

and proteases [143]. It may be possible that another component in PRP may work together

with iRBCs to activate platelets which is likely to occur in an in vivo setting.

To parallel the PRP experiment, the CHRONO LOG® Model 700 Whole Blood/Optical

Lumi-Aggregometer was used to measure Adenosine 5'-triphosphate (ATP) release from

dense granules of activated platelets. Activated platelets release ATP from their dense

62

granules [144] and the Chronolog is based on the luciferin-luciferase assay which binds ATP,

generating light which is proportional to the amount of ATP released by the platelets when

stimulated with an appropriate agonist .

In this chapter, PRP incubated with thrombin was used as a positive control and had a ATP

reading of >0.5nm which fell into the expected range. When intact purified trophozoites were

incubated with PRP, no ATP was released indicating that purified RBCs at trophozoite stage

were unable to activate PRP, which further strengthens the previous experiment by flow

cytometry where purified human platelets in the presence of intact trophozoites were not

activated.

A contributing factor to the lack of platelet activation observed could be the time between

Percoll purification and subsequent use of parasites in the platelet assay. Purified RBCs at

trophozoite stage were used shortly after purification although it may be possible that the

time between purifying and use in the assay was too long thus making the parasites no longer

viable and therefore the molecule(s) of interest unable to activate platelets.

A number of attempts and protocol variations were carried out to test if Plasmodium iRBCs

can cause platelet activation. Whole protein lysate from trophozoite stage infected cells was

observed to activate platelets based on the anti-PAC1 marker. However, subsequent parasite

preparations, including other lysate preparations and intact parasitised cells produced

inconsistent effects and generally no activity against platelets. It remains unclear whether

this was due to technical reasons, variations in the parasite strain as various cultures were

used over the course of these trials (more than 12 months), or other unknown reasons.

63

Chapter 4 Thrombocytopenia in Mouse

Models of Plasmodium chabaudi

It was reported as far back as 1924 that platelet numbers were reduced in the peripheral blood

of patients with acute malaria [145]. This decrease in platelet number is termed

'thrombocytopenia', with clinical thrombocytopenia cited in the literature as platelet

concentration less than <150,000/mm3 [146]. During a malaria infection, up to 80% of

patients have thrombocytopenia, with a distinct negative correlation between platelet

concentration and parasitaemia. The causes of malaria-induced thrombocytopenia are wide

and varied, and not well understood, but platelet consumption or destruction is thought to

play a vital role [147]. Other possible mechanisms that have been proposed include splenic

pooling, liver sequestration and endothelial activation.

It has been previously shown in human [124], mouse [70] and monkey [125] models of

malaria that a loss of platelets accompanies an increase in parasitaemia. In a study conducted

in the McMorran lab, thrombocytopenia was investigated in a cohort of mice infected with

Plasmodium chabaudi. Whilst thrombocytopenia was observed it was also noted here that

platelets may bind preferentially to infected iRBCs over uRBCs [70].

Whilst cohorts of mice were sacrificed in that study [70], the present mouse model used to

measure platelet concentration levels was based on Alugupalli and colleagues [148]. When

compared to human malaria infections such as that by de Mast and colleagues, both models

show that thrombocytopenia occurs before the onset of symptoms such as anaemia, therefore

showing platelet loss is a good indicator of a suspected malarial infection. The mouse model

used shows that although platelet concentration returns to normal they usually die as a result

64

of anaemia whilst human studies show that with anti-malarials, platelet concentrations also

return to normal [149].

The research reported in this chapter builds upon previous mouse malaria studies and

describes the relationship between platelet bound iRBCs and platelet bound uRBCs in

thrombocytopenia.

The aims of this chapter were to

1. Develop protocols to quantify platelet, parasite and platelet-RBC complex

concentrations in mice on a daily basis, and determine the time taken of platelet

clearance in mice infected with P. chabaudi to occur.

2. Investigate the relationship between the concentration of unbound platelets and

platelet complexes and assess the contribution of complexes towards the loss of

platelets in malaria-induced thrombocytopenia.

3. Determine if platelet loss was a result of inhibition of platelet production or due to

platelet consumption, by measuring the time for platelet clearance to occur during the

course of infection

In order to address these aims, experiments were first conducted to develop a protocol to

allow continuous monitoring of parasite, platelet, RBC and platelet-RBC complexes using

flow cytometry to observe and quantify thrombocytopenia and platelet-RBC complex

formation over the course of an infection in the same mice. This allowed confounding factors

such as inter/intra-mouse variation to be monitored. Several pilot experiments were

conducted to optimise the daily collection of blood samples from the tail, as well as

optimising the use of flow cytometry with counting beads and antibodies specific for platelets

65

and RBCs (refer to Section 2.4.1 and 2.4.3) section for the optimised protocol). To measure

the rates of platelet clearance a protocol was developed using IV injected EZ-Link NHS-

Biotin (biotin) to label all circulating cells and quantify the relative proportions of biotin-

labelled and unlabeled platelets over time. This involved optimising the volumes of blood

and concentrations of streptavidin-fluorophore conjugate to efficiently label all biotinylated

cells in the samples.

Male mice were infected with 1x105 parasites (P. chabaudi) and during the course of

infection, platelet, RBC, iRBC concentrations, and frequencies of uRBC and iRBC bound to

platelets were measured (plt:uRBC and plt:iRBC respectively). Three separate infection

experiments were conducted, consisting of between three and seven infected mice as well as

between three and nine uninfected controls. The first experiment was conducted to observe

thrombocytopenia whilst in the other two experiments, infected animals were IV injected

with biotin when parasite levels were between 1% & 3% to monitor clearance of circulating

platelets. RBCs were measured using the RBC cell surface marker TER-119 antibody which

recognizes a molecule associated with glycophorin A and marks all stages of murine RBCs.

Platelets were measured using anti-CD41 as CD41 is a glycoprotein expressed by platelets.

To measure infected RBCs, Hoechst dye (which stains DNA, of which mature RBCs have

none) and anti-TER-119 were used. Table 2.1 provides a summary of the markers used and

the parameters each measured.

66

4.1 Challenge #1: Platelets, Red Blood Cells and Platelet Complexes during a

Plasmodium chabaudi infection in male mice

The major aim of this challenge was to confirm that the optimized platelet, RBC and parasite

quantification protocol could be used to successfully observe malaria-induced

thrombocytopenia and the formation of platelet-RBC complexes. A total of eight mice were

investigated, five uninfected and five infected with 1x105 P. chabaudi parasites via

intraperitoneal (IP) injection. Of the infected, only three mice had detectable parasites in their

bloodstream.

The development of parasitaemia was monitored in the infected mice by Giemsa staining as

described in section 2.4.2. Blood samples for platelet and RBC quantification were collected

each day beginning seven days post-infection, and sampling continued until the infections

had resolved (Figure 4.1a). Quantifiable levels of parasites were observed between days ten

and 17 following infection, and peak parasitaemia occurred on day 13 (30% +/- 6.2%).

Measured platelet concentrations in the uninfected mice were relatively stable for the first

two days of sampling although they were approximately 70% of the levels expected for this

strain. There was an unusual spike in platelet numbers measured on day nine (for both

infected and uninfected groups) that was probably due to unidentifiable technical reasons or

perhaps a FACS calibration error rather that representing a true physiological change. Platelet

concentrations in the infected mice were the same as the uninfected mice between days

seven-ten, and then began decreasing markedly on day 11. Levels reached a nadir on day 13

(4.7x107 +/- 5.5x106cells/mL) and began increasing and reached the same levels as the

uninfected group on day 16. The lower levels of platelets in the infected group were

statistically significant on days 12-15.

67

RBC concentration was also measured using flow cytometry (Figure 4.1b). From the first

three days of sampling (days seven-nine), uninfected mice had relatively consistent RBC

concentrations although this was 70% of what was expected for C57BL/6 mice

(1.0x1010cells/mL). By the fourth day (day ten post-infection) of sampling, RBC

concentrations dropped by 15% (5.7x109 +/- 5.5x109cells/mL) of the original RBC

concentration and continued to decline until the end of the experiment. The first five days

decreased slowly and thereafter, a large decrease was seen by the end of experiment where

RBC concentration was 3.6x109 +/- 8.8x108cells/mL. There was a spike in RBC

concentrations on day 15 possibly due to a technical rather than biological reason. This

decrease in RBC concentration may have been a result of daily sampling and therefore mild

anaemia may be occurring as a result of lost RBCs. In infected mice, days seven and eight

had consistent RBC concentrations whilst on day nine the spike in RBCs may have been due

to technical reasons rather than biological. Although from day nine there is a decrease in

RBC concentrations a significant difference is only observed between infected and

uninfected mice RBC concentrations on day 14 and 15. RBC concentration reached a nadir

on days 15 and started to increase to uninfected concentrations thereafter. One mouse died

on day 15 which was most likely due to anaemia and all remaining mice recovered with

decreasing parasite load.

68

7 8 9 10 11 12 13 14 15 16 17 180.0

5.0×1008

1.0×1009

1.5×1009

0

10

20

30

40

Infected (3)

Uninfected (5)

Parasitaemia

Day post infection

1 mouse dead

*

**

**

*

a

pla

tele

ts/m

L%

para

sita

em

ia

Figure 4.1 Platelet and RBC concentration compared to parasitaemia in male C57BL/6

mice. 5 µL blood was collected from infected mice (3) and uninfected mice (5), mixed with

ACD and labeled with anti-TER-119 and anti-CD41 to measure RBC and platelets

respectively. Y-axis indicates cells/mL and X-axis indicates day post parasite infection. a)

Platelet concentration compared to parasitaemia – platelet levels reached a nadir on day 13

b) RBC concentrations compared to parasitaemia – RBC levels reached a nadir on day 15.

Error bars depict SEM. *p <0.05, **p <0.01, ***p <0.001. Red box indicates expected

values from published data. 2way ANOVA was used to measure differences between

infected and uninfected mice on various days.

7 8 9 10 11 12 13 14 15 16 17 18

0.0

2.0×1009

4.0×1009

6.0×1009

8.0×1009

1.0×1010

0

10

20

30

40

Infected (3)

Uninfected (5)

Parasitaemia

Day post infection

***

1 mouse dead

*

b

RB

C/m

L%

para

sita

em

ia

69

4.1.1 Preferential Binding of Platelets to Red Blood Cells During a Malaria Infection

The percent of platelets bound to iRBCs and uRBCs is shown in Figure 4.2a. Platelet binding

to RBCs in uninfected mice remained stable throughout the course of infection (~0.4%) and

there was no difference between platelets bound to uRBCs in infected mice compared to

uninfected mice. This shows that growth of parasites in the bloodstream and the platelet loss

does not apparently affect the frequency of the platelets bound to uRBCs during infection. In

infected mice, plt:iRBC complexes were first observed and quantifiable on Day 11. There

was a significantly greater difference between platelet binding to iRBCs compared to uRBCs

on days 11 and 17. On day 11, platelet binding was 7-fold higher to iRBCs compared to

uRBCs. On day 17, platelet binding was 11-fold higher to iRBCs compared to uninfected

RBCs.

The days on which preferential binding was observed coincided with the times when parasite

levels were high enough to be quantified and platelet numbers had not yet reached a nadir

(day 11), or had recovered (day 17). Therefore, the ability to observe preferential binding

may depend on there being sufficient numbers of parasites and platelets in the circulation.

Figure 4.2b describes the relationship between parasitaemia, platelet concentration and

plt:iRBC binding in infected mice where days 11 and 17 show preferential binding when

platelet concentration is still high and parasitaemia is quantifiable for the first time.

70

7 8 9 10 11 12 13 14 15 16 17

0.0

5.0×1008

1.0×1009

1.5×1009

0

20

40

60

80

Plt:iRBC (infected mice)

Platelets (infected)

Parasitaemia

Day post infection

pla

tele

ts/m

L

% p

ara

sita

em

ia a

nd

(plt:iR

BC

s x

10)

7 8 9 10 11 12 13 14 15 16 170

2

4

6

8

10 Plt:iRBC (infected mice)

Plt:RBC (infected mice)

Plt:uRBC (uninfected mice)

***

***

Day post infection

% o

f p

late

let

bo

un

d R

BC

s

Figure 4.2 Relationship between platelet binding to iRBC and uRBCs in male C57BL/6

mice. Flow cytometry was used to measure platelet bound RBC populations from infected

mice (3) using anti-TER-119 and Hoechst to distinguish iRBCs from uRBCs, and anti-CD41

for platelets. a) The percentage of platelets bound to RBCs is shown for each day post-

infection. b) Relationship between parasitaemia, platelet concentration and plt:iRBC binding

a

a

b

a

a

71

in infected mice. Error bars represent SEM. **p <0.01, ***p < 0.005 2way ANOVA was

carried out to determine differences in platelet binding.

4.2 Challenge 2: Platelet Clearance in Male Mice

A preliminary study consisting of a small cohort of mice was used in the next malaria

infection to measure the clearance of platelets during a malarial infection. To determine

clearance of platelets during a P. chabaudi infection, biotin was used to label all cells in the

blood including platelets by IV injection to track the original platelets in circulation over the

course of infection along with newly produced platelets which would therefore not be

biotinylated. This allowed us to determine whether thrombocytopenia was a result of platelet

consumption or inhibition of platelet production by measuring the proportion of biotinylated

and nonbiotinylated platelets.

4.2.1 Platelet and Red Blood Cell Concentration during a Plasmodium chabaudi

infection in male mice

During this challenge parasites were much more virulent than previous challenges, with

parasitaemia reaching approximately 80% resulting in all mice dying by the end of challenge.

Measured platelet concentrations in the uninfected mice were at expected levels of C57BL/6

mice on the first two days of sampling (1x109cells/mL). There was an unusual decrease in

platelet numbers measured on day eight, probably due to unidentifiable technical reasons

rather that representing a true physiological change. For the rest of the infection, platelet

concentrations remained at expected levels. Platelet concentration in infected mice were at

similar levels pre-infection and not significant compared to uninfected mice platelet

concentrations for the first two days of sampling. Platelet concentration between infected and

uninfected mice were significantly different on days 9-12. Due to the unidentifiable decrease

72

in platelet concentrations on day eight, due to an error, in infected mice, it is not possible to

tell if there was no significant difference in platelet concentrations that day.

RBC concentration was also measured using flow cytometry. RBC concentrations in

uninfected mice were approximately 50% of the expected value for C57BL/6 mice and stayed

relatively consistent throughout the course of infection. The fluctuations in RBC

concentration in infected mice make it difficult to determine direct differences, although

anaemia is clearly observed on days 11-12 of infection suggesting that all mice died of

anaemia.

73

Pre

infe

ctio

n 1 8 9 10 11 12

0.0

2.0×1009

4.0×1009

6.0×1009

8.0×1009

0

20

40

60

80

100

Infected (5)

Uninfected (3)

Day post infection

Parasitaemia

one dead three more dead

b

RB

C/m

L

% p

ara

sita

em

ia

Pre

infe

ctio

n 1 8 9 10 11 12

0.0

5.0×1008

1.0×1009

1.5×1009

0

20

40

60

80

100

Infected (5)

Uninfected (3)

parasitaemia

Day post infection

******

*** ***

one dead three more dead

a

pla

tele

ts/m

L

% p

ara

sita

em

ia

Figure 4.3 Platelet and RBC concentration compared to parasitaemia in male C57BL/6

mice. 5 µL blood was collected from infected mice (5) and uninfected mice (3), mixed with

ACD and labeled with anti-TER-119 and anti-CD41 to measure RBC and platelets

respectively. Y-axis indicates cells/mL and X-axis indicates day post parasite infection. a)

Platelet concentration compared to parasitaemia b) RBC concentrations compared to

parasitaemia. Error bars depict SEM. ***p <0.001. Red box indicates expected values from

published data. 2way ANOVA was used to measure differences between infected and

uninfected mice on various days.

74

4.2.2 Platelet Clearance During a Plasmodium chabaudi Infection in Male Mice

To determine if platelet clearance was affected during a malaria infection, biotin was injected

into the lateral vein of the mouse tail via IV injection when parasites were detected in the

bloodstream. Blood was sampled approximately four hours after IV injection on the day of

biotinylation, and initial percentage of biotinylated platelets designated as 100%.

Biotinylated platelets were gated as CD41+Biotin+ (Section 2.4.4) by flow cytometry.

Samples were taken approximately every 24 hours to measure biotinylated and non-

biotinylated platelets over the course of infection.

The time taken for 50% of the original population of biotinylated platelets to be cleared was

calculated using linear regression on GraphPad Prism as described in section 2.4.6. There

was a significantly shorter time of platelet consumption in infected mice (1.6 +/- 0.02 days)

compared to uninfected mice (2.3 +/- 0.1 days) to reach 50%, indicating platelet consumption

was playing a role in malaria-induced thrombocytopenia. However, whether the inhibition

of platelet production was a factor was uncertain, and so, was addressed next.

75

0 1 2 3 40

20

40

60

80

100 Infected (5)

Uninfected (3)

Day ofbiotinylation

uninfected

infected

Day post biotinylation

% b

ioti

nyla

ted

pla

tele

ts

Figure 4.4 Clearance of biotinylated platelets. A 1/2000 dilution of blood was labeled with

anti-CD41 and streptavidin to measure the percentage of biotinylated platelets in infected

mice (5) and uninfected mice (3) using flow cytometry. Error bars represent SEM. Arrows

indicate day post-biotinylation where 50% biotinylated platelets remain in circulation. Red

(infected) and blue (uninfected). *p 0.0357 Linear regression was used to calculate the

difference between biotinylated platelets in infected and uninfected mice.

76

4.2.3 Platelet Consumption or Inhibition of Production

The concentration of biotinylated and nonbiotinylated platelets over the course of infection

is shown in Figure 4.5. On the day of biotinylation, there was no significant difference in the

concentration of biotinylated platelets and nonbiotinylated platelets between infected and

uninfected mice.

Due to a possible artefact/experimental error, the platelet concentration on the day of

biotinylation is much lower than expected, however on subsequent days platelet

measurements were of expected numbers.

On days one-three post biotinylation, there was a significant difference in biotinylated

platelets between infected and uninfected mice. There was a significant difference between

day one-four post biotinylation in the concentration of non-biotinylated platelets between

infected and infected mice, which suggests that platelet consumption but also possibly an

inhibition of platelet production plays a role in malaria induced thrombocytopenia.

77

Figure 4.5 Biotinylated and nonbiotinylated platelets in infected and uninfected mice

over the course of infection. Biotin was injected at 1.5mg into the lateral vein of infected

mice (5) and uninfected mice (3) and samples collected on the day of biotinylation and 24

hours later on subsequent days in order to measure the proportion of biotinylated (red) and

nonbiotinylated (blue) platelets in infected (shaded) and uninfected mice (unshaded) over the

course of infection. Error bars represent SEM. **p <0.01, ***p <0.001. 2way ANOVA was

carried out to measure differences between infected and uninfected mice. Red statistical lines

are comparing differences between biotinylated platelets in infected and uninfected mice.

Blue statistical lines are comparing differences between nonbiotinylated platelets in infected

and uninfected mice.

Day

of b

iotin

ylat

ion 1 2 3 4

0.0

5.0×1008

1.0×1009

1.5×1009

Biotinylated (infected)

nonbiotinylated (infected)

Biotinylated (uninfected)

nonbiotinylated (uninfected)

Day post biotinylation

*** *** **

** ****** ***

pla

tele

ts/m

L

78

4.2.4 Preferential Binding of Platelets to Red Blood Cells

The percent of platelets which were bound to iRBCs and uRBCs is shown in Figure 4.6a.

Platelet binding to RBCs in uninfected mice was not significantly different throughout the

duration of infection. There was no difference between platelets bound to uRBCs in infected

mice compared to uninfected mice. In infected mice plt:iRBC complexes were first observed

and quantifiable on day eight. There was a significantly greater difference between platelets

binding to iRBCs compared to uRBCs only on day eight. On day eight platelet binding was

3.5-fold higher to iRBCs compared to uninfected RBCs.

The day on which preferential binding was observed (day eight) coincides again with the

times when parasites levels were again high enough to be quantified and platelet

concentrations were not yet significantly different between infected and uninfected mice.

4.6b describes the relationship between parasitaemia, platelet concentration and plt:iRBC

binding in infected mice where on day eight preferential binding is observed when platelet

concentration is still high and parasitaemia is quantifiable for the first time.

79

8 9 10 11 12

0.0

0.5

1.0

1.5

2.0

2.5

Plt:iRBC (infected mice)

Plt:uRBC (infected mice)

Plt:uRBC (uinfected mice)

biotinylation

**

Day post infection

% p

late

let

bo

un

d R

BC

s

8 9 10 11 12

0.0

2.0×1008

4.0×1008

6.0×1008

8.0×1008

0

20

40

60

80

100

Platelets (infected)

Plt:iRBCs (infected)

Parasitaemia

post infection

pla

tele

ts/m

L%

para

sita

em

ia a

nd

(plt:iR

BC

s x

10)

Figure 4.6 Relationship between platelet binding to iRBC and uRBCs in male C57BL/6

mice. Flow cytometry was used to measure platelet bound RBC populations using anti-TER-

119 and Hoechst to distinguish iRBCs from uRBCs and anti-CD41 for platelets in infected

mice (5). a) The percentage of platelets bound to RBCs is shown for each day post-infection.

b) Relationship between free platelets, plt:iRBCs and parasitaemia in infected mice. Error

bars represent SEM. **p <0.01 2way ANOVA was carried out to determine differences in

platelet binding.

b

a

a

b

a

a

a

a

a

80

4.2.5 Rate of Loss of Free Platelets vs Platelet Bound Red Blood Cells

In order to determine the proportion of platelets bound to iRBC and uRBCs contributing to

thrombocytopenia in infected mice, the total unbound platelets and total biotinylated platelets

verses plt:iRBCs and plt:uRBCs were compared in Figure 4.7. The contribution complexes

have in proportion to total platelets (platelet bound iRBCs + platelet bound uRBCs +

biotinylated + nonbiotinylated platelets) was 3.6% on the day of biotinylation, 3.4% day one,

3.3% day two, 2.1% day three, and 2.6% on day four post biotinylation. However, due to the

inability to distinguish what part of the complex is biotinylated (the platelet or RBC since IV

biotinylation in vivo stains all blood cell types) the concentration of complexes cannot

accurately determine newly formed complexes or platelets which may have become detached

over the course of infection. The concentration of free biotinylated platelets decreases over

the duration of infection whilst the total platelet pool also decreases but takes into the account

newly synthesized platelets.

81

0 1 2 3 41.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

Biotinylated unbound platelets

Plt:iRBC

Plt:uRBC

Total Platelets

Day ofbiotinylation

Day of biotinylation

pla

tele

t co

mp

lexes/m

L

Figure 4.7 Preferential binding of platelets to infected and uninfected cells vs free

platelets. Concentration total platelets (blue), free biotinylated platelets (black), plt:iRBC

(red) and plt:uRBC (green) in five infected mice was measured via flow cytometry. Total

platelets include plt:iRBC/plt:uRBC/biotinylated platelets and nonbiotinylated platelets. Red

arrow indicates time when 50% biotinylated platelets remain. Error bars represent SEM.

**p <0.01

82

4.3 Challenge #3: Platelet Clearance in Male Mice

The previous experiment was used as a preliminary study and as such had a low number of

mice in both the infected and uninfected groups. The last malaria infection of this chapter,

had a larger cohort of mice to increase the statistical power.

4.3.1 Platelet and Red Blood Cell Concentration During a Plasmodium chabaudi

Infection in Male Mice

A total of 16 mice (seven infected and nine uninfected) were used in the last challenge of this

chapter. 1x105 P. chabaudi parasites were injected by IP into mice, and platelet, RBC and

iRBC concentrations were measured using flow cytometry. The markers used to investigate

this were anti-CD41, anti-TER-119 and Hoechst respectively. Parasitaemia was verified with

Giemsa stained slides and parasites were first detected in the bloodstream from day ten until

the end of challenge on day 17, with peak parasitaemia reached on day 14 (53.8 +/- 10.4%).

Three mice died by the end of the challenge.

Figure 4.8a shows platelet concentrations and the corresponding parasitaemia in male

C57BL/6 mice infected with P. chabaudi over the course of infection.

Platelet concentrations in uninfected mice were at the expected levels and were stable

throughout the infection (1x109cells/mL). Platelet concentrations in infected mice were

similar to uninfected mice concentrations pre-infection and days one to nine, then started to

decrease markedly from day ten. Platelet concentration reached a nadir on day 13 in infected

mice and started thereafter to increase.

There was a significant difference in platelet concentrations between infected and uninfected

mice between days 11-15.

83

RBC concentration was also measured using flow cytometry. In uninfected mice,

concentrations were at approximately 60% of expected levels of C57BL/6 mice at the start

of infection but fluctuated on a daily basis, although there appears to be anaemia by the end

of the challenge as RBC concentration by day 17 had dropped by 40% compared to the

starting concentration. Infected mice RBCs were similar to uninfected concentrations for the

first two days of sampling, then spiked (day seven), most likely due to technical aspects

rather than biological. RBC concentrations for the next four days fluctuate, however there is

a clear decrease in RBC concentration from day 12 onwards with a significant difference

between iRBC and uRBC concentrations on day 16, although due to the fluctuating nature

of concentrations it is difficult to make a definite comparison on this day.

84

Pre

finfe

ctio

n 1 7 8 9 10 11 12 13 14 15 16 17

0.0

5.0×1008

1.0×1009

1.5×1009

2.0×1009

0

20

40

60

80Infected (7)

Uninfected (9)

Parasitaemia**

Day post infection

*** ******

***

one deadtwo dead

pla

tele

ts/m

l

% p

ara

sita

em

ia

Pre

finfe

ctio

n 1 7 8 9 10 11 12 13 14 15 16 17

0.0

2.0×1009

4.0×1009

6.0×1009

8.0×1009

1.0×1010

0

20

40

60

80

Infected (7)

Uninfected (9)

Parasitaemia

one deadtwo dead

Day post infection

*

RB

Cs/m

L

% p

ara

sita

em

ia

Figure 4.8 Platelet and RBC concentration compared to parasitaemia in male C57BL/6

mice. Nine uninfected mice were used as control mice whilst seven mice were infected with

1x105 parasites and samples collected daily and measured on flow cytometry. a) Platelet

concentration over the course of infection with parasitaemia curve. b) RBC concentrations

over the course of infection with parasitaemia curve. Error bars depict SEM. *p <0.05, ** p

< 0.01 ***p <0.001. Red box indicates published values. 2way ANOVA was used to measure

differences between infected and uninfected mice on various days.

a

a

a

b

a

a

85

4.3.2 Platelet Clearance During a Plasmodium chabaudi Infection in Male Mice

To determine if platelet clearance was affected during a malaria infection, biotin was injected

into the lateral vein of the mouse tail by IV when parasites were detected in the bloodstream.

Blood was sampled approximately four hours after IV injection on the day of biotinylation,

and initial percentage of biotinylated platelets designated as 100%. Biotinylated platelets

were gated as CD41+Biotin+ by flow cytometry. Samples were taken approximately every 24

hours to measure biotinylated and non-biotinylated platelets over the course of infection.

The time taken for 50% of the original population of biotinylated platelets to be cleared was

calculated by using linear regression on GraphPad Prism as described in section 2.4.6. There

was a significantly shorter time in infected mice (1.5 +/- 0.05 days) compared to uninfected

mice

(2.1 +/- 0.1 days) to reach 50% of original platelet levels, indicating platelet consumption is

playing a role in malaria-induced thrombocytopenia. However the inhibition of platelet

production was again addressed in the next Section 4.3.3.

86

0 1 2 3 40

20

40

60

80

100Infected (5)

Uninfected (4)

Day ofbiotinylation

infected uninfected

Day post biotinylation

% b

ioti

nyla

ted

pla

tele

ts

Figure 4.9 Clearance of biotinylated platelets. Biotin was injected into the lateral tail vein of five

infected mice and four uninfected mice. Blood samples were collected on the day of biotinylation and

four consecutive days thereafter. Percent of biotinylated platelets was measured via flow cytometry.

Arrows indicate day post-biotinylation where 50% biotinylated platelets remain in

circulation. Red (infected) and blue (uninfected). Error bars represent SEM. P 0.0159. Linear

regression was used to calculate the difference between biotinylated platelets in infected and

uninfected mice.

87

4.3.3 Platelet Consumption or Inhibition of Production

The concentration of biotinylated and nonbiotinylated platelets over the course of infection.is

shown in Figure 4.10. From the day of biotinylation and the next two days, there was a

significant difference in the remaining concentration of biotinylated platelets between

infected and uninfected mice. From days three to six post biotinylation, there was also a

significant difference in the concentration of nonbiotinylated platelets between infected and

uninfected mice suggesting that platelet production may also possibly be playing a role.

88

Figure 4.10 Biotinylated and nonbiotinylated platelets in infected and uninfected mice

during infection. Concentration of biotinylated and nonbiotinylated during infection in

infected mice (5) and uninfected mice (4). Error bars represent SEM. ***p <0.001. 2way

ANOVA was carried out to measure differences between infected and uninfected mice.

Day

of b

iotin

ylat

ion 1 2 3 4 5 6

0.0

5.0×10 08

1.0×10 09

1.5×10 09

2.0×10 09

Biotinylated (infected)

Unbiotinylated (infected)

Biotinylated (uninfected)

Unbiotinylated (uninfected)

Day post biotinylation

** *** ***

***

******

* **

pla

tele

ts/m

l

89

4.3.4 Preferential Binding of Platelets

The percent of platelets bound to iRBCs and uRBCs is shown in Figure 4.11a. Platelet

binding to RBCs in uninfected mice was not significantly different throughout the duration

of infection. There was no difference between platelets bound to uRBCs in infected mice

compared to uninfected mice. In infected mice plt:iRBC complexes were first observed and

quantifiable on day ten. There was a significantly greater difference between platelet binding

to iRBCs compared to uRBCs only on day ten. On day ten platelet binding was 2.3-fold

higher in iRBCs compared to uRBCs.

The day on which preferential binding was observed (day ten) coincides again with the times

when parasites levels were high enough to be quantified and platelet concentrations were not

yet significantly different between infected and uninfected mice.

90

9 10 11 12 13 14 15 16 17

0.0

5.0×1008

1.0×1009

1.5×1009

0

20

40

60

Plt:iRBC (infected mice)

Platelets (infected)

Parasitaemia

Day post infection

pla

tele

ts/m

l

% p

ara

sita

em

ia a

nd

(plt:iR

BC

s x

10)

9 10 11 12 13 14 15 16 17

0.0

0.5

1.0

1.5

2.0

2.5plt:iRBC (infected mice)

plt:uRBC (infected mice)

plt:uRBC (uninfected mice)

***

Day post infection

% b

ind

ing

pla

tele

ts t

o R

BC

s

Figure 4.11 Relationship between platelet binding to iRBC and uRBCs in male

C57BL/6 mice. Flow cytometry was used to measure platelet bound RBC populations using

anti-TER-119 and Hoechst to distinguish iRBCs from uRBCs, and CD41 for platelets in

infected mice (5). a) The percentage of platelets bound to RBCs is shown for each day post-

infection. b) Relationship between free platelets, plt:iRBCs and parasitaemia in infected mice

Error bars represent SEM. ***p <0.001 2way ANOVA was carried out to determine

differences in platelet binding.

b

a

a

a

a

a

91

4.3.5 Rate of Loss of Free Platelets vs Platelet Bound Red Blood Cells

In order to determine the proportion of platelets bound to iRBC and uRBCs contributing to

thrombocytopenia in infected mice, the total unbound platelets and biotinylated platelets

were compared to platelet bound iRBCs and platelet bound uRBCs in Figure 4.12. The

contribution complexes have in proportion to total platelets (platelet bound iRBCs + platelet

bound uRBCs + biotinylated + nonbiotinylated platelets) was 2.5% on the day of

biotinylation, 3.3% day one, 2.8% day two, 1.5% day three, and 1.1% on day four post

biotinylation. However, again due to the inability to distinguish what part of the complex is

biotinylated (the platelet or RBC) the concentration of complexes measured does not take

into account newly formed complexes or platelets that may have become detached over the

course of infection. The concentration of free biotinylated platelets decreases over the

duration of infection whilst the total platelet pool also decreases, but takes into the account

newly synthesized platelets. Taken together with data from the previous experiment, these

results suggest that platelet complexes contribute a very small proportion of platelet loss in

a mouse model of malaria-induced thrombocytopenia.

92

Day

of b

iotin

ylat

ion 1 2 3 4

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

Biotinylated platelets

plt:iRBCs

plt:uRBCs

Day post biotinylation

Total Platelets

pla

tele

ts/m

L

Figure 4.12 Preferential binding of platelets to infected and uninfected cells vs free

platelets. Concentration total platelets (black), free biotinylated platelets (green), plt:iRBC

(red) and plt:uRBC (blue) in infected mice (5) was measured via flow cytometry. Total

platelets include plt:iRBC/plt:uRBC/biotinylated platelets and nonbiotinylated platelets. Red

arrow indicates time taken for 50% of biotinylated platelets to remain in circulation. Error

bars represent SEM. **p <0.01

93

Discussion

It has been previously reported in a P. chabaudi mouse model of malaria, mice experience

thrombocytopenia, with platelets preferentially binding to infected iRBCs rather than uRBCs

[70]. The aim of this chapter was to determine if the thrombocytopenia that is observed in

acute malaria may be a result of this platelet binding to RBCs and subsequent clearance of

these platelet-RBC complexes.

Reported platelet numbers in C57BL/6 mice are 1.0x109cells/mL [150]. These numbers

correspond with the observed platelet concentrations in all three mouse experiments

described in 4.1a, 4.3a and 4.8a. Various parameters such as sampling method and frequency

of sampling could potentially cause platelet numbers to fluctuate, hence care was taken to

avoid platelet activation during blood collection by using plastic pipette tips to decrease

platelet activation. A small tail snip on a daily basis did not seem to cause platelet loss in

uninfected mice as platelet concentrations remained stable throughout the course of the

infection. However, as can be noted in Figures 4.1b, 4.3b and 4.8b there was a decrease in

RBC numbers from the start of infection. For unknown reasons, RBC concentration was

always lower in mice than the expected value, however control mice were sampled on the

same schedule as their infected counterparts, and as such, they still serve as an appropriate

control for platelet activation and RBC loss that could be caused by this sampling.

There were statistically significant differences in platelet concentrations in infected mice

compared to uninfected mice as parasitaemia increased, with peak parasitaemia coinciding

with the lowest concentration of platelets. This occurred in all three P. chabaudi challenges

(Figures 4.1a, 4.3a and 4.8a) which has previously been shown in a P. chabaudi model of

94

malaria [70]. This chapter demonstrates that, as reported in published work,

thrombocytopenia occurs in mice with increasing parasitaemia and before clinical symptoms

appear, such as anaemia. In previous studies in the McMorran lab, malaria challenges were

carried out with cohorts of mice that were sacrificed each day [70]. Tracking the same mice

over the course of infection, as in the current study, allows for fewer mice to be used and the

measurement of platelet number in relation to parasitaemia, individually, limiting

confounding factors. The concentrations of RBCs measured in this study were lower than

published numbers (1x1010/mL) due to unidentified reasons. It is important to note that in all

three challenges presented in this chapter, it appeared that daily bleeding from the mice may

have resulted in a lower number of RBCs when compared to the post-infection RBC

concentrations. The time taken for the bleeding to stop may further contribute to the loss of

RBCs.

During a malaria infection, the loss of platelets can either be attributed to platelet

consumption or a decrease in platelet production. The first aim in this chapter was to measure

clearance of platelets (or time it takes for 50% of the original biotinylated population to be

consumed). Both Figures 4.4 (p 0.0357) and 4.9 (p 0.0159) show a significant decrease in

platelet half-life in infected mice; this happens approximately 1.5 days in infected mice

compared to 2.3 days in uninfected mice post-biotinylation. This increase in clearance

suggests that during a malaria infection in mice, platelet consumption is higher than in

uninfected mice. Figures 4.5 and 4.10 show between infected and uninfected mice on the

same day, when the proportion of biotinylated platelets is compared, there was a significantly

lower proportion of biotinylated platelets remaining during the infection. A difference in the

concentration of non-biotinylated platelets suggests that the rate of consumption may have

95

exceeded the rate of production. This is shown by way of uninfected mice platelet

concentrations staying in equilibrium, but infected mice platelet concentrations increasing

more slowly. Taken together, platelet consumption plays a role but inhibition of platelet

production may also be occurring. Future aims to measure the role of platelet production

could include measuring megakaryocyte (platelet precursors) concentration in the bone

marrow of mice over the course of infection, though this would mean having to sacrifice

cohorts of mice daily.

It has previously been shown that platelets bind preferentially to iRBCs compared to the

proportion binding to uRBCs [70]. When calculating the proportion of platelet binding, it is

important to consider two factors; 1) the concentration of platelets at a given time and 2) the

proportion of iRBCs and uRBCs at the same given time.

In all three experiments, the days which experienced preferential binding of platelets to

iRBCs compared to uRBCs had both factors likely contribute to the positive result. It seems

feasible that there is a significant difference in binding of platelets to infected cells than

uninfected cells where there is a small population of infected cells in circulation but a high

number of platelets, which is what occurs on the days which show preferential binding.

Platelets may become activated on binding to RBCs which in turn could contribute to more

platelets becoming activated and binding to uRBCs or iRBCs.

To determine the effect platelet bound complexes had on thrombocytopenia, when the

concentration of free biotinylated platelets and the total platelet pool was plotted against the

concentration of platelet bound iRBCs and uRBCs (Figures 4.7 and 4.12), it was observed

that over the course of infection, a very small percent of platelets were bound compared to

96

the total platelet pool. Platelet-bound- complex clearance is impossible to measure in the

above experiments, due to the inability to distinguish which part of the complex is

biotinylated (ie the RBC or the platelet, due to IV injection of biotin which labels all blood

cells). This factor also makes it impossible to see the exact concentration of complexes being

lost over time as it is impossible to determine when the complex was made.

The concentration of complexes graphed in Figure 4.7 and 4.12 takes into account new

complexes that are being formed thus making it difficult to directly compare free platelet loss

with complex loss. However, what it does show is that the concentration of platelet

complexes may only contribute to a small portion of malaria-induced thrombocytopenia as

the proportion of platelets lost between days is greater than what complexes contribute. It is

interesting to note that the concentration of platelets bound to iRBCs stays fairly constant

across the duration of infection thus suggesting that platelets are either binding to iRBCs at

the same rate they are detaching or not detaching at all and therefore not being cleared away.

Future Directions:

Future directions to further analyze the role of platelet binding could involve labeling

platelets and RBCs ex vivo and injecting them back into the mouse, thus being able to

distinguish clearly what part of the complex is biotinylated and hence the proportion of

binding that is being lost over the course of infection. This in turn would allow the time taken

for platelet complexes to be cleared from circulation and determine if they are being cleared

away at a faster rate than free platelets which in turn could contribute to malaria-induced

thrombocytopenia

Organ sequestration studies could also be carried out by observing if plt:RBC complexes are

sequestered in organs such as liver or spleen using in vivo live imaging technologies.

97

With platelets playing a potential protective role in malaria, mitigating platelet loss early on

infection may help enhance the role of platelets in killing the malarial parasite.

98

Chapter 5 General Discussion

The aim of this thesis was divided into two main parts; (i) discover molecules responsible

for platelet activation when purified human platelets were incubated with P. falciparum with

the subsequent release of PF4 and, (ii) observe thrombocytopenia in a mouse model of

malaria using P. chabaudi and see the contribution platelets binding to red blood cells have

in malaria-induced thrombocytopenia.

It has been previously shown that platelets can bind to both iRBCs and uRBCs, but they

release PF4 onto the surface of the iRBC only. Binding of iRBCs to platelets can occur via

the platelet molecule CD36 and the iRBC ligand PfEMP1 but the molecule(s) unique to the

iRBCs which allows subsequent PF4 release was to be determined (chapter 3). Using infected

lysate via hypotonic lysis activation of platelets was inconsistent and incubation of platelets

with trophozoite stages RBCs proved unsuccessful unlike previous studies. While previous

studies have shown PF4 release [100, 111] but with different platelet:trophozoite ratios, no

PF4 was released in the current study. It is possible that parasites were not viable from the

time of purification to the time of use in the platelet assay, or that at a given point in

trophozoite development, that parasites express or secrete certain platelet activating

molecules which was not expressed at the time of the current studies parasite purification.

Due to unsuccessful attempts at activating platelets, it was decided to move to the second

aim of which focused on platelets in an in vivo setting and more specifically during malaria-

induced thrombocytopenia (Chapter 4).

99

Thrombocytopenia is seen in various other parasites, bacterial and viral infections. In dengue

virus infections for example, thrombocytopenia is observed in infected individuals and relies

on two events; decreased platelet production in the bone marrow and/or increased destruction

or clearance of platelets from peripheral blood [151]. In the early stages of dengue infection,

studies have shown platelet activation (as determined by increased levels of anti-CD62P) and

apoptosis (as shown by increased caspases and phosphotidylserine expression) [152, 153].

Activation of complement factor C3, followed by binding of C5b-9 complex to the platelet

surface is also linked to thrombocytopenia in individuals [154, 155]. In malaria-induced

thrombocytopenia, no exact mechanism has been elucidated although numerous mechanisms

have been suggested, such as immune-mediated [156] and splenic sequestration [129] . The

second part of this chapter was to observe thrombocytopenia in mice infected with P.

chabaudi and to measure how much of platelet binding to RBCs contributed to

thrombocytopenia.

Platelets have been shown to preferentially bind to iRBCs over uRBCs and it is this binding

that was hypothesised to contribute to thrombocytopenia through complex removal from the

host's blood stream. Binding depends on two factors; the concentration of platelets at any

given time and the percent of iRBCs. In all three infections in chapter four, there was a

significant difference in binding to iRBCs compared to uRBCs the day before there was a

significant difference in platelet concentration between infected and uninfected mice. This

also coincided with when parasites were first observed in the bloodstream and may suggest

that complexes are being cleared away. However, by comparing free total platelets over the

course of infection to platelet bound iRBCs and uRBCs, it is seen that bound platelets

contribute only a small percentage of total platelet concentration at a given time, although

100

without knowing the life-span of these platelets it is impossible to make this conclusion

decisively.

A number of methods have been described for platelet survival studies including isotopic

labelling using 111In of 51Cr [157] whereas the duel isotopic labelling methods used in

humans also allow the simultaneous measurement of survival for two platelet populations in

animals. Nonisotopic labelling such as fluorescent platelet labelling allows the investigator

to identify platelet populations using flow cytometry with biotinylation being the most

popular. Although platelet specific markers could be used to study platelet survival such as

X488, in the current study both RBC was to be measured alongside platelets. However, RBC

biotinylation rates fluctuated over the days and was never consistent even in the initial days

of biotinylation where % biotinylation should remain constant hence biotin over a platelet

specific label was chosen.

Biotinylation of platelets did however, show that platelet consumption occurs, and

interestingly, that it is possible that platelet consumption exceeds platelet production.

Biotinylation of platelets allows tracking of the original platelet population which was carried

out in infections two and three of Chapter 4. It was observed that platelets in infected mice

cleared platelets by approximately one day faster which suggests the role of platelet

consumption in a mouse malaria model of thrombocytopenia. Direct comparison of

biotinylated complexes and free biotinylated complexes was unable to be carried out due to

the inability to distinguish what part of the complex is biotinylated.

Overall, this thesis was unable to replicate the previous finding of platelet activation which

was a prerequisite to finding the molecule(s) responsible for PF4 deposition onto the surface

101

of the iRBC and not uRBC. Platelets exposed to infected lysate of trophozoite stage parasites

were able to be activated as based on anti-PAC1 expression but not consistently.

Malaria induced thrombocytopenia was measured by developing a flow cytometry staining

protocol allowing individual mice to be tracked over the course of infection.

Platelet clearance was significantly faster in infected mice as determined by the percentage

of biotinylated platelets remaining in circulation. A possible suppression of platelet

production as indicated by a significant decrease of nonbiotinylated platelets in infected mice

was also observed.

Prior to the onset of thrombocytopenia, when platelet concentration was high and

parasitaemia was quantifiable, there was preferential binding to iRBCs compared to uRBCs;

however, due to inability to track platelet complexes over the course of infection and measure

clearance, future evaluation of clearance of platelet binding may provide more insight into

the role they play towards malaria-induced thrombocytopenia.

102

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