The Impact of Aspirin Dose Modification on the Innate Immune Response - Will Lower Dose Aspirin Therapy Reduce the Response to Endotoxin
Phase: PHASE4
Conditions: Acute Coronary Syndrome
Sponsor: Sheffield Teaching Hospitals NHS Foundation Trust

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
 1
Clinical Study Protocol
The impact of aspirin dose 
modification, with or without 
ticagrelor, on the innate immune 
response:
WILL 
lOWer 
dose 
aspirin 
Therapy ReducE the response to 
Endotoxin? 
(WILLOW TREE)

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
 2
RESEARCH REFERENCE NUMBERS
STH20370
TRIAL REGISTRY NUMBER
Clinicaltrials.gov: TBC
OTHER RESEARCH REFERENCE NUMBERS
EudraCT: (2018-004285-35)
MHRA: 21304/0268/001-0001
IRAS ID: 254420 
SPONSOR
Sheffield Teaching Hospitals NHS Foundation Trust
This protocol has regard for the NHS Health Research Authority guidance and order of content

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
 3
SIGNATURE PAGE
The undersigned confirm that the following protocol has been agreed and accepted and that the Chief 
Investigator agrees to conduct the trial in compliance with the approved protocol and will adhere to the 
principles outlined in the Medicines for Human Use (Clinical Trials) Regulations 2004 (SI 2004/1031), 
amended regulations (SI 2006/1928) and any subsequent amendments of the clinical trial regulations, 
(and any other relevant) SOPs, and other regulatory requirements as 
amended. 
I agree to ensure that the confidential information contained in this document will not be used for any other 
purpose other than the evaluation or conduct of the clinical investigation without the prior written consent 
of the Sponsor 
I also confirm that I will make the findings of the trial publicly available through publication or other 
dissemination tools without any unnecessary delay and that an honest accurate and transparent account of 
the trial will be given; and that any discrepancies and serious breaches of GCP from the trial as planned in 
this protocol will be explained. 
For and on behalf of the Trial Sponsor:
Signature: 
......................................................................................................
Date: 
....../....../......
Name (please print):
......................................................................................................
Position: 
......................................................................................................
Chief Investigator:
Signature: 
.....................................................................................................
Date: 
....../....../......
Name: (please print): Professor Robert Storey

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
 4
KEY TRIAL CONTACTS
Chief Investigator/Principal 
Investigator (Single site)
Professor Robert F. Storey
Professor and Honorary Consultant in Cardiology
Department of Infection, Immunity and Cardiovascular Disease
University of Sheffield
Beech Hill Road, Sheffield
S10 2RX
Telephone 0114 2261124
Fax 0114 3052008 Email r.f.storey@sheffield.ac.uk
Co-Investigator
Dr William Parker
Clinical Research Fellow in Cardiology
Cardiovascular Research Unit
Northern General Hospital
Barnsley Road Drive, Sheffield
S5 7AU
Telephone 0114 2266159
Fax 0114 3052008 Email w.parker@sheffield.ac.uk
Trial Co-ordinator
Dr Hannah McMellon
Senior Research Co-Ordinator
Cardiovascular Research Unit
Centre for Biomedical Research
Northern General Hospital
Barnsley Road Drive, Sheffield
S5 7AU
Telephone 0114 2266159
Fax 0114 3052008 Email h.mcmellon@sheffield.ac.uk
Sponsor
Sheffield Teaching Hospitals NHS Foundation Trust
Clinical Research & Innovation Office
D Floor, Royal Hallamshire Hospital
Glossop Road, Sheffield
S10 2JF
Tel. 0114 2712763
Funder
British Heart Foundation: BHF Clinical Research Training Fellowship no. 
FS/18/49/33752 - William Parker
Clinical Research Facility
Sheffield Clinical Research Facility
Centre for Biomedical Research
Northern General Hospital
Barnsley Road Drive, Sheffield
S5 7AU
Pharmacy Co-ordinator
Mrs Kim Ryalls
Pharmacy Department
Northern General Hospital
Herries Road, Sheffield
S5 7AU

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
 5
i. LIST of CONTENTS
GENERAL INFORMATION
Page No.
TITLE PAGE
1
RESEARCH REFERENCE NUMBERS
2
SIGNATURE PAGE
3
KEY TRIAL CONTACTS
4
i. LIST of CONTENTS
5
ii. LIST OF ABBREVIATIONS
6
iii. TRIAL SUMMARY
8
iv. FUNDING
9
v. ROLE OF SPONSOR AND FUNDER
9
vi. ROLES & RESPONSIBILITIES OF TRIAL MANAGEMENT COMMITTEES, GROUPS AND
INDIVIDUALS 
9
vii, PROTOCOL CONTRIBUTORS
9
viii. KEYWORDS
10
ix. TRIAL FLOW CHART
10
SECTION
1. BACKGROUND
11
2. RATIONALE
11
3. OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS
16
4. TRIAL DESIGN
17
5. TRIAL SETTING
18
6. PARTICIPANT ELIGIBILITY CRITERIA
18
7. TRIAL PROCEDURES
19
8. STUDY MEDICATIONS
32
9. PHARMACOVIGILANCE
38
10. STATISTICS AND DATA ANALYSIS
42
11. DATA MANAGEMENT
50
12. MONITORING, AUDIT & INSPECTION
50
13  ETHICAL AND REGULATORY CONSIDERATIONS
51
14. DISSEMINATION POLICY
53
15. REFERENCES
54
APPENDIX 1: Sample collection plan
57

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
6
ii. LIST OF ABBREVATIONS
AA
Arachidonic Acid
ACE
Angiotensinogen Converting Enzyme
ACS
Acute Coronary Syndrome
ADP
Adenosine Diphosphate
AE
Adverse Event
ANOVA
Analysis of Variance
APR
Annual Progress Report
AR
Adverse Reaction
AUC
Area Under the Curve
BD
Twice-daily
BHF
British Heart Foundation
BLT1
Leukotriene B receptor
BMI
Body Mass Index
CI
Chief Investigator
COX
Cyclo-oxygenase
CRF
Case Report Form
CRP
C-reactive Protein
CTA
Clinical Trial Authorisation
CYP
Cytochrome P450
DAPT
Dual Antiplatelet Therapy
DSUR
Development safety update report
EP
Prostanglandin E receptor
EU
European Union
EudraCT
European Clinical Trials Database
GCP
Good Clinical Practice
GP
General Practitioner
HRA
Health Research Authority
ICF
Informed Consent Form
IL-1
Interleukin 1
IL-6
Interleukin 6
IMP
Investigation Medicinal Product
IV
Intravenous
kg
Kilograms
l 
Litres
LTB4
Leukotriene B4
m2
Metres Squared
MACE
Major Adverse Cardiovascular Events
mg
Milligrams
MHRA
Medicines and Healthcare Regulatory Authority

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
7
MI
Myocardial Infarction
ml
Millilitres
mmHg
Millimetres of Mercury
ng
Nanograms
NGH-CRF
Northern General Hospital Clinical Research Facility
NHS
National Health Service
NIHR
National Institute of Healthcare Research
NIMP
Non-investigational medicinal product
NR
Not Recorded
NSAID
Non-Steroidal Anti-Inflammatory Drug
OD
Once-daily
P2Y12
Platelet adenosine diphosphate receptor
PCI
Percutaneous Coronary Intervention
PGE2
Prostaglandin E2
PGI2
Prostacyclin
PGI-M
Prostacyclin Metabolite
PI 
Principal Investigator
PIS
Patient Information Sheet
PLATO
PLATelet inhibition and patient Outcomes
REC
Research Ethics Committee
SAE
Serious Adverse Event
SAR
Serious Adverse Reaction
SmPC
Summary of Product Characteristics
SOP
Standard Operating Procedure
STH
Sheffield Teaching Hospitals
SUSAR
Suspected unexpected serious adverse reaction
TLR4
Toll-like Receptor 4
TMF
Trial Master File
TMG
Trial Management Group
TNF-
Tumour Necrosis Factor 
TXA2
Thromboxane A2
µg
Micrograms
UK
United Kingdom
µL
Microlitres
µmol
Micromoles
USA
United States of America

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
 8
iii. TRIAL SUMMARY
Trial Title
The impact of aspirin dose modification, with or without ticagrelor, on the innate 
immune response:
WILL lOWer dose aspirin Therapy ReducE the response to Endotoxin? 
(WILLOW 
TREE)
Internal ref. no.
STH20370
Clinical Phase 
Phase IV
Trial Design
Randomised controlled hybrid parallel-group and crossover study
Trial Participants
Healthy volunteers aged 18-65 years, male or women of non-childbearing potential
Planned Sample Size
72
Treatment duration
2 x 10 (to 14) day periods
Follow up duration
Until 10 days after last IMP/endotoxin dose
Planned Trial Period
January 2019
February 2022
Objectives
Outcome Measures
Primary
Assess inflammatory response to 
intravenous endotoxin
Plasma tumour necrosis 
Secondary
Assess inflammatory response to 
intravenous endotoxin
Plasma interleukin 6
Serum C-reactive protein
Circulating leukocyte count (and subsets)
Assess effects on key arachidonic 
acid metabolites during 
endotoxaemia
Serum thromboxane B2
Serum prostaglandin E2
Urine prostacyclin metabolite
Assess effects on platelet function 
and haemostasis during 
endotoxaemia
Bleeding time
Platelet aggregation responses to 
arachidonic acid, collagen and adenosine 
diphosphate
Investigational 
Medicinal Product(s)
1. Aspirin (aspirin lysine)
2. Ticagrelor
Additionally, sterile bacterial endotoxin will be administered to model inflammation 
(regarded as a NIMP for the purpose of this protocol)
Formulation, Dose, 
Route of 
Administration
1. 100 mg sachets for dissolution, administered orally as 20 mg twice-daily,
75 mg once-daily or 300 mg once-daily
2. 90 mg oro-dispersible tablets administered as a single loading dose of 180
mg

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
9
iv. FUNDING AND SUPPORT IN KIND
FUNDERS
FINANCIAL AND NON FINANCIAL SUPPORT GIVEN
British Heart Foundation
(BHF)
Funding of the study: BHF Clinical Research Training Fellowship no. 
FS/18/49/33752 - William Parker: 
on 
Northern General 
Hospital Clinical 
Research Facility (NGH-
CRF)
Provision of space and services within the NGH-CRF in kind to conduct the 
study
v. ROLE OF TRIAL SPONSOR AND FUNDER
The sponsor, Sheffield Teaching Hospitals NHS Foundation Trust, will contribute to study design and 
arrangements before approving the protocol and associated study documents. The sponsor will handle site 
initiation, monitoring and close-out. They will also provide pharmacy and clinical support to the study.
The funder, British Heart Foundation, reviewed and approved an application for this study, and will provide 
monetary resources to complete it.
vi. ROLES AND RESPONSIBILITIES OF TRIAL MANAGEMENT COMMITTEES/GROUPS
The trial management group, chaired by the CI and including the sub-investigators, co-ordinators and 
laboratory team, will meet regularly to discuss a documented agenda. Minutes will be kept and shared with 
the sponsor. A trial management plan, agreed with the sponsor, will be written and a copy kept in the trial 
master file (TMF).
Given this is an exploratory study utilising healthy volunteers rather than patients, and will not change usual 
clinical care, there will not be a formal data monitoring committee. However, procedures defined in this 
protocol will be followed with regards to safety monitoring, including discussion with the sponsor in the 
event of any serious adverse event occurring.
vii. PROTOCOL CONTRIBUTORS
Dr William Parker led the writing of this protocol, under the supervision of the CI (Professor Rob Storey), as 
well as Professor Ian Sabroe, Professor Bianca Rocca and Dr Ramzi Ajjan.
Dr Heather Judge assisted with designing laboratory methods.
Dr Erica Wallis, Dr Nana Theodorou and Mrs Sam Walmsley critically reviewed the protocol on behalf of the 
sponsor.
Mrs Kim Ryalls provided input into the pharmacy arrangements detailed in the protocol.
Representatives of the British Heart Foundation reviewed the protocol from a scientific and statistical point-
of-view.
At an early stage in development, the study design and patient information sheet were reviewed by the 
Sheffield Cardiovascular Patient Panel and suggestions for changes incorporated into further revisions.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
10
viii. KEY WORDS:
Aspirin, ticagrelor, inflammation, arachidonic acid 
metabolites, platelets, haemostasis
ix. TRIAL FLOW CHART

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
11
1
BACKGROUND
Acute coronary syndromes (ACS) are responsible for over 33,000 deaths a year in the UK alone. 
Atherothrombosis is the central pathological process in the development of ACS, driven by inflammation 
which leads to formation of lipid-rich plaques that may then rupture or erode, leading to activation of 
platelets and coagulation factors, vessel occlusion and distal ischaemia [1]. Levels of cytokines, such as 
-
interleukin 6 (IL-6) , strongly correlate with outcomes in ACS [2].
The PLATelet inhibition and patient Outcomes (PLATO) study showed that dual antiplatelet therapy (DAPT) 
with the irreversible cyclo-oxygenase (COX) inhibitor aspirin (acetylsalicylic acid) and the platelet P2Y12
receptor antagonist ticagrelor was superior to the previous standard regimen of aspirin and clopidogrel in 
ACS, reducing the incidence of major adverse cardiovascular events (MACE). Consequently, the 
combination of aspirin 75 mg OD and ticagrelor 90 mg BD now represents a first-line recommended 
regimen of DAPT in ACS [3, 4]. Despite such potent antiplatelet therapy, the MACE risk remains around 10% 
in 1 year [5]. 
Recently, reducing inflammation has been identified as a rewarding strategy for tackling residual risk and 
further improving outcomes after ACS. Significantly, chronic administration of a monoclonal antibody 
against a pro-inflammatory cytokine, interleukin-1 (IL-1), reduced the incidence of MACE in patients with a 
history of myocardial infarction (MI) and evidence of ongoing systemic inflammatory response [6]. However, 
this approach was associated with an increased risk of fatal infection and so other strategies for reducing 
systemic inflammation are a priority for future research.
We found that platelet P2Y12 inhibitors attenuate the inflammatory response in an established in vivo
endotoxaemia model [7] and have used this technique again to study the effect of hypoglycaemia on 
inflammatory response [8]. 
Aspirin is a non-selective, irreversible inhibitor of cyclooxygenase (COX) 1 and 2 enzymes, at lower doses 
exhibiting relative pharmacokinetic selectivity for platelet COX1 [9], which is responsible for the synthesis of 
thromboxane A2 (TXA2), a pro-aggregant and vasoconstrictive prostanoid. At doses >325 mg/daily, based 
on the higher systemic concentration, aspirin can also significantly inhibit endothelial-derived COX2, leading 
to reduced release of the antithrombotic and vasodilatory compound prostacyclin (PGI2). Aspirin is able to 
act as an antiplatelet drug by inhibiting TXA2-mediated platelet activation without concurrent inhibition of 
PGI2 entirely counteracting this [10].
Aspirin has established benefits of preventing recurrent thrombotic events after acute MI [11] but, despite 
this, trials of long-term aspirin therapy carried out in high-risk patients with conditions associated with 
chronic inflammation, such as diabetes mellitus or peripheral vascular disease, have been unable to show 
convincing beneficial effects on the frequency of MACE [12-16].
2
RATIONALE
In addition to its antiplatelet effects, aspirin has immunomodulatory effects. On the one hand, including at 
a dose of 75 mg OD, it may have anti-inflammatory effects mediated via generation of 15-epi-lipoxin A4
[17], whilst, on the other hand, there is growing evidence that aspirin may increase certain factors key to 
the progression of atherothrombosis. As well as observed effects in vitro and ex vivo, it has now been 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
12
shown that aspirin, tested at a dose of 80 mg OD, potentiates both in vivo TNF-
IL-6 release in 
response to endotoxaemia and that, although a standard regimen of ticagrelor appears to counteract these 
effects to an extent, significant augmentation of inflammatory response by aspirin persists despite this 
[18].
The mechanism for this effect has not been fully elucidated, but may be related to inhibition of platelet-
derived prostaglandin E2 (PGE2), another eicosanoid synthesised by platelets and leukocytes in response to 
endotoxin [18, 19] that can inhibit the synthesis and release of pro-inflammatory cytokines from monocytes 
via its action on prostaglandin receptors EP2 and EP4 [20, 21]. Endotoxin binds to toll-like receptor 4 
(TLR4) on the surface of monocytes, resulting in signalling including upregulation of nuclear factor kappa B 
(NF B), with an increase in synthesis and release of pro-inflammatory cytokines, including TNF-
and IL-6, 
mediating vascular inflammation [22, 23]. The action of PGE2 on EP2 can also reduce the translation and 
surface expression of TLR4 [24]. In addition to binding endotoxin, TLR4 also functions as a damage 
receptor, activated by a range of factors released during atherogenesis, endothelial injury and thrombosis, 
including fibrin degradation products and damage-associated molecular patterns [25, 26]. An established 
link also exists between bacterial infection, for example pneumonia, which results in inflammation via the 
endotoxin-TLR4 pathway, and elevated risk of MACE [27]. This makes endotoxaemia a highly relevant 
model of inflammation to use in the study of atheroinflammation, in contrast to other models that may not 
be so pathophysiologically pertinent to cardiovascular disease and may not be potentiated by aspirin [28, 
29]. Aspirin, a COX inhibitor, may reduce release of PGE2, therefore potentiating the pro-inflammatory 
response to endotoxin. COX inhibition may also hypothetically result in increases in other non-COX derived 
eicosanoids such as leukotriene B4 (LTB4), which antagonises the effect of PGE2 via action on the 
leukotriene BLT1 receptor [30]. 
It has been proposed that aspirin could safely be stopped in ticagrelor-treated patients with a history of
percutaneous coronary intervention (PCI), however this approach was not superior in a recent large 
randomised clinical trial [31], although another large study is ongoing [32]. Although ticagrelor 
monotherapy may indeed be sufficient to prevent stent thrombosis, there are likely to remain significant 
numbers of patients at high-risk of ongoing native plaque rupture events in whom DAPT is needed to 
optimise protection against future MACE, since aspirin and ticagrelor have additive antithrombotic effects 
[33, 34]. Aspirin, in the setting of DAPT, may therefore provide advantages of augmenting the 
antithrombotic effect of P2Y12 inhibition but may hypothetically result in disadvantageous effects upon 
progression of atherothrombosis. If this profile could be optimised, then outcomes may be improved. It is 
also plausible that imbalance in these factors might contribute to the lack of benefit of ticagrelor over 
clopidogrel in patients treated with higher doses of aspirin in the PLATO study [35], although this remains to 
be fully explored. 
We completed a pilot study of patients with a history of ACS receiving aspirin and ticagrelor, and have 
shown that a novel regimen of very-low-dose twice-daily aspirin (Aspirin lysine) (20 mg BD), when compared 
to the standard regimen of 75 mg OD, may lead to lower circulating levels of IL-6 and leukocyte count. We 
also observed that, compared to standard dosing, the novel regimen had reduced peak antithrombotic 
effect but maintained a similar trough effect, and this was associated with an improvement in haemostasis 
when assessed by bleeding time, which is a useful measure for assessing the impact of antithrombotic 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
13
drug combinations (in contrast to limited sensitivity for effects of antithrombotic monotherapy). There was 
also a trend towards increased levels of prostacyclin metabolite, suggesting reduced COX2 inhibition [36].
2.1
Assessment and management of risk
Aspirin and ticagrelor are both widely used as antiplatelet drugs for the treatment of cardiovascular 
disease. Combining aspirin and ticagrelor increases the risk of bleeding over that of either treatment when 
ence) to do so, and we will limit 
Interactions with other drugs will not be an 
issue as those receiving any regular medication at screening will be excluded from participation.
Known side effect profiles
Aspirin (Aspirin lysine)
Reported side effects of aspirin (Aspirin lysine), taken from the current SmPC, include:
More common:
Increased bleeding tendency (e.g. nose bleed, bleeding gums, bruises, intestinal bleeds and 
bleeding due to trauma). It is normal to note increased bleeding with cuts or spontaneous bruising 
with any dose of aspirin.
Stomach irritation. This is usually mild and, if necessary, can often be treated with acid-lowering 
medications. There is some evidence that lower doses of aspirin have a lower chance of causing 
irritation than high doses.
Less common side effects of aspirin:
Rhinitis, shortness of breath and itching have been reported uncommonly (up to 1 in 100 patients).
Blood count abnormalities, allergic reactions, bleeding into the brain, inflammation of the blood 
vessels, asthma, heavy periods, nausea and vomiting have been reported rarely (up to 1 in 1000).
Other cases of renal failure, water retention, stomach or intestinal ulceration, headache, ringing in 
the ears, reduced hearing, vertigo and diarrhoea have been reported (exact chance not known).
Ticagrelor
Reported side effects of ticagrelor include the following (reproduced from the current SmPC [37]):

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
14
Category
Very common
Common
Uncommon
Neoplasms benign, 
malignant and 
unspecified (including 
cysts and polyps)
Tumour bleedinga
Blood and lymphatic 
system disorders
Blood disorder bleedingb
Immune system disorders
Hypersensitivity including 
angioedemac
Metabolism and nutrition 
disorders
Hyperuricaemiad
Gout/Gouty Arthritis
Psychiatric disorders
Confusion
Nervous system disorders
Dizziness, syncope, 
headache
Intracranial haemorrhage
Eye disorders
Eye haemorrhagee
Ear and labyrinth 
disorders
Vertigo
Ear haemorrhage
Vascular disorders
Hypotension
Respiratory, thoracic and 
mediastinal disorders
Dyspnoea
Respiratory system 
bleedingf
Gastrointestinal disorders
Gastrointestinal 
haemorrhageg, diarrhoea, 
nausea, dyspepsia, 
constipation
Retroperitoneal haemorrhage
Skin and subcutaneous 
tissue disorders
Subcutaneous or dermal 
bleedingh, rash, pruritus
Musculoskeletal 
connective tissue and 
bone
Muscular bleedingi
Renal and urinary 
disorders
Urinary tract bleedingj
Reproductive system and 
breast disorders
Reproductive system bleedingk
Investigations
Blood creatinine 
increasedd
Injury, poisoning and 
procedural complications
Post procedural 
haemorrhage, traumatic 
bleedingl

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
15
a e.g. bleeding from bladder cancer, gastric cancer, colon cancer
b e.g. increased tendency to bruise, spontaneous haematoma, haemorrhagic diathesis
c Identified in post-marketing experience
d Frequencies derived from lab observations (Uric acid increases to >upper limit of normal from baseline below or within reference range. 
Creatinine increases of >50% from baseline.) and not crude adverse event report frequency.
e e.g. conjunctival, retinal, intraocular bleeding
f e.g. epistaxis, haemoptysis
g e.g. gingival bleeding, rectal haemorrhage, gastric ulcer haemorrhage
h e.g. ecchymosis, skin haemorrhage, petechiae
i e.g. haemarthrosis, muscle haemorrhage
j e.g. haematuria, cystitis haemorrhagic
k e.g. vaginal haemorrhage, haematospermia, postmenopausal haemorrhage
l e.g. contusion, traumatic haematoma, traumatic haemorrhage
Endotoxin
Escherichia coli endotoxin at a dose of 2 ng/kg induces a controlled, safe inflammatory response peaking 
at 2 hours and subsiding by 6 hours [38]. This can typically be associated with symptoms of fever, 
headache, myalgia/arthralgia and occasionally rigors. On average, body temperature rises to 38oC. Rare 
cases of bradycardia and hypotension have been reported. These symptoms and signs typically peak at 2-3 
hours after administration then return to normal by 6 hours. 
Over the past 20 years, over a thousand healthy volunteers have taken part in similar studies of 
endotoxaemia and it is exceptionally rare for it to have any serious effects.
Mitigation of risks
Any risks in this study are mitigated by:
a robust informed consent procedure, followed before any study-related activities take place
ensuring at screening that potential participants are in good general health
excluding those with significant co-morbidity or receiving any concomitant medication
as short as feasible exposure to study drugs (eg. using a single dose of ticagrelor rather than a 
more prolonged period of dual antiplatelet therapy) whilst maintaining scientific credibility 
intravenous hydration before and after endotoxin administration, and nursing of participants supine 
during challenges
performing endotoxin challenges in the Clinical Research Facility within a large acute hospital 
(including 24 hour emergency medical and critical care cover)
supervision of endotoxin challenges by a study physician
frequent measurement of vital signs during endotoxin challenges

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
16
continuous cardiac monitoring during endotoxin challenges
rigorous safety monitoring by the study team and sponsor
a track record of safely performing similar studies including antiplatelet medication and endotoxin 
administration to healthy volunteers by our group and others
3
OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS
3.1
Primary objective
The primary objective of this study is to assess, in healthy volunteers, the effects of 3 regimens of aspirin 
(Aspirin lysine), or no aspirin, on the release of TNF- during endotoxaemia.
It is hypothesised that TNF-
will be significantly lower when receiving aspirin 20 mg BD compared to 75 
mg OD and 300 mg OD.
3.2
Secondary objectives
The secondary objectives of this study are to assess, during endotoxaemia, the effects of 3 regimens of 
aspirin (Aspirin lysine) or no aspirin, with and without a loading dose of ticagrelor, on:
1. Plasma TNF-
(comparisons other than those stated in the primary objective)
2. Plasma IL-6
3. Serum CRP
4. Leukocyte count (and subsets)
5. Serum TXB2
6. Serum PGE2
7. Urine PGI-M
8. Bleeding time
9. Platelet aggregation responses to arachidonic acid (AA), collagen and adenosine diphosphate 
(ADP).
3.3
Outcome measures/endpoints
Primary endpoint/outcome
The primary endpoint will be plasma TNF-
at 2 hours post-injection of 2 ng/kg endotoxin compared 
between participants receiving no IMP, aspirin (Aspirin lysine) 20 mg BD, aspirin (Aspirin lysine) 75 mg OD 
and aspirin (Aspirin lysine) 300 mg OD.
Secondary endpoints/outcomes
1.
Plasma levels from 0 to 6 hours after endotoxin administration, of TNF-
2.
Plasma levels from 0 to 6 hours after endotoxin administration, of plasma TNF-
3.
Change in serum CRP from 0 to 6 hours after endotoxin administration
4.
Leukocyte count, from 0 to 6 hours after endotoxin administration
5.
Serum TXB2 from 0 to 6 hours after endotoxin administration

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
17
6.
Serum PGE2, from 0 to 6 hours after endotoxin administration
7.
AUC of the graph, from 0 to 6 hours after endotoxin administration, of urine PGI-M
8.
AUC of the graph, from 0 to 3 hours after endotoxin administration, of bleeding time
9.
Mean platelet aggregation responses to arachidonic acid (AA), collagen and adenosine diphosphate 
(ADP), from 0 to 6 hours after endotoxin administration.
Secondary endpoints will be compared between:
1. Participants receiving no IMP, aspirin (aspirin lysine) 20 mg BD, aspirin (aspirin lysine) 75 mg OD 
and aspirin (aspirin lysine) 300 mg OD.
2. Participants receiving no aspirin and a 180 mg loading dose of ticagrelor, aspirin (aspirin lysine) 20 
mg BD plus a 180 mg loading dose of ticagrelor, aspirin (aspirin lysine) 75 mg OD plus a 180 mg 
loading dose of ticagrelor, and aspirin (aspirin lysine) 300 mg OD plus a 180 mg loading dose of 
ticagrelor.
3. Participants receiving no IMP and a 180 mg loading dose of ticagrelor. 
4. Participants receiving aspirin (aspirin lysine) 20 mg BD, and aspirin (aspirin lysine) 20 mg BD plus a 
180 mg loading dose of ticagrelor. 
5. Participants receiving aspirin (aspirin lysine) 75 mg OD, and aspirin (aspirin lysine) 75 mg OD plus a 
180 mg loading dose of ticagrelor.
6. Participants receiving aspirin (aspirin lysine) 300 mg OD, and aspirin (aspirin lysine) 300 mg OD 
plus a 180 mg loading dose of ticagrelor.
7. Participants receiving no IMP, aspirin (aspirin lysine) 20 mg BD, aspirin (aspirin lysine) 75 mg OD 
and aspirin (aspirin lysine) 300 mg OD, no aspirin and a 180 mg loading dose of ticagrelor, aspirin 
(aspirin lysine) 20 mg BD plus a 180 mg loading dose of ticagrelor, aspirin (aspirin lysine) 75 mg OD 
plus a 180 mg loading dose of ticagrelor, and aspirin (aspirin lysine) 300 mg OD plus a 180 mg 
loading dose of ticagrelor.
Analyses 2-7 will also be performed for plasma TNF-
(as secondary endpoints). For further details see 
section 10 of this protocol.
4
TRIAL DESIGN
The trial is a pharmacodynamic study to determine the effect of aspirin dose modification on immune 
response, compared to control and compared between the presence and absence of potent P2Y12
inhibition with ticagrelor. Healthy volunteers will receive either no aspirin (control group) or one of three 
doses of aspirin (aspirin lysine) with or without a loading dose of ticagrelor on the last day of the first 
medication period, as specified in this protocol, for 10-14 days. They will then receive E. coli endotoxin to 
induce an immune response. Serial measurements of inflammatory markers, cytokines, leucocyte function, 
prostanoids and platelet function will be taken over 6 hours. Bleeding time will be measured before 
endotoxin administration and 3 hours after it to assess haemostasis. After a washout period of at least 5 
weeks, and not more than 18 weeks, subjects will then crossover to receive the same regimen of aspirin 
(aspirin lysine) (or no aspirin in the control group) without or with a loading dose of ticagrelor on the last day 
of the second medication period. The dose-dependent effects of aspirin and the impact of ticagrelor will be 
compared between the groups.
A total of 72 eligible subjects are planned to be randomised. There will be three regimens of aspirin (aspirin 
lysine) included in the study, in addition to a control group (no aspirin), each tested with and without a 180 
mg loading dose of ticagrelor. Aspirin (aspirin lysine) 75 mg OD represents the current standard regimen in 
ACS and for primary and secondary prevention of cardiovascular disease. Aspirin (aspirin lysine) 20 mg BD 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
18
is a novel regimen hypothesised to provide a better profile of inflammatory and antithrombotic effect than 
75 mg OD. Lastly, aspirin 300 mg OD represents a regimen commonly given after coronary artery bypass 
grafting. 
Randomisation will be in a 1:1:1:1:1:1:1:1 fashion and managed by a commercially available service for 
this purpose, SealedEnvelope, with whom the sponsor will have a study-specific contract.
E. coli endotoxin will be administered at the end of each treatment period according to the protocol. 
Subjects will be recruited from the area local to the Northern General Hospital, Sheffield. For example, we 
will recruit members of staff and students from universities in Sheffield and from Sheffield Teaching 
Hospitals. Volunteers will be given as much time as they need to consider taking part in the study prior to 
signing informed consent.
5
TRIAL SETTING
This will be a single-centre open-label study, conducted by the Cardiovascular Research Unit, University of 
Sheffield within the Clinical Research Facility at the Northern General Hospital, Sheffield.
Participants will be healthy volunteers, recruited primarily from local higher education establishments, but 
also staff from local healthcare institutions.
6
PARTICIPANT ELIGIBILITY CRITERIA
6.1
Inclusion criteria
To participate in this trial, subjects must meet all of the following criteria:
1. Healthy male subjects, or female subjects not of childbearing potential (either surgically sterile or 
post-menopausal)
2. Age between 18 and 65 years inclusive
3. Non-smokers
4. Body mass index (BMI) between 18 and 28 kg/m2 inclusive, with a body weight between 60-100 
kg
5. In good health as determined by a medical history, physical examination, vital signs and clinical 
laboratory test results, including renal and liver function, and full blood count
6. Provision of informed consent before any trial-related activity
6.2
Exclusion criteria
Subjects meeting any of the following criteria will be excluded from participation in the study:
1.
Any history of cancer, diabetes or, in the opinion of the investigator, clinically-significant 
cardiovascular, 
respiratory, 
metabolic, 
renal, 
hepatic, 
gastrointestinal, 
haematological, 
dermatological, neurological, psychiatric or other major disorders.
2.
Any history of either significant multiple drug allergies or known allergy to the study drugs or any 
medicine chemically related to the study drugs.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
19
3.
A clinically-significant illness within the preceding 2 weeks.
4.
Any clinically-significant abnormal laboratory test result (full blood count, urea & electrolytes 
[sodium, potassium, urea and creatinine], liver function tests, clotting screen, urinalysis), at 
screening (visit 1), in the opinion of the investigator.
5.
A supine blood pressure at screening, after resting for 5 minutes, higher than 150/90 mmHg or 
lower than 105/65 mmHg.
6.
A supine heart rate at screening, after resting for 5 minutes, outside the range of 50-100 
beats/min.
7.
Receipt of any prescribed or over-the-counter systemic or topical medication within the preceding
48 hours.
8.
Planned or expected requirement, during the next 3 months (at randomisation, or 3 weeks at the 
start of period 2), for any non-study systemic or topical prescribed drug, or for systemic or topical 
over-the-counter NSAID, corticosteroid, antihistamine or any other drug that could affect 
inflammation, thrombosis or haemostasis in the opinion of the investigator.  
9.
Receipt of an investigational medicinal product, excluding those for the purposes of this study,
within the previous four month period (new chemical entity) or three month period (licensed 
product) or subjects who have received a vaccine within the previous three weeks.
10. Any donation of blood or plasma in the preceding one month period, excluding for the purposes of 
this study.
11. A history of alcohol or drug abuse.
12. Mental incapacity or language barriers that preclude adequate understanding.
13. Any other factor that, in the opinion of the investigator
and reliably complete the study, or would affect the scientific validity of the results obtained.
7
TRIAL PROCEDURES
Details of analysis of blood & urine samples
The aim of the analysis of the study samples will be to: (i) show the dose-dependent effects of aspirin on 
the immune response to endotoxaemia including on inflammatory markers, cytokines, prostanoids, 
leucocytes, haemostasis and platelet function, and (ii) show whether the addition of ticagrelor modifies 
these effects.
We will measure inflammatory markers and cytokines that include, but are not limited to, TNF , IL-6 and 
CRP.
To determine the detailed effects of aspirin dose modification on circulating prostanoids in the 
unstimulated and endotoxin-stimulated states, we will measure a panel of eicosanoids and/or their 
metabolites, including, but not limited to, PGE2, PGI2 and TXB2 and will additionally assess the effect of 
ticagrelor on endotoxin-induced responses. 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
20
Haemostasis will be assessed by measuring bleeding time pre- and 3 hours post-endotoxin using a method 
shown to be sensitive to additive effects of antiplatelet agents [33]. The procedure for measuring bleeding 
time is described in the study-specific SOP relating to this.
Finally, in order to confirm the efficacy of the antiplatelet aspects of the drug regimens being studied, the 
activity of a broad range of pathways of platelet activation and aggregation will be assessed by light 
transmittance aggregometry pre-medication, post-medication at peak inflammation and during the 
resolution phase of inflammation, using arachidonic acid 0.3 and 1 mmol/l, collagen 4 and 16 µg/ml, and 
ADP 20 µmol/l as agonists.
Study samples will either be measured immediately after collection (eg. light transmittance aggregometry) 
or stored for assays at a later time (other endpoints).
We will also store acellular samples of serum, plasma, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) 
and urine within the Cardiovascular Research Unit for future, as yet unplanned, studies. Consent will be 
sought for this at study enrolment.
7.1
Recruitment
Potential participants will self-identify through established channels of advertisement to local Universities 
and NHS staff and students, using documents approved for this purpose by the relevant research 
authorities. 
7.1.1 Participant identification
Potential participants will initially self-identify, based on our advertisement, and medically-qualified 
members of the research team will then determine eligibility.
7.1.2
Screening
Screening will occur at visit 1. The following study procedures will be performed, after obtaining written 
consent for the study:
Medical history
Physical examination
Collection of demographic data
Vital signs (pulse, blood pressure and temperature
Weight and BMI
Recording of any concomitant medication
Safety blood tests: 12.5 ml blood sample for full blood count, urea and electrolytes (sodium, 
potassium, urea, creatinine), liver function tests and clotting screen

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
21
Urinalysis
7.1.3
Payment
Volunteers who receive at least one endotoxin injection will receive £100 per endotoxin injection (maximum 
£200) to reimburse them for their time, inconvenience and any discomfort caused. Transportation to and 
from the Clinical Research Facility will be provided if necessary.
7.2 Consent
Written, informed consent, using the current version of the approved designated form for this study, will be 
obtained prior to any study procedures being carried out. This will be explained and obtained by a 
medically-qualified member of the research team, listed on the delegation log. Participants will have the 
chance to read the ICF/PIS for as long as they need, and will be able to ask any questions, prior to signing. 
Minors and those judged to be without the mental capacity to provide informed consent will not be enrolled 
into the study.
Participants will remain free to withdraw at any time from the trial without giving reasons and without 
prejudicing his/her further treatment and will be provided with a contact point where he/she may obtain 
further information about the trial. Samples collected up to the point of withdrawal will only be used after 
withdrawal if the participant consents for this, otherwise they will be destroyed. However, data collected up 
to that point will be used for analysis, and this will be explicitly stated in the participant information sheet 
and consent form.
7.2.1
Additional consent provisions for collection and use of participant data and biological specimens in
ancillary studies, if applicable
As described above, we will also store acellular samples of serum, plasma, DNA, RNA and urine for future, 
as yet unplanned, studies. Explicit consent for this will be sought on the consent form at enrolment. Any 
cellular samples taken will be destroyed before the end of the study.
7.3
The randomisation scheme
Participants will be randomised to one of the following eight treatment sequences, in a 1:1:1:1:1:1:1:1 fashion:
o
No IMP for 10-14
no aspirin for 10-14 days, but a 
loading dose of ticagrelor (180 mg) given one hour before endotoxin injection (sequence 1).
o
No aspirin for 10-14 days, but a loading dose of ticagrelor (180 mg) given one hour before 
endotoxin injection, 
10-14 days (sequence 
2).
o
Aspirin (aspirin lysine) 20 mg BD for 10-14
aspirin (aspirin lysine) 20 mg BD for 10-14 days plus a loading dose of ticagrelor (180 mg)
given one hour before endotoxin injection (sequence 3).

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
22
o
Aspirin (aspirin lysine) 20 mg BD for 10-14 days plus a loading dose of ticagrelor (180 mg)
given one hour before endotoxin injection
aspirin 
(aspirin lysine) 20 mg BD for 10-14 days (sequence 4).
o
Aspirin (aspirin lysine) 75 mg OD for 10-14
week washout period, then 
aspirin (aspirin lysine) 75 mg OD for 10-14 days plus a loading dose of ticagrelor (180 mg)
given one hour before endotoxin injection.
o
Aspirin (aspirin lysine) 75 mg OD for 10-14 days plus a loading dose of ticagrelor (180 mg)
given one hour before endotoxin injection
aspirin 
(aspirin lysine) 75 mg OD for 10-14 days (sequence 6).
o
Aspirin (aspirin lysine) 300 mg OD for 10-14
aspirin (aspirin lysine) 300 mg OD for 10-14 days plus a loading dose of ticagrelor (180 mg)
given one hour before endotoxin injection (sequence 7).
o
Aspirin (aspirin lysine) 300 mg OD for 10-14 days plus a loading dose of ticagrelor (180 mg)
given one hour before endotoxin injection
aspirin 
(aspirin lysine) 300 mg OD for 10-14 days (sequence 8).
7.3.1
Method of implementing the randomisation/allocation sequence
Randomisation 
will 
be 
handled 
by 
an 
online 
interactive 
web-based 
randomisation 
service, 
sealedenvelope.com, using a simple randomisation method.
Participants will be allocated a three-digit number at enrolment (starting at 001)
01
E01001), then if they proceed to randomisation they will be allocated a separate three-digit randomisation 
number (starting at 001)
.
The system will generate an immediate email to the investigators stating the treatment allocation and this will 
Hospital Pharmacy with, if appropriate, a study-specific prescription for study medication for period 1. A 
separate prescription will be issued for period 2 but using the same randomisation document as evidence of 
the allocation.
7.4
Blinding
This study will be open-label i.e. unblinded to participants and investigators throughout. However, those 
performing the laboratory assessments will be blinded to treatment allocation in order to reduce bias.
7.5
Emergency Unblinding
Procedures for emergency unblinding are not relevant to this study.
7.6
Baseline data
At visit 1
Medical history

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
23
Physical examination
Demographic data
Vital signs (pulse, blood pressure and temperature
Weight and BMI
Concomitant medication
Lab safety parameters: full blood count, serum urea & electrolytes (sodium, potassium, urea and 
creatinine), liver function tests, clotting screen; and urinalysis
At visit 2
Vital signs: pulse, blood pressure and temperature
Physical examination
TNF-
IL-6, serum TXB2, urine PGI-M, platelet aggregation responses to arachidonic acid, ADP and 
collagen
Bleeding time
7.7
Trial assessments
Visit 1 - Screening (Day -21 to 0)
Screening of subjects and all study-related procedures will take place in the Sheffield Clinical Research 
Facility, a specialist environment for the conduct of clinical research. The following assessments and 
procedures will be performed:
Full informed consent, including completion of the informed consent form
Inclusion/exclusion criteria (see section 6)
Medical history
Physical examination
Demographic data
Vital signs: pulse, blood pressure and temperature
Weight and BMI
Concomitant medication

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
24
Lab safety: 12.5 ml blood sample for full blood count, urea & electrolytes (sodium, potassium, urea 
and creatinine), liver function tests, clotting screen; and urinalysis
If eligible, subjects will be asked to return to the Clinical Research Facility, Northern General Hospital, on 
Day 1 for the experimental part of the study. They will be randomised to receive a two-period medication 
regimen (Figure 3), with each period consisting of 10 to 14 days of aspirin (aspirin lysine) or no aspirin prior 
to the experimental days, with ticagrelor loading dose at the end of one period, 1 hour prior to endotoxin 
administration, and without ticagrelor loading dose at the end of the other.
There will be a washout period of at least 5 weeks between the first experimental day and the second 
Volunteers who complete the study will receive £100 per medication period/experimental day to reimburse 
them for their time, inconvenience and any discomfort caused. Transportation to and from the Clinical 
Research Facility will be provided if necessary. Subjects who fail screening will be recorded on a screen 
failure log with the reason for failure.
Visit 2 (Day 0)
Vital signs
Physical examination
Reconfirm eligibility criteria met (by a medically qualified member of the study team, see section 6)
and no withdrawal criteria met (section 7.10)
Randomisation
27 ml venous blood samples for baseline cytokines, prostanoids, inflammatory markers, platelet 
function, and plasma, serum, DNA and RNA for storage
Bleeding time measurement
Urine sample (around 20 ml will be taken for study purposes) for baseline prostanoid measurement
Provided with supply of study antiplatelet medication for period 1 as determined by randomisation
Dosing training for use of aspirin lysine if required
Period 1: Day 1 - Day 10 (to 14)
Participants will receive one of the following antiplatelet medication regimens for 10 to 14 days:
o
No IMP
o
No aspirin but a loading dose of ticagrelor (180 mg) taken on the last day of the medication 
period, 1 hour prior to endotoxin injection
o
Aspirin (aspirin lysine) 20 mg BD (only)
o
Aspirin (aspirin lysine) 20 mg BD, and a loading dose of ticagrelor (180 mg) taken on the 
last day of the medication period, 1 hour prior to endotoxin injection
o
Aspirin (aspirin lysine) 75 mg OD (only)

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
25
o
Aspirin (aspirin lysine) 75 mg OD, and a loading dose of ticagrelor (180 mg) taken on the 
last day of the medication period, 1 hour prior to endotoxin injection
o
Aspirin (aspirin lysine) 300 mg OD (only)
o
Aspirin (aspirin lysine) 300 mg OD, and a loading dose of ticagrelor (180 mg) taken on the 
last day of the medication period, 1 hour prior to endotoxin injection
Visit 3  - Period 1: Day 11 (to 15)
On arrival
Vital signs
Physical examination
Adverse event recording
Concomitant medication recorded
IMP compliance check for period 1
Medically-qualified member of the research team to confirm that the withdrawal criteria (section 
7.10) are not met, prior to proceeding further with visit
-1 hour (1 hour before endotoxin injection)
IV cannula insertion (x2, one in each arm)
35.5 ml venous blood sample for baseline lab safety (full blood count, urea & electrolytes [sodium, 
potassium, urea and creatinine], liver function tests, clotting screen), cytokines, prostanoids, 
inflammatory markers and platelet function
Bleeding time measurement
Urine sample for prostanoid assessment at baseline
Receive last dose of aspirin of period 1, or last dose of aspirin plus a loading dose of ticagrelor (180 
mg), or no IMP, as directed by randomisation allocation
Collect unused study medication and perform drug accountability check (sachet/pill count)
Infusion of 250 ml 0.9% saline over 30 minutes
23 ml venous blood at 60 minutes after last dose of oral antiplatelet medication or equivalent time 
if not receiving IMP during period 1 (just before endotoxin administration)
0 hour (time of endotoxin injection)

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
26
Continuous cardiac monitoring starts
E.coli endotoxin 2 ng/kg intravenously administered over 1 minute
Vital signs at 5, 15 and 30 minutes post-endotoxin
Continuous IV infusion of 250 ml 0.9% saline over 1 hour
14 ml venous blood sample at 30 minutes post endotoxin
1 hour post endotoxin injection
Vital signs at 1 hour and 1.5 hours post endotoxin injection
Continuous IV infusion of 250 ml 0.9% saline over 1 hour
14 ml venous blood sample at 1 hour post endotoxin
Urine sample at 1 hour post endotoxin
14 ml venous blood sample at 1½ hours post endotoxin
2 hours post-endotoxin injection
Vital signs at 2 hours post endotoxin
Continuous IV infusion of 250 ml 0.9% saline over 1 hour
23 ml venous blood sample at 2 hours post endotoxin
Urine sample at 2 hours post endotoxin
3 hours post-endotoxin injection
Vital signs at 3 hours post endotoxin
23 ml venous blood sample at 3 hours post endotoxin
Bleeding time measurement at 3 hours post endotoxin
4 hours post-endotoxin injection
Vital signs at 4 hours post endotoxin
14 ml venous blood sample at 4 hours post endotoxin
Urine sample at 4 hours post endotoxin

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
27
6 hours post-endotoxin injection
Vital signs at 6 hours post endotoxin
14 ml venous blood sample at 6 hours post endotoxin
Urine sample at 6 hours post endotoxin
Continuous cardiac monitoring ends
Visit 4 (Period 1, 1 day after visit 3)
Telephone follow-up for adverse events
Facility to convert to in-person visit if any concerns raised
There will then follow a washout period of at least 5 weeks, and not more than 18 weeks, between the first 
endotoxin challenge day and the beginning of the second medication period. This is to avoid reliably any 
tolerance of the response to endotoxin, a phenomenon absent by 5 weeks [39].
Visit 5 (10 to 14 days after visit 3)
Telephone follow-up for adverse events
Visit 6 (Period 2, Day 0)
Vital signs (BP, pulse, temperature)
Physical examination
Adverse event recording
Concomitant medication recording
Medically qualified investigator to confirm eligibility criteria again (see section 6) and that no 
withdrawal criteria (section 7.10) are met at this point prior to proceeding to medication period 2
Laboratory safety blood tests: 12.5 ml venous blood for full blood count, urea & electrolytes 
(sodium, potassium, urea and creatinine), liver function tests, clotting screen
Medication dispensed for period 2
Dosing training for use of aspirin lysine if required
Participants will be advised to begin antiplatelet medication, allocated as follows, for period 2 from the 
morning after visit 6:
o
If received no IMP in period 1, to receive no aspirin, but a loading dose of ticagrelor (180
mg) 1 hour before endotoxin injection in period 2.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
28
o
If received no aspirin but a loading dose of ticagrelor (180 mg) 1 hour before endotoxin 
injection in period 1, to receive no IMP in period 2.
o
If received aspirin (aspirin lysine) 20 mg BD (only) in period 1, to receive aspirin (aspirin 
lysine) 20 mg BD plus a loading dose of ticagrelor (180 mg) 1 hour before endotoxin 
injection in period 2.
o
If received aspirin (aspirin lysine) 20 mg BD plus a loading dose of ticagrelor (180 mg) 1 
hour before endotoxin injection in period 1, to receive aspirin (aspirin lysine) 20 mg BD 
(only) in period 2.
o
If received aspirin (aspirin lysine) 75 mg OD (only) in period 1, to receive aspirin (aspirin 
lysine) 75 mg OD plus a loading dose of ticagrelor (180 mg) 1 hour before endotoxin 
injection in period 2.
o
If received aspirin (aspirin lysine) 75 mg OD plus a loading dose of ticagrelor (180 mg) 1 
hour before endotoxin injection in period 1, to receive aspirin (aspirin lysine) 75 mg OD 
(only) in period 2.
o
If received aspirin (aspirin lysine) 300 mg OD (only) in period 1, to receive aspirin (aspirin 
lysine) 300  mg OD plus a loading dose of ticagrelor (180 mg) 1 hour before endotoxin 
injection in period 2.
o
If received aspirin (aspirin lysine) 300 mg OD plus a loading dose of ticagrelor (180 mg) 1 
hour before endotoxin injection in period 1, to receive aspirin (aspirin lysine) 300 mg OD 
(only) in period 2.
Period 2, Days 1 to 10 (to 14)
Participants take antiplatelet medication for period 2 as determined by the randomisation 
allocation (figure 3)
Visit 7 (Period 2, Day 11 [to 15])
On arrival
Vital signs
Physical examination
Adverse event recording
Concomitant medication recorded
IMP compliance check for period 2
Medically-qualified member of the research team to confirm that the withdrawal criteria (section 
7.10) are not met, prior to proceeding further with visit

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
29
-1 hour (1 hour before endotoxin injection)
IV cannula insertion (x2, one in each arm)
35.5 ml venous blood sample for baseline lab safety (full blood count, urea & electrolytes [sodium, 
potassium, urea and creatinine], liver function tests, clotting screen), cytokines, prostanoids, 
inflammatory markers and platelet function
Bleeding time measurement
Urine sample for prostanoid assessment at baseline
Receive last dose of aspirin of period 1, or last dose of aspirin plus a loading dose of ticagrelor (180 
mg), or no IMP, as directed by randomisation allocation
Collect unused study medication and perform drug accountability check (sachet/pill count)
Infusion of 250 ml 0.9% saline over 30 minutes
23 ml venous blood at 60 minutes after last dose of oral antiplatelet medication or equivalent time 
if not receiving IMP during period 1 (just before endotoxin administration)
0 hour (time of endotoxin injection)
Continuous cardiac monitoring starts
E.coli endotoxin 2 ng/kg intravenously administered over 1 minute
Vital signs at 5, 15 and 30 minutes post-endotoxin
Continuous IV infusion of 250 ml 0.9% saline over 1 hour
14 ml venous blood sample at 30 minutes post endotoxin
1 hour post-endotoxin injection
Vital signs at 1 hour and 1.5 hours post endotoxin injection
Continuous IV infusion of 250 ml 0.9% saline over 1 hour
14 ml venous blood sample at 1 hour post endotoxin
Urine sample at 1 hour post endotoxin
14 ml venous blood sample at 1½ hours post endotoxin
2 hours post-endotoxin injection
Vital signs at 2 hours post endotoxin

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
30
Continuous IV infusion of 250 ml 0.9% saline over 1 hour
23 ml venous blood sample at 2 hours post endotoxin
Urine sample at 2 hours post endotoxin
3 hours post-endotoxin injection
Vital signs at 3 hours post endotoxin
23 ml venous blood sample at 3 hours post endotoxin
Bleeding time measurement at 3 hours post endotoxin
4 hours post-endotoxin injection
Vital signs at 4 hours post endotoxin
14 ml venous blood sample at 4 hours post endotoxin
Urine sample at 4 hours post endotoxin
6 hours post-endotoxin injection
Vital signs at 6 hours post endotoxin
14 ml venous blood sample at 6 hours post endotoxin
Urine sample at 6 hours post endotoxin
Continuous cardiac monitoring ends
Visit 8 (Period 2, 1 day after visit 6)
Telephone follow-up for adverse events
Facility to convert to in-person visit if any concerns raised
Visit 9 (Period 2, 10 to 14 days after visit 6)
Telephone follow-up for adverse events
Study investigations will be performed at the following time points:*

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
31
*Baseline refers to visit 2, and the pre- and post-endotoxin samples are to be taken during both experimental periods.
Inflammatory markers and cytokines
TNF- : Baseline, pre-last medication dose, pre-endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 hours post-
endotoxin
IL-6: Baseline, pre-last medication dose, pre-endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 hours post-
endotoxin
CRP: Baseline, pre-endotoxin and 6 hours post-endotoxin
Prostanoids and Leukotrienes
Serum TXB2: Baseline, pre-last medication dose, pre-endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 hours 
post-endotoxin
Urinary PGI-M: Baseline, pre-endotoxin and 1, 2, 4 and 6 hours post-endotoxin
Urinary creatinine: Baseline, pre-endotoxin and 1, 2, 4 and 6 hours
Serum PGE2: Baseline, pre-endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 hours post-endotoxin
Leucocyte phenotype and activation status
White blood cell count with differential leukocyte count: Baseline, pre-last medication dose, pre-
endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 hours post-endotoxin
Haemostasis
Bleeding time: Pre-endotoxin and 3 hours post-endotoxin
Platelet function tests
Light transmittance aggregometry (agonists: arachidonic acid 0.3 and 1 mmol/l, collagen 4 and 16 
mg/ml, and ADP 20 µmol/l): Baseline, pre-endotoxin and 3 hours post-endotoxin
Samples for storage
Plasma for storage: Baseline, pre-last medication dose, pre-endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin
Serum for storage: Baseline, pre-last medication dose, pre-endotoxin and 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin
DNA/RNA for storage: Baseline
Urine for storage: Baseline, pre-endotoxin and 1, 2, 4 and 6 hours post-endotoxin
Details of fluid management
We anticipate that the total fluid load per volunteer will be 1000 ml over the 8 hours of the day at the NGH-
CRF (250 ml preload and 750 ml infusion). The volunteers will also receive fluid boluses if required, which 
would be typically be in the range of 250-500 ml normal saline, given if mean arterial blood pressure is 
reduced by 20% or more or to maintain a systolic blood pressure over 100 mmHg and/or to relieve 
symptoms related to hypotension. Under normal circumstances, we would give a maximum of 1000 ml of 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
32
0.9% saline over 1 hour and a maximum of 2000 ml of 0.9% saline over the 8 hours of the day at the NGH-
CRF.
7.8
Long term follow-up assessments
Participants will be followed up 10 (to 14) days after the second endotoxin injection day (Period 2, Day 21) 
by telephone. Any new adverse events will be recorded. Participants will be thanked for their involvement in 
the study and arrangements made to provide the monetary reimbursement due to them. In the event of a 
participant remaining uncontactable at the end of the follow up window, we will interrogate the hospital 
records of the participant to clarify vital status and any evidence of untoward events. Participants will be 
xin injection day. A file 
note will be made in this instance.
7.9
Qualitative assessments
Not applicable to this study
7.10
Withdrawal or postponement criteria
Subjects may be withdrawn or may discontinue from the trial if the following occur:
Inability to insert intravenous cannula and/or obtain venous blood samples
Withdrawal of consent
Development of an intolerable adverse event due to study participation as determined by the 
investigator and/or subject
Development of an intercurrent illness, condition or procedural complication that would interfere 
, unless further study activities can be appropriately postponed 
(see section 7.10.1)
At the time of planned endotoxin injection, receipt of a non-study medication in the preceding 48
hours that, in the opinion of the investigator, is likely to affect inflammation, thrombosis or haemostasis,
unless the endotoxin injection visit can be appropriately postponed (see section 7.10.1)
Poor compliance (<80% of doses taken) with study aspirin (aspirin lysine) during the preceding 
medication period, unless the endotoxin visit can be appropriately postponed (see section 7.10.1) to 
achieve 80 % compliance
Violation of the protocol
The investigator feels it
participation in the study
Previously unknown data becoming available raising concern about the safety of the study drugs, so 
that continuation could cause potential risks to the subjects
The reason for withdrawal/discontinuation will be documented in the Case Report Form (CRF).
Withdrawn participants who receive at least one dose of IMP or endotoxin will be followed up by telephone 
at 10-14 days after withdrawal, and additionally all who discontinue due to adverse events (AEs) will be 
followed up until resolution or stabilisation. All outcomes of AEs will be recorded in the CRF.
Withdrawn participants who have proceeded to randomisation will not be replaced.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
33
7.10.1 Provision for postponement of visit in the event of intercurrent illness
In the event of the following occurring in relation to a study participant:
-
an intercurrent illness, for example a viral upper respiratory tract infection;
-
requirement for a course of medication that, in the opinion of the investigators, is likely to affect 
measures of inflammation, thrombosis or haemostasis; or
-
compliance with aspirin (aspirin lysine) is <80% during the preceding study medication period
then if the subsequent endotoxin injection visit cannot be postponed to a time within the permitted window 
of 10-14 days into the medication period at which the illness or relevant effect of medication has deemed 
to have resolved or compliance has increased to at least 80% for the medication period, study medication 
may be stopped and the medication period restarted once the intercurrent illness or requirement for 
medication is deemed to have resolved, or when compliance is likely to be at least 80%. In this instance, an 
unscheduled visit will be arranged, the existing supply of aspirin (aspirin lysine) will be collected from the 
participant and returned to pharmacy for counting and destruction, and a new supply of aspirin (aspirin 
lysine) will be issued, dispensed after study-specific prescription request has been made to the pharmacy 
at the Northern General Hospital. A file note will be made in the TMF.
7.11
Storage and analysis of clinical samples
The samples will be stored and analysed according to the study-specific laboratory manual.
7.12
End of study
The study will end when the last laboratory analyses are completed. The sponsor will notify the MHRA of the 
end of the study within 90 days of its completion. In the event of early termination, this will be reported within 
15 days. 
8
STUDY MEDICATIONS
8.1
Name and description of investigational medicinal product(s)
The following medications will be used for experimental purposes during the study:
Aspirin lysine (
Aspegic [Sanofi-Aventis
-Aventis] 
)
o
Each aspirin lysine 100 mg sachet contains 180 mg aspirin lysine, equivalent to 100 mg 
acetylsalicyclic acid (aspirin). In this protocol the stated dose refers to the aspirin content.
o
Participants will be asked to prepare doses of 20 mg twice daily orally, where directed by 
the protocol, by dissolving 100 mg (1 sachet) in 100ml of drinking water and ingesting 20 
ml of the solution using a graduated syringe, discarding the remainder.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
34
o
Participants will be asked to prepare doses of 75 mg once daily orally, where directed by the 
protocol, by dissolving 100 mg (1 sachet) in 100 ml of drinking water, removing 25 ml of 
the solution using a graduated syringe, and ingesting the remainder.
o
Participants will be asked to prepare doses of 300 mg once daily orally, where directed by 
the protocol, by dissolving 300 mg (3 sachets) in 100 ml of drinking water and ingesting the 
solution.
o
A new sachet (or sachets) will be used for each dose of study medication
Ticagrelor 90 mg oro-dispersible tablets (AstraZeneca, 10 tablets per pack)
o
Participants will be asked to take a loading dose of 180 mg (two orodispersible 90 mg 
tablets) 1 hour before endotoxin injection where specified by the randomisation allocation. 
This will be directly observed by the research team. 
8.2
Regulatory status of the drug
Aspirin (aspirin lysine) 100 mg sachets are not licensed in the UK but are licensed in other EU 
countries (e.g. Belgium, Italy) for the purposes of analgesia and antipyresis. Other preparations of 
aspirin are licensed in the UK for similar indications, as well as an antithrombotic agent after ACS.
As there is no marketing authorisation in the UK for aspirin lysine, we will import this from another 
EU country facilitated by an existing relationship with Mawdsleys Ltd.
Ticagrelor 90 mg orodispersible tablets are licensed in the UK, in combination with aspirin, for the 
treatment of ACS.
We will retain the original presentation of drugs as supplied without modification, but will be asking 
participants to vary the doses of aspirin (aspirin lysine) as detailed elsewhere in this protocol.
8.3
Product Characteristics
Aspirin (aspirin lysine) 100 mg sachets have a current summary of product characteristics (SmPC) that has 
been translated and notarised from the original French. A copy will be kept in the trial management file.
The current version of the SmPC for ticagrelor 90 mg orodispersible tablets will also be available for 
reference in the trial management file.
The study team will check for updates to these documents at regular intervals throughout the development 
and conduct of the study, and will review and file these as needed, if necessary obtaining further notarised 
translations. If an update to section 4.8 (the reference safety information) is involved, the revised SmPC will 
be submitted and approved as a substantial amendment before the revised SmPC is filed as the new 
reference safety information for the trial for the assessment of expectedness of adverse events.
8.4
Drug storage and supply
Aspirin (aspirin lysine) 100 mg sachets will be sourced by the Northern General Hospital Pharmacy through 
a study-specific arrangement with Mawdsleys Ltd at market price.
Ticagrelor 90 mg orodispersible tablets will be sourced by the Northern General Hospital Pharmacy locally 
at market price.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
35
The initial shipment will be requested on site initiation (manual ordering) and re-ordering will occur 
manually as necessary until the end of the study.
Prior to study medication dispensing, all study medications will be kept in a secure location in the pharmacy 
at Northern General Hospital, Sheffield, under appropriate storage conditions with temperature excursions 
permitted between 15ºC and 30ºC.  Both aspirin (aspirin lysine) and ticagrelor will be segregated as clinical 
trial stock for the purposes of this study.
Aspirin (aspirin lysine) will be supplied to the investigators upon receipt of a suitable signed study-specific 
prescription at the start of each period. Only medically-qualified members of the study team will be able to 
complete the prescription. Where participants are required to take aspirin (aspirin lysine) by the 
randomisation allocation, one box of 30 sachets will be dispensed per period for each participant 
randomised to receive either aspirin 20 mg BD or aspirin 75 mg OD and two boxes of 30 sachets will be 
dispensed per period for each participant randomised to receive aspirin 300 mg OD.
Packs of ten 90 mg oro-dispersible ticagrelor tablets will be dispensed from the pharmacy to the 
investigators, on request, for storage in a dedicated area within the NGH-CRF. The pack will then be split 
and used to administer doses to a number of participants. Prescription, which will only be able to be made 
by a medically-qualified member of the study team, and administration will be recorded on a Sheffield 
Teaching Hospitals Drug Administration Record. A drug accountability log and temperature monitoring 
records will be kept for the ticagrelor stored in the NGH-CRF.
There are no specific post-dispensing storage instructions.
Unused medication and used packaging will be returned to pharmacy at the end of each treatment period, 
who will perform pill/sachet count for accountability and will supervise destruction.
In the event of postponing visit 3 or 7 because of intercurrent illness and restarting a medication period 
from the beginning, unused medication from the period prior to the postponement will be returned to 
pharmacy and a new study-specific prescription will be made by a medically-qualified investigator for a new 
supply of aspirin (aspirin lysine).
8.5
Preparation and labelling of Investigational Medicinal Product
Labels for aspirin (aspirin lysine) and ticagrelor will be prepared in accordance with Good Manufacturing 
Practice and regulatory guidelines of the Medicines Healthcare and Regulatory Agency. The label will 
include the following information: drug, formulation, dose, and dosing frequency as well as other 
information to comply with annex 13.
Participants will also be given written, illustrated dose-preparation instructions for aspirin (aspirin lysine) as 
described elsewhere in this protocol.
8.6
Dosage schedules
The dosage schedules are described in section 7.3, 7.7 and the trial flow chart within this protocol.
When taking once-daily doses of study medication, this will be on waking, with the exception of the 
endotoxin injection days when they will be asked to delay taking until the appropriate point within the study 
visit. When taking twice-daily, this should be on waking (with the exception of the endotoxin injection days 
when they will be asked to delay taking until the appropriate point within the study visit) and then as close 
to 12 hours later as possible.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
36
In the case of a missed dose, the participant will be advised to take the dose when they remember if within 
6 hours (twice-daily regimens) or 12 hours (once-daily regimens) of the intended time, otherwise to wait to 
take the next dose on time. In the event of vomiting after a dose, they should wait for the next dose before 
taking the study medication again in order avoid accidental overdose.
The doses of aspirin (aspirin lysine) and ticagrelor will not be adjusted for any parameter. The maximum 
duration that a participant will receive aspirin (aspirin lysine) will be 28 days and ticagrelor 1 day.
8.7
Dosage modifications
Discontinuation of aspirin (aspirin lysine) and/or withholding ticagrelor may be considered in the following 
circumstances:
Major bleeding, including life-threatening bleeding
Intolerable adverse reaction such as minor bleeding that cannot be controlled by local measures
Discovery of severe thrombocytopenia (platelet count < 50,000/ L)
No dose adjustment beyond that specified by the randomisation schedule will be permitted.
8.8
Known drug reactions and interaction with other therapies
Details are to be found in section 8.9 below, in the current SmPCs for aspirin (aspirin lysine) and ticagrelor
orodispersible tablets.
8.9
Concomitant medication
Regular concomitant medication will lead to exclusion from randomisation. Recording of any prescribed or 
over-the-counter medication after randomisation will be made at each subsequent visit. If at visits 3 and 7,
any of the criteria in section 7.10 are met, the participant will be withdrawn if the visit cannot be 
postponed. Similarly, if at visit 6 the criteria in section 6 are not met, the participant will be withdrawn if the 
visit cannot be postponed. All individual medications, prescription and over-the-counter, will be recorded 
peri-event for any SAE or discontinuation due to AE. The following should be observed if any participant 
receives concomitant medication after randomisation:
Oral antiplatelet therapies
Aspirin (acetylsalicylic acid): Aspirin use, with the exception of the study medication as prescribed, is 
prohibited during the study period. At enrolment, patients will be asked to not use aspirin as an analgesic 
and they will be made aware of the range of over-the-counter products that contain it. If no contraindication 
exists, paracetamol will be recommended if the need for analgesia arises, but endotoxin injection should 
not be performed if paracetamol has been received in the preceding 24 hours. Patients will be asked about 
extra aspirin use, including over the counter supplies, at all visits. If, during the course of study treatment, a 
participant develops a clinical indication for regular antiplatelet therapy, a clinically appropriate regimen will 
be prescribed and they will be followed up but withdrawn from the study. Participants who receive at least 
one dose of IMP or endotoxin will be followed up by telephone 10-14 days after withdrawal.
Other oral antiplatelet therapies: Treatment with any other oral antiplatelet therapy apart from the study 
medication (e.g. clopidogrel, prasugrel, dipyridamole, cilostazol) is prohibited during the course of study 
medication. However, if during the course of study treatment, a participant develops a contraindication to 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
37
aspirin or ticagrelor, these will be discontinued/withheld and they will be withdrawn. Participants who 
receive at least one dose of IMP or endotoxin will be followed up by telephone 10-14 days after withdrawal.
As all participants will be taking ticagrelor at some stage during the study, drugs that interact with its 
metabolism should be avoided. Strong inhibitors of CYP3A4 substantially increase plasma ticagrelor levels 
whereas strong inducers of CYP3A4 have the opposite effects. Consequently, strong CYP3A4 inhibitors (eg, 
ketoconazole, itraconazole, voriconazole, telithromycin, clarithromycin [but not erythromycin or 
azithromycin], nefazadone, ritonavir, saquinavir, nelfinavir, indinavir, atanazavir, or over 1 litre daily of 
grapefruit juice) should not be co-administered with ticagrelor as plasma levels of ticagrelor would be 
substantially increased. If regular treatment with such therapies is essential, then ticagrelor should be 
withheld and they should be withdrawn. Participants who receive at least one dose of IMP or endotoxin will 
be followed up by telephone 10-14 days after withdrawal.
Concomitant therapy with simvastatin or lovastatin at doses higher than 40 mg daily is not permitted since 
ticagrelor significantly increases the levels of these statins and theoretically therefore may increase the risk 
mg daily or any dose of any other statin is permitted) but if a participant fails to meet the eligibility criteria 
for the study (section 6) at the next check (visit 3, 6 or 7), they will be withdrawn if the visit cannot be 
postponed. Standard monitoring of patients for possible statin-associated myopathy should be carried out.
Co-administration of ticagrelor with CYP3A substrates with a narrow therapeutic index (e.g. cyclosporine 
and quinidine) should be avoided.
Co-administration of ticagrelor with strong inducers of CYP3A should also be avoided (e.g. 
rifampin/rifampicin, rifabutin, phenytoin, carbamazepine, phenobarbital). 
If regular treatment with such therapies becomes essential during the study medication period then they 
will be withdrawn from the study. Participants who receive at least one dose of IMP or endotoxin will be 
followed up by telephone 10-14 days after withdrawal.
Non-steroidal anti-inflammatory drugs (NSAIDs)
NSAIDs may affect the antiplatelet and immunomodulatory effects of aspirin whilst increasing the risk of 
gastric irritation/ulceration and renal impairment. Levels of arachidonic acid metabolites may also be 
affected. Requirement for regular treatment with an NSAID at enrolment meets the exclusion criteria of the 
study. Treatment with NSAIDs during the study period is discouraged. COX2 inhibitors are prohibited in 
combination with study medication. Paracetamol is safe in combination with both aspirin and ticagrelor and 
therefore will be the recommended analgesic/antipyretic agent if required. In the case of a participant 
requiring treatment with an NSAID/paracetamol/COX2 inhibitor, if they fail to meet the eligibility criteria for 
the study (section 6) at the next check (visit 3, 6 or 7), they will be withdrawn if the visit cannot be 
postponed. Participants who receive at least one dose of IMP or endotoxin will be followed up by telephone 
10-14 days after withdrawal.
Other anti-inflammatory/immunomodulatory drugs
Oral, topical or inhaled corticosteroids; disease-modifying anti-rheumatic drugs; immunosuppressants; 
chemotherapy drugs; oral or topical antihistamines: In the case of a participant requiring regular treatment 
with these agents, if they fail to meet the eligibility criteria for the study (section 6) at the next check (visit 3, 
6 or 7), they will be withdrawn if the visit cannot be postponed Participants who receive at least one dose of 
IMP or endotoxin will be followed up by telephone 10-14 days after withdrawal.
Diuretics

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
38
Diuretics, including loop (eg. furosemide, bumetanide), thiazide (eg. bendroflumethiazide, indapamide) and 
potassium-sparing agents (eg. spironolactone, eplerenone, amiloride), exert effects on renal prostaglandin 
synthesis and therefore may interfere with urinary prostaglandins. If a requirement for regular diuretic 
treatment develops during the study, a participant who fails to meet the eligibility criteria for the study 
(section 6) at the next check (visit 3, 6 or 7), will be withdrawn if the visit cannot be postponed . Those who 
receive at least one dose of IMP or endotoxin will be followed up by telephone 10-14 days after withdrawal.
ACE inhibitors/Angiotensin receptor blockers
ACE inhibitors and angiotensin receptor blockers can affect renal prostaglandin production. If a participant 
fails to meet the eligibility criteria for the study (section 6) at the next check (visit 3, 6 or 7), they will be 
withdrawn if the visit cannot be postponed but followed up by telephone at 10-14 days after withdrawal.
Parenteral anticoagulants
In the event of an indication for parenteral anticoagulation developing, the participant will be withdrawn 
from the study but participants who receive at least one dose of IMP or endotoxin will be followed up by 
telephone 10-14 days after withdrawal.
Glycoprotein IIb/IIIa antagonists
In the event of an indication for an agent in this group developing, the participant will be withdrawn from 
the study. Participants who receive at least one dose of IMP or endotoxin will be followed up by telephone 
10-14 days after withdrawal.
Oral anticoagulants
The use of oral anticoagulants (e.g. warfarin, apixaban, rivaroxaban, dabigatran) in combination with study 
medication is prohibited. If oral anticoagulation is considered essential during the intended course of the 
study, the participant will be withdrawn from the study. Participants who receive at least one dose of IMP or 
endotoxin will be followed up by telephone 10-14 days after withdrawal.
Fibrinolytic agents
Treatment with fibrinolytic agents should be avoided whenever possible during treatment with study 
medication. If a participant is to receive a fibrinolytic agent the participant will be withdrawn from the study. 
Participants who receive at least one dose of IMP or endotoxin will be followed up by telephone 10-14 days 
after withdrawal.
Other drugs
If regular treatment with any other prescribed or over-the-counter systemic or topical medication becomes 
required after randomisation, if a participant fails to meet the eligibility criteria for the study (section 6) at 
the next check (visit 3, 6 or 7), they will be withdrawn if the visit cannot be postponed.
8.10
Trial restrictions

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
39
Whilst undergoing sampling during visits 3 and 7, subjects will take a light breakfast before 8 am then 
remain nil by mouth until 2 hours post-endotoxin. 
Surgery and other invasive procedures
If surgery or another procedure becomes necessary during the study medication period, study medication 
should be stopped and the participant will be withdrawn from the study. Participants who receive at least 
one dose of IMP or endotoxin will be followed up by telephone 10-14 days after withdrawal.
The effects of aspirin, but not ticagrelor, may be reversed by platelet transfusion, which might be 
considered in the event of life-threatening bleeding. 
Intercurrent illness
In the event of a participant developing an intercurrent illness after randomisation, for example a viral 
upper respiratory tract infection, medication periods or endotoxin injections may be postponed, at the 
discretion of the investigator, until resolution of the illness. A file note should be generated and placed in 
the trial master file.
8.11
Assessment of compliance with treatment
Compliance will be assessed at the end of each treatment period by counting the remaining sachets of 
aspirin (aspirin lysine) returned by the participant, and by direct observation of the participant taking the 
loading dose of ticagrelor and last dose of aspirin in each period, where applicable. Sachet counts will be
carried out by the investigators and then cross-checked by the Pharmacy Department. Compliance will be 
recorded in the CRF.
8.12
Name and description of each Non-Investigational Medicinal Product (NIMP)
Although not a medicinal product, in this study we are additionally administering sterile bacterial endotoxin,
2 ng/kg intravenously. As a challenge agent, this is classified as a NIMP, as per European Commission 
Guidance Document SANCO/C/8/SF/cg/a.5.001(2011)332855. This will be reconstituted with water for 
Endotoxin specifically designed for human challenge 
studies will be obtained from Dr Anthony Suffredini, National Institutes of Health, Bethesda, USA. Endotoxin 
will be securely stored separately in cold storage within the Cardiovascular Research Unit, Northern General 
Hospital. The temperature of the storage environment will be monitored by recording a daily maximum and 
minimum temperature log (with the exception of weekends). The allowable temperature range will be 2 to 8 
degrees Celsius. An accountability log to track each vial will be kept in the trial master file. Endotoxin will 
be prescribed by a medically-qualified investigator on a Sheffield Teaching Hospitals Drug Administration 
Record. Administration will be recorded on the same chart.
9
PHARMACOVIGILANCE
9.1
Definitions
Term
Definition
Adverse Event (AE)
Any untoward medical occurrence in a participant to whom a medicinal 
product has been administered, including occurrences which are not 
necessarily caused by or related to that product.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
40
Adverse Reaction (AR)
An untoward and unintended response in a participant to an 
investigational medicinal product which is related to any dose 
administered to that participant.
The phrase "response to an investigational medicinal product" means 
that a causal relationship between a trial medication and an AE is at 
least a reasonable possibility, i.e. the relationship cannot be ruled out.
All cases judged by either the reporting medically qualified professional 
or the Sponsor as having a reasonable suspected causal relationship to 
the trial medication qualify as adverse reactions. It is important to note 
that this is entirely separate to the known side effects listed in the SmPC. 
It is specifically a temporal relationship between taking the drug, the half-
life, and the time of the event or any valid alternative aetiology that would 
explain the event.
Serious Adverse Event
(SAE)
A serious adverse event is any untoward medical occurrence that:
results in death
is life-threatening
requires inpatient hospitalisation or prolongation of existing hospitalisation
results in persistent or significant disability/incapacity
consists of a congenital anomaly or birth defect
Other 
jeopardise the participant or require an intervention to prevent one of the 
above consequences.
NOTE: The term "life-threatening" in the definition of "serious" refers to an 
event in which the participant was at risk of death at the time of the 
event; it does not refer to an event which hypothetically might have 
caused death if it were more severe.
Serious Adverse
Reaction (SAR)
An adverse event that is both serious and, in the opinion of the reporting 
Investigator, believed with reasonable probability to be due to one of the 
trial treatments, based on the information provided.
Suspected Unexpected
Serious Adverse
Reaction (SUSAR)
A serious adverse reaction, the nature and severity of which is not 
consistent with the information about the medicinal product in question 
set out in the reference safety information:
SmPC for aspirin (aspirin lysine)
SmPC for ticagrelor
9.2
Operational definitions for (S)AEs
Adverse events and reactions will be recorded and reported from randomisation to 10 days after visit 3 
(first endotoxin injection day), and from visit 6 (start of period 2) to 10 days after visit 7 (second endotoxin 
injection day). Thus, adverse events occurring in the break between the first and second medication 
periods, excepting those originating in the 10 days after endotoxin injection, will not be recorded or 
reported.
All SAEs will be reported to the sponsor within 24 hours of the research team becoming aware of them. This 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
41
specifically for this purpose. A copy will be kept in the trial master file and the AE log will be completed. All 
SAEs will be reviewed at the regular trial management group meetings.
All other AEs not meeting the criteria for reporting as serious will be recorded in the CRF, with the exception 
of the following events which will not be reported, as they can be expected as a normal response to 
endotoxin:
Abnormal body temperature, excepting if >39oC and <34oC, unless this persists at 6 hours post-
endotoxin or meets the criteria for reporting as an SAE.
Change in pulse rate, excepting pulse rates of >140 bpm and <45 bpm or if meeting the criteria for 
SAE reporting.
Change (increase or decrease) in systolic or diastolic blood pressure from baseline of <40 mmHg 
unless meeting the criteria for SAE reporting.
Self-limiting symptoms wholly due to endotoxaemia, for example fever, rigors, general malaise, 
nausea, vomiting or headache, at the discretion of the investigator, unless meeting the criteria for 
SAE reporting.
Alterations in leukocyte (or subset) counts, platelet count or C-reactive protein unless meeting the 
criteria for SAE reporting.
The investigators will assess the relatedness of adverse events to the IMPs (aspirin [aspirin lysine], ticagrelor) 
and NIMP (endotoxin). 
9.3
Recording and reporting of SAEs, SARs AND SUSARs
SAEs/SUSARs will be recorded and reported from randomisation to 10 days after visit 3 (first endotoxin 
injection day), and from visit 6 (start of period 2) to 10 days after visit 7 (second endotoxin injection day).
Thus, adverse events occurring in the break between the first and second medication periods, excepting 
those originating in the 10 days after endotoxin injection, will not be recorded or reported.
These must be recorded on the Sheffield Teaching Hospitals NHS Foundation Trust SAE reporting 
Form and emailed to the Sponsor
within 24 hours of the 
research staff becoming aware of the event.
For each SAE or SUSAR the following information will be collected:
full details in medical terms and case description
event duration (start and end dates, if applicable)
action taken
outcome
seriousness criteria
causality (i.e. relatedness to trial drug / investigation), in the opinion of the investigator
whether the event would be considered anticipated.
Any change of condition or other follow-up information should be emailed to the Sponsor as soon as 
it is available or at least within 24 hours of the information becoming available. Events will be 
followed up until the event has resolved or a final outcome has been reached.
All SAEs assigned by the PI or delegate as both suspected to be related to IMP-treatment and 
unexpected will be classified as SUSARs and will be subject to expedited reporting to the Medicines 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
42
and Healthcare Products Regulatory Agency (MHRA). The sponsor will inform the MHRA, the REC and 
the relevant Marketing Authorisation Holder(s) of SUSARs within the required expedited reporting 
timescales.
SAEs related to the NIMP (endotoxin) will be reported by the investigators to the sponsor, but further 
forwarding to other agencies will not be required, as per European Directive 2011/C 172/01. NIMP-
related SAEs will be evaluated by the sponsor and investigator team with regard to ongoing safety
implications.  If there are ongoing safety implications for the trial then these will be addressed via urgent 
safety measure, substantial amendment or termination of the trial.
If, on adjudication by the investigators, there is a possibility that an SAE is related to interaction between 
the NIMP and at least one of the IMPs, the process will be followed for reporting this as an IMP-related 
event, including any required onward notification by the sponsor concerning the relevant IMP.
If an SAE is judged as possibly related to both NIMP and an IMP then it will be assessed with regard to 
expectedness with regard to the IMP and reported onwards as a SUSAR if it is judged to be unexpected in 
relation to the IMP
9.4
Responsibilities
Principal Investigator (PI) or delegate: 
1. Checking for AEs and ARs when participants attend for treatment / follow-up.
2. Using medical judgement in assigning seriousness, causality and whether the event/reaction 
was anticipated using the Reference Safety Information approved for the trial.
3. Using medical judgement in assigning seriousness and causality and providing an opinion on 
whether the event/reaction was anticipated using the Reference Safety Information 
approved for the trial. 
4. Ensuring that all SAEs are recorded and reported to the sponsor within 24 hours of becoming 
aware of the event and provide further follow-up information as soon as available. Ensuring 
that SAEs are chased with Sponsor if a record of receipt is not received within 2 working days 
of initial reporting. 
5. Ensuring that AEs and ARs are recorded and reported to the sponsor in line with the 
requirements of the protocol. 
Chief Investigator (CI, in this single-site study the same individual as the PI) or delegate:
1. Clinical oversight of the safety of patients participating in the trial, including an ongoing 
review of the risk / benefit.
2. Using medical judgement in assigning whether and event/reaction was anticipated or 
expected in line with the Reference Safety Information.
3. Immediate review of all SUSARs. 
4. Assigning Medical Dictionary for Regulatory Activities (MedDRA) or Body System coding to all 
SAEs and SARs.
5. Preparing the clinical sections and final sign off of the Development Safety Update Report 
(DSUR).
Sponsor: (NB where relevant these can be delegated to CI)
1. Collection and verification of AEs, ARs, SAEs, SARs and SUSARs according to the trial 
protocol. 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
43
2. Expedited reporting of SUSARs to the Competent Authority (MHRA) and REC within required 
timelines.
3. Checking for (annually) and notifying PIs of updates to the Reference Safety Information for 
the trial.
4. Preparing standard tables and other relevant information for the DSUR in collaboration with 
the CI and ensuring timely submission to the MHRA and REC.
9.5
Notification of deaths
o
Any deaths will be treated as an SAE and reported accordingly to the sponsor using the SAE 
reporting procedures within 24 hours of becoming aware, irrespective of whether the death 
is related to the IMP, endotoxin administration or an unrelated event.
9.6
Pregnancy reporting
Pregnancy is not expected to occur as women of childbearing potential will be excluded from study 
involvement. Nevertheless, any pregnancy occurring in a study participant should be reported to the 
Chief Investigator and the Sponsor using the relevant sponsor-provided Pregnancy Reporting Form 
within 24 hours of notification. Any pregnant participant will be withdrawn from the study. Follow-up 
of the pregnant participant and child born to a pregnant trial participant, if this becomes necessary, 
will be discussed with the sponsor in this unlikely event. 
There are no restrictions nor requirements for follow up of the children of male participants in the 
study.
9.7
Overdose
An overdose will be defined as any amount taken, above that prescribed, that in the opinion of a medically-
qualified investigator has the potential to cause significant harm. If an overdose of a study drug occurs, 
then investigators or other site personnel will inform the Sponsor immediately that they become aware of it, 
and in any case within 24 hours. Overdoses may be observed from sachet/tablet counts or patient 
comment.
In case of overdose of aspirin (aspirin lysine), this will be considered equivalent to standard preparations of 
aspirin.  Aspirin is an established agent and standard guidelines for the assessment and management of 
overdose should be followed.
Similarly, ticagrelor is an established drug and usual clinical procedures will be followed in the event of 
overdose.
Participants who receive an overdose of medication may be withdrawn from the study at the discretion of 
the investigators.
If an SAE is associated with the overdose, the investigators will ensure the overdose is fully described in the
SAE report form.
9.8
Reporting urgent safety measures
If any urgent safety measures are taken, the CI/Sponsor shall immediately and, in any event, no later than 
3 days from the date the measures are taken, give written notice to the MHRA and the relevant REC of the 
measures taken and the circumstances giving rise to those measures.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
44
9.9
The type and duration of the follow-up of participants after adverse reactions.
Adverse drug reactions will be followed up by telephone or in person, as necessary, until resolved or stable.
Follow up of existing SUSARs (i.e. those which start within the reporting period) will need to be reported to 
the sponsor indefinitely until resolved.
9.10
Development safety update reports
The CI will provide (in addition to the expedited reporting above) DSURs once a year throughout the clinical 
trial, or as necessary, to the Competent Authority (MHRA), where relevant the Research Ethics Committee
and the sponsor. 
The report will be submitted within 60 days of the Developmental International Birth Date (DIBD) of the trial 
each year until the trial is declared ended.
10
STATISTICS AND DATA ANALYSIS
10.1
Sample size calculation
we saw, after log transformation, a mean 2 hour post-endotoxin plasma TNF- 1.88 pg/ml with a standard 
deviation of 0.410 pg/ml.
There is no available raw data and no reliable estimate of the means or standard deviations of the log 
transformed TNF-
three aspirin doses using the same methods. However, a study using a 
different endotoxin regimen has shown that aspirin 80 mg OD (which is not expected to be significantly 
different in effect to 75 mg OD) increased peak plasma TNF- by around 40% compared to those receiving no 
drug.
We have a clear hypothesis that increasing aspirin dose increases TNF- , therefore a one-sided test will
suffice.
We estimate that clinically-relevant differences in plasma TNF- at 2 hours post-endotoxin between the dosing 
regimens might be a 20% increase with 20 mg BD vs. no aspirin, 40% increase with 75 mg OD vs. no aspirin 
and 60% with 300 mg OD vs. no aspirin, based on the raw data.
These assumptions provide the following sample size estimate:
 Test significance level, 
 0.050
     Number of groups, G
4
     Variance of means, V=S(mi-m)2 / G
   0.031
     Common standard deviation, s
   0.410
 Effect size, D2 = V/s2
  0.1828
 Power ( % )
    80
 n per group
16
Therefore, when the sample size in each of the 4 groups is 16, a one-way analysis of variance will have 80% 
power to detect at the 0.050 level a difference in means characterised by a Variance of means, V= (
i- )2 / G
of 0.031, assuming that the common standard deviation is 0.410.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
45
To allow for up to 10% drop-out, the sample size per group will be 18. Drop-outs will not be replaced but it is 
expected that there will be at least 16 per group after withdrawals.
This sample size calculation was performed by Mrs Kathleen Baster CStat, Senior Consultant, Statistical 
Services Unit, University of Sheffield, to whom the investigators are grateful.
10.2
Planned recruitment rate
It is aimed to recruit participants at the minimum 
experience of similar studies, this is sensibly feasible.
10.3
Statistical analysis plan
10.3.1 Summary of baseline data and flow of patients
The following baseline data will be collected and reported:
From visit 1
Demographic data (age, sex, ethnicity)
Vital signs: pulse, blood pressure and temperature
Weight and BMI
Full blood count, urea & electrolytes, liver function tests
At visit 2
TNF-
IL-6, serum TXB2, urine PGI-M, platelet aggregation responses to arachidonic acid, ADP and 
collagen
Bleeding time
Categorical data will be reported as proportions and percentages. Differences between the groups will be 
assessed
contingency test. Continuous data will be reported as mean and standard 
deviation if normally distributed otherwise median and interquartile range. Differences between the parallel 
groups will be assessed with ANOVA, with Bonferroni pairwise comparisons made if any ANOVA reaches 
p<0.05.
A CONSORT flow diagram will be prepared for inclusion in the report of study findings.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
46
10.3.2 Primary outcome analysis
The primary endpoint will be plasma TNF-
at 2 hours following endotoxin administration assessed between 
aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg OD, without ticagrelor) by one-way ANOVA 
with treatment as a factor.
10.3.3 Secondary outcome analysis
Secondary analyses will be as follows:
Between aspirin dose regimens
1. Plasma TNF-
over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 
6 hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, without ticagrelor) by two-way mixed ANOVA with treatment as a between-subjects 
factor and timepoint as a within-subjects factor. 
2. Plasma IL-6 at 2 hours following endotoxin administration assessed between aspirin groups (control 
vs. 20 mg BD vs. 75 mg OD vs. 300 mg OD, without ticagrelor) by one-way ANOVA with treatment as 
a factor. 
3. Plasma IL-6 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, without ticagrelor) by two-way mixed ANOVA with treatment as a between-subjects 
factor and timepoint as a within-subjects factor. 
4. Change in serum CRP over time following endotoxin administration (measured at 0 and 6 hours 
post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg 
OD, without ticagrelor) by one-way ANOVA.
5. Serum TXB2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, without ticagrelor) by two-way mixed ANOVA with treatment as a between-subjects 
factor and timepoint as a within-subjects factor. 
6. Urinary PGI-M (measured at 0, 2, 4 and 6 hours, each adjusted for [divided by] urinary creatinine)
over time following endotoxin administration assessed between aspirin groups (control vs. 20 mg 
BD vs. 75 mg OD vs. 300 mg OD, without ticagrelor) by two-way mixed ANOVA with treatment as a 
between-subjects factor and timepoint as a within-subjects factor. 
7. Serum PGE2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, without ticagrelor) by two-way mixed ANOVA with treatment as a between-subjects 
factor and timepoint as a within-subjects factor. 
8. Circulating leukocyte count over time following endotoxin administration (measured at 0, 0.5, 1, 
1.5, 2, 3, 4 and 6 hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 
75 mg OD vs. 300 mg OD, without ticagrelor) by two-way mixed ANOVA with treatment as a 
between-subjects factor and timepoint as a within-subjects factor. 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
47
9. AUC of the graph of bleeding time following endotoxin administration (measured at 0 and 6 hours
post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg 
OD, without ticagrelor) by one-way ANOVA.
10. AUC of the graphs of platelet aggregation to arachidonic acid (0.3 and 1 mmol/L), collagen (4 and 
16 g/ml) and ADP (20
mol/L) over time following endotoxin administration (measured at 0 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, without ticagrelor) by one-way ANOVA.
Between DAPT regimens:
11. Plasma TNF-
at 2 hours following endotoxin administration assessed between aspirin groups 
(control vs. 20 mg BD vs. 75 mg OD vs. 300 mg OD, given in combination with ticagrelor) by one-
way ANOVA with treatment as a factor.
12. Plasma TNF-
over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 
6 hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, given in combination with ticagrelor) by two-way mixed ANOVA with treatment as a 
between-subjects factor and timepoint as a within-subjects factor. 
13. Plasma IL-6 at 2 hours following endotoxin administration assessed between aspirin groups (control 
vs. 20 mg BD vs. 75 mg OD vs. 300 mg OD, given in combination with ticagrelor) by one-way ANOVA 
with treatment as a factor.
14. Plasma IL-6 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, given in combination with ticagrelor) by two-way mixed ANOVA with treatment as a 
between-subjects factor and timepoint as a within-subjects factor. 
15. Change in serum CRP over time following endotoxin administration (measured at 0 and 6 hours 
post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg 
OD, given in combination with ticagrelor) by one-way ANOVA.
16. Serum TXB2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, given in combination with ticagrelor) by two-way mixed ANOVA with treatment as a 
between-subjects factor and timepoint as a within-subjects factor. 
17. Urinary PGI-M (measured at 0, 2, 4 and 6 hours, each adjusted for [divided by] urinary creatinine) 
over time following endotoxin administration assessed between aspirin groups (control vs. 20 mg 
BD vs. 75 mg OD vs. 300 mg OD, given in combination with ticagrelor) by two-way mixed ANOVA 
with treatment as a between-subjects factor and timepoint as a within-subjects factor. 
18. Serum PGE2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, given in combination with ticagrelor) by two-way mixed ANOVA with treatment as a 
between-subjects factor and timepoint as a within-subjects factor. 
19. Circulating leukocyte count over time following endotoxin administration (measured at 0, 0.5, 1, 
1.5, 2, 3, 4 and 6 hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
48
75 mg OD vs. 300 mg OD, given in combination with ticagrelor) by two-way mixed ANOVA with 
treatment as a between-subjects factor and timepoint as a within-subjects factor. 
20. AUC of the graph of bleeding time following endotoxin administration (measured at 0 and 3 hours 
post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg 
OD, given in combination with ticagrelor) by one-way mixed ANOVA.
21. AUC of the graphs of platelet aggregation to arachidonic acid (0.3 and 1 mmol/L), collagen (4 and 
16 g/ml) and ADP (20 mol/L) over time following endotoxin administration (measured at 0 and 3 
hours post-endotoxin) assessed between aspirin groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, given in combination with ticagrelor) by one-way ANOVA.
To compare aspirin monotherapy with aspirin and ticagrelor in combination:
1. Plasma TNF-
at 2 hours following endotoxin administration) compared between each aspirin 
regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each 
corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 
mg BD) by paired t-tests. 
2. AUC of the graph of plasma TNF-
over time following endotoxin administration (measured at 0, 
0.5, 1, 1.5, 2, 3, 4 and 6 hours post-endotoxin) compared between each aspirin regimen (control, 
20 mg BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests. 
3. Plasma IL-6 at 2 hours following endotoxin administration, compared between each aspirin regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT 
regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) by paired t-
tests. 
4. AUC of the graph of plasma IL-6 over time following endotoxin administration (measured at 0, 0.5, 
1, 1.5, 2, 3, 4 and 6 hours post-endotoxin) compared between each aspirin regimen (control, 20 mg 
BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests.
5. Change in serum CRP over time following endotoxin administration (measured at 0 and 6 hours 
post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 mg 
OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 
300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests.
6. AUC of the graph of serum TXB2 over time following endotoxin administration (measured at 0, 0.5, 
1, 1.5, 2, 3, 4 and 6 hours post-endotoxin) compared between each aspirin regimen (control, 20 mg 
BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests.
7. AUC of the graph of urinary PGI-M (measured at 0, 2, 4 and 6 hours, each adjusted for [divided by] 
urinary creatinine) over time following endotoxin administration compared between each aspirin 
regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each 
corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 
mg BD) by paired t-tests.
8. AUC of the graph of serum PGE2 over time following endotoxin administration (measured at 0, 0.5, 
1, 1.5, 2, 3, 4 and 6 hours post-endotoxin) compared between each aspirin regimen (control, 20 mg 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
49
BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests.
9. AUC of the graph of circulating leukocyte count over time following endotoxin administration 
(measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 hours post-endotoxin) compared between each aspirin 
regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each 
corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 
mg BD) by paired t-tests.
10. AUC of the graph of bleeding time following endotoxin administration (measured at 0 and 3 hours 
post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 mg 
OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 
300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests.
11. AUC of the graphs of platelet aggregation to arachidonic acid (0.3 and 1 mmol/L), collagen (4 and 
16 g/ml) and ADP (20 mol/L) over time following endotoxin administration (measured at 0 and 3
hours post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 
mg OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg 
OD, 300 mg OD, each with ticagrelor 90 mg BD) by paired t-tests.
And to model the effects of all variables (considering parallel groups):
12. Plasma TNF-
over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 
6 hours post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 
300 mg OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 
mg OD, 300 mg OD, each with ticagrelor 90 mg BD) ) with a mixed three-way ANOVA with aspirin-
dose and presence or absence of ticagrelor as between-subjects factors and timepoint as a within-
subject factor.
13. Plasma IL-6 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 
mg OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg 
OD, 300 mg OD, each with ticagrelor 90 mg BD) with a mixed three-way ANOVA with aspirin-dose 
and presence or absence of ticagrelor as between-subjects factors and timepoint as a within-
subject factor.
14. Change in serum CRP over time following endotoxin administration (measured at 0 and 6 hours 
post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 mg 
OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 
300 mg OD, each with ticagrelor 90 mg BD) with a mixed three-way ANOVA with aspirin-dose and 
presence or absence of ticagrelor as between-subjects factors and timepoint as a within-subject 
factor.
15. Serum TXB2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 
mg OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg 
OD, 300 mg OD, each with ticagrelor 90 mg BD) ) with a mixed three-way ANOVA with aspirin-dose 
and presence or absence of ticagrelor as between-subjects factors and timepoint as a within-
subject factor.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
50
16. Urinary PGI-M (measured at 0, 2, 4 and 6 hours, each adjusted for [divided by] urinary creatinine) 
over time following endotoxin administration compared between each aspirin regimen (control, 20 
mg BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) with a mixed three-way 
ANOVA with aspirin-dose and presence or absence of ticagrelor as between-subjects factors and 
timepoint as a within-subject factor.
17. Serum PGE2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 
mg OD, each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg 
OD, 300 mg OD, each with ticagrelor 90 mg BD) with a mixed three-way ANOVA with aspirin-dose 
and presence or absence of ticagrelor as between-subjects factors and timepoint as a within-
subject factor.
18. Circulating leukocyte count over time following endotoxin administration (measured at 0, 0.5, 1, 
1.5, 2, 3, 4 and 6 hours post-endotoxin) compared between each aspirin regimen (control, 20 mg 
BD, 75 mg OD, 300 mg OD, each without ticagrelor) and each corresponding DAPT regimen 
(control, 20 mg BD, 75 mg OD, 300 mg OD, each with ticagrelor 90 mg BD) ) with a mixed three-way 
ANOVA with aspirin-dose and presence or absence of ticagrelor as between-subjects factors and 
timepoint as a within-subject factor.
19. Bleeding time following endotoxin administration (measured at 0 and 3 hours post-endotoxin) 
compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each without 
ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, each 
with ticagrelor 90 mg BD) with a mixed three-way ANOVA with aspirin-dose and presence or 
absence of ticagrelor as between-subjects factors and timepoint as a within-subject factor.
20. Platelet aggregation to arachidonic acid (0.3 and 1 mmol/L), collagen (4 and 16 g/ml) and ADP 
(20 
mol/L) over time following endotoxin administration (measured at 0 and 3 hours post-
endotoxin) compared between each aspirin regimen (control, 20 mg BD, 75 mg OD, 300 mg OD, 
each without ticagrelor) and each corresponding DAPT regimen (control, 20 mg BD, 75 mg OD, 300 
mg OD, each with ticagrelor 90 mg BD) with a mixed three-way ANOVA with aspirin-dose and 
presence or absence of ticagrelor as between-subjects factors and timepoint as a within-subject 
factor.
For all endpoints (primary and secondary) tested with ANOVA, pairwise comparisons with Bonferroni 
correction will be used to explore relationships further where the prespecified test suggests significant 
differences between the groups (p<0.05).
10.4
Subgroup analyses
No pre-specified subgroup analyses are planned.
10.5
Adjusted analysis
If variables are found to be of skewed distribution, logarithmic transformation will be performed.
10.6
Interim analysis and criteria for the premature termination of the trial

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
51
After discussion between the investigators and the Sponsor, face-to-face participant study visits were 
halted on 17th March 2020 due to the unprecedented circumstances arising during the coronavirus 
disease 2019 (Covid-19) pandemic. It was considered by the investigators that the results of the study may 
have important implications for the management of cardiovascular disease patients who contracted Covid-
19 in terms of optimal antiplatelet medication, in view of the morbidity and mortality in Covid-19 associated 
with dysregulated inflammatory response. It was noted that the recruitment and study activities to date had 
reached the level that had originally been planned for the study, as per the application funded by the British 
Heart Foundation. Given the uncertainty regarding restarting the trial, the need to preserve healthcare 
resources and to limit face-to-face encounters, the investigators and Sponsor decided that an interim 
analysis should be performed. 
Sample size for interim analysis
Data from all participants who have completed visit 3 (first endotoxin day) will be included in the interim 
analysis. This is expected to include 46 participants who have completed visit 3, 37 of whom have 
completed the whole study giving a total of 83 endotoxin day visits, anticipated to be split into 
approximately equal numbers receiving each of the eight study treatment regimens.
Statistical procedure for interim analysis
The primary endpoint of the interim analysis will be plasma TNF-
measured at 2 hours after endotoxin 
administration assessed between aspirin treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg 
OD, without ticagrelor) using one-way ANOVA.
Should this demonstrate a significant difference between the groups (p<0.05), the following pre-specified 
pairwise comparisons will be performed, in hierarchical fashion:
300 mg OD vs. no aspirin
then, if significant (p<0.05)
75 mg OD vs. no aspirin
then, if significant (p<0.05)
300 mg OD vs 20 mg BD
then, if significant (p<0.05)
75 mg OD vs 20 mg BD
then, if significant (p<0.05)

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
52
20 mg BD vs no aspirin
Other analyses will be exploratory.
Criteria for premature termination
The trial shall be prematurely terminated if the primary interim analysis demonstrates a statistically 
significant (p<0.05) effect of aspirin dosing on plasma TNF-
two hours after endotoxin injection.
Additionally, continuing the study will be considered futile, and therefore discontinued, if the lower limit of 
the 95 % confidence interval of the mean change in TNF-
at 2 hours after endotoxin from the  300 mg 
aspirin OD group to no aspirin group is less than (more negative than) -2000 pg/mL.
Should neither of these criteria be met, the investigators will meet to review the results of the interim 
analysis and, in discussion with the Sponsor, decide on whether to continue the study, taking into account 
feasibility, including consideration of local and national restrictions.
Secondary analyses to be carried out in the event of premature termination
If the trial is prematurely terminated, data concerning other endpoints will then be analysed as follows, 
using methods taking best into account the cases of participant data missing from one period:
1. Plasma TNF-
over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 
6 hours post-endotoxin) assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD 
vs. 300 mg OD, with or without ticagrelor) using a mixed effect linear model with patient as a 
random effect, aspirin dose, presence or absence of ticagrelor treatment and treatment period (first 
or second) as fixed effects, and baseline value as covariate.
2. Plasma IL-6 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, with or without ticagrelor) using a mixed effect linear model with patient as a random 
effect, aspirin dose, presence or absence of ticagrelor treatment and treatment period (first or 
second) as fixed effects, and baseline value as covariate.
3. Change in serum CRP over time following endotoxin administration (measured at 0 and 6 hours 
post-endotoxin) assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 
mg OD, with or without ticagrelor) using a mixed effect linear model with patient as a random effect, 
aspirin dose, presence or absence of ticagrelor treatment and treatment period (first or second) as 
fixed effects, and baseline value as covariate.
4. Serum TXB2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, with or without ticagrelor) using a mixed effect linear model with patient as a random 
effect, aspirin dose, presence or absence of ticagrelor treatment and treatment period (first or 
second) as fixed effects, and baseline value as covariate.
5. Urinary PGI-M (measured at 0, 2, 4 and 6 hours, each adjusted for [divided by] urinary creatinine) 
over time following endotoxin administration, assessed between treatment groups (control vs. 20 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
53
mg BD vs. 75 mg OD vs. 300 mg OD, with or without ticagrelor) using a mixed effect linear model 
with patient as a random effect, aspirin dose, presence or absence of ticagrelor treatment and 
treatment period (first or second) as fixed effects, and baseline value as covariate.
6. Serum PGE2 over time following endotoxin administration (measured at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 
hours post-endotoxin) assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 
300 mg OD, with or without ticagrelor) using a mixed effect linear model with patient as a random 
effect, aspirin dose, presence or absence of ticagrelor treatment and treatment period (first or 
second) as fixed effects, and baseline value as covariate.
7. Circulating leukocyte count over time following endotoxin administration (measured at 0, 0.5, 1, 
1.5, 2, 3, 4 and 6 hours post-endotoxin) assessed between treatment groups (control vs. 20 mg BD 
vs. 75 mg OD vs. 300 mg OD, with or without ticagrelor) using a mixed effect linear model with 
patient as a random effect, aspirin dose, presence or absence of ticagrelor treatment and 
treatment period (first or second) as fixed effects, and baseline values as covariate.
8. Bleeding time following endotoxin administration (measured at 0 and 3 hours post-endotoxin) 
assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg OD, with or 
without ticagrelor) using a mixed effect linear model with patient as a random effect, aspirin dose, 
presence or absence of ticagrelor treatment and treatment period (first or second) as fixed effects,
and baseline values as covariate.
9. Platelet aggregation to arachidonic acid (0.3 and 1 mmol/L), collagen (4 and 16 g/ml) and ADP 
(20 
mol/L) over time following endotoxin administration (measured at 0 and 6 hours post-
endotoxin) assessed between treatment groups (control vs. 20 mg BD vs. 75 mg OD vs. 300 mg 
OD, with or without ticagrelor) using a mixed effect linear model with patient as a random effect, 
aspirin dose and presence or absence of ticagrelor treatment and treatment period as fixed effects, 
and baseline values as covariate.
10.7 Participant population
The pharmacodynamic analysis set will include all participants who achieve at least 80% 
compliance with study medication during each of the two periods and who complete both 
endotoxin injection days.
The safety analysis set (for the purposes of adverse event reporting etc.) will include any 
participant randomised into the trial that received at least one dose of trial drug or one dose of 
intravenous endotoxin.
10.8
Procedure(s) to account for missing or spurious data
relevant section of the CRF
Where analysis is performed using ANOVA, missing data will be estimated by multiple imputation 
using the IBM SPSS software package. Sensitivity analyses will be carried out to report the
robustness of this approach. 

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
54
10.9
Other statistical considerations.
Not applicable to this study
10.10 Economic evaluation
Not applicable to this study.
11
DATA MANAGEMENT
11.1
Data collection tools and source document identification
Source data will be recorded on the study-specific paper case report form (CRF). Source data will feed from 
the CRF to the trial master database..
A paper trial master file will be kept within the Cardiovascular Research Unit at the University of Sheffield, 
maintained by the Research Co-ordinator.
11.2
Data handling and record keeping
The investigators will maintain SOPs for the use of the CRF and database, maintain an audit trail of data 
changes ensuring that there is no deletion of entered data, maintain a security system to protect against 
unauthorized access, maintain a list of the individuals authorized to make data changes, maintain 
adequate backup of the data, and archiving of any source data (i.e. hard copy and electronic). This will 
include a clear record of data changes if transformed during processing. The investigators will use an 
unambiguous participant identification code that allows identification of all the data reported for each 
participant. All electronic data will be kept securely stored on University of Sheffield systems, or external 
handlers contracted by the University of Sheffield for this purpose. The Sponsor will be responsible for 
ensuring compliance with the requirements outlined above. 
11.3
Access to Data
Direct access will be granted to authorised representatives from the Sponsor, host institution and the 
regulatory authorities to permit trial-related monitoring, audits and inspections in line with participant 
consent.
11.4 Archiving
Archiving will be authorised by the Sponsor following submission of the end of trial report. The sponsor will 
archive all study documents through existing SOPs and external contracts for a minimum of 15 years after 
the end of the study, as per local protocols. Destruction of essential documents will require authorisation 
from the Sponsor. The trial database will be kept by the investigators for at least 15 years, at which point 
storage arrangements will be reviewed, in electronic format on University of Sheffield file storage systems.
This will be stated in the PIS.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
55
12
MONITORING, AUDIT & INSPECTION
A Trial Monitoring Plan will be developed and agreed by the Trial Management Group (TMG) and 
CI based on the trial risk assessment, which may include on-site monitoring. This will be 
dependent on a documented risk assessment of the trial by the Sponsor.
It is anticipated that monitoring audits will take place after the first participant first visit and last 
participant last visit.
The monitoring plan will be kept in the trial master file.
The monitoring personnel will be determined by the Sponsor.
The processes reviewed will include participant enrolment, consent, eligibility, and allocation to 
trial groups; adherence to trial interventions and policies to protect participants, including 
reporting of harm and completeness, accuracy, and timeliness of data collection.
Monitoring will be performed through site visit to review original documentation.
The investigators will host the site visits.
13
ETHICAL AND REGULATORY CONSIDERATIONS
13.1
Research Ethics Committee (REC) review and report, and Health Research Authority (HRA) approval
Before the start of the trial, approval will be sought from a REC for the trial protocol, informed 
consent forms and other relevant documents e.g. advertisements and GP information letters.
HRA approval will also be obtained.
Substantial amendments that require review by REC will not be implemented until the REC grants 
a favourable opinion for the amendment and HRA amendment approval is received. 
Amendments may also need to be reviewed and accepted by the MHRA and/or NHS R&D 
departments before they can be implemented in practice at the site.  
All correspondence with the REC, MHRA and HRA will be retained in the Trial Master File.
An annual progress report (APR) will be submitted to the REC within 30 days of the anniversary 
date on which the favourable opinion was given, and annually until the trial is declared ended.
I
The Chief Investigator will notify the REC of the end of the trial.
if the trial is ended prematurely, the Chief Investigator will notify the REC, including the reasons 
for the premature termination.
Within one year after the end of the trial, the Chief Investigator will submit a final report with the 
results, including any publications/abstracts, to the REC.
13.2
Peer review
The study design has been reviewed and approved by multiple independent scientific/medical personnel 
on behalf of the British Heart Foundation, who also reviewed the statistical aspects.
13.3
Public and Patient Involvement

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
56
Members of the Sheffield Cardiovascular Patient Panel have been involved in reviewing the design of the study 
and key participant documents during development of this protocol and associated documentation.
13.4
Regulatory Compliance
The trial will not commence until a Clinical Trial Authorisation (CTA) is obtained from the MHRA
and Favourable REC opinion. 
The protocol and trial conduct will comply with the Medicines for Human Use (Clinical Trials) 
Regulations 2004 and any relevant amendments.
No ionising radiation will be used during this study.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
57
13.5
Protocol compliance
Prospective, planned deviations or waivers to the protocol are not allowed under the UK 
regulations on Clinical Trials and must not be used e.g. it is not acceptable to enrol a participant if 
they do not meet the eligibility criteria or restrictions specified in the trial protocol.
Accidental protocol deviations must be adequately documented on the relevant Sponsor-provided 
forms and reported to the Chief Investigator and Sponsor immediately. 
Deviations from the protocol which are found to frequently recur will require immediate action 
and could potentially be classified as a serious breach, at the discretion of the Sponsor.
13.6
Notification of Serious Breaches to GCP and/or the protocol
(a) the safety or physical or mental integrity of the participants of the trial; or
(b) the scientific value of the trial
The Sponsor will be notified immediately of any case where the above definition applies during the trial 
conduct phase
The Sponsor will notify the licensing authority in writing of any serious breach of
(a) the conditions and principles of GCP in connection with that trial; or 
(b) the protocol relating to that trial, as amended from time to time, within 7 days of becoming 
aware of that breach
13.7
Data protection and patient confidentiality
All investigators and trial site staff must comply with the requirements of the Data Protection Act 2018 with 
regards to the collection, storage, processing and disclosure of personal information and will uphold the 
Personal information will be collected by the investigators, kept secure within a room in the 
Cardiovascular Research Unit that is kept locked and alarmed out-of-hours, and will be
maintained by the staff of the Cardiovascular Research Unit. This will involve:
o
Within the master trial database the creation of coded, depersonalised data whereby the 
will be replaced by the study enrolment number.
o
Secure maintenance of the data and the linking code in separate locations using encrypted 
digital files within password protected folders and storage media on NHS or University of 
Sheffield computing systems.
o
Limiting access to the minimum number of individuals necessary for quality control, audit, 
and analysis.
Monitoring visits will take place at site to avoid any data breaches. Transmission of information 
relating to safety eg. SAE reports will occur by secure NHS email channels. Any sharing of data 
with collaborators will be anonymised.
Study source documents will be kept for a minimum of 15 years as per Sponsor protocols.
The data custodian will be the CI.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
58
13.8
Financial and other competing interests for the chief investigator, PIs at each site and committee
members for the overall trial management
Robert F. Storey: institutional research grants/support from AstraZeneca, and PlaqueTec; consultancy fees 
from Actelion, AstraZeneca, Avacta, Bayer, Bristol Myers Squibb, Novartis, PlaqueTec, Haemonetics and 
Thromboserin; speaker fees from AstraZeneca, and Bayer. Nominated by University of Sheffield as inventor 
on patent application related to concepts explored in this study.
William A. E.  Parker: nominated by University of Sheffield as inventor on patent application related to 
concepts explored in this study. 
13.9
Indemnity
The NHS indemnity scheme will apply for harm arising from management of research and research 
conduct. Additionally, The University of Sheffield will provide insurance against liabilities for which it may be 
legally liable and this cover will include any such liabilities arising out of this research project/study.
13.10 Amendments
Any changes to the protocol or informed consent form will be assessed by the Sponsor to determine the 
necessary approvals to be obtained (MHRA, HRA and/or REC). The Sponsor will then approve the amendment 
when the necessary approvals have been granted.
13.11 Post-trial care
Whilst not able to offer study-
investigators will ensure that participants are signposted to the relevant NHS services should these be 
needed.
13.12 Access to the final trial dataset
The investigators identified on the delegation log will have access to the full dataset, at the 
discretion of the CI.
14
DISSEMINIATION POLICY
14.1
Dissemination policy
The data arising from the trial will be owned by University of Sheffield.
On completion of the trial, the data will be analysed and tabulated and a Final Trial Report 
prepared.
The trial report will be accessible via the EudraCT system, in a medical journal and on request 
from the CI.
The CI will retain the sole right to publish any of the trial data.
The British Heart Foundation and the Sheffield NIHR Clinical Research Facility will be 
acknowledged within any publications but will not have review/publication rights of the data from 
the trial.
Participants will be able to obtain a short summary of the results by letter on request.
It will not be possible for the participant to specifically request results from the investigators.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
59
The disclosure of the trial protocol, full trial report, anonymised participant level dataset, and 
statistical code for generating the results may be made available to interested parties at the 
discretion of the CI, not before 1 year after study completion.
14.2
Authorship eligibility guidelines and any intended use of professional writers
Those individuals who contribute significantly to the development, conduct and writing up of the study will be 
offered authorship of the final trial report. Individually-named authors will meet the authorship criteria of The 
International Committee of Medical Journal Editors. No professional medical writers will be involved in the 
preparation of reports or publications.
15
REFERENCES
1.
Jackson, S.P., Arterial thrombosis--insidious, unpredictable and deadly. Nat Med, 2011. 
17(11): p. 1423-36.
2.
Luc, G., et al., C-reactive protein, interleukin-6, and fibrinogen as predictors of coronary 
heart disease: the PRIME Study. Arterioscler Thromb Vasc Biol, 2003. 23(7): p. 1255-61.
3.
Roffi, M., et al., 2015 ESC Guidelines for the management of acute coronary syndromes in 
patients presenting without persistent ST-segment elevation. Eur Heart J, 2015. 37: p. 267-315.
4.
Steg, P.G., et al., ESC Guidelines for the management of acute myocardial infarction in 
patients presenting with ST-segment elevation. Eur Heart J, 2012. 33(20): p. 2569-619.
5.
Wallentin, L., et al., Ticagrelor versus clopidogrel in patients with acute coronary syndromes.
N Engl J Med, 2009. 361(11): p. 1045-57.
6.
Ridker, P.M., et al., Antiinflammatory Therapy with Canakinumab for Atherosclerotic 
Disease. N Engl J Med, 2017. 377(12): p. 1119-1131.
7.
Thomas, M.R., et al., Platelet P2Y12 Inhibitors Reduce Systemic Inflammation and Its 
Prothrombotic Effects in an Experimental Human Model. Arterioscler Thromb Vasc Biol, 2015. 
35(12): p. 2562-70.
8.
Iqbal, A., et al., Hypoglycaemia increases platelet reactivity and promotes formation of 
proatherogenic
platelet-leucocyte aggregates. Diabetologia, 2017. 60: p. S532.
9.
Patrono, C., et al., Antiplatelet Agents for the Treatment and Prevention of Coronary 
Atherothrombosis. J Am Coll Cardiol, 2017. 70(14): p. 1760-1776.
10.
Patrono, C., Aspirin as an antiplatelet drug. N Engl J Med, 1994. 330(18): p. 1287-94.
11.
investigators, I.-. Randomised trial of intravenous streptokinase, oral aspirin, both, or 
neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. ISIS-2 (Second 
International Study of Infarct Survival) Collaborative Group. Lancet, 1988. 2(8607): p. 349-60.
12.
Belch, J., et al., The prevention of progression of arterial disease and diabetes (POPADAD) 
trial: factorial randomised placebo controlled trial of aspirin and antioxidants in patients with 
diabetes and asymptomatic peripheral arterial disease. BMJ, 2008. 337.
13.
Pignone, M., et al., Aspirin for Primary Prevention of Cardiovascular Events in People With 
Diabetes: A position statement of the American Diabetes Association, a scientific statement of the 
American Heart Association, and an expert consensus document of the American College of 
Cardiology Foundation. Diabetes Care, 2010. 33(6): p. 1395-402.
14.
Ogawa, H., et al., Low-dose aspirin for primary prevention of atherosclerotic events in 
patients with type 2 diabetes: a randomized controlled trial. Jama, 2008. 300(18): p. 2134-41.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
60
15.
Ikeda, Y., et al., Low-dose aspirin for primary prevention of cardiovascular events in 
Japanese patients 60 years or older with atherosclerotic risk factors: a randomized clinical trial.
Jama, 2014. 312(23): p. 2510-20.
16.
Gaziano, J.M., et al., Use of aspirin to reduce risk of initial vascular events in patients at 
moderate risk of cardiovascular disease (ARRIVE): a randomised, double-blind, placebo-controlled 
trial. Lancet, 2018.
17.
Paul-Clark, M.J., et al., 15-epi-lipoxin A4-mediated induction of nitric oxide explains how 
aspirin inhibits acute inflammation. J Exp Med, 2004. 200(1): p. 69-78.
18.
Kiers, D., et al., A randomised trial on the effect of anti-platelet therapy on the systemic 
inflammatory response in human endotoxaemia. Thromb Haemost, 2017. 117(9): p. 1798-1807.
19.
McAdam, B.F., et al., Effect of regulated expression of human cyclooxygenase isoforms on 
eicosanoid and isoeicosanoid production in inflammation. J Clin Invest, 2000. 105(10): p. 1473-82.
20.
Birrell, M.A., et al., Anti-inflammatory effects of PGE2 in the lung: role of the EP4 receptor 
subtype. Thorax, 2015. 70(8): p. 740-7.
21.
Na, Y.R., et al., Endogenous prostaglandin E2 potentiates anti-inflammatory phenotype of 
macrophage through the CREB-C/EBP-beta cascade. Eur J Immunol, 2015. 45(9): p. 2661-71.
22.
Chow, J.C., et al., Toll-like receptor-4 mediates lipopolysaccharide-induced signal 
transduction. J Biol Chem, 1999. 274(16): p. 10689-92.
23.
Brasier, A.R., The nuclear factor-kappaB-interleukin-6 signalling pathway mediating 
vascular inflammation. Cardiovasc Res, 2010. 86(2): p. 211-8.
24.
Degraaf, A.J., et al., Prostaglandin E2 reduces Toll-like receptor 4 expression in alveolar 
macrophages by inhibition of translation. Am J Respir Cell Mol Biol, 2014. 51(2): p. 242-50.
25.
Lee, K.M. and S.Y. Seong, Partial role of TLR4 as a receptor responding to damage-
associated molecular pattern. Immunol Lett, 2009. 125(1): p. 31-9.
26.
Smiley, S.T., J.A. King, and W.W. Hancock, Fibrinogen stimulates macrophage chemokine 
secretion through toll-like receptor 4. J Immunol, 2001. 167(5): p. 2887-94.
27.
Smeeth, L., et al., Risk of myocardial infarction and stroke after acute infection or 
vaccination. N Engl J Med, 2004. 351(25): p. 2611-8.
28.
Morris, T., et al., Effects of low-dose aspirin on acute inflammatory responses in humans. J 
Immunol, 2009. 183(3): p. 2089-96.
29.
Layne, K., et al., Anti-platelet drugs attenuate the expansion of circulating CD14highCD16+ 
monocytes under pro-inflammatory conditions. Cardiovasc Res, 2016. 111(1): p. 26-33.
30.
Serezani, C.H., et al., Leukotriene B4 amplifies NF-kappaB activation in mouse 
macrophages by reducing SOCS1 inhibition of MyD88 expression. J Clin Invest, 2011. 121(2): p. 
671-82.
31.
Vranckx, P., et al., Ticagrelor plus aspirin for 1 month, followed by ticagrelor monotherapy 
for 23 months vs aspirin plus clopidogrel or ticagrelor for 12 months, followed by aspirin 
monotherapy for 12 months after implantation of a drug-eluting stent: a multicentre, open-label, 
randomised superiority trial. Lancet, 2018.
32.
Baber, U., et al., Ticagrelor with aspirin or alone in high-risk patients after coronary 
intervention: Rationale and design of the TWILIGHT study. Am Heart J, 2016. 182: p. 125-134.
33.
Teng, R., J. Maya, and K. Butler, Evaluation of the pharmacokinetics and 
pharmacodynamics of ticagrelor co-administered with aspirin in healthy volunteers. Platelets, 2013. 
24(8): p. 615-624.
34.
Scavone, M., et al., Inhibition of the platelet P2Y12 receptor for adenosine diphosphate 
does not impair the capacity of platelet to synthesize thromboxane A2. Eur Heart J, 2015.
35.
Mahaffey, K.W., et al., Ticagrelor Compared With Clopidogrel by Geographic Region in the 
Platelet Inhibition and Patient Outcomes (PLATO) Trial. Circulation, 2011. 124(5): p. 544-554.

     STH20370 WILLOW TREE Clinical Study Protocol v5.0, 26 Jul 2021
61
36.
Parker, W., et al., Ultra-low-dose twice-daily aspirin improves hemostasis and maintains 
platelet inhibition in acute coronary syndrome patients receiving ticagrelor. Journal of the American 
College of Cardiology, 2018. 71(11 Supplement): p. A30.
37.
Brilique 90 mg film coated tablets - Summary of Product Characteristics (SmPC) - (eMC). 
2018 
 
[cited 
2018 
14 
May 
2018]; 
Available 
from: 
https://www.medicines.org.uk/emc/product/5767/smpc.
38.
Suffredini, A.F., H.D. Hochstein, and F.G. McMahon, Dose-related inflammatory effects of 
intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 
endotoxin. J Infect Dis, 1999. 179(5): p. 1278-82.
39.
Rittig, N., et al., Hormone and Cytokine Responses to Repeated Endotoxin Exposures-No 
Evidence of Endotoxin Tolerance After 5 Weeks in Humans. Shock, 2015. 44(1): p. 32-5.