Clinical trial NCT02931045
Conditions: Myocardial Infarction
Keywords: extracellular vesicles inflammation thrombosis antiplatelet drugs
Sponsor: Medical University of Warsaw
Start date: 2017-12-30; Completion date: 2019-12-30
Enrollment: 60 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Platelet activation and aggregation leads to myocardial infarction. Platelet P2Y12 receptors are essential for platelet activation. Antagonists against the P2Y12 receptor, which are established in secondary prevention of myocardial infarction, have unexplained anti-inflammatory effects. A novel P2Y12 receptor antagonist ticagrelor reduced infection-related mortality compared to clopidogrel, previous standard treatment for patients with myocardial infarction. Activated platelets release pro-inflammatory and procoagulant platelet extracellular vesicles. The investigators assume that decrease in infection-related mortality in patients treated with ticagrelor may be explained by greater inhibition of the release of platelet vesicles by ticagrelor, compared to clopidogrel. This study is expected to identify an additional mechanism of action of ticagrelor, which might contribute to the observed clinical benefits in patients treated with ticagrelor.

Detailed description:

Arms: arm 1: Ticagrelor: oral, 180 mg once (loading dose) followed by 90 mg twice daily (maintenance dose) arm 2: Clopidogrel: oral, 300 mg or 600 mg once (loading dose) followed by 75 mg once daily (maintenance dose)
Interventions: intervention 1: Ticagrelor intervention 2: Clopidogrel
Intervention descriptions: intervention 1: Comparison of ticagrelor with another antiplatelet drug (clopidogrel) intervention 2: Comparison of clopidogrel with another antiplatelet drug (ticagrelor)

Protocol documents:
Prot_SAP_000.pdf: 
Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
Clinical Study Protocol  
Drug Substance 
ticagrelor 
Study Code 
KB/112/2016 
Edition Number 
1 
Date 
21.12.2016 
Antiplatelet therapy effect on platelet extracellular vesicles in acute 
myocardial infarction (AFFECT EV) 
Sponsor: Medical University of Warsaw 
The following Amendment(s) and Administrative Changes have been made to this protocol since the date of 
preparation: 
Amendment No. 
Date of Amendment 
Local Amendment No: 
Date of Local Amendment 
Administrative 
Change No. 
Date of Administrative 
Change 
Local Administrative 
Change No. 
Date of Local 
Administrative Change 
 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
PROTOCOL SYNOPSIS 
 
Antiplatelet therapy effect on platelet extracellular vesicles in acute 
myocardial infarction (AFFECT EV) 
 
 
Principal Investigator 
Aleksandra Gąsecka, MD 
Study site(s) and number of subjects planned 
1st Chair and Department of Cardiology, Medical University of Warsaw, 60 subjects 
Study period 
 
Phase of development 
Estimated date of first subject enrolled 
28.12.2016 
4 
 
Estimated date of last subject completed 
28.12.2017 
4 
 
Study design 
This is a prospective, single-centre, randomised, open-label, parallel group design, phase 4, 
proof-of-concept study conducted at the 1st Chair and Department of Cardiology, Medical 
University of Warsaw, Poland. The analytical part will be conducted in the Vesicle 
Observation Centre, Academic Medical Centre, University of Amsterdam, The Netherlands. 
The duration of this study is expected to be 12 months. 
 
Objectives 
Primary Objective: 
Outcome Measure: 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
To compare the effects of ticagrelor and 
clopidogrel on the concentration of PEVs 
in patients with acute myocardial 
infarction.  
Concentration of PEV exposing P-selectin and 
PS (flow cytometry). 
 
Secondary Objective: 
Outcome Measure : 
To compare the effects of ticagrelor and 
clopidogrel on the concentration of EVs 
exposing fibrinogen, exposing 
phosphatidylserine, EVs from endothelial 
cells, EVs from leukocytes. 
Concentration of EV exposing fibrinogen, 
exposing phosphatidylserine, EVs from 
endothelial cells, EVs from leukocytes. 
 
Safety Objective: 
Outcome Measure : 
To evaluate the safety and tolerability of 
ticagrelor in relation to clopidogrel. To be 
reported descriptively. 
- Vital signs 
- Abnormal blood tests values (haematology and 
clinical chemistry) 
- Abnormal values in urinalysis 
- Abnormalities in 12-lead ECG 
- AEs/ SAEs 
 
 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
Target subject population 
Subjects with the first ST-elevation acute myocardial infarction (STEMI) or non-STEMI 
(NSTEMI) who underwent percutaneous coronary intervention (PCI) with stent implantation 
and received a loading dose of clopidogrel (600 mg) prior to PCI.  
Duration of treatment 
6 months 
Investigational product, dosage and mode of administration 
All drugs, including the investigational product ticagrelor, will be administered orally. All 
subjects will be administered a loading dose of clopidogrel (600 mg) prior to PCI. Subjects 
randomised to ticagrelor will receive a loading dose of ticagrelor (180 mg), followed by a 
maintenance dose (90 mg twice daily). Subjects randomised to clopidogrel will continue the 
treatment with a maintenance dose of clopidogrel (75 mg once daily). According to the 
guidelines of European Society of Cardiology, double antiplatelet therapy with acetylsalicylic 
acid (ASA) and a P2Y12 receptor antagonist will be continued for 12 months. The study 
treatment will last for 6 months until the last visit at the end of the treatment (LSLV). After 
termination of the study patients will be prescribed double antiplatelet therapy for the 
remaining 6 months. Ticagrelor will be recommended over clopidogrel. Subjects who choose 
to switch to clopidogrel due to financial constraints will be prescribed clopidogrel. 
Statistical methods 
Categorical variables will be presented as number (%); continuous variables will be presented 
as mean and standard deviation (SD) or median with inter-quartile range. Shapiro–Wilk test 
will be used to test for non-Gaussian distribution of continuous variables. Student's two-sided 
t-test or ANOVA-repeated measures will be used for normally distributed values. Mann–
Whitney U test or Friedman test will be used for variables without normal distribution. A p-
value below 0.05 will be considered significant. 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
TABLE OF CONTENTS 
PAGE 
 
TITLE PAGE ............................................................................................................................ 1 
PROTOCOL SYNOPSIS ......................................................................................................... 2 
TABLE OF CONTENTS ......................................................................................................... 5 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ........................................... 10 
1. 
INTRODUCTION .................................................................................................................. 11 
1.1 
Background and rationale for conducting this study .............................................................. 11 
1.2 
Rationale for study design, doses and control groups ............................................................ 12 
1.3 
Study Design .......................................................................................................................... 13 
2. 
STUDY OBJECTIVES .......................................................................................................... 14 
2.1 
Primary objective .................................................................................................................... 14 
2.2 
Secondary objectives .............................................................................................................. 14 
2.3 
Safety objectives ..................................................................................................................... 14 
2.4 
Exploratory objectives ............................................................................................................ 15 
3. 
SUBJECT SELECTION, ENROLMENT, RANDOMISATION, RESTRICTIONS, 
DISCONTINUATION AND WITHDRAWAL ..................................................................... 15 
3.1 
Inclusion criteria ..................................................................................................................... 15 
3.2 
Exclusion criteria .................................................................................................................... 15 
3.3 
Subject enrolment and randomization .................................................................................... 16 
3.4 
Procedures for handling incorrectly enrolled or randomized subjects ................................... 16 
3.5 
Methods for assigning treatment groups ................................................................................ 16 
3.6 
Methods for ensuring blinding ............................................................................................... 17 
3.7 
Methods for unblinding .......................................................................................................... 17 
3.8 
Restrictions ............................................................................................................................. 17 
3.9 
Discontinuation of investigational product............................................................................. 17 
3.9.1 
Procedures for discontinuation of a subject from investigational product ............................. 17 
3.10 
Criteria for withdrawal ........................................................................................................... 17 
3.10.1 
Screen failures ........................................................................................................................ 17 
3.10.2 
Withdrawal of the informed consent ...................................................................................... 18 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
3.11 
Discontinuation of the study ................................................................................................... 18 
4. 
STUDY PLAN AND TIMING OF PROCEDURES ............................................................. 18 
4.1 
Enrolment/screening period .................................................................................................... 23 
4.2 
Treatment period..................................................................................................................... 23 
4.3 
Follow-up period .................................................................................................................... 23 
5. 
STUDY ASSESSMENTS ...................................................................................................... 23 
5.1 
Efficacy assessments .............................................................................................................. 23 
5.2 
Safety assessments .................................................................................................................. 23 
5.2.1 
Laboratory safety assessments ............................................................................................... 23 
5.2.2 
Physical examination .............................................................................................................. 24 
5.2.3 
ECG ........................................................................................................................................ 24 
5.2.3.1 
Resting 12-lead ECG .............................................................................................................. 24 
5.2.4 
Vital signs ............................................................................................................................... 25 
5.2.4.1 
Pulse and blood pressure ........................................................................................................ 25 
5.2.4.2 
Body temperature ................................................................................................................... 25 
5.2.5 
Other safety assessments ........................................................................................................ 25 
5.3 
Other assessments ................................................................................................................... 25 
5.3.1 
Patient reported outcomes ...................................................................................................... 25 
5.3.1.1 
<<Name of PRO method or questionnaire>> ......................................................................... 26 
5.4 
Pharmacokinetics .................................................................................................................... 26 
5.4.1 
Collection of samples ............................................................. Error! Bookmark not defined. 
5.4.2 
Determination of drug concentration ...................................... Error! Bookmark not defined. 
5.4.3 
Storage and destruction of pharmacokinetic samples............. Error! Bookmark not defined. 
5.5 
Pharmacodynamics ................................................................................................................. 26 
5.5.1 
Collection of samples ............................................................. Error! Bookmark not defined. 
5.5.2 
Storage, re-use and destruction of pharmacodynamic samplesError! Bookmark not defined. 
5.6 
Pharmacogenetics ................................................................................................................... 26 
5.6.1 
Collection of pharmacogenetic samples ................................. Error! Bookmark not defined. 
5.6.2 
Storage, re-use and destruction of pharmacogenetic samples Error! Bookmark not defined. 
5.7 
Biomarker analysis ................................................................................................................. 26 
5.7.1 
Storage, re-use and destruction of biological samples ........................................................... 27 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
5.7.2 
Labelling and shipment of biological samples ....................................................................... 27 
5.7.3 
Chain of custody of biological samples .................................................................................. 27 
5.7.4 
Withdrawal of Informed Consent for donated biological samples ......................................... 27 
6. 
SAFETY REPORTING AND MEDICAL MANAGEMENT ............................................... 28 
6.1 
Definition of adverse events ................................................................................................... 28 
6.2 
Recording of adverse events ................................................................................................... 28 
6.2.1 
Time period for collection of adverse events ......................................................................... 28 
6.2.2 
Follow-up of unresolved adverse events ................................................................................ 29 
6.2.3 
Variables ................................................................................................................................. 29 
6.2.4 
Causality assessment .............................................................................................................. 30 
6.2.5 
Disease progression ................................................................................................................ 30 
6.3 
Reporting of serious adverse events ....................................................................................... 31 
6.4 
Overdose ................................................................................................................................. 31 
6.5 
Pregnancy ............................................................................................................................... 31 
6.5.1 
Maternal exposure .................................................................................................................. 31 
6.5.2 
Paternal exposure .................................................................................................................... 31 
6.6 
Management of toxicities <<Dose Reductions>> .................................................................. 31 
6.7 
Study governance and oversight ............................................................................................. 32 
6.7.1 
Steering Committee ................................................................................................................ 32 
6.7.2 
Data Monitoring Committee ................................................................................................... 32 
6.7.3 
Scientific Advisory Committee .............................................................................................. 32 
7. 
INVESTIGATIONAL PRODUCT AND OTHER TREATMENTS ..................................... 32 
7.1 
Identity of investigational product(s) ..................................................................................... 32 
7.2 
Dose and treatment regimens ................................................................................................. 33 
7.3 
Labelling ................................................................................................................................. 33 
7.4 
Storage .................................................................................................................................... 33 
7.5 
Compliance ............................................................................................................................. 34 
7.6 
Accountability ........................................................................................................................ 34 
7.7 
Concomitant and other treatments .......................................................................................... 34 
7.7.1 
Other concomitant treatment .................................................................................................. 35 
7.8 
Post Study Access to Study Treatment ................................................................................... 35 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
8. 
STATISTICAL ANALYSES ................................................................................................. 35 
8.1 
Statistical considerations ........................................................................................................ 35 
8.2 
Sample size estimate ............................................................... Error! Bookmark not defined. 
8.3 
Definitions of analysis sets ..................................................................................................... 36 
8.3.1 
Efficacy analysis set ............................................................................................................... 36 
8.3.2 
Safety analysis set ................................................................................................................... 36 
8.3.3 
PK analysis set ........................................................................................................................ 37 
8.3.4 
PRO analysis set ..................................................................................................................... 37 
8.4 
Outcome measures for analyses ............................................. Error! Bookmark not defined. 
8.5 
Methods for statistical analyses .............................................. Error! Bookmark not defined. 
8.5.1 
Analysis of the primary variable(s) ........................................................................................ 37 
8.5.2 
Analysis of the secondary variable(s) ..................................................................................... 37 
8.5.3 
Subgroup analysis (if applicable) ........................................................................................... 38 
8.5.4 
Interim analysis ...................................................................................................................... 38 
8.5.5 
Sensitivity analysis (if applicable) .......................................................................................... 38 
8.5.6 
Exploratory analysis (if applicable)) ...................................................................................... 38 
9. 
STUDY AND DATA MANAGEMENT ............................................................................... 38 
9.1 
Training of study site personnel ............................................................................................. 38 
9.2 
Monitoring of the study .......................................................................................................... 38 
9.2.1 
Source data ............................................................................................................................. 38 
9.2.2 
Study agreements .................................................................................................................... 39 
9.2.3 
Archiving of study documents ................................................................................................ 39 
9.3 
Study timetable and end of study ........................................................................................... 39 
9.4 
Data management ................................................................... Error! Bookmark not defined. 
10. 
ETHICAL AND REGULATORY REQUIREMENTS ......................................................... 41 
10.1 
Ethical conduct of the study ................................................................................................... 41 
10.2 
Subject data protection ........................................................................................................... 41 
10.3 
Ethics and regulatory review .................................................................................................. 41 
10.4 
Informed consent .................................................................................................................... 41 
10.5 
Changes to the protocol and informed consent form .............................................................. 42 
10.6 
Audits and inspections ............................................................................................................ 42 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
11. 
LIST OF REFERENCES ....................................................................................................... 42 
 
LIST OF TABLES 
< 
Table x 
Laboratory Safety Variables ..................................................................... 23 
LIST OF FIGURES 
Figure 1 
Study flow chart ....................................................................................... 13 
 
LIST OF APPENDICES 
Appendix A 
Signatures 
Appendix B 
Bioethical Committee Approval and Amendment (in Polish) 
Appendix C 
Patient Information Sheet and Consent Form (in Polish) 
Appendix D 
Protocol for blood collection and handling 
Appendix E  
Conference abstract and sample size calculation 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
The following abbreviations and special terms are used in this study Clinical Study Protocol. 
Abbreviation or 
special term 
Explanation 
AE 
Adverse event 
CRF 
Case Report Form (electronic/paper) 
CSA 
Clinical Study Agreement 
CSR 
Clinical Study Report 
CTCAE 
Common Terminology Criteria for Adverse Event 
DAE 
Discontinuation of Investigational Product due to Adverse Event 
DNA 
Deoxyribonucleic acid  
EC 
Ethics Committee, synonymous to Institutional Review Board (IRB) and 
Independent Ethics Committee (IEC) 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonisation 
IP 
Investigational Product  
IVRS 
Interactive Voice Response System 
IWRS 
Interactive Web Response System 
LSLV 
Last Subject Last Visit 
OAE 
Other Significant Adverse Event 
PGx 
Pharmacogenetic research 
PI 
Principal Investigator  
SAE 
Serious adverse event  
WBDC 
Web Based Data Capture 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
1. 
INTRODUCTION 
1.1 
Background and rationale for conducting this study 
Platelet activation and aggregation in response to atherosclerotic plaque rupture is a 
key event in the pathogenesis of acute myocardial infarction (AMI) [1]. Platelet P2Y12 
receptors for adenosine diphosphate are essential for platelet activation [2], and from this 
reason antagonists against the P2Y12 receptor are established in secondary prevention of 
cardiovascular events [3]. Beyond blocking platelet activation, antagonists of the P2Y12 
receptor have hitherto unexplained anti-inflammatory effects [4,5]. Findings from the 
PLATelet inhibition and patient Outcomes (PLATO) study demonstrated that platelet 
inhibition with a novel and potent P2Y12 receptor antagonist ticagrelor reduced mortality 
following pulmonary infections and sepsis compared to clopidogrel, previous standard 
treatment for patients with AMI [5].  
 
Activated platelets release platelet extracellular vesicles (PEV) [6]. PEV differ from 
other biomarkers of inflammation and platelet activation, because (i) PEV are released from 
activated platelets at the early stage of developing arterial thrombosis [7], (ii) PEV expose 
specific platelet antigens and thus can be identified as being platelet-specific [8], (iii) PEV 
participate both in inflammation and in coagulation due to exposure of P-selectin and 
phosphatidylserine (PS) [9,10]. P-selectin initiates binding of platelets and/ or PEV to 
leukocytes which leads to leukocyte activation and secretion of cytokines [9], and (ii) PS and 
other negatively charged phospholipids bind positively charged clotting factors thereby 
propagating thrombin generation [10]. Additionally, PEV serve as pro-inflammatory stimuli in 
acute lung injury [11], which is one of the leading causes of death in sepsis [12]. Because 
identification and characterization of PEV directly in human plasma has recently become 
feasible [13], and because PEV can be measured in biorepositories thereby facilitating 
validation in multicentre clinical trials [14], it is likely that the requirements for PEV to 
become a reliable biomarker can now be fulfilled. 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
1.2 
Rationale for study design, doses and control groups 
Rationale for study design 
Clinical studies suggest that platelet P2Y12 receptor antagonists exert anti-
inflammatory effects [4,5], but the underlying mechanisms have not been clearly defined in 
vivo. This study is expected to identify an additional mechanism of action of the P2Y12 
antagonist ticagrelor (inhibition of PEV release), which might partly explain the lower rate of 
infection-related mortality in patients treated with ticagrelor, compared to clopidogrel [5]. 
Regarding the exponentially growing interest in the field of EV in the last decade [6,13,14], as 
well as the crucial role of PEV in inflammation and thrombosis [9,10], determining the effect 
of ticagrelor on PEV release is an important step in cardiovascular pharmacology research. 
Although the study is preliminary in the design and not powered for mortality, the results are 
expected to pave the road for further studies designed to investigate the association between 
concentrations of PEV and infection-releated mortality in patients treated with ticagrelor, and 
facilitate the development of PEV-based pharmaceuticals with applications in atherosclerosis-
related cardiovascular diseases. 
The randomised and investigator-blinded allocation to the study groups along with 
state-of-the art methods of PEV analysis will account for the reliability of results. The study 
will be conducted in the 1st Chair and Department of Cardiology, Medical University of 
Warsaw, Poland. The analytical part will be conducted in the Vesicle Observation Centre, 
Academic Medical Centre, University of Amsterdam, The Netherlands. The involvement of 
experts with a track record on extracellular vesicles from the Netherlands (Dr. Rienk 
Nieuwland, Prof. Auguste Sturk) will ensure a scientifically sound project and optimize 
dissemination of results.  
Doses 
All drugs will be administered orally, in doses recommended by European Society of 
Cardiology in the Guidelines on Myocardial Revascularization [3]. If not already administered 
prior to hospital admission, patients will receive a loading dose of clopidogrel (600 mg) prior 
to percutaneous coronary intervention (PCI). Patients randomised to ticagrelor will receive a 
loading dose of ticagrelor (180 mg), followed by a maintenance dose (90 mg twice daily). 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition Number 1 
Date 21.12.2016 
Patients randomised to clopidogrel will continue the treatment with a maintenance dose of 
clopidogrel (75 mg once daily).  
Control group 
• Patients randomised to clopidogrel. 
Benefit/risk and ethical assessment 
All study drugs are administered on-label. According to the Guidelines on Myocardial 
Revascularization, both in subjects with STEMI and NSTEMI ticagrelor is recommended over 
clopidogrel, and clopidogrel is recommended when ticagrelor is not available or is 
contraindicated [3]. Because ticagrelor is not reimbursed in Poland, it is not available for the 
majority of Polish patients due to financial constraints. Participation in the study ensures the 
free access to ticagrelor for patients randomized to the study group during the first 6 months 
after AMI, which is the period of the highest risk of recurrent major adverse cardiovascular 
events (MACE) [15,16]. Participation in the study ensures also the free access to clopidogrel 
for patients randomized to the control group during the first 6 months after AMI. All patients 
will undergo an additional cardiovascular investigation at the last visit at 6 months. 
1.3 
Study Design  
Figure 1. Study flow chart. 
 
Blood: Platelet extracellular vesicles, aggregometry 
Abbreviations: AMI – acute myocardial infarction; PCI – percutaneous coronary intervention; 
ASA – acetylsalicylic acid; R – randomization, QD – once daily; BID – twice daily


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
2. 
STUDY OBJECTIVES 
2.1 
Primary objective 
Primary Objective: 
Outcome Measure: 
To compare the effects of ticagrelor and 
clopidogrel on the concentration of PEVs 
in patients with acute myocardial 
infarction.  
Concentration of PEV exposing P-selectin and 
PS (flow cytometry). 
 
2.2 
Secondary objectives 
Secondary Objective: 
Outcome Measure : 
To compare the effects of ticagrelor and 
clopidogrel on the concentration of EVs 
exposing fibrinogen, exposing 
phosphatidylserine, EVs from endothelial 
cells, EVs from leukocytes. 
Concentration of EV exposing fibrinogen, 
exposing phosphatidylserine, EVs from 
endothelial cells, EVs from leukocytes. 
 
2.3 
Safety objectives 
Safety Objective: 
Outcome Measure : 
To evaluate the safety and tolerability of 
ticagrelor in relation to clopidogrel. To be 
reported descriptively. 
- Vital signs 
- Abnormal values in blood tests ( haematology 
and clinical chemistry) 
- Abnormal values or in urinalysis 
- Abnormalities in 12-lead ECG 
- AEs/ SAEs 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
2.4 
Exploratory objectives   
Exploratory Objective: 
Outcome Measure : 
To identify an additional mechanism of 
action of ticagrelor, which may at least in 
part explain the lower rate of infection-
related mortality in patients treated with 
ticagrelor, compared to clopidogrel. 
Concentration of PEV exposing P-selectin and 
phosphatidylserine (PS). 
 
 
3. 
SUBJECT SELECTION, ENROLMENT, RANDOMISATION, 
RESTRICTIONS, DISCONTINUATION AND WITHDRAWAL  
3.1 
Inclusion criteria 
• Age > 18 years 
• Informed consent to participate in the study 
• PCI due to first ST-elevation myocardial infarction (STEMI) or non-STEMI (NSTEMI) 
• Administration of the loading dose of clopidogrel (600 mg) prior to PCI 
3.2 
Exclusion criteria 
• Known coagulopathy 
• Active pathological bleeding 
• Known history of bleeding disorder 
• Suspicion of intracranial haemorrhage 
• Need for oral anticoagulation therapy 
• Administration of GPIIb-IIIa antagonists 
• Cardiogenic schock 
• Severe chronic renal failure (eGFR < 30 mL/min) 
• Severe liver insufficiency 
• Infectious disease 
• Autoimmune disease 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
• Neoplasm 
• Chronic dyspnea 
• Increased risk of bradycardia 
• Known pregnancy, breast-feeding, or intention to become pregnant during the study period 
• Study drug intolerance 
• Co-administration of ticagrelor or clopidogrel with strong CYP3A4 inhibitors 
• Participation in any previous study with ticagrelor or clopidogrel 
3.3 
Subject enrolment and randomization 
Subjects will be enrolled by the PI at the 1st Chair and Department of Cardiology, Medical 
University of Warsaw. Subjects will be randomized by an independent operator at 1st 
Department of Cardiology, Medical University of Warsaw. Randomisation will be carried out 
in 1:1 ratio using block randomization via the mobile application “Randomizer for Clinical 
Trial” (Medsharing, France). 
3.4 
Procedures for handling incorrectly enrolled or randomized subjects 
Subjects incorrectly enrolled or randomized subjects will be withdrawn from the study. These 
subjects: 
• will be apologized, 
• will be administered double antiplatelet therapy with ASA and a P2Y12 antagonist, as well as 
other medications, if required, 
• will be asked to return the study medication, if identified after the study medication has 
already been dispensed, 
• will be monitored in the same way as subjects continuing the study until the last visit at 6 
months. 
The PI will maintain a list of incorrectly enrolled or randomized subjects in the Investigator's 
Study File.  
3.5 
Methods for assigning treatment groups 
Upon successful randomization, the subject will be assigned a randomization number and 
treatment. The subject’s identification data and ID number, and randomization number will be 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
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Date  
listed on the Patient Identification List and Patient Enrolment List, respectively, and stored in 
the Investigator's Study File. 
3.6 
Methods for ensuring blinding 
Not applicable to study subjects. The PI will be blinded to the randomization arm and 
treatment of study subjects. The operator analyzing the samples will be blinded to all subject-
related baseline and clinical data.  
3.7 
Methods for unblinding  
Not applicable for patients. The PI will be unblinded by combining the Patient Identification 
List  including subjects identification data and Patient Enrolment List including data on 
subjects randomization  
3.8 
Restrictions. 
Not stated. 
3.9 
Discontinuation of investigational product  
The investigational product (IP) may be discontinued at any time at the discretion of the PI or 
the study subject. Criteria for discontinuing the investigational product include: 
• Withdrawal of informed consent. 
• Development of exclusion criteria, pregnancy or other safety reasons during the study 
(requirement of unacceptable concomitant medication, adverse event, intolerance of the IP) 
• Protocol non-compliance. 
• Incorrect enrolment or randomization of the subject. 
 
3.9.1 
Procedures for discontinuation of a subject from investigational product  
For subjects discontinued from the IP, the same measurements and assessments will be 
performed as in patients continuing the study until the last visit at 6 months, including 
monitoring of the adverse events. The subjects will be asked to return the study medication. 
3.10 
Criteria for withdrawal  
Criteria for withdrawal from the study include: 
• Withdrawal of informed consent. 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
• Development of exclusion criteria, pregnancy or other safety reasons during the study 
(requirement of unacceptable concomitant medication, adverse event, intolerance of the 
investigationa l product). 
• Protocol non-compliance. 
• Incorrect enrolment or randomization of the subject. 
• Lost to the last visit at 6 months. 
• Any other reason, in the investigator's opinion, that would impede the subject's participation in 
the study 
The withdrawn subjects will be replaced by subjects subsequently recruited for the study, until 
the estimated sample size of the study participants will be achieved. 
3.10.1 
Screen failures  
Patients who fail to meet the inclusion or meet the exclusion criteria will be defined as 
screening failures. The PI will maintain a Screening Failure List which includes screen 
failures in the Investigator's Study File. The list will document the subject number, subject 
initials, demographics and the reason(s) for excluding the patient from the study. It will be 
used to determine systematic bias in selection of patients for entry into the study. 
3.10.2 
Withdrawal of the informed consent 
Subjects are free to withdraw from the study at any time for any reason. Should a subject 
withdraw a consent, every effort will be made to complete and report the observations as 
thoroughly as possible.  
3.11 
Discontinuation of the study 
The Sponsor or PI may discontinute the study prematurely at any time due to the following 
reasons: 
• the safety of the participants is doubtful or at risk,  
• alterations in accepted clinical practice that make the continuation of a clinical trial unwise,  
• early evidence of benefit or harm of the experimental intervention  
• insufficient participant recruitment. 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
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4. 
STUDY PLAN AND TIMING OF PROCEDURES  


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
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Visit 
Visit window 
  Day –1 to 0 for Visit 1 
  ±1 day for Visit 2 
  ±3 days for Visit 3   
  ±5 days for Visit 4 
  ± 6 months for Visit 5 
1  
 
2 
3 
4 
5 
 
 
 
No follow-up  
For details 
see Protocol 
Section 
Week 
 
0 
1 
 
 
24 
  
 
 
 
Day 
- 
0 
1 
3 
5 
168 
 
 
 
 
Written informed consent 
X 
 
 
 
 
 
 
 
 
 
Demographics  
X 
 
 
 
 
 
 
 
 
 
Physical examination, 
height, and weight 
X 
 
X 
X 
X 
X 
 
 
 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
Visit 
Visit window 
  Day –1 to 0 for Visit 1 
  ±1 day for Visit 2 
  ±3 days for Visit 3   
  ±5 days for Visit 4 
  ± 6 months for Visit 5 
1  
 
2 
3 
4 
5 
 
 
 
No follow-up  
For details 
see Protocol 
Section 
Week 
 
0 
1 
 
 
24 
  
 
 
 
Day 
- 
0 
1 
3 
5 
168 
 
 
 
 
Medical/surgical history 
X 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
X 
 
 
 
 
 
 
 
 
12-lead ECG 
X 
 
X 
X 
X 
X 
 
 
 
 
Vital signs 
X 
X 
X 
X 
X 
X 
 
 
 
 
Randomisation to study 
treatment 
 
X 
 
 
 
 
 
 
 
 
Treatment 
dispensed/returned 
 
X 
 
 
 
X 
 
 
 
 
Concomitant medication  
X 
X 
X 
X 
X 
X 
 
 
 
 
Adverse event review (AEs 
and SAEs) 
 
X 
X 
X 
X 
X 
 
 
 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
Visit 
Visit window 
  Day –1 to 0 for Visit 1 
  ±1 day for Visit 2 
  ±3 days for Visit 3   
  ±5 days for Visit 4 
  ± 6 months for Visit 5 
1  
 
2 
3 
4 
5 
 
 
 
No follow-up  
For details 
see Protocol 
Section 
Week 
 
0 
1 
 
 
24 
  
 
 
 
Day 
- 
0 
1 
3 
5 
168 
 
 
 
 
Blood samples for 
haematology and clinical 
chemistry 
X 
 
 
X 
 
X 
 
 
 
 
Urinalysis 
X 
 
 
X 
 
X 
 
 
 
 
Blood sampling for 
pharmacokinetics 
 
n.a. 
n.a. 
n.a. 
n.a. 
n.a. 
 
 
 
 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
4.1 
Enrolment/screening period 
6 months  
4.2 
Treatment period 
6 months 
4.3 
Follow-up period 
No follow-up period after the end of the study treatment. 
5. 
STUDY ASSESSMENTS   
5.1 
Efficacy assessments 
The efficacy of ticagrelor will be assessed as a decrease in the concentration of EVs, in 
comparison with clopidogrel. 
  
5.2 
Safety assessments 
5.2.1 
Laboratory safety assessments 
Table x 
Laboratory Safety Variables 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
Haematology/Haemostasis (whole blood) 
Clinical Chemistry (serum or plasma) 
B-Haemoglobin (Hb) 
S/P-Creatinine 
B-Leukocyte count 
S/P-Aspartate transaminase (AST) 
B-Platelet count 
S/P-Alanine transaminase (ALT) 
 
S/P-Potassium 
 
S/P-Sodium 
Urinalysis (dipstick) 
U-Hb/Erythrocytes/Blood  
 
U-Protein/Albumin  
 
U-Glucose  
 
 
 
5.2.2 
Physical examination 
A comprehensive physical examination consisting of observation, percussion, palpation and 
auscultation will be conducted with state-of-the art questionnaire commonly used at 1st  Chair 
and Department of Cardiology. The following will be examined: general appearance, skin 
(colour, hydration, oedema, scars, spots, haemorrhagic manifestations) , head and neck (eyes, 
ears, nose, mucous membranes, lymph nodes, thyroid gland), cardiovascular system (heart, 
carotid arteries, peripheral arteries), respiratory tract (chest, lungs), abdomen (peristaltic 
sounds, palpation of the abdominal organs including liver and spleen), neurological system 
(cerebral functions, cerebellum tests, motor and sensory nerves, cranial nerves, reflexes), 
musculoskeletal system (skeletal abnormalities, mobility). 
5.2.3 
ECG 
The ECG report will include heart rhythm, heart rate, heart axis, P wave, PR interval, QRS 
complex, ST segment, T wave, and QT interval. 
5.2.3.1 
Resting 12-lead ECG 
Resting 12-lead ECG will be performed using state-of-the-art ECG instrument available at 1st 
Chair and Department of Cardiology. 


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Drug Substance ticagrelor  
Study Code KB/112/2016 
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5.2.4 
Vital signs 
The following vital signs will be examined: body temperature, respiratory rate, pulse rate, 
blood pressure. 
5.2.4.1 
Pulse and blood pressure 
Pulse and blood pressure will be measured at both arms during the first visit. If there is a 
difference less than 10 mmHg in systolic blood pressure between the arms, blood pressure will 
be measured at the non-dominant arm. Otherwise blood pressure will be measured at the arm 
with the higher blood pressure. The measurement will be done digitally using the instruments 
available at 1st Chair and Department of Cardiology. 
5.2.4.2 
Body temperature 
Body temperature will be measured at the forehead using digital thermometer available at 1st 
Chair and Department of Cardiology. 
5.2.5 
Other safety assessments 
Chest x-ray and echocardiography will be conducted as other safety assessments. 
5.3 
Other assessments 
5.3.1 
Patient reported outcomes (PRO) 
PROs will be collected via paper forms and kept in the Investigator's Study File. The following 
measures will be collected as PRO: 
• Symptoms (cardiovascular, respiratory, gastrointestinal, urogenital, neurological, 
musculoskeletal),  
• Health-related quality of life (HRQL)  


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Drug Substance ticagrelor  
Study Code KB/112/2016 
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5.3.1.1 
Name of PRO method or questionnaire 
• State-of-the art used at 1st Chair and Department of Cardiology to assess symptoms. 
• SF-12 Health Survey validated in patients after myocardial infarction to assess quality of life 
[17]. 
5.4 
Pharmacokinetics 
Not applicable 
5.5 
Pharmacodynamics 
Not applicable. 
5.6 
Pharmacogenetics 
Not applicable 
5.7 
Biomarker analysis  
Concentrations of PEV and other biomarkers will be analysed in citrate-anticoagulated plasma 
prepared according to recently standardized protocols for handling and storage of human body 
fluids for analysis of extracellular vesicles [14,18]. Venous blood will be collected from 
fasting individuals to measure concentrations of EVs and platelet aggregation. The protocol 
for blood collection and handling is enclosed in Appendix D. Blood sampling will be 
conducted in all subjects three times: (i) 24 hours following administration of loading dose of 
clopidogrel, (ii) 48 hours following randomisation to ticagrelor or clopidogrel group, and (iii) 
6 months following the index hospitalization. PEV analysis 
➢ Concentrations of PEVs will be determined by state-of-the art flow cytometry (Apogee 
A60 Micro, UK) [14].  A selection of typical PEV samples will be isolated by size-
exclusion chromatography [19] and analysed by transmission electron microscopy to 
confirm the presence of PEV and to study PEV morphology.  
➢ Platelet function 
Platelet function will be assessed by Multiplate impedance aggregometry (Multiplate 
Analyzer, Roche Diagnostics, Germany) using adenosine diphosphate (ADP; 6.5 µM) and 
thrombin receptor-activating peptide (TRAP, 32  µM) as agonists. Methodology applied to 
measure platelet function enables rapid and reliable on-site evaluation of platelet function, 


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including monitoring of platelet inhibition in patients treated with the P2Y12 receptor 
antagonists [20].  
5.7.1 
Storage, re-use and destruction of biological samples 
All samples will be stored at 1st Chair and Department of Cardiology in a freezer, at the 
temperature of -80°C, under continuous temperature monitoring. In case of the freezer 
breakdown, the samples will be immediately transported to the nearby Centre for Preclinical 
Research and Technology (5 minutes walking distance). Only one freeze-thaw cycle will be 
permitted to analyse biomarkers. The re-use of thawed samples will not be permitted in course 
of the study. Every effort will be made to avoid destruction of stored samples. 
5.7.2 
Labelling and shipment of biological samples 
All collected samples will be labelled with a unique code number prepared using Brady Label 
Printer (Brady, US) and analysed in one block by an operator blinded to patient- and 
treatment-related data. All samples will be transported on dry ice to Vesicle Observation 
Centre, Academic Medical Centre, University of Amsterdam, The Netherlands for analysis. 
5.7.3 
Chain of custody of biological samples  
The PI or the study nurse will withdraw the blood with strict adherence to the protocol. The PI 
will prepare the plasma and transport the samples for storage (2 minutes walking distance). 
The PI will be the only person handling the samples to minimize the risk of sample handling 
variability, thereby maximizing the reliability of obtained results. The samples will be stored 
as described in the point 5.7.1. of the protocol until shipped to the Vesicle Observation Centre, 
Academic Medical Centre, University of Amsterdam for analysis. 
5.7.4 
Withdrawal of Informed Consent for donated biological samples  
Subjects are free to withdraw the Informed Consent for donated biological samples at any 
time for any reason. The donated biological samples from subjects who withdrew a consent 
will be excluded from analysis.  


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
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6. 
SAFETY REPORTING AND MEDICAL MANAGEMENT 
6.1 
Definition of adverse events 
Adverse events (AE) and serious adverse events (SAE) will be defined according to the 
guidelines of Food and Drug Association. AE will be defined as the development of an 
undesirable medical condition or the deterioration of a pre-existing medical condition 
occurring during the study in a subject administered a pharmaceutical product which does not 
necessarily has a causal relationship with the product. SAE will be defined as any AE which 
fulfil one or more of the following criteria: 
• Results in death; 
• Is immediately life-threatening; 
• Requires hospitalization;  
• Results in persistent or significant disability or incapacity. 
• Is a congenital anomaly/ birth defect. 
The following hospitalisations will not be considered to be SAE: 
• 
Those planned before entry into the study; 
• 
 Elective treatment for a condition unrelated to study indication or study treatment; 
• 
Occurred on an emergency outpatient basis and do not result in admission (unless 
fulfilling other criteria in SAE definition); 
• 
Part of normal treatment or monitoring of the study indication and are not associated with 
any deterioration in condition. 
6.2  
Recording of adverse events 
AE will be collected by means of a standard question:  “Have you had any health problems 
since the previous visit?”  Reported AE’s and/ or observed AE’s will be recorded on the AE 
Form. The AE Forms will be stored in Investigator's Study File. 
6.1.1 
Time period for collection of adverse events 
AE and SAE will be analyzed at each visit during index hospitalization and at the last visit at 
6-months. 


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Drug Substance ticagrelor  
Study Code KB/112/2016 
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6.1.2 
Follow-up of unresolved adverse events 
Every effort will be made to follow-up and resolve the unresolved AE. 
6.1.3 Variables  
 The following variables will be collected for each AE; 
• 
Description of AE. 
• 
The date and time when the AE started and stopped. 
• 
The intensity of the AE according as assessed by study subjects according to the 
following scale: 
➢ Mild  = awareness of sign or symptom, but easily tolerated 
➢ Moderate  = discomfort sufficient to cause interference with normal activities 
➢ Severe =  incapacitating, with inability to perform normal activities.  
• 
The intensity of the AE assessed by PI as CTCAE grade 1-5. 
• 
Whether the AE is serious or not. The AE assessed by the study subject as severe AE is 
not equivalent to serious AE. 
• 
Causality assessment in relation to study treatment. 
• 
Action taken with regard to investigational product. 
• 
Withdrawal of the subject from the study due to AE. 
• 
Outcome. 
In addition, the following variables will be collected for SAEs: 
• 
Date when AE met criteria for serious AE. 
• 
Date Investigator became aware of serious AE. 
• 
The reason why AE is serious. 
• 
Date of hospitalization. 
• 
Date of discharge or death. 
• 
Probable cause of death. 
• 
Autopsy performed. 


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Drug Substance ticagrelor  
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Date  
6.1.4 
Causality assessment 
The causality of AE in relation to study treatment will be assessed by the PI, who in 
completing the relevant AE Report Form will answer ‘yes’ or ‘no’ to the question “Do you 
consider that there is a reasonable possibility that the event may have been caused by the 
study medication?” Causality will be assessed using the following definitions: 
• Very likely 
➢ The AE follows a reasonable temporal sequence from study treatment 
administration; 
➢ Abates upon discontinuation of study treatment; 
➢ Reappears on repeated exposure (re-challenge). 
• Probable 
➢ The AE follows a reasonable temporal sequence from study treatment 
administration; 
➢ Abates upon discontinuation of study treatment; 
➢ Cannot reasonably be explained by known characteristics of the subject's 
clinical state. 
• Possible 
➢ The AE follows a reasonable temporal sequence from study treatment 
administration, but could have been produced by the subject's clinical state or 
other mode of therapy administered to the subject. 
• Doubtful 
➢ The temporal association between study treatment and AE is such that the 
study treatment is not likely to have any reasonable association with the 
observed event. 
• Very unlikely 
➢ The AE is definitely produced by the subject's clinical state or other mode of 
therapy administered to the subject. 
 
6.1.5 
Disease progression 
Not applicable for the purpose of the study. 


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Drug Substance ticagrelor  
Study Code KB/112/2016 
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6.2 
Reporting of serious adverse events 
Information about all SAE will be recorded on the SAE Form in Investigator’s Study File. All 
events documented in the SAE Form will be reported within 24 hours to the Sponsor 
(personally or via fax) and to the Office of Medicinal Products, Medicinal Devices and 
Biological Products (via the online form available at www.urpl.gov.pl). All SAEs are to be 
submitted to the AstraZeneca Product Safety mailbox: 
AEMailboxClinicalTrialTCS@astrazeneca.com 
6.3 
Overdose 
A subject with confirmed or suspected overdose of the study medications will undergo 
thorough clinical and laboratory investigation. In case of any abnormalities upon the 
investigation, the subjects will be admitted to 1st Chair and Department of Cardiology for 
further assessment and monitoring.  
6.4 
Pregnancy 
6.4.1 
Maternal exposure 
The female subject will be instructed to inform the PI if she becomes pregnant during the 
study and seek advice regarding continuation of the study treatment. The PI will report the 
pregnancy to the Sponsor within 24 hours of being notified of the pregnancy. The pregnancy 
will be followed up until the outcome is known. 
6.4.2 
Paternal exposure  
Pregnancy occurring in the partner of a male subject participating in the study will also be will 
be counselled as described above, reported to the Sponsor and followed up until the outcome 
is known. 
6.5 
Management of toxicities <<Dose Reductions>> 
A subject with confirmed or suspected toxicity of the study medications will be admitted to 1st 
Chair and Department of Cardiology for assessment and monitoring. After considering of 


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benefit to risk ratio the drug will be temporarily withheld. If the toxicity continues after re-
initation of the treatment, the drug will be permanently withheld. 
6.6 
Study governance and oversight 
6.6.1 
Steering Committee 
The Steering Committee will regularly discuss the course and the quality of the study. The 
Steering Committee includes Prof. Filipiak (Vice-Rector of Medical University of Warsaw 
and a Study Coordinator), Prof. Opolski (Head of the 1st Chair and Department of Cardiology 
of Medical University of Warsaw), Prof. Auguste Sturk (Head of the Department of Clinical 
Chemistry of Academic Medical Centre of University of Amsterdam), Dr. Rienk Nieuwland 
(Head of the Vesicle Observation Centre of Academic Medical Centre of University of 
Amsterdam), and PI (cardiology resident and PhD student at the 1st Chair and Department of 
Cardiology of Medical University of Warsaw). 
6.6.2 
Data Monitoring Committee 
There is a Monitoring Committee at the study site which monitors the Investigator’s Study 
File to verify adherence to the protocol and the completeness, consistency and accuracy of the 
data. The investigator agrees to cooperate with the Monitoring Committee to ensure that any 
problems detected in the course of these monitoring visits are resolved. 
 
6.6.3 
Scientific Advisory Committee 
The Scientific Advisory Committee consists of the same members as the Steering Committee 
excluding PI. The PI will regularly discuss the questionable issues related to the study with the 
Scientific Advisory Committee. 
7. 
INVESTIGATIONAL PRODUCT AND OTHER TREATMENTS 
7.1 
Identity of investigational product(s) 
 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
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Investigational product 
Dosage form and strength 
Manufacturer 
Ticagrelor 
Tablets, 90 mg 
AstraZeneca 
Clopidogrel 
Tablets, 75 mg 
Synoptis Pharma 
 
7.2 
Dose and treatment regimens 
The investigational product ticagrelor will be administered orally in a  loading dose 180 mg, 
followed by a maintenance dose of 90 mg twice daily for 6 months. Clopidogrel will be 
administered orally in a  loading dose 600 mg, followed by a maintenance dose of 75 mg 
twice daily. 
7.3 
Labelling 
Each study drug box will be labelled with the following information: 
• Sponsor identification 
• Manufacturer's identification 
• Protocol number 
• Caution statement 
• 'For Clinical Trial Use Only' statement 
• Investigational product identification and batch number 
• Quantity of bottles 
• Storage conditions 
• Expiry date 
7.4 
Storage 
The investigational product will be stored in accordance with the manufacturers' instructions. 
Required conditions for storage will be printed on the medication label. The temperature 
within the load will be measured at least twice daily and recorded in the Investigator’s Study 
File. Investigational product will be kept under adequate security by the PI and only accessible 
to authorised study personnel. 


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7.5 
Compliance 
Each subject will be dispensed sufficient medication for 24 weeks of therapy. Compliance will 
be checked by counting of blisters at the last visit at 6 months. 
7.6 
Accountability 
The PI will maintain current record of the receipt, inventory and dispensing, including 
shipping invoices, of all study supplies. The Investigational Product Accountability File will 
include: 
• Date received 
• Reference number and amount received and placed in storage 
• Name of study medication and dosage 
• Label ID number or batch number 
• Name of person responsible for each investigational product inventory entry 
• Amount dispensed to and returned by each subject, including unique subject identifiers 
• Non-study disposition (e.g. lost, wasted, broken) 
• Amount returned to Sponsor 
Accountability files will be available for inspection by the monitor at any time. Upon 
completion/ termination of the study, unused investigational product will be returned to the 
Sponsor reconciliation. 
7.7 
Concomitant and other treatments  
Resticted Medication/Class of drug: 
 
Non-steroid anti-inflammatory drugs 
Oral anticoagulants 
 
 
 
 
Prohibited Medication/Class of drug: 
 


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Ketoconazole/ antifungal drugs 
Clarithromycin/ antibiotics 
Ritonavir/ antiretroviral medications 
Other strong CYP3A4 inhibitors 
 
 
 
 
7.7.1 
Other concomitant treatment 
Subjects will receive a loading dose of aspirin (300 mg) prior to PCI. Unfractionated 
heparin will be administered during PCI in the dosage left at the discretion of interventional 
cardiologist. On discharge, all subjects will receive treatment with ASA 75 mg once daily, 
atorvastatin at least 10 mg once daily, and angiotensin-converting enzyme (ACE)-inhibitor or 
angiotensin II receptor antagonist (ARB). If indicated, subjects will receive treatment with β-
receptor antagonist and mineralocorticoid-receptor antagonist. 
 
7.8 
Post Study Access to Study Treatment 
According to the guidelines, double antiplatelet therapy with ASA and a P2Y12 
receptor antagonist will be continued for 12 months. Because the study duration is 6 months, 
the study treatment will be continued until last visit at 6 months. After termination of the study 
patients will be prescribed double antiplatelet therapy for the remaining 6 months. Ticagrelor 
will be recommended over clopidogrel. Patients who choose to switch to clopidogrel due to 
financial constraints will be prescribed clopidogrel. 
 
8. 
STATISTICAL ANALYSES  
8.1 
Statistical considerations 
A single statistical analysis will be performed at the end of the study by an independent 
professional statistician from the Epidemiology Department of Medical University o Warsaw 
using IBM SPSS Statistics 20 and GraphPad Prism 5. An intention to treat (ITT) approach 


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will followed, i.e. statistical analysis will be based on data from all patients who were 
randomized and from whom meaningful data were collected. Data will be displayed 
graphically for visual inspection. Categorical variables will be presented as number and 
percent; continuous variables will be presented as mean  and standard deviation (SD) or 
median with inter-quartile range. Shapiro–Wilk test will be used to test for non-Gaussian 
distribution of continuous variables.  
8.2 
Sample size 
Because there is no sufficient data to assess the impact of ticagrelor on the concentrations of 
PEV in patients with AMI, the SD and mean difference between the groups was estimated 
based on the preliminary experiments, which we conducted in during preparation for this 
study. The required sample size was calculated by a two-sided t-test at a significance level of 
0.05 with the following assumptions: (i) SD in each group ± 1, (ii) mean difference between 
the groups = 1, (iii) nominal test power = 0.9. Based on this sample size estimation, it is 
calculated that a total of 60 patients will be enrolled in the trial. The conference abstract [15], 
as well as the explanation on how the sample size has been calculated are included in 
Appendix E. 
8.3 
Definitions of analysis sets 
The following sets of analysis will be performed: analysis of baseline characteristics, efficacy 
analysis, PRO analysis. 
8.3.1 
Efficacy analysis set 
The efficacy of ticagrelor will be assessed as decrease in concentration of PEVs (flow 
cytometry), in comparison with clopidogrel. 
8.3.2 
Safety analysis set 
AE reported throughout the course of the study including pre-study, study and post-study 
findings of physical examination, vital signs, laboratory variables, and 12-lead ECG will be 
listed individually per treatment group and analyzed. Due to the fact that the study is not 


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powered for clinical endpoints, AE will be analyzed as safety events and reported 
descriptively. 
8.3.3 
PK analysis set 
Not applicable. 
8.3.4 
PRO analysis set 
PRO reported throughout the course of the study including findings of the standard symptoms 
questionnaire used at 1st Chair and Department of Cardiology and SF-12 questionnaire will be 
listed individually per treatment group and analyzed.  
8.3.5 
Analysis of the primary variable(s) 
Primary variable is the concentration of PEV exposing P-selectin and PS. Student's two-sided 
t-test or Mann–Whitney U test will be used to compare the concentrations between the two 
treatment arms. ANOVA-repeated measures or Friedman test will be used to compare the 
concentrations between the three blood samplings for PEV analysis within each group. A p-
value below 0.05 will be considered significant. 
8.3.6 
Analysis of the secondary variable(s) 
Secondary variables are the concentration of EVs exposing fibrinogen, exposing 
phosphatidylserine, EVs from endothelial cells, EVs from leukocytes (flow cytometry( and 
platelet aggregation (Multiplate impedance aggregometry). Student's two-sided t-test or 
Mann–Whitney U test will be used to compare the concentrations between the two treatment 
arms. ANOVA-repeated measures or Friedman test will be used to compare the concentrations 
between the three blood samplings for PEV analysis within each group. A p-value below 0.05 
will be considered significant. 
 
 
 


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8.3.7 
Subgroup analysis (if applicable) 
Not applicable. 
8.3.8 
Interim analysis 
Not applicable. 
8.3.9 
Sensitivity analysis (if applicable) 
Not applicable. 
8.3.10 
Exploratory analysis (if applicable))  
Not applicable. 
9. 
STUDY AND DATA MANAGEMENT  
9.1 
Training of study site personnel 
The PI attented a 4-week-long internship at the Vesicle Observation Centre of Academic 
Medical Centre of University of Amsterdam, where she was trained in protocols of collection 
and handling of body fluids for analysis of extracellular vesicles, as well as methods to isolate 
and detect EV with EV-dedicated flow cytometry (Apogee A60-Micro), enzyme-linked 
immunosorbent assay (ELISA), single particle detection methods such as Nanoparticle 
Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). The PI will transfer 
her knowledge and skills to the study site personnel, which includes two other investigators 
and a study nurse. All samples throughout the study will be handled by a PI exclusively to 
minimize the investigator-related variability and maximize the reliability of the results. 
9.2 
Monitoring of the study 
9.2.1 
Source data 
Source data include original hospital records, clinical charts, original laboratory reports, 
memoranda, pharmacy dispensing records, recorded data from automated instruments, x-rays, 
magnetic or electronic media, and all other documents stored in subjects’ individual files.The 


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PI will maintain source documents for each patient in the study. The information in the 
Investigator’s Study File will be traceable to these source documents. 
9.2.2 
Study agreements 
The Sponsor declare to sign the confidential agreement and the study agreement with 
AstraZeneca Pharma Poland and to conduct the study according to signed agreements. 
9.2.3 
Archiving of study documents 
The source data will be archived according to the local legal and regulatory requirements of 1st 
Chair and Department of Cardiology, Medical University of Warsaw. 
9.3 
Study timetable and end of study 
Project IEC approval  
 
 
May 10, 2016 
First Subject In                 
 
 
December 28, 2016 
50% Enrollment           
 
 
March 28, 2017 
Last Subject In (100% enrollment)      
June 28, 2017 
Last Subject Last Visit (Treatment end)  
December 28, 2018 
Publication                      
 
 
June 28, 2019 
Data management will be conducted using database created for the study and stored on the 
validated platform for data protection of Academic Medical Centre, University of Amsterdam. 
Accurate and reliable data collection will be assured by verification and cross-check of the 
Investigator’s Study File against the source documents. Investigator’s Study File will be 
maintained including at least the following documents/information: 
• Signed protocol and amendments 
• 
Sample case report forms (CRF) 
• 
Current informed consent form and all revisions 
• 
Current patient information sheet and all revisions 
• Financial aspects of the study 


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• All signed agreements/contracts 
• 
Dated, documented approval of ethics committee and regulatory authorities 
• 
Monitoring reports 
• 
Reports to ethics committee and regulatory authorities 
• 
Notification by sponsor of safety information 
• Screening Failure List 
• Incorrectly enrolled or randomized subjects list 
• Patient Enrolment List.   
• Patient Identification List  
• Patient reported outcomes 
• AE Forms and SAE Forms 
• Investigational Product Accountability File 
 
Adverse Event (AE) Reconciliation 
After termination of the study the AE Reconciliation will be performed. 
Data Management of genotype data 
No genotype data will be collected throughout the study. 
 
Data associated with human biological samples 
Only the PI, study site personnel and statistician will have access to data associated with 
human biological samples. The server hosting the platform is recurred with personal 
passwords. 
Management of external data 
Only the PI, study site personnel, statistician, and treating physician will have access to 
external data. The external data will be protected and archived according to the local data 
policy. 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
10. 
ETHICAL AND REGULATORY REQUIREMENTS 
10.1 
Ethical conduct of the study 
The study will be conducted in compliance with the protocol, GCP, the ethical principles 
described in the Declaration of Helsinki, the requirements of the European Medicines Agency, 
and local legal and regulatory requirements.  
10.2 
Subject data protection 
The PI will assure that the subjects' anonymity will be maintained  and that the confidentiality 
of records and documents that could identify subjects will be protected, respecting the privacy 
and confidentiality rules in accordance with applicable regulatory requirements. Before 
unblinding of the PI, subjects will be identified only by their assigned identification number 
and initials. A co-investigator will keep a Patient Identification List with complete 
identification information (name, address, contact number).   
 
10.3 
Ethics and regulatory review 
The study protocol was approved by the IEC of Medical University of Warsaw in May 2016 
(approval number: KB/112/2016, amendment: KB/79/A/2016, Appendix B), and is registered 
on ClinicalTrials. The PI will inform IEC of Medical University of Warsaw about any serious 
and/or unexpected events occurring during the study, and any new information that may 
adversely affect the safety of the subjects or the conduct of the study. All correspondence with 
the IEC of Medical University of Warsaw will be stored by the PI in the Study. 
 
10.4 
Informed consent 
The PI will inform every subject in detail about the nature of the study, its purpose, the 
treatments and the probability of random assignment to treatment groups. Every subject will 
receive information sheet and provide written informed consent prior to participation the 
study. A copy of the Patient Information Sheet and signed Consent Form will be given to the 
subject. The original will be stored by the PI in the Investigator's Study File. A sample of the 


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
Patient Information Sheet and Consent Form can be found in the Appendix C of this protocol 
(in Polish). The study subjects will also be informed that: 
• Participation in this study is voluntary and the informed consent to participate in the study 
may be withdrawn at any time for any reason, and that withdrawal of consent will not affect 
the subsequent medical treatment or relationship with the treating physician. 
• They will be notified if information becomes available that may be relevant to their 
willingness to continue participation in the study. 
• They will be notified if alternative treatment becomes available, as well as about the potential 
benefits and risks of this alternative treatment. 
• They will be strongly encouraged to contact the PI to report on any potentially study-related 
adverse events, as well as for further information regarding the study. 
 
10.5 
Changes to the protocol and informed consent form 
The PI will inform IEC of Medical University of Warsaw about any amendment to the 
protocol, informed consent changes or revisions of other documents originally submitted for 
review. 
 
10.6 
Audits and inspections 
Auditors and inspectors from Sponsor and representatives of IEC or other regulatory agencies 
will be granted direct access to subject medical records and other study documents for 
verification of study procedures and data without violating the confidentiality of the subject. 
The subject should be informed that by signing a written informed consent form that he or she 
is authorizing such access.  
 
11. 
LIST OF REFERENCES 
1. Linden MD, Jackson DE. Platelets: pleiotropic roles in atherogenesis and 
atherothrombosis. Int J Biochem Cell Biol. 2010;42:1762-1766.  


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
2. Hechler B, Gachet C. Purinergic Receptors in Thrombosis and Inflammation. 
Arterioscler Thromb Vasc Biol. 2015;35:2307-2315.  
3. Windecker S, et al. 2014 ESC/EACTS Guidelines on myocardial revascularization: 
The Task Force on Myocardial Revascularization of the European Society of 
Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery 
(EACTS)Developed with the special contribution of the European Association of 
Percutaneous Cardiovascular Interventions (EAPCI). Eur Heart J. 2014;35):2541-
2619.  
4. Thomas MR, et al. Platelet P2Y12 inhibitors reduce systemic inflammation and its 
prothrombotic effects in an experimental human model. Arterioscler Thromb Vasc 
Biol. 2015; 35: 2562–257.  
5. Storey RF, et al. Lower mortality following pulmonary adverse events and sepsis with 
ticagrelor compared to clopidogrel in the PLATO study. Platelets 2014; 25: 517–525.  
6. van der Pol E, et al. Classification, functions, and clinical relevance of extracellular 
vesicles. Pharmacol Rev;64:676-705.  
7. Owens AP 3rd, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 
2011; 108: 1284-1297.  
8. van der Pol E, et al. Recent developments in the nomenclature, presence, isolation, 
detection and clinical impact of extracellular vesicles. J Thromb Haemost 2016;14:48-
56. 
9. Vajen T, et al. Microvesicles from platelets: novel drivers of vascular inflammation. 
Thromb Haemost. 2015;114:228-236  
10. Van Der Meijden PE, et al. Platelet- and erythrocyte-derived microparticles trigger 
thrombin generation via factor XIIa. J Thromb Haemost. 2012;10:1355-1362.  
11. Xie RF, et al. Platelet-derived microparticles induce polymorphonuclear leukocyte-
mediated damage of human pulmonary microvascular endothelial cells. Transfusion. 
2015; 55:1051-1057.  
12. Erickson SE, et al. Recent trends in acute lung injury mortality: 1996–2005. Critical 
Care Medicine. 2009;37:1574–1579.  


Clinical Study Protocol 
Drug Substance ticagrelor  
Study Code KB/112/2016 
Edition1 
Date  
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methods in extracellular vesicle research. J Extracell Vesicles 2013; 2. 
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syndromes: implications for clinical practice from the GRACE registry. Nat Clin Pract 
Cardiovasc Med. 2008 ;5: 580-589.  
16. Thune JJ, Signorovitch JE, Kober L, et al. Predictors and prognostic impact of 
recurrent myocardial infarction in patients with left ventricular dysfunction, heart 
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153. 
17. Failde I, Medina P, Ramirez C, Arana R. Construct and criterion validity of the SF-12 
health questionnaire in patients with acute myocardial infarction and unstable angina. J 
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chromatography. J Extracell Vesicles 2014; 3. 
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