Pharmacokinetics and Pharmacodynamics of Platelet P2Y12 Inhibitors in Patients Undergoing Percutaneous Coronary Intervention (PCI) for Acute Myocardial Infarction: A Pilot Study
Phase: PHASE4
Conditions: Heart Attack
Sponsor: The Royal Wolverhampton Hospitals NHS Trust

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Pharmacokinetics and 
Pharmacodynamics of Platelet P2Y12 
Inhibitors in Patients Undergoing 
Percutaneous Coronary Intervention 
(PCI) for Acute Myocardial Infarction: A 
Pilot Study 
Version 2.2 Dated 21/5/15 
 
 
 
 
 
SPONSOR: The Royal Wolverhampton NHS Trust 
FUNDERS: Part funded by The Royal Wolverhampton Hospital NHS Trust Pharmacy Department 
 
 
 
 
NHS REC reference: 14/WM/1236 
 
EudraCT number. 2014-004238-25 
Sponsor Ref. 2012-004-0402-CARD. 
 
Protocol authorised by: 
 
 
 

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Study Management Group 
 
Chief Investigator:  
 
Dr J Cotton 
 
Co-investigators:  
 
Dr V Amoah,  
Ms N Khan,  
Dr J Vickers,  
Dr K Wilmer,  
Dr S Khogali,  
Dr J Martins,  
Dr A Armesilla 
 
Statistician:   
 
Prof A Nevill 
 
Study Management:  Dr V Amoah 
 
 
Clinical Queries 
 
Clinical queries should be directed to Dr James Cotton who will direct the query to the 
appropriate person. 
 
 
Sponsor 
 
The Royal Wolverhampton NHS Trust is the research sponsor for this study.  For further 
information regarding the sponsorship conditions, please contact R&D Department, The 
Royal Wolverhampton NHS Trust– Tel: 01902 695065 or email: 
lorraine.jacques@nhs.net  
 
 
Funder 
 
This study has been part funded by the Royal Wolverhampton Hospitals NHS Trust 
Pharmacy Department.  
 
 
This protocol describes the above study and provides information about procedures for 
entering participants.  Every care was taken in its drafting, but corrections or amendments 
may be necessary. These will be circulated to investigators in the study. 
Problems relating to this study should be referred, in the first instance, to the Chief 
Investigator.  
 
This study will adhere to the principles outlined in the NHS Research Governance 
Framework for Health and Social Care (2nd edition). It will be conducted in compliance 

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with the protocol, the Data Protection Act and other regulatory requirements as 
appropriate. 
 
 
 

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CONTENTS 
Glossary and Technical Terms 
 
 
 
 
 
 
6 
Keywords 
 
 
 
 
 
 
 
 
 
6 
1 
Lay Summary  
 
 
 
 
 
 
 
7 
2 
Introduction 
 
 
 
 
 
 
 
 
8 
 
2.1 
Background 
 
 
 
 
 
 
 
8 
 
2.2 
Rationale for Current Study  
 
 
 
 
10 
3 
Study Objectives 
 
 
 
 
 
 
 
11 
4 
Study Design  
 
 
 
 
 
 
 
12 
 
4.1 
Study Outcome Measures 
 
 
 
 
 
12 
5 
Participant Entry 
 
 
 
 
 
 
 
13 
 
5.1 
Consent 
 
 
 
 
 
 
 
13 
 
5.2 
Pre-registration Evaluation 
 
 
 
 
 
13 
 
5.3 
Inclusion Criteria 
 
 
 
 
 
 
14 
 
5.4 
Exclusion Criteria 
 
 
 
 
 
 
14 
 
5.5 
Withdrawal Criteria  
 
 
 
 
 
14 
6 
Assessment and Follow-up 
 
 
 
 
 
 
15 
 
6.1 
Subjects 
 
 
 
 
 
 
 
15 
 
6.2 
Sampling 
 
 
 
 
 
 
 
15 
 
 
6.2.1 STEMI patients 
 
 
 
 
 
15 
 
 
6.2.2 NSTEMI patients 
 
 
 
 
 
15 
 
6.3 
Pharmacodynamic and Pharmacokinetic Analysis 
 
 
15 
 
 
6.3.1 VerifyNow for Aspirin and P2Y12 analysis  
 
15 
 
 
6.3.2 VASP Flow Cytometry 
 
 
 
 
16 
 
 
6.3.3 Liquid Chromatography with Tandem Mass Spectrometry 16 

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7 
Statistics and Data Analysis Adverse Events  
 
 
 
18 
8 
End of Study  
 
 
 
 
 
 
 
18 
9 
Adverse Events 
 
 
 
 
 
 
 
20 
 
9.1 
Definitions 
 
 
 
 
 
 
 
20 
 
 
9.1.1 Adverse Events 
 
 
 
 
 
20 
 
 
9.1.2 Serious Adverse Events 
 
 
 
 
20 
 
9.2 
Reporting Procedures  
 
 
 
 
 
20 
 
 
9.2.1 Non-serious Adverse Events  
 
 
 
20 
 
 
9.2.2 Serious Adverse Events 
 
 
 
 
20 
10 
Regulatory Issues 
 
 
 
 
 
 
 
22 
 
10.1 
Ethics Approval 
 
 
 
 
 
 
22 
 
10.2 
Consent 
 
 
 
 
 
 
 
22 
 
10.3 
Confidentiality 
 
 
 
 
 
 
22 
 
10.4 
Indemnity 
 
 
 
 
 
 
 
22 
 
10.5 
Sponsor 
 
 
 
 
 
 
 
22 
 
10.6 
Funding 
 
 
 
 
 
 
 
22 
 
10.7 
Audits and Inspections 
 
 
 
 
 
22 
11 
Study Management 
 
 
 
 
 
 
 
22 
12 
Publication Policy 
 
 
 
 
 
 
 
23 
13 
References 
 
 
 
 
 
 
 
 
24 
14 
Appendices 
 
 
 
 
 
 
 
 
28 
 
14.1 
Appendix 1 
 
 
 
 
 
 
 
29 
 
14.2 
Appendix 2 
 
 
 
 
 
 
 
30 
 
14.3 
Appendix 3 
 
 
 
 
 
 
 
31 
 
 

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GLOSSARY OF ABBREVIATIONS AND TECHNICAL TERMS 
ACS 
Acute coronary syndrome 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
CABG 
Coronary artery bypass graft 
ECG 
Electrocardiogram 
HTPR 
High on-treatment platelet reayctivity 
NSTEMI 
Non-ST segment elevation myocardial infarction 
PPCI 
Primary percutaneous coronary intervention 
SAE 
Serious adverse event 
STEMI 
ST-segment elevation myocardial infarction 
TIMI 
Thrombolysis in myocardial infarction 
UA 
Unstable angina 
VASP 
Vasodilator stimulated phosphoprotein phosphorylation 
 
 
KEYWORDS 
 
Pharmacodynamic 
Pharmacokinetic 
Antiplatelet 
Myocardial infarction 
Clopidogrel 
Prasugrel 
Ticagrelor 
STEMI 
NSTEMI 
 
 
 

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1. 
LAY SUMMARY 
 
Major heart attacks are caused by a number of factors, the two major of which are furring 
up of a coronary artery with atheroma and then sudden clot formation on this area leading 
to a blockage and interruption of blood flow. The clots that lead to heart attacks are 
largely made of clotting blood cells (platelets) that in health repair blood vessels and 
inhibit spontaneous bleeding. One of the main treatment strategies for heart attacks is to 
make these cells less “sticky”. Aspirin is a main stay of treatment at our institution and in 
addition one of three other antiplatelet agents acting on the same platelet activation 
pathway (P2Y12 receptor) are used (clopidogrel, prasugrel or ticagrelor). When a patient 
is admitted with a major heart attack, they are treated with emergency primary 
percutaneous coronary intervention (PPCI) a technique where a wire and balloon are used 
to reopen the coronary artery and then usually a stent (a slotted metal tube) is placed to 
keep the artery open. Aspirin and one of the P2Y12 inhibitor agents are given to prevent 
further clots and all have been shown to reduce negative events following heart attacks 
and angioplasty with stent insertion. 
All three P2Y12 inhibitor agents are taken in tablet form immediately before the 
emergency PPCI procedure. It appears that these agents in healthy stable patients take at 
least 30 min to 2 hours to have an adequate effect. In PPCI patients, often the procedures 
are performed well within this timescale. Furthermore patients who are having a heart 
attack may not have normal drug absorption with blood being diverted away from the 
stomach and gut activity being suppressed by other drugs such as morphine. 
In this study patients with major heart attacks will be given one of the three anti-platelet 
agents (being treated as per normal clinical pathways). Blood samples will be taken to 
assess the effect of the anti-platelet agents when the balloon opens the artery and after 20 
minutes, 60 min and 4 hours after taking the drugs. 
A variety of tests will be used to check the activity of the drugs including measuring the 
concentration of the active component of each of the three anti-platelet agents in the 
blood. 
In total the patients in the study will require up to 4 extra blood tests, each using 15 ml of 
blood (approximately 3 teaspoons of blood). This will not cause any ill effects but may 
involve up to 3 extra needle pricks.  
One of the major negative occurrences after PPCI is clotting within the stent- this occurs 
in 0-2% of cases and in other patients is associated with a poor response to these anti-
platelet drugs. It is possible that this study will identify which, if any, of the agents used 
are working during the procedure, given the very short timescales involved. In the future 
it might be that an intravenous agent will be more valuable in the setting of PPCI. 

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2. 
INTRODUCTION  
 
2.1 
BACKGROUND 
The success of modern treatment for ST-segment elevation myocardial infarction 
(STEMI) is dependent, in part, on the adequate suppression of platelet activity. Primary 
percutaneous coronary intervention (PPCI) for STEMI management has led to marked 
reductions in morbidity and mortality (1). Aspirin is the mainstay anti-platelet agent (2) 
which acts by irreversible acetylation and inhibition of the COX-1 enzyme, thereby 
reducing the production of thromboxane A2, a potent platelet activator. The advent of the 
platelet P2Y12 inhibitors led to further improvement in outcomes in patients with acute 
coronary syndromes (ACS) undergoing PCI with reduced ischaemic complications (3) 
albeit at an increase in rate of bleeding complications. Both of these agents have a 
synergistic effect on platelet function with the addition of clopidogrel to aspirin leading 
to approximately 20% reduction in negative cardiovascular events (4). Activation of 
platelets leads to a conformational change in the surface glycoprotein IIb/IIIa receptor 
that allows cross bridging with fibrinogen and rapid aggregation (platelet thrombus 
formation). 
During PPCI thrombus formation and dispersion can have a major impact on outcome. 
Intravenous platelet IIb/IIIa receptor antagonists such as abciximab were initially shown 
to have a positive impact on primary angioplasty outcomes leading to a class IIa 
indication within the European Society of Cardiology (ESC) guidelines for use in the 
treatment of STEMI with angioplasty (5). 
As oral antiplatelet inhibition has improved (specifically with the addition of clopidogrel, 
prasugrel or ticagrelor) there has been a concomitant reduction in the use of intravenous 
IIb/IIIa agents during PCI for patients with ACS. This has been partly informed by later 
studies suggesting that the additive value of these agents to dual anti-platelet therapy is 
questionable (6). In addition, the extra bleeding complications are seen by some to be 
excessive (7).  
 
Clopidogrel, the most commonly used P2Y12 inhibitor has been shown to reduce 
ischaemic complications in patients with ACS both with and without PCI (8), but there 
are significant issues regarding both the uniformity of action across patients (9) and also 
the speed of onset of action of this agent (10). There are a variety of causes for the 
variable response to clopidogrel amongst subjects, leading to high on-treatment platelet 
reactivity (HTPR), but key is the two stage hepatic conversion to its active metabolite 
with variances in the CYP 450 isoenzymes (particularly 2C19) playing a major role.  
As many as 40 % of treated patents may have a suboptimal response to clopidogrel and 
these patients are more likely to suffer recurrent ischaemic events and in particular stent 
thrombosis (11). Notably one of the first papers demonstrating this problem involved 
PPCI patients (12). Further large scale trials have identified the risk of poor clopidogrel 
activity with negative outcomes in PPCI patients (13).  
 
Prasugrel, a newer thienopyridine has a greater uniformity of action and a more rapid 
onset of action than clopidogrel. This appears to lead to improved outcomes in patients 

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treated with PCI for ACS and in particular a reduction in stent thrombosis (TRITON-
TIMI 38).(14). NICE guidelines (TA 182) now recommend the use of prasugrel in 
treatment of patients either over the age of 75 or over 60kg undergoing PPCI.  
The TIMI-TRITON 38 trial was however biased toward prasugrel in that the majority of 
drug dosing was performed at the end of the PCI procedure, and therefore there would 
have been less P2Y12 activity for a number of hours after the administration of the agent 
with the slower onset of action. Also the majority of patients included were Non-STEMI 
(NSTEMI) or convalescent STEMI patients undergoing PCI. In the STEMI subgroup 
(again with a 2:1 convalescent to acute ratio) there was no advantage of the newer agent 
seen, possibly due to issues with drug absorption and metabolism (14). 
 
Ticagrelor differs from the thienopyridine class of antiplatelet agents (clopidogrel, 
prasugrel, and ticlopidine) as it does not exist as a prodrug and thus does not require 
biotransformation by hepatic enzymes before becoming active  Also ticagrelor binds 
reversibly to P2Y12 receptors leading to a faster onset and offset than clopidogrel (15). 
The PLATO trial which compared ticagrelor with clopidogrel, demonstrated an 
improvement in clinical outcome in the ticagrelor group. (16). Moreover, in the 
RESPOND study, ticagrelor therapy was associated with uniform and superior platelet 
inhibition in both previously identified clopidogrel responders and nonresponders, and 
that inhibition, in turn, was associated with an extremely low prevalence of high on-
treatment platelet reactivity (17). Ticagrelor has recently received recommendation in the 
ESC guideline as a front-line treatment option for STEMI and NSTEMI (18,19) 
 
The synthetic hirudin analogue Bivalirudin is a reversible direct thrombin inhibitor with 
important effects on the coagulation pathways and also inhibits the activity of circulating 
and clot bound thrombin, thus also having a partial antiplatelet effect. This agent, when 
used with dual antiplatelet therapy in patients undergoing PPCI for STEMI, appears to 
confer a survival benefit compared to heparin, IIb/IIIa receptor antagonism and dual 
antiplatelet therapy (with aspirin and clopidogrel) (20). This benefit is largely due to a 
reduction in major bleeding. In the Horizon-AMI trial, however, there was a marked 
increase in early stent thrombosis in the bivalirudin group (from 1.4% vs 0.3 %). 
 In view of these positive findings many operators now use bivalirudin routinely (as 
recommended as a possible treatment by NICE guidance TA 230) but to mitigate against 
the higher risk of stent thrombosis are giving one of the newer P2Y12 antagonists 
(prasugrel or ticagrelor) which have been shown to lead to lower stent thrombosis rates in 
other ACS groups (14, 21). Whilst both of these agents have been shown to have a faster 
onset of action than clopidogrel, there are currently no data on the onset of action 
required in the setting of PPCI. Ideally a full antiplatelet effect would be evident at the 
start of the PPCI, whereas in practice dosing of clopidogrel, prasugrel or ticagrelor 
typically occurs on admission to hospital (possible only 10-20 minutes prior to PPCI).  
 
During PPCI (particularly using bivalirudin rather than i.v. IIb/IIIa antagonists) it is likely 
therefore that patient’s would be advantaged by rapid P2Y12 antagonism. Currently there 
are no data concerning the comparative rapidity of clinical effect of these agents in the 
setting of an acute STEMI when compared to other groups. 
 

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2.2 
RATIONALE FOR CURRENT STUDY 
It is unlikely that gastro intestinal absorption during a STEMI is equivalent to that of a 
resting patient. Hypotension and or diversion of splanchnic blood flow is likely to slow 
drug absorption and the intrinsic increase in platelet activity seen during STEMI (22, 23) 
may also inhibit the antiplatelet effect of drugs. A previous pharmacokinetic study has 
shown reduced drug absorption during STEMI (24). In the current study we aim to 
demonstrate whether prasugrel, or ticagrelor are having a significant clinical effect during 
the primary angioplasty (STEMI patients), and investigate the pharmacodynamic 
properties of these agents in the setting of STEMI. By also studying patients with 
unstable angina (UA)/NSTEMI we will investigate whether the condition of STEMI per 
se affects the pharmacodynamics of these agents. 
A number of methods have been developed to assess the effect of P2Y12 receptor 
inhibition. Their effect is probably best measured using techniques that specifically 
measure P2Y12 ADP receptor activation, such as vasodilator stimulated phosphoprotein 
phosphorylation (VASP) and the VerifyNow P2Y12 system (Accumetrics, San Diego, 
CA) (24). We will utilize both of these methods in this study. We will also measure 
aspirin activity using the VerifyNow aspirin test. 
 
 

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3. 
STUDY OBJECTIVES 
 
1) To determine the degree and time-course of platelet inhibition in patients admitted 
with NSTEMI treated with prasugrel within the first 4 hours of loading. These 
data will be compared with data already collected on STEMI patients treated with 
prasugrel to determine whether the condition of STEMI per se reduces the 
efficacy of prasugrel treatment. 
 
2) To demonstrate the degree and time-course of platelet inhibition by ticagrelor 
given acutely before emergency primary angioplasty for STEMI, during the 
procedure and in the following 4 hours. This will be compared with a cohort of 
patients presenting with NSTEMI treated with ticagrelor to determine whether the 
condition of STEMI per se reduces the efficacy of ticagrelor treatment. 
 
 
 
 

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4. 
STUDY DESIGN 
This is a pilot study which is both non-randomised and observational in nature.  
We plan on recruiting a total of 45 participants into the study who will comprise of 
patients admitted to New Cross Hospital suffering from an acute coronary syndrome. The 
study will complete an on-going project (REC ref no. 13/WM/0025 ethical approval 
granted from South Birmingham Regional Ethics Committee). Whole blood samples will 
be taken from participants following a single loading dose of an antiplatelet agent once 
the diagnosis of an ACS has been made. We anticipate the conclusion of subject 
recruitment in 3 months from the study start date  
 
4.1 
STUDY OUTCOME MEASURES 
 
a) Degree of platelet inhibition measured with VerifyNow TM rapid platelet function 
analyser  
b) Degree of platelet inhibition measured with VASP flow cytometry 
c) Concentration of prasugrel and ticagrelor active metabolite in plasma measured with 
liquid chromatography with tandem mass spectrometry. 
This study is not powered for clinical outcomes; however clinical data will be recorded. 
Clinical endpoints to be recorded for each participant will include:  
 
d) Angiographic variables –  
 thrombolysis in myocardial infarction (TIMI) flow at end of case,  
 slow flow/no reflow  
 
e) ECG variables –  
 ST-segment resolution at end of case 
 
f) Safety endpoints –  
 Death 
 CABG 
 TIMI major and minor bleed (see appendix 1 for bleeding criteria) 
 
 

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5. 
PARTICIPANT ENTRY  
 
5.1 
CONSENT 
Consent will be a 2 stage process for patients undergoing PPCI for STEMI. Because the 
drugs given will be as per local and national recommended guidelines, consent will be 
verbal prior to the PPCI and first 3 blood samples. A shortened patient information sheet 
will be read to the patient (so as not to delay urgent treatment) and if verbal consent is 
gained it will be recorded in the clinical notes and the site file consent record. This is in 
line with NRES guidelines that suggest that patients admitted with STEMI are considered 
as unconscious patients initially. 
Following emergency PCI when the patient is pain free and has been able to rest, a full 
patient information sheet will be proffered and written consent sought. If the patient does 
not want to be part of the trial the previous samples will be discarded and the data not 
used for analysis. Treatment will continue in accordance with clinical guidelines. The 
patients’ participation in the trial will end with the 4hr sample. 
For patients with UA/NSTEMI full written consent will be taken after the patient has read 
the full patient information sheet. Blood tests will be taken at time-points which coincide 
with STEMI time-points (20 minutes, 60 minutes, and 4 hours post dosage) 
Patient Data will be anonymised and stored on RWHT trust fully encrypted computers 
 
 
5.2 
PRE-REGISTRATION EVALUATIONS  
All research subjects will be identified from STEMI patients admitted for PPCI at the 
Heart and Lung Centre in Wolverhampton and also UA/NSTEMI acute coronary 
syndrome patients admitted to New Cross Hospital, Wolverhampton. Patient groups 
(below) are carefully selected so that all patients will receive anti-platelet drugs as per 
current accepted National and local guidelines and as per the manufacturers` datasheets. 
 
 
1) 
Group 2: Patients with NSTEMI who are under 75 years of age and greater 
than 60Kg in weight receiving Prasugrel loading (60mg) however: -  
i. After sample collection patients treated with intracoronary stent 
placement on the same day as loading will receive prasugrel 
maintenance dose (10mg per day) as per licensing agreement for 
prasugrel 
ii. After sample collection patients who are not stented after loading 
will receive clopidogrel maintenance dose (75mg per day). 
 
2) 
Group 5: Patients admitted with STEMI receiving ticagrelor loading (180 mg) 
and then maintenance (90mg bd per day). 

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3) 
Group 6: Patients Admitted with NSTEMI receiving ticagrelor loading (180 
mg) and then maintenance (90mg bd per day). 
 
We plan to recruit 15 patients into each group.  
 
5.3 
INCLUSION CRITERIA 
1) Patients presenting with STEMI for PCI (characterized by chest discomfort, and 
prominent ST-segment elevation) 
2) Patients presenting with NSTEMI (characterized by chest discomfort, raised 
levels of myocardial enzymes and/or ST-segment depression or prominent T wave 
inversion) 
3) Able to give verbal consent (STEMI patient’s pre procedure) and/or written 
consent (STEMI after procedure and NSTEMI patients prior to enrolment). 
4) Age>18 years of age 
5) Able to take Aspirin and either prasugrel or ticagrelor. 
6) Have no concurrent septic or inflammatory illness 
7) Thienopyridine naive 
 
5.4 
EXCLUSION CRITERIA 
1) Be unable to provide verbal and written consent 
2) Allergic to aspirin or any of the P2Y12 antagonists in the trial 
3) Have pre-existing cardiogenic shock 
4) Have a concurrent septic or inflammatory disease e.g. rheumatoid arthritis, lupus, 
pneumonia. 
5) Already taking a P2Y12 inhibitor 
6) Known bleeding diathesis 
7) Patients over 75 years of age or under 60 kg or those who have had a previous 
stroke/transient ischaemic attack, will not be eligible for prasugrel but rather 
ticagrelor. 
8) Patients with a history of intracranial haemorrhage will not receive prasugrel or 
ticagrelor but rather will receive treatment with clopidogrel. 
 
5.5 
WITHDRAWAL CRITERIA 
Patients who decide to withdraw their consent to participate in the study will have all 
samples collected up to the time of withdrawal disposed of in appropriate manner. All 
information collected on the patient for the purpose of the study shall be erased. The 
withdrawal shall be recorded in the study patient identification log. The treatment of the 
patient will continue in accordance with clinical guidelines should the participant chose 
to withdraw or not. 
 
 

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6. 
ASSESSMENT AND FOLLOW-UP  
 
6.1 Subjects: A total of 45 subjects will be included in the study of which 15 patients 
will undergo PPCI as part of the treatment for STEMI.  
6.2 Sampling 
6.2.1 STEMI patients will have a P2Y12 inhibitor administered (ticagrelor as per 
current local and national guidance) following verbal consent. The time of loading dose 
will be recorded and the patient transferred to the cardiac catheterization suite. After the 
insertion of the radial or femoral sheath, 15 ml of whole blood will be drawn from this 
sheath at 20 minutes post dosing (or as close to this time point as practicable) and also at 
the first balloon inflation time. A further 15 ml will be taken at 60 minutes post dosing 
(or as close as is practicable). Further sampling on the ward will take place after 4 hours 
post dosing from an anticubital vein using a 21 gauge needle (see appendix 2 for a flow 
chart summarizing procedure for these groups). 
6.2.2 NSTEMI patients will have a P2Y12 inhibitor administered (prasugrel or 
ticagrelor) following full written consent. The time of loading dose will be carefully 
recorded. 15 ml of blood will be taken at 20 minutes, 60 minutes and then 4 hours post 
dosing solely from an anticubital vein using a 21 gauge needle (see appendix 3 for a flow 
chart summarizing procedure for these groups).  
6.3 PHARMACODYNAMIC AND PHARMACOKINETIC ANALYSIS 
6.3.1 VerifyNow for aspirin and P2Y12 analysis 
VerifyNow ™ (Accumetrics, San Diego, California, USA) is a near patient test 
comprising a turbidimetric based optical detection system that measures platelet 
aggregation as an increase in light transmittance.  
We are already using this system for clinical research in our department and it has FDA 
approval for the assessment of Aspirin and P2Y12 activity. VerifyNow has also been used 
in the assessment of ticagrelor activity in a number of studies (17, 25, 26). 2 ml of whole 
blood is transferred into a Greiner Bio-One Vacuette containing 3.2% sodium citrate 
(Accumetrics, San Diego, California, USA) and inverted carefully, after which the tube is 
left at room temperature for a minimum of 15 and 30 minutes for both P2Y12 and aspirin 
assays respectively. The tube is then loaded into a specific assay device and analysed as 
per the manufacturers’ instructions (Accumetrics). The system is calibrated daily 
electronically and full quality control is performed with each batch of assay devices. This 
system uses a similar principle to formal platelet aggregometry. Arachidonic acid is used 
in the aspirin assay, ADP in the P2Y12 assay. Aspirin will already have been given in the 

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ambulance and its antiplatelet effect will be assessed at 20 minutes and 60 minutes after 
the P2Y12 inhibitor loading dose 
Results are shown as aspirin response units (ARU) and P2Y12 reaction units (PRU) for 
aspirin and P2Y12 assays respectively.  
6.3.2 VASP flow cytometry 
VASP, an intracellular actin regulatory protein, is a substrate of both cyclic adenosine 
monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent 
protein kinases (27). Dephosphorylation of VASP follows P2Y12 stimulation. Conversely, 
inhibition of the P2Y12 receptor by clopidogrel and stimulation of prostaglandin E1-
activated adenylyl cyclase induce phosphorylation of VASP by cAMP-dependent protein 
kinases. Thus levels of VASP phosphorylation/dephosphorylation reflect P2Y12 
inhibition/activation. 
Despite using a commercially available assay (Biocytex, Asnieres, France), VASP flow 
cytometry is primarily a research based assessment owing to the complexity of the 
procedure and the requirement for significant technical expertise. It is seen by some as a 
gold standard test for P2Y12 inhibition.  
Whole blood transferred to tubes containing 3.2% sodium citrate is incubated with PGE1 
or PGE1 and ADP for 10 minutes then fixed with paraformaldehyde, then platelets are 
permeabilized with non – ionic detergent. The cells are than labelled with a monoclonal 
antibody against 239-phosphorylated VASP and then a secondary fluoroscein 
isothicyanate-conjurgated polyclonal antibody. Analysis will be performed on a Becton 
Dickinson (Plymouth, UK) FACS Calibur flow cytometer, and 10,000 platelets will be 
gated per assessment as per the manufacturer’s instructions. Results are expressed as 
percentage platelet reactivity index (% PRI). VASP flow cytometry will be performed 
within 48 hours of sampling.  
VASP Flow cytometric analysis measures activity at the P2Y12 receptor very effectively 
and thereby indirectly the activity of P2Y12 inhibitor and has been shown to be of value in 
a number of clinical trials including one carried out by our own research group (28, 29). 
6.3.3 Liquid chromatography with tandem mass spectrometry. 
We will be using this technique to quantify the active metabolites of prasugrel, and 
ticagrelor. The active metabolite of prasugrel are not stable in blood, therefore 3’-
methoxy-phenacylbromide (an alkylating reagent) will be added within 30 seconds of 
sampling to whole blood collected in EDTA tubes to derivatize and stabilize the 
metabolites. Derivatized blood samples will be centrifuged at 1500g for 10 minutes. The 
plasma will be extracted and frozen at -80C until analysis.  
Blood samples for ticagrelor active metabolite quantification will be collected in lithium 
heparin tubes. The active metabolites of ticagrelor are stable in blood, thus require no 

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stabilization. The blood samples will undergo the same processing and storage protocol 
as highlighted above. 
The derivatized and ticagrelor plasma samples then undergo solid phase extraction and 
are then separated using liquid chromatography and quantified using mass spectrometry. 
The primary pharmacokinetic parameters (i.e. the active metabolites of prasugrel, and 
ticagrelor) are calculated using the maximum observed concentration (Cmax) and area 
under the plasma concentration curve (AUC). This technique has already been developed 
and validated used previously to detect the active metabolite of either prasugrel and 
ticagrelor in a number of publicized studies (30-34). 
 
 
 

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To summarize, each sample will be used for 
1) P2Y12 inhibition estimation using the near patient Accumetrics VerifyNow test 
2) P2Y12 inhibition will also be estimated by Flow Cytometry using VASP 
phosphorylation test. 
3) Aspirin effect using near patient Accumetrics VerifyNow test. 
4) The active metabolites of prasugrel and ticagrelor will be measured using liquid 
chromatography with mass spectrometry.  
 
Time 
Pre Dosing 
20 Min post 
dosing 
Balloon 
Time (PPCI) 
60 min post 
dosing 
4 hrs post 
dosing 
Procedure 
Consent 
15 ml blood  
15 ml blood 
15 ml blood 
15 ml blood 
VN Aspirin 
 
x 
X 
x 
 
VN P2Y12 
 
x 
X 
x 
X 
VASP 
Phosphorylation 
 
x 
X 
x 
X 
Estimation of 
active metabolite 
 
x 
X 
x 
X 
 
7. 
STATISTICS AND DATA ANALYSIS 
All statistical analysis will be performed in collaboration with Professor Nevill, Professor 
of biomedical statistics at University of Wolverhampton. 
Continuous variables will be compared using parametric and non-parametric tests as 
appropriate. Dichotomous variables will be compared by Fishers exact test. Logistic 
regression analysis will be used to predict the significant independent predictors of poor 
outcome post angiography. 
 
8. 
END OF STUDY 
The study will come to an end when the participant recruitment target has been met, all 
samples have been analysed and the data has been scrutinised, and finally an article 

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summarising the study written and submitted to a peer reviewed journal for consideration 
of publication.  
 
 
 
 

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9. 
ADVERSE EVENTS 
 
9.1 
DEFINITIONS   
 
9.1.1 Adverse Event (AE): any untoward medical occurrence in a patient or clinical 
study subject.   
 
9.1.2 Serious Adverse Event (SAE): any untoward and unexpected medical 
occurrence or affect that: 
 
Results in death 
 
Is life-threatening – refers to an event in which the subject 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 
 
Requires hospitalisation, or prolongation of existing inpatients’ 
hospitalisation 
 
Results in persistent or significant disability or incapacity 
 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other 
situations. Important AEs that are not immediately life-threatening or do not result in 
death or hospitalisation but may jeopardise the subject or may require intervention to 
prevent one of the other outcomes listed in the definition above, should also be 
considered serious. 
 
9.2 
REPORTING PROCEDURES 
 
All adverse will be reported.  Depending on the nature of the event the reporting 
procedures below will be followed. Any questions concerning adverse event reporting 
will be directed to the Chief Investigator in the first instance. 
 
9.2.1 Non serious AEs 
 
All such events, whether expected or not, will be recorded. 
 
9.2.2 Serious AEs 
 
An SAE form will be completed and faxed to the Chief Investigator within 24 hours.  
However hospitalisations for elective treatment of a pre-existing condition do not need 
reporting as SAEs. 
 
Information about all serious adverse events will be collected and recorded on the Serious 
Adverse Event Report Form and faxed to the R&D Directorate at New Cross Hospital 
within 24 hours of learning of its occurrence.  
 
All serious adverse events will be reported to the appropriate regulatory and ethical 
authorities. 

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21
 
 
Contact details for reporting SAEs 
Fax: 01902 695646, attention Dr James Cotton 
Please send SAE forms to: Dr James Cotton 
Tel: 01902 694200 (Mon to Fri 09.00 – 17.00) 
 

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22
10. 
REGULATORY ISSUES 
 
10.1 
ETHICS APPROVAL 
The Chief Investigator has previously obtained approval from the South Birmingham 
Research Ethics Committee (REC ref. 13/WM/0025).   
 
10.2 
CONSENT  
Fully informed consent will be taken from the patient.  
 
10.3 
CONFIDENTIALITY 
 
Blood samples will anonymised with members of the research team at New Cross 
Hospital having access to the patient’s details. All data collected on the patient will be 
stored in an anonymised fashion on and stored on password protected computer at New 
Cross Hospital. Data and all appropriate documentation will be stored for a minimum of 
5 years after the completion of the study, including the follow-up period. 
 
10.4 
INDEMNITY 
 
Standard NHS Indemnity is in place for this study only. 
 
10.5 
SPONSOR 
 
The Royal Wolverhampton NHS Trust has agreed to act as Sponsor for this Study.  
 
10.6 
FUNDING 
 
This study has been part funded by the United Kingdom Clinical Pharmacy Association.  
 
10.7 
AUDITS AND INSPECTIONS  
 
The study will be audited and monitored by the sponsor’s monitoring team. 
 
11. 
STUDY MANAGEMENT 
 
The day-to-day management of the study will be co-ordinated by Dr Vincent Amoah. 
 
Trial Management Committee: 
 
Due to the nature of the study in that this is not a randomised controlled trial, and the 
drugs being used for the study are licenced for this indication and are in use, a Data 
Monitoring Committee will not be required. In this study the Trial Management 
Committee will protect patient safety. If adverse events of a particularly serious type are 
occurring frequently, then the TMC would have to strongly consider termination of the 
study. This evaluation will be made in consideration of risk/benefit.  
 

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12. 
PUBLICATION POLICY 
 
We intend to submit an article detailing the results of the study to a peer review journal for 
consideration of publication. 
 
 
 
 

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24
13. 
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responders and ReSPONDers and the Effect of Switching Therapies: the RESPOND 
Study. Circulation 2010; 121:1188-99. 
 18. Steg PG, James SK, Atar D, et al. ESC Guidelines for the management of acute myocardial 
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 19. Hamm CW, Bassand JP, Agewall S, et al. ESC Guidelines for the management of  
 
acute coronary syndromes in patients presenting without persistent ST-segment  
 
elevation. Eur Heart J 2011; 32: 2999-3054. 

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 20. Stone GW, Witzenbichler B, Guagliumi G, et al. Bivalirudin during primary PCI in acute 
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 22. Frelinger AL, III, Michelson AD, Wiviott SD, et al. Intrinsic platelet reactivity before P2Y12 
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prasugrel or high-dose clopidogrel. Results from PRINCIPLE-TIMI 44. Thromb 
Haemost 2011; 106:219-26. 
 23. Mathur A, Robinson MS, Cotton J, Martin JF, Erusalimsky JD. Platelet reactivity in acute 
coronary syndromes: evidence for differences in platelet behaviour between unstable 
angina and myocardial infarction. Thromb Haemost 2001; 85:989-94. 
 24. Kumana CR, Rambihar VS, Willis K, et al. Absorption and antidysrhythmic activity of oral 
disopyramide phosphate after acute myocardial infarction. Br J Clin Pharmacol 1982; 
14:529-37. 
 25. Jeong YH, Bliden KP, Antonino MJ, et al. Usefulness of the VerifyNow P2Y12 assay to 
evaluate the antiplatelet effects of ticagrelor and clopidogrel therapies. Am Heart J 2012; 
164: 35-42. 
 26. Trenk D, Stone GW, Gawaz M, Kastrati M, Angiolillo DJ, Müller U, Richardt G, 
Jakubowski JA, Neumann FJ. A randomized trial of prasugrel versus clopidogrel in 
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(Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on 
Clopidogrel to Guide Alternative Therapy With Prasugrel) study. J Am Coll Cardiol. 
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 27. Waldmann R, Nieberding M, Walter U. Vasodilator-stimulated protein phosphorylation in 
platelets is mediated by cAMP- and cGMP-dependent protein kinases. Eur J Biochem 
1987; 167:441-8. 
 28. Aleil B, Ravanat C, Cazenave JP, Rochoux G, Heitz A, Gachet C. Flow cytometric analysis 
of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in 
patients with ischemic cardiovascular diseases. J Thromb Haemost 2005; 3:85-92. 
 29. Cotton JM, Worrall AM, Hobson AR, et al. Individualised assessment of response to 
clopidogrel in patients presenting with acute coronary syndromes: a role for short 
thrombelastography? Cardiovasc Ther 2010; 28:139-46. 

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 30. Farid NA, McIntosh M, Garofolo F, et al. Determination of the active and inactive 
metabolites of prasugrel in human plasma by liquid chromatography/tandem mass 
spectrometry. Rapid Commun Mass Spectrom 2007; 21:169-79. 
 31. Payne CD, Li YG, Small DS, et al. Increased active metabolite formation explains the greater 
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Pharmacol 2007; 50:555-62. 
 
 32. Takahashi M, Pang H, Kikuchi A, et al. Stabilization of the clopidogrel active metabolite in 
whole blood and its assay in human plasma by LC-MS/MS. J Am Soc Mass Spectrom 
2006; 17:27S. 
 33. Butler K, Teng R. Pharmacokinetics, pharmacodynamics, safety and tolerability 
 
  of multiple ascending doses of ticagrelor in healthy volunteers. Br J  
 
  Pharmacology 2010; 70; 65-77. 
 34. Sillén H, Cook M, Davis Patty. Determination of ticagrelor and two metabolites in plasma 
samples by liquid chromatography and mass spectrometry. J. Chromatogr. B 2010; 878: 
2299-2306. 
 
 
 
 35. Mehran R, Rao SV, Bhatt DL, et al. Standardized bleeding definitions for cardiovascular 
clinical trials: a consensus report from the Bleeding Academic Research Consortium. 
Circulation 2011; 123:2736-47. 
 36. Bovill EG, Terrin ML, Stump DC, et al. Hemorrhagic events during therapy with 
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APPENDICES: 
 
 

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Appendix 1 – Criteria of major and minor TIMI bleeding (35-37) 
 
1. Major 
 
Any intracranial bleeding (excluding microhaemorrhages <10 mm evident only on 
gradient-echo MRI)  
 
Clinically overt signs of hemorrhage associated with a drop in hemoglobin of ≥5 g/dL 
or a ≥15% absolute decrease in haematocrit  
 
Fatal bleeding (bleeding that directly results in death within 7 days)  
2. Minor 
 
Clinically overt (including imaging), resulting in hemoglobin drop of 3 to <5 g/dL or 
≥10% decrease in haematocrit  
 
No observed blood loss: ≥4 g/dL decrease in the haemoglobin concentration or ≥12% 
decrease in haematocrit  
 
Any overt sign of hemorrhage that meets one of the following criteria and does not 
meet criteria for a major or minor bleeding event, as defined above:  
 
Requiring intervention (medical practitioner-guided medical or surgical 
treatment to stop or treat bleeding, including temporarily or permanently 
discontinuing or changing the dose of a medication or study drug)  
 
Leading to or prolonging hospitalization  
 
Prompting evaluation (leading to an unscheduled visit to a healthcare 
professional and diagnostic testing, either laboratory or imaging)  
 

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Patient admitted with STEMI
Verbal consent given by patient
Group 5
Ticagrelor 
loading dose
                Transfer patient to catheter suite to undergo PPCI procedure
1st blood sample (15 ml)
2nd blood sample (15 ml)
 Transfer patient back to cardiology ward
Written consent given by patient
3rd blood sample (15 ml)
4th blood sample (15 ml)
20 minutes post 
dosing
First balloon inflation
4 hours post dosing
YES
YES
NO
NO
1St, 2nd, and 3rd samples will be 
discarded and the data not 
used for analysis. Treatment 
will continue in accordance 
with clinical guidelines.
Treatment will continue in 
accordance with clinical 
guidelines. 
Each blood sample taken will be subjected to 
analysis using VerifyNow, Flow Cytometry using 
VASP phosphorylation, and liquid 
chromatography with mass spectrometry.
60 minutes post 
dosing
Appendix 2

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     Patient admitted with NSTEMI
Written consent given by 
patient
Group 2 
Group 6
Prasugrel 
loading dose
Ticagrelor 
loading dose
1st blood sample (15 ml)
2nd blood sample (15 ml)
3rd blood sample (15 ml)
20 minutes post 
dosing
60 minutes post 
dosing
4 hours post dosing
YES
NO
Treatment will continue in 
accordance with clinical 
guidelines. 
Each blood sample taken will be subjected to 
analysis using VerifyNow, Flow Cytometry using 
VASP phosphorylation, and liquid 
chromatography with mass spectrometry.
Appendix 3