Clinical trial NCT02486367
Conditions: Aortic Stenosis Inflammation Thrombosis
Sponsor: University Hospitals Cleveland Medical Center
Start date: 2015-06-01; Completion date: 2019-01-01
Enrollment: 60 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The central hypothesis of this study is that TAVR leads to platelet deposition and inflammatory cell activation that can be attenuated by the potent anti-platelet and/or pleiotropic effects of ticagrelor.

This single center, prospective randomized trial addresses the following specific aims:

1. To determine whether high-potency ADP receptor blockade reduces measures of platelet activation in patients after TAVR.
2. To determine whether high-potency ADP receptor blockade mitigates the pro-thrombotic inflammatory response observed after TAVR.

Detailed description:
BACKGROUND

Transcatheter Aortic Valve Replacement (TAVR) has emerged as an important alternative to surgical aortic valve replacement. While this technology represents an important advance over medical therapy or surgical AVR in poor operative candidates, the absolute mortality rates remain high, even in the great majority in whom an optimal hemodynamic result is achieved. In the randomized literature, the majority of these patients die within two years and two thirds of these deaths are due to cardiovascular (CV) events.

The mechanisms responsible for this limited survival are unclear from the clinical trials completed to date. While persistent valve disease undoubtedly plays a role in a subset of patients, particularly in patients with significant aortic regurgitation, the majority of events are due to non-valve related co-morbidities.

The hypothesis of this study is that TAVR results in at least three simultaneous CV insults: 1) the abrupt release of severely elevated left ventricular pressure into a non-compliant systemic vasculature leads to generalized endothelial cell activation, 2) the exposure of the pro-thrombotic and neo-antigenic contents of a degenerated aortic valve (known to histologically resemble atherosclerosis), and 3) the exposure of the replacement valve (bovine valve, stainless steel frame, polyester wrap). The investigators propose that these proximate events lead to platelet activation. Given the important link between thrombosis and inflammation governed by platelet-derived mediators and leukocyte-platelet interactions, they further hypothesize that monocyte activation is mediated, at least in part, by platelet-monocyte interactions, which has been shown to induce the expansion of inflammatory monocytes. Given the pro-thrombotic nature of inflammatory monocytes, they suspect a positive feedback loop may exist via the interplay of these thrombotic -inflammatory mechanisms, which may be abrogated via high potency ADP-receptor blockade.

TRIAL DESIGN Primary Objective of the Study This trial is designed to determine whether high-potency ADP-receptor blockade with ticagrelor, compared to standard care with clopidogrel, affects platelet responsiveness and the pattern of prothrombotic monocyte activation seen early after TAVR.

Primary and Secondary Outcomes The primary endpoint will be platelet responsiveness: platelet function will be measured one day after TAVR using the VerifyNow P2Y12 assay, and expressed in platelet reactivity units. The key secondary outcome measure will be the percentage of inflammatory monocytes, measured one day after TAVR. Inflammatory monocytes will be determined by flow cytometry, and expressed as a percentage of total monocytes.

Arms: arm 1: 300mg load followed by 75mg daily. arm 2: 180mg load followed by 90mg twice daily for 30 days.
Interventions: intervention 1: Clopidogrel intervention 2: Ticagrelor
Intervention descriptions: intervention 1: Standard ADP receptor blockade intervention 2: High potency ADP receptor blockade

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
Inflammation and Thrombosis in Patients With Severe Aortic Stenosis After 
Transcatheter Aortic Valve Replacement (TAVR) 
 
 
NCT02486367 
 
January 31, 2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Supplementary Clinical Study Protocol 
 
Title: Platelet and Monocyte Activation After Transcatheter Aortic Valve Replacement 
(POTENT-TAVR) 
Subtitle: POTENT-TAVR: A Mechanistic Randomized Trial of Ticagrelor versus 
Clopidogrel 
 
Table of Contents 
 
1. Cover page …………………………………………… ……………………………….………2 
2. Synopsis………………………………………………… ……………………………….…….3 
3. Introductory Background…………………………… ………………………………..……..4 
4. Study objectives………………………………………… ………………………………..…..6 
5. Study design………………………………………………… ………………………….……..7 
6. References………………………………………… ………………………………..………...12 
 
 
 
 
 


1. Cover page  
 
Title: Platelet and Monocyte Activation After Transcatheter Aortic Valve Replacement 
(POTENT-TAVR): POTENT-TAVR: A Mechanistic Randomized Trial of Ticagrelor versus 
Clopidogrel 
 
Acronym:  POTENT_TAVR 
Test drug: Ticagrelor 
Active Control: Clopidogrel 
Clinical study phase: Phase 4  
Sponsor: AstraZeneca 
 
Principal Investigator: 
David A. Zidar MD, PhD 
Associate Professor of Medicine 
Case Western Reserve School of Medicine 
Cleveland, OH 44106 
Daz21@case.edu 
 
Compliance Statement: 
The study will be conducted in compliance with the protocol and any applicable regulatory 
requirements. 
 
Confidentiality 
The information provided in this document is strictly confidential and is intended solely for the 
guidance of clinical investigation. Reproduction or disclosure of this document - whether in part 
or in full - to parties not associated with the clinical investigation, or its use for any other 
purpose, without the prior written consent is not permitted.  
 
 
 
 
 


2. Synopsis 
 
Title: Platelet and Monocyte Activation After Transcatheter Aortic Valve Replacement 
(POTENT-TAVR) 
 
Clinical study phase: Phase 4 
Study objective(s): 1. To determine whether high-potency ADP receptor blockade reduces 
measures of platelet activation in patients after TAVR.  2. To determine whether high-potency 
ADP receptor blockade mitigates the pro-thrombotic inflammatory response observed after 
TAVR.   
Test drug: Ticagrelor 
Dose: 90mg twice daily 
Route of administration: Oral 
Duration of treatment: The first dose of Ticagrelor will be started (180mg load) the morning of 
planned TAVR. Treatment will continue for 30 days.   
Active Comparator: Clopidogrel 
Dose: 75 mg once daily 
Route of administration: Oral 
Duration of treatment: The first dose of Clopidogrel will be given (300 mg load if P2Y12 naïve 
or 75mg if P2Y12 non-naive) the morning of planned TAVR. Treatment will continue for 30 days.   
Background treatment: Cardiovascular medications will be continued without planned dose 
titration as per standard after TAVR.  Low dose aspirin 81mg once daily will be initiated on the 
day of TAVR or continued as pre-existing therapy.   
Indication:  Aortic Stenosis and planned transcatheter aortic valve replacement (TAVR). 
Primary Endpoints: P2Y12 platelet responsiveness (PRU) using the VerfiyNow assay at 1 day 
post procedure and the percentage of inflammatory (CD14+CD16+) monocytes (compared to 
total monocytes) 1 day after TAVR.  
Key inclusion criteria: 1) Valvular heart disease and a clinical indication for TAVR, 2) Age of 
18 years or older, 3) Capable of informed consent.  4) Planned transfemoral TAVR. 
Key exclusion criteria: 1) Prior history of stroke, transient ischemic attack (TIA), or intracranial 
hemorrhage, 2) Established bleeding diathesis or thrombocytopenia (<150k/dl), 3) End-stage 
renal disease, 4) Severe hepatic impairment or liver cirrhosis, 5) Pregnancy, 6) Current 
infection, 7) History of autoimmune disease, 8) Established allergy to contrast agents, 
thienopyridines, aspirin, or ticagrelor, 9) History of solid organ transplantation, 10) Atrial 
Fibrillation, DVT, PE or other indication for long term anti-coagulation, 11) Plan for direct aortic 
access or trans-apical TAVR, 12) Enrollment in another clinical trial, 13) Recent (< 12 months) 
or active excessive bleeding. 
Type of control:  Active treatment 
Clinical Event Committee: No 
Data Safety Monitoring Board Yes 
Number of subjects: A total of 60 subjects will be randomized. 
 
 
 
 
 
 
 


3. Introductory Background 
Transcatheter Aortic Valve Replacement (TAVR) has emerged as an important 
alternative to surgical aortic valve replacement.  While this technology represents an important 
advance over medical therapy or surgical AVR in poor operative candidates, the absolute 
mortality rates remain high, even in the great majority in whom an optimal hemodynamic result 
is achieved.  In the randomized literature, the majority of these patients die within two years and 
two thirds of these deaths are due to cardiovascular (CV) events1.    
The mechanisms responsible for this limited survival are unclear from the clinical trials 
completed to date.  While persistent valve disease undoubtedly plays a role in a subset of 
patients, particularly in patients with significant aortic regurgitation, the majority of events are 
due to non-valve related co-morbidities.   
Our multi-disciplinary group includes members with broad expertise in cardiovascular 
and immunologic clinical research.  The over-arching aim of our collaborative work to date is to 
identify shared mechanisms of inflammation and thrombosis that correlate with cardiovascular 
disease.   Recent work from our group has focused on the prothrombotic phenotype of activated 
inflammatory monocytes which are preferentially expanded in both HIV and ACS2.   
Clopidogrel treated patients following TAVR have a 64.3% rate of platelet 
hyporesponsiveness (VerifyNow assay, hyporesponsiveness defined as PRU <208)3.  In this 
post-TAVR population (n=28) the median PRU was 244.5 (interquartile range 164.8-290.3).  In 
preliminary experiments, we find a profile 
of immune activation after TAVR 
characterized by the expansion of 
CD14+CD16+ inflammatory monocytes, a 
marker we and others have found 
correlates with adverse cardiovascular 
events 2,4.  Of note, we have previously 
demonstrated that inflammatory 
monocytes express high levels of tissue 
factor and this feature also correlates with 
ACS2.  Thus, post-TAVR patients 
represent a patient population in which 
high-potency ADP receptor blockade may 
provide protection against MACE. 
 
Hypothesis 
 
Our hypothesis is that TAVR results in at least three simultaneous CV insults: 1) the 
abrupt release of severely elevated left ventricular pressure into a non-compliant systemic 
vasculature leads to generalized endothelial cell activation, 2) the exposure of the pro-
thrombotic and neo-antigenic contents of a degenerated aortic valve (known to histologically 
resemble atherosclerosis), and 3) the exposure of the replacement valve (bovine valve, 
stainless steel frame, polyester wrap).  We propose that these proximate events lead to platelet 
activation.  Given the important link between thrombosis and inflammation governed by platelet-
derived mediators and leukocyte-platelet interactions, we further hypothesize that monocyte 
activation is mediated, at least in part, by platelet-monocyte interactions, which has been shown 
to induce the expansion of inflammatory monocytes5.  Given the pro-thrombotic nature of 
inflammatory monocytes, we suspect a positive feedback loop may exist via the interplay of 
these thrombotic –inflammatory mechanisms, which may be abrogated via high potency ADP-
receptor blockade (Figure 1).  
If our study results indicate an improved thrombotic and immune convalescence with 
Ticagrelor, we would conclude that the mechanism of inflammation after TAVR is mediated in 
Figure 1. Mechanistic hypothesis of TAVR leading to 
platelet activation and inflammation  
TAVR
Vascular Injury
Endothelial Activation
Native Aortic Valve Disruption
Neoantigen Exposure
Platelet Activation
Immune Activation
P2Y12


part by platelet activation regulated by the P2Y12 receptor.  This would form the basis for 
additional study of the clinical effects of P2Y12 blockade post TAVR, powered for clinical 
events.  Importantly, the findings of this study might also provide mechanistic proof of principle 
for additional study of P2Y12 blockade in other disorders that carry elevated CV risk in the 
setting of chronic immune activation.  One such population is the vast majority of HIV patients, 
who achieve controlled viremia but are left with chronic immune activation and elevated CV 
risk6.      
 
Study Drug Background: Ticagrelor 
FDA Label: https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/022433s015lbl.pdf
 
Ticagrelor is approved by the United States Federal Drug Administration (FDA) to 
reduce the rate of cardiovascular death, myocardial infarction, and stroke in pateints with acute 
coronary syndrome or a history of myocardial infarction. Ticagrelor was studied in the PLATO 
trial which included patients with acute coronary syndrome, randomized to Ticagrelor or 
Clopidogrel for 12 months.  At 12 months, a composite of death from vascular causes, 
myocardial infarction, or stroke (the primary endpoint) had occurred in 9.8% of patients 
receiving ticagrelor as compared with 11.7% of those receiving clopidogrel (hazard ratio, 0.84; 
95% confidence interval [CI], 0.77 to 0.92; P<0.001). No significant difference in the rates of 
major bleeding was found between the ticagrelor and clopidogrel groups (11.6% and 11.2%, 
respectively; P=0.43), but ticagrelor was associated with a higher rate of major bleeding not 
related to coronary-artery bypass grafting (4.5% vs. 3.8%, P=0.03), including more instances of 
fatal intracranial bleeding and fewer of fatal bleeding of other types. 
 
  
 
 


4. Study objectives 
Transcatheter aortic valve replacement (TAVR) has been associated with reductions in 
CV morbidity and death in extreme risk/surgery inadvisable patients, but cardiovascular events 
rates remain unacceptably high, with mortality rates of 30% and 50% at 1 and 2 years, 
respectively1,7.  Thus, even after successful treatment of aortic stenosis, this population persists 
with a risk of CV mortality that is 5-fold greater than cohorts studied in contemporary myocardial 
infarction trials such as PLATO8.  
 
The mechanisms and predictors of adverse outcomes following TAVR are poorly 
understood.  Among the most important predictor of poor outcomes is peri-procedural stroke, 
which is associated with a hazard ratio of 3.5 for mortality at 30 days9.  Since 45% of cerebral-
vascular accidents (CVA) occur beyond 24 hours post procedure10, optimizing anti-thrombotic 
therapies in the immediate post-procedure period may be a feasible strategy to reduce CVA 
following TAVR.  However, the majority of major adverse cardiovascular events (MACE) 
following TAVR remains unexplained and thus, a better understanding of the mediators of CV 
risk is urgently needed in this high risk population. The role of anti-platelet therapy in this patient 
population is also uncertain.  While dual anti-platelet therapy with aspirin and clopidogrel is 
typically recommended for a period of 6 months, its effectiveness in the prevention of adverse 
outcomes post-TAVR is unknown11. 
In patients with cardiovascular disease, among the strongest and most consistent 
surrogates of poor long term outcome is clopidogrel non-responsiveness.  This is typically 
identified in 20-30% of patients with acute coronary syndromes (ACS) and is associated with an 
increased risk of MACE12-15.  Although the prevalence of clopidogrel non-responsiveness in 
patients with aortic stenosis has not been previously reported, preliminary data from our group 
indicates a high rate of clopidogrel hyporesponsiveness (64.3%) in our post-TAVR population. 
Another marker of poor prognosis in patients with cardiovascular disease is elevated 
proportions of CD14+CD16+ inflammatory monocytes.  In an unselected cardiac catheterization 
population, elevated CD14+CD16+ inflammatory monocytes were associated with a hazard 
ratio of 3.1 for future death, MI, or stoke4.  Our studies have recently shown that these 
monocytes express high levels of tissue factor and are dynamically regulated in the setting of 
both HIV and ACS2.  Interestingly, we will now report that TAVR is associated with a robust, 
inflammatory response characterized by a marked expansion of CD14+CD16+ inflammatory 
monocytes, evident even within 24 hours of an uncomplicated procedure.    
The central hypothesis of our proposal is that TAVR-induced injury of the 
diseased native aortic valve leads to platelet deposition and inflammatory cell activation 
that can be attenuated by the potent anti-platelet and/or pleiotropic effects of ticagrelor.  
We propose a single center, prospective randomized trial to test the following specific aims: 
1.  To determine whether high-potency ADP receptor blockade reduces measures 
of platelet activation in patients after TAVR.   
2. To determine whether high-potency ADP receptor blockade mitigates the pro-
thrombotic inflammatory response observed after TAVR.   
The co-primary study endpoints will be 1) platelet responsiveness as measured by the 
VerifyNow assay (platelet reactivity units, PRU) and 2) the proportion of inflammatory 
monocytes by polychromatic flow cytometry 1 day after TAVR.  Secondary endpoints will 
include other measures of thrombotic and immunologic activation.   
 
 


5. Study design 
 
This is a single center, prospective, randomized, open label trial with blinded endpoint 
evaluation.  
 
5.1 Screening, entry, and randomization:  
 
Patients will be screened for 
inclusion/exclusion criteria as part of their pre-
TAVR clinical evaluation (See Table 1).  We 
plan to screen 150 patients per year, of whom 
100 are expected to go on to TAVR.  We will 
continue enrollment until 60 patients are 
randomized.  All patients will be required to sign 
informed consent prior to planned TAVR.  The 
scheme for a participant’s movement through 
the trial is shown in figure 2.  
 
Patients will be randomized in a 1:1 
fashion (n=60) to either continue Clopidogrel 
(300mg load, 75mg daily) or begin Ticagrelor.  
Patients will receive a 180mg load followed by 
90mg twice daily for one month.  This will be an 
open label design given the different frequency 
of dosing.  Decisions regarding the 
discontinuance of dual anti-platelet therapy after 
randomization will be at the discretion of the 
treating physician.   
 
Patients will be seen at 30 days post 
TAVR per routine clinical practice.   At this time, 
the final blood draw will be obtained.  The 
occurrence of MACE and bleeding events will be determined by history and corroborated by 
examining relevant medical records.  Patients will revert to clopidogrel therapy at this time and 
the duration of dual anti-platelet therapy will be left to the discretion of the treating physician.   
 
5.2 Blood Biomarker Procurement:   
 
Patients will receive a baseline blood draw prior to TAVR.  Patients will then have blood 
draws at 24 hours, 7 days, and 30 days post procedure.  Patients will be seen in followup 
according routine clinical practice at 7 and 30 days.  All blood samples will be obtained using a 
30 mL syringe which is then transferred into tubes containing 3.2% sodium citrate (blue top) or 
K2-EDTA (purple top).  Aliquots of whole blood will be used for platelet responsiveness studies, 
monocyte flow cytometry, leukocyte-platelet interaction, and T cell activation studies.  Plasma 
and Peripheral blood mononuclear cells (PBMC) will be cryopreserved at -80 and -150 degrees, 
respectively.  Plasma based assays will be batched and performed at intervals using freshly 
thawed cryopreserved samples. 
 
5.3 Study Endpoints and Statistical Plan   
 
All endpoints will be determined according to an intention to treat analysis.  The primary 
thrombotic endpoint will be platelet responsiveness (PRU) using the VerfiyNow assay at 1 day 
post procedure.   Platelet reactivity in the Clopidogrel and Ticagrelor groups with be compared 
TABLE 1. Inclusion/Exclusion Criteria 
 
Inclusion criteria:  
1) Valvular heart disease and a clinical indication for 
TAVR,  
2) Age of 18 years or older,  
3) Capable of informed consent.   
4) Planned transfemoral TAVR. 
  
Exclusion criteria:  
1) Prior history of stroke, transient ischemic attack 
(TIA), or intracranial hemorrhage,  
2) Established bleeding diathesis or 
thrombocytopenia (<150k/dl),  
3) End-stage renal disease,  
4) Severe hepatic impairment or liver cirrhosis,  
5) Pregnancy,  
6) Current infection,  
7) History of autoimmune disease,  
8) Established allergy to contrast agents, 
thienopyridines, aspirin, or ticagrelor,  
9) History of solid organ transplantation,  
10) Atrial Fibrillation, DVT, PE or other indication for 
long term anti-coagulation,  
11) Plan for direct aortic access or trans-apical 
TAVR,  
12) Enrollment in another clinical trial,  
13) Recent (< 12 months) or active excessive 
bleeding. 
  


using an independent samples t-test (or non-parametric test 
as appropriate), with significance declared for p-values 
<0.025.  The primary inflammatory endpoint is the 
percentage of inflammatory monocytes (compared to total 
monocytes) as a continuous variable 1 day after TAVR.   
The percentage of inflammatory monocytes in the 
Clopidogrel and Ticagrelor groups with be compared using 
an independent samples t-test (or non-parametric test as 
appropriate), with significance declared for p-values <0.025.  
The percentage of inflammatory monocytes will also be 
compared over multiple time points to determine the 
trajectory and duration of the immune response, to guide 
duration of treatment in future studies.  The values at 
baseline, 1 day, and 7 day will be compared within group 
using exploratory longitudinal mixed models to determine 
the kinetics of convalescence and between groups to 
establish whether treatment effects exist.  Models that 
include the 30-day values also will be fit.  Time period will 
be considered both as a continuous covariate (in days), as 
well as a categorical variable.  Inferential interest will focus 
on treatment by time interaction.  These models will also be 
used to explore the relationship between the level of P2Y12 
inhibition and the reduction in inflammatory monocytes 
between treatments. This relationship could be different 
given that Ticagrelor is not only a more potent P2Y12 
antagonist as compared with clopidogrel but may also have 
a unique dual mode of action; P2Y12 antagonism complemented with adenosine uptake 
inhibition.  
 
Secondary measures of thrombotic and immune status will be crucial for the 
determination of mechanism and to guide future studies.  These will include: platelet-leukocyte 
aggregates, platelet P-selectin and activated GPIIb/IIIa expression, markers of T-cell activation 
(i.e., HLA-DR/CD38 co-expression), and plasma markers of inflammation (CRP, IL-6, TNF-a, IL-
6, sCD14, CD40L, oxidized LDL) and thrombosis (thrombin-antithrombin complexes, F1.2, 
soluble P-selectin, S100A8/A9, D-dimers).  These secondary measures will be analyzed as 
continuous variables at baseline, 1 day, 7 days, and 30 days.  PBMC will be isolated and will be 
used to assess T cell activation as continuous variables using the percentage of HLA-
DR+/CD38+ CD4 and CD8 T-cells.  These values at the specified time points will also be 
compared within group using longitudinal mixed models to determine their kinetics and between 
groups to establish whether a treatment by time interactions exists.   
 
5.4 Time point/frame of measurement for primary variable(s) 
The primary endpoints will be assessed 1 day after study drug initiation and TAVR. 
Measurements taken at 7 and 30 days after TAVR will be considered secondary/exploratory.   
 
5.5. Study variable(s) 
5.5.a Clinical/Demographic Variables:  Demographic and clinical variables will be captured by 
interview and electronic chart review at study entry. 
Patients screened and enrolled
TAVR
Pre-procedural blood draw
Randomization
Ticagrelor
Clopidogrel
1 day post procedural blood draw
7 day post procedural blood draw
Study Completion
30 day post procedural blood draw
Figure 2. Trial Design Schematic 


5.5.b P2Y12 Platelet Reactivity: Platelet reactivity to adenosine diphosphate (ADP) will be 
measured as platelet reactivity units (PRU) using the VerifyNow assay (Accriva Diagnostics, 
San Diego, California) using whole blood collected in a device- compatible citrated tube.   
 
5.5.c Whole Blood Flow Cytometric Analysis of Patient Samples: 
 Blood samples will be lysed using BD FACS lysing solution (BD, Franklin Lakes, NJ), washed, 
and stained as Table 2.  Compensation will be performed using standard compensation beads 
(BD Biosciences) and appropriate isotype control antibodies.  Flow cytometry will be performed 
using a MACSQUANT analyzer. Monocyte phenotypes (classical, non-classical inflammatory 
CD14+CD16+, 
non-classical 
patrolling 
CD14lowCD16++) 
will be analyzed by 
whole blood flow 
cytometry using 
antibodies to CD14 
and CD16 as we 
have previously 
described16. 
Specifically, the % 
inflammatory 
monocytes is the 
proportion of 
inflammatory 
monocyte to the 
sum of classical, inflammatory and patrolling monocytes.  The definition of CD16 positive versus 
negative gating will be based upon a pan-isotype panel.  Final values for % inflammatory 
monocytes will be the mean of the duplicates obtained using panel A and panel B.  Monocyte-
CD62P+ platelet aggregation will be analyzed as the mean fluorescence intensity (MFI) of P-
selection (Monocyte Panel A) and Monocyte- GP IIb/IIIa+ platelet aggregation will be analyzed 
as the percentage of monocytes which exhibit positive staining for activated IIb/IIIa (PAC-1 
antibody- Monocyte Panel B), with total monocytes as the denominator. 
 
5.5.d Blood Collection and Isolation of Peripheral Blood Mononuclear Cells and Plasma: 30 mL 
blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA; BD Vacutainer, 
Franklin Lakes, NJ) and 2 mL in tubes containing 3.2% sodium citrate (BD Vacutainer, Franklin 
Lakes, NJ). Samples were then maintained at room temperature until processing. Plasma was 
isolated from the EDTA blood through centrifugation and preserved at -80 degrees Celsius.   
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque Plus (Sigma, St. 
Louis, MO) density gradient centrifugation, washed, re-suspended in 90% fetal bovine serum 
(FBS; Gemini, West Sacramento, CA) and 10% dimethylsulfoxide (DMSO; Sigma, St. Louis, 
MO), and cryopreserved at -150 degrees F. 
5.5.e Plasma Cytokine and Immune Biomarkers: Plasma based assays are done using 
commercially available ELISA and cytokine bead array technology (BD Biosciences).  
 
5.6 Power Calculation and Sample Size Estimate 
Monocyte Panel A 
Target 
Fluorochrome 
Vendor 
Catalog Number 
Clone 
Dilution 
CD14 
Pacific Blue 
BD Bioscience 
558121 
M5E2 
1:50 
Tissue Factor 
FITC 
Biomedica Diagnostics 
4507CJ 
23C11 
1:50 
CD16 
PE 
BD Bioscience 
555407 
3G8 
1:50 
CD62p 
Pe-Cy5 
BD Bioscience 
551142 
AK-4 
1:50 
CD69 
Pe-Cy7 
BD Bioscience 
335792 
L78 
1:50 
CX3CR1 
APC 
BioLegend 
341610 
29A-1 
1:50 
HLA-DR 
APC-Cy7 
BD Bioscience 
335796 
L243 
1:50 
Monocyte Panel B 
Target 
Fluorochrome 
Source / Vendor 
Catalog Number 
Clone 
Dilution 
CD14 
Pacific Blue 
BD Bioscience 
340507 
M5E2 
1:50 
GP IIb/IIIa 
FITC 
BD Bioscience 
347363 
PAC-1 
1:50 
MRP8/14 
PE 
Novus Biologicals  
NBP2-25269PE 
48M7C7 
1:250 
CD41 
Pe/Cy5 
BioLegend 
303708 
HIP8 
1:50 
CD16 
Pe-Cy7 
BD Bioscience 
557744 
3G8 
1:50 
CX3CR1 
APC 
BioLegend 
341610 
29A-1 
1:50 
CD62l 
APC-eFluor780 
eBioscience 
47-0629-42 
DREG-56 
1:50 


Our power calculation is based upon the inflammatory co-primary endpoint, using our 
preliminary data.  In preliminary studies of 25 patients undergoing TAVR, the mean proportion of 
inflammatory monocytes 1 days after TAVR was 20.0% with a standard deviation of 5.0 %.  
Thus, a total of 60 patients, randomized 30:30 would be expected to have 80% power to detect 
an absolute effect of 4% (i.e. ticagrelor treated patients having 16 % percent inflammatory 
monocytes), using a two sided t-test with Type I error of 0.025.  Note that we are using 
Bonferonni correction, to account for two primary outcomes.  We expect dropout of randomized 
patients to be less than 10% prior to the 1 day blood draw.  All randomized patients will be 
analyzed using an intention to treat analysis.  We will assess the missing completely at random 
assumption by comparing results with analyses derived by assuming that the data is missing at 
random (MAR), and use of multiple imputation approaches. 
 
5.7 Interruption or Discontinuation of treatment 
Neither erroneous randomization of subject in the presence of exclusion criteria or non-fulfilment 
of inclusion criteria nor a post-randomization change in health status that results in 
the subject meeting one or more of the exclusion criteria should automatically lead to the 
interruption or permanent discontinuation of the assigned therapies, unless continuing the 
medications places the subject at undue risk as determined by the investigator. Such 
situations though very rare, may occur and should be handled on a case-by-case basis. 
Compliance with study drug medication will be assessed at day 1, 7, and 30.  Drug supplies for 
each subject will be inventoried and reconciled throughout the study. Any discrepancies 
between actual and expected amount of study medication will be discussed with the subject at 
the time of the visit, and any explanation will be documented in the source records and captured 
in the eCRF. Subjects will return empty study drug containers and unused study drug visits. 
Study assigned therapies may be temporarily discontinued at the discretion of the treating 
physician. Subjects can resume the assigned therapy as soon as the treating physician 
considers it safe to do so. The reason for discontinuation of the assigned therapy will be 
recorded in the eCRF and in the subject's medical records. The decision to restart or 
permanently withdraw study medication is at the discretion of the investigator. 
 
5.8 Treatment assignment 
Randomization will be performed via seal envelop system. Opaque envelopes (60) will be filled 
with a sheet of paper in which Ticagrelor (30) or Clopidogrel (30) is written and envelops are to 
be opened after informed consent is received and on or before the start of the TAVR such that 
study drug initiation occurs prior to TAVR.  Randomization envelops are stored securely at the 
Clinical trials office at University Hospitals (UH).  
 
5.9 Blinding 
This trial is an open label trial and patients are not blinded. The laboratory staff will receive 
study samples containing patient study identification numbers but without any identifiers with 
regard to treatment assignment.  Similarly, the analysis of flow cytometric data will be done by 
laboratory staff blinded to treatment assignment.   
 
5.10. Drug logistics and accountability 
Ticagrelor and Clopidogrel will be stored at the investigational pharmacy at University Hospitals 
Cleveland Medical Center in accordance with GCP and GMP requirements and according to the 


product label. Receipt, distribution, return, and destruction (if any) of study medications will be 
properly documented. 
 
5.11 Prior and concomitant therapy 
All ongoing medications at screening and used during the study (prescription and over-the 
counter), including adjunct therapy or medical devices, will be recorded in the appropriate 
section of the eCRF throughout the study. Use of ASA and clopidogrel or any other P2Y12 
inhibitor prior and during TAVR procedure should also be recorded. It is advised that the 
appropriate local and national guideline recommendations are followed for any concomitant 
medications. Concomitant medication will be evaluated when considering AE causal 
relationship.  
 
5.12 End of study 
The end of the study as a whole will be reached 2 years after the last subject has 
been enrolled.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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