The Thromboxane Receptor Antagonist to Block the Effects of Non-Platelet Thromboxane Generation and Improve Endothelial Function (TRAP) Trial
Phase: PHASE2
Conditions: Cardiovascular Diseases, Vascular Dilation
Sponsor: Jeffrey Rade

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 1 
 
 
 
CLINICAL STUDY PROTOCOL 
 
The Thromboxane Receptor Antagonist to Block the Effects of Non-Platelet 
Thromboxane Generation and Improve Endothelial Function (TRAP) Trial 
 
 
Protocol Number: 
H00017901 
 
Principal Investigator: 
Jeffrey J. Rade, MD 
University of Massachusetts Medical School 
65 Lake Avenue North, Room H2-46, 
Worcester, MA 01655 
Phone: 774-441-6310 
FAX: 774-441-6305 
jeffrey.rade@umassmed.edu 
 
Investigational Drug: 
Ifetroban Oral Capsule 
IND Number: 
138088 
 
ClinicalTrials.gov Identifier: 
NCT03962855 
 
Study Sponsor: 
American Heart Association 
7272 Greenville Avenue 
Dallas, TX 75231 
 
Protocol Version Date: 
 August 16, 2022 
 
 
 
 
THIS DOCUMENT IS CONFIDENTIAL AND FOR THE SOLE INFORMATION AND USE OF 
THE INVESTIGATOR’S RESEARCH TEAM AND THE APPOINTED INSTITUTIONAL 
REVIEW BOARD. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 2 
 
 
Approvals and Revision History 
 
 
 
Approved By 
Name 
Title 
Signature 
Date 
Jeffrey J. Rade, MD 
Professor of Medicine 
October 31, 2020 
 
 
 
 
 
 
Revision History 
Version Number 
Date 
01 
May 20, 2019 
Initial Release 
02 
July 29, 2019 
Amendment 1 
03 
February 3, 2020 
04 
October 31, 2020 
05 
August 16, 2022 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 3 
 
 
Table of Contents 
Approvals and Revision History ..................................................................................................... 2 
Table of Contents ............................................................................................................................ 3 
PROTOCOL SUMMARY .................................................................................................................... 6 
ABBREVIATIONS ............................................................................................................................... 9 
1 
Background ............................................................................................................................11 
1.1 
Overview of Thromboxane Biology ..................................................................................11 
1.2 
Thromboxane Receptor Biology ........................................................................................11 
1.3 
TXA2  Generation and Clinical Outcome .......................................................................... 12 
1.4 
Factors Associated with Non-platelet TXA2 Generation .................................................. 13 
1.5 
Oxidative Stress Stimulates TXA2  Generation in Endothelial Cells ................................. 14 
1.6 
Non-platelet Thromboxane Generation and Atherothrombosis ........................................ 15 
1.7 
Renal Effects of TXA2  and TPr Inhibition ........................................................................ 16 
1.8 
Summary of Mechanistic Hypothesis Tested in this Study ............................................... 16 
2 
Ifetroban. ............................................................................................................................... 17 
2.1 
Pharmacology.................................................................................................................... 17 
2.2 
Pre-Clinical and Human Studies ....................................................................................... 17 
2.3 
Bleeding Risks .................................................................................................................. 17 
2.4 
Effect on Blood Pressure and Renal Function .................................................................. 18 
2.5 
Heart Failure ..................................................................................................................... 18 
3 
Study Objectives.................................................................................................................... 18 
4 
Study Design ......................................................................................................................... 19 
4.1 
Screening Phase ................................................................................................................ 19 
4.2 
Randomization .................................................................................................................. 19 
4.3 
Study Interventional Phase ................................................................................................ 19 
4.3.1 
Study Visit 1 (Baseline Day 0, Outpatient) ....................................................................19 
5 
Study Population and Eligibility Criteria .............................................................................. 20 
5.1 
Study Population ............................................................................................................... 20 
5.2 
Inclusion Criteria ............................................................................................................... 20 
5.3 
Exclusion Criteria ............................................................................................................. 20 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 4 
 
 
6 
Recruitment and Screening Procedures ................................................................................. 21 
6.1 
Common Recruitment Procedures .................................................................................... 21 
6.2 
Estimated Enrollment Period ............................................................................................ 21 
6.3 
Informed Consent Procedures ........................................................................................... 21 
6.4 
Confidentiality and HIPAA Requirements ........................................................................ 21 
6.5 
Protection of Human Subjects ........................................................................................... 21 
6.6 
Summary of Risks and Benefits ........................................................................................ 21 
6.6.1 
Risks ...............................................................................................................................21 
7 
Study Drug Handling ............................................................................................................. 22 
7.1 
Drug Dispensing ............................................................................................................... 22 
7.2 
Drug Storage, Accountability and Destruction ................................................................. 22 
7.3 
Emergency Unblinding ..................................................................................................... 22 
8 
Outcome Determinations....................................................................................................... 22 
8.1 
Primary Endpoint .............................................................................................................. 22 
8.2 
Secondary Endpoints ......................................................................................................... 22 
8.3 
Exploratory Endpoints ...................................................................................................... 23 
9 
Subject Safety ........................................................................................................................ 23 
9.1 
Institutional Review Board ............................................................................................... 23 
9.2 
Food and Drug Administration ......................................................................................... 23 
9.3 
Independent Clinical Event Adjudicator ........................................................................... 23 
9.4 
Adverse Events ................................................................................................................. 24 
9.4.1 
Definitions ......................................................................................................................24 
9.4.2 
Recording and Reporting of Adverse Events .................................................................25 
9.4.3 
Suspected Unexpected Serious Adverse Reaction .........................................................26 
10 Statistical Considerations ...................................................................................................... 26 
10.1 
Sample Size Calculations .................................................................................................. 26 
10.1.1 
Primary Endpoint ...........................................................................................................26 
10.1.2 
Secondary Endpoints ......................................................................................................26 
10.2 
Statistical Analysis Plan .................................................................................................... 27 
11 Data Management ................................................................................................................. 27 
12 Regulatory Issues .................................................................................................................. 27 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 5 
 
 
12.1 
Ethics and Good Clinical Practice .................................................................................... 27 
12.2 
Institutional Review Board ............................................................................................... 27 
12.3 
Informed Consent .............................................................................................................. 27 
12.4 
Quality Control and Assurance ......................................................................................... 28 
13 References ............................................................................................................................. 29 
14 Appendix A: Table of Study Events ...................................................................................... 34 
15 Appendix B: Brachial Reactivity .......................................................................................... 35 
15.1 
Introduction ....................................................................................................................... 35 
15.2 
Protocol ............................................................................................................................. 35 
15.2.1 
Subject preparation ........................................................................................................35 
15.2.2 
Conduct of the Brachial Reactivity Study ......................................................................36 
15.3 
Representative Results ...................................................................................................... 37 
16 Appendix C: Peripheral Arterial Tonometry ......................................................................... 37 
16.1 
Introduction ....................................................................................................................... 37 
16.2 
Protocol ............................................................................................................................. 38 
16.2.1 
Subject Preparation ........................................................................................................38 
16.2.2 
Prepare the Endo-PAT System for Study ........................................................................39 
16.2.3 
Conduct of the Endo-PAT Study ....................................................................................39 
16.2.4 
Review and Analysis ......................................................................................................40 
16.3 
Representative Results ...................................................................................................... 41 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 6 
 
 
PROTOCOL SUMMARY 
 
 
Title of Study 
The Thromboxane Receptor Antagonism to Blunt the Effects of Non- 
Platelet Thromboxane Generation and Improve Endothelial Function 
(TRAP) Trial 
Protocol Number 
H00017901 
Investigational Drug Ifetroban Oral Capsules 
Drug Manufacturer 
Cumberland Pharmaceuticals Inc. 
Objectives 
The objectives of this study are: 
To determine if thromboxane receptor antagonism improves 
endothelial function in subjects with non-platelet thromboxane 
generation. 
To determine if thromboxane receptor antagonism reduces non- 
platelet thromboxane generation. 
To determine if thromboxane receptor antagonism reduces oxidative 
stress, inflammation and thrombotic potential. 
To determine if thromboxane receptor antagonism alters renal 
prostaglandin secretion and improves renal function. 
Study Design 
This is a phase 2b, single center, prospectively randomized, double- 
blinded, placebo-controlled clinical trial conducted at the UMass 
Medical School/UMass Memorial Medical Center. 
Subjects with cardiovascular disease on standard aspirin therapy with 
elevated non-platelet thromboxane generation defined by urine 
thromboxane B2 metabolites >1145 pg/mg creatinine will be 
randomized to receive either ifetroban 250 mg capsules once daily or 
matching placebo. 
Number of Subjects 
57 subjects randomized 1:1 to ifetroban or placebo. 
Treatment Duration 
4 weeks 
Study Endpoints 
The primary trial endpoint will be the change from baseline in 
Reactive Hyperemia Index (RHI) measured by peripheral arterial 
tonometry (PAT). 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 7 
 
 
 
Secondary trial endpoints will be the change from baseline in on- 
treatment: 1) % flow-mediated vasodilation (FMD),and; 2) urine 11- 
dhTXB2. 
Exploratory trial endpoints will be the change from baseline in on- 
treatment: 1) urine 8-iso-PFG2α; 2) plasma tissue factor; 3) plasma 
ICAM-1; 4) plasma activated protein C; 5) hs-CRP; 6) NT pro-BNP; 
7) urine 2,3-dinor-6-ketoPGF1α and ratio of 11-dhTXB2 to 2,3-dinor- 
6-ketoPGF1α; 8) urine TXB2, 6-ketoPGF1α and the ratio of TXB2 to 6- 
ketoPGF1α, and; 9) glomerular filtration rate. 
Inclusion Criteria 
Males and females 18-80 years of age with established cardiovascular 
disease who take ≥81 mg daily of aspirin and have thromboxane B2 
metabolites >1145 pg/mg creatinine on screening. 
Able to provide written consent and comply with protocol-specific 
procedures. 
Exclusion Criteria 
Chronic oral anticoagulation with non-vitamin K antagonist. 
Anticipated change or interruption in aspirin therapy during the study 
period. 
ST segment myocardial infarction within the past 30 days. 
Cardiac surgery within the past 30 days. 
Stage 4-5 renal failure or on renal replacement therapy. 
An ongoing uncontrolled severe inflammatory condition. 
Pregnant, intending to become pregnant or breast feeding. 
Known ifetroban or aspirin sensitivity 
Inability to perform vascular testing. 
Participation in another investigational drug trial within 30 days of 
randomization. 
Safety Evaluation 
Safety assessments will be performed at all study visits and will 
include physical examination and vital signs (blood pressure, pulse 
rate, respiration rate). 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 8 
 
 
 
Statistical Methods 
Descriptive statistics of patient characteristics will be generated using 
conventional methods. Continuous data will be compared between 
treatment groups using ANCOVA. Dichotomous data will be 
compared using chi-square analysis with a Yate’s correction. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 9 
 
 
 
 
Abbreviation Definition 
ABBREVIATIONS 
8-iso-PGF2α 
isoprostane generated by free radical peroxidation of arachidonate 
AA 
arachidonic acid 
ACS 
acute coronary syndrome 
ADP 
adenosine diphosphate 
AE 
adverse event 
AERD 
aspirin exacerbated respiratory disease 
ANCOVA 
analysis of covariance 
ASA 
acetylsalicylic acid, aspirin 
BMS 
Bristol Myers Squibb 
BUN 
blood urea nitrogen 
CABG 
coronary artery bypass graft 
CAD 
coronary arterial disease 
CEA 
CHARISMA 
Clinical Events Adjudicator 
Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, 
Management and Avoidance 
COX 
cyclooxygenase one (prostaglandin-endoperoxide synthase or PTGS) 
CPI 
Cumberland Pharmaceuticals Inc. 
CVD 
cardiovascular disease 
EC 
ECC 
endothelial cell 
endocardial endothelial cell 
ELISA 
enzyme-linked immunosorbant assay 
FDA 
Food and Drug Administration 
FHS 
FMD 
Framingham Heart Study 
flow mediated dilation 
FRIMS 
Flexible Randomization and Inventory Management System 
GFR 
glomerular filtration rate 
HOPE 
Heart Outcomes Prevention Evaluation 
HR 
hazard ratio 
hs-CRP 
high sensitivity C-reactive protein 
HUVEC 
human umbilical vein endothelial cells 
ICAM-1 
intracellular adhesion molecule one 
ID 
identification 
ife-AG 
ifetroban acylglucuronide 
IND 
Investigational New Drug 
INR 
International Normalized Ratio 
IPr 
IRB 
prostaglandin I2 (prostacyclin) receptor 
institutional review board 
IsoP 
isoprostane 
IUPAC 
International Union of Pure and Applied Chemistry 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 10 
 
 
IV 
intravenous 
kd 
dissociation constant 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 11 
 
 
Abbreviation Definition 
kg 
kilogram 
L 
liter 
LC-MS/MS 
Liquid chromatography-tandem mass spectrometry 
LnRHI 
log transformed Reactive Hyperemia Index 
LVEF 
µg 
left ventricular ejection fraction 
microgram 
µM 
micromole 
mg 
milligram 
MI 
myocardial infarction 
min 
minute 
mL 
milliliter 
mM 
millimolar 
N 
number 
nM 
nanomolar 
NO 
nitric oxide 
NT pro-BNP 
N terminal pro-brain natriuretic protein 
NYHA 
New York Heart Association 
PAH 
pulmonary arterial hypertension 
PCI 
percutaneous coronary intervention 
PGF1α 
prostaglandin F1α 
PGF2α 
prostaglandin F2α 
PGH2 
prostaglandin H2 
PGI2 
prostaglandin I2, prostacyclin 
PVD 
peripheral vascular disease 
REDCap 
Research Electronic Data Capture 
RHI 
RIGOR 
ROS 
Reactive Hyperemia Index 
Reduction in Graft Occlusion Rate study 
reactive oxygen species 
SAE 
serious adverse event 
SAR 
serious adverse reaction 
SMC 
smooth muscle cell 
STEMI 
ST segment elevation myocardial infarction 
t½ 
plasma terminal elimination half-life 
TNF-α 
tumor necrosis factor--α 
TPr 
thromboxane prostanoid receptor 
TXA2 
thromboxane A2 
TXAS 
TXB2 
TXB2-M 
thromboxane synthase 
thromboxane B2 
thromboxane B2-metbolites 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 12 
 
 
1 
Background 
1.1 
Overview of Thromboxane Biology 
Thromboxane A2 (TXA2) is a signal-activated 
eicosanoid generated from the metabolism of 
arachidonic acid (AA) via the actions of the 
cyclooxygenase (COX) and downstream 
thromboxane synthase (TXAS) enzymes.4 In 
healthy adults, the vast majority of TXA2 is 
produced in platelets, where it not only mediates 
activation of the platelet in which it is formed, but 
is also released to locally amplify thrombotic 
stimuli by directly activating adjacent platelets 
and causing potent vasoconstriction.5 Aspirin 
(ASA) exerts its antiplatelet effect by irreversibly 
inhibiting the COX-1 enzyme for the life of the 
platelet, thereby suppressing platelet TXA2 
generation and activation. In pathologic conditions such as cardiovascular disease (CVD), TXA2 
generation increases substantially in non-platelet tissues such as endothelial cell (EC) and in 
inflammatory cells (Figure 1). Unlike platelet-derived TXA2, standard doses of ASA are 
ineffective at suppressing non-platelet-derived TXA2 due to the short circulating half-life of ASA 
(t1/2 ~20 min), the ability of nucleated cells to regenerate COX-1 and formation of TXA2 mediated 
by COX-2, which unlike COX-1 is not effectively inhibited by ASA. 
 
TXA2 is a short-lived molecule (t1/2 ~30 sec) that undergoes aqueous nucleophilic attack to form 
thromboxane B2 (TXB2), an inert eicosanoid that circulates in the bloodstream. TXB2 itself is 
extensively metabolized via enzyme-mediated β-oxidation and dehydrogenation to dozens of 
inactive end-order metabolites (TXB2-M) that are concentrated in the urine (Figure 1).6 
Measurement of one of these stable urine metabolites, 11-dehydroTXB2 (11-dhTXB2) was initially 
developed as a clinical indicator of ASA responsiveness based on data showing that ASA 
suppressed urine 11-dhTXB2 to <400 pg/mg creatinine in >90% of healthy adults.7 With the 
realization that non-platelet sources generate substantial amounts of TXA2 in pathologic 
conditions, urine 11-dhTXB2 is now considered a reflection of systemic non-renal TXA2 
generation. (TXA2 generated by the renal glomeruli undergoes limited metabolism prior to 
secretion, thus urine TXB2 is considered reflective of renal-specific TXA2 generation.8) 
 
1.2 
Thromboxane Receptor Biology 
The biologic activity of TXA2 is mediated through the binding to the thromboxane-prostanoid 
receptor (TPr), a G-protein coupled transmembrane protein present on a diverse array of cell types, 
including EC, vascular smooth muscle cells (SMC) and cardiac myocytes.9-12 Two isoforms exist 
that can form heterodimers, TPα, which is widely distributed, and TPβ, which is restricted to limited 
cell types such as EC.13 High-affinity ligands for TPr are TXA2 and PGH2 (Kd 6.19-16 nM), but 
isoprostanes such as 8-isoPGF2α that form non-enzymatically from arachidonic acid under 
conditions of oxidative stress, are also physiologically important ligands (Kd 31.8 nM).14 Key 
cellular effects of TPr activation are shown in Table 1. In whole blood vessel preparations, TPr 
activation inhibits nitric oxide (NO) production and endothelial-dependent vasodilation.15 Several 
Figure 1. Sources of TXA2 generation. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 13 
 
 
reports have shown that TPr activation not only 
increases superoxide generation but oxidative stress 
increases transport and stabilization of both TPr 
isoforms on cell membranes.16-18 In general, the TXA2- 
TPr pathway’s pro-coagulant, pro-inflammatory and 
vasoconstrictor effects counterbalance and in some 
cases directly antagonize the anti-coagulant, ant- 
inflammatory and vasodilating effects of prostacyclin 
(PGI2) and NO. 
Table 1. Cellular effects of TPr 
activation.9, 11, 15-26 
 
1.3 
TXA2 Generation and Clinical 
Outcome 
Several studies have shown that elevated urine levels 
of TXA2 metabolites in patients with CVD on standard 
ASA therapy is associated with an increased risk of 
atherothrombotic events and death.3, 27-30 Sub-studies 
from the HOPE and CHARISMA trials found that in 
patients with/high risk for CVD on ASA, those with 
urine TXB2-M in the highest quartile had a ~1.7-fold 
increased risk of death, myocardial infarction (MI) or 
stroke compared those in the lowest quartile.27, 29 Early 
interpretation of these results was that ASA failed to adequately inhibit platelet COX-1 activity in 
a subset of patients with CVD, leading to persistent TXA2 generation, increased platelet reactivity 
and atherothrombosis. It is now understood that ASA is actually very effective at inhibiting platelet 
TXA2 generation and that the observed persistent TXA2 generation originates from non-platelet 
tissue that cannot be inhibited by standard ASA therapy. 
 
This concept was definitively demonstrated in the Reductions in Graft Occlusion Rates (RIGOR) 
study.3 Given that ASA reduces the rate of vein graft (VG) thrombosis by 50% after coronary artery 
bypass graft (CABG) surgery31, the RIGOR study hypothesized that failure of ASA to inhibit 
platelet activation would increase the prevalence of VG occlusion 6 months after CABG surgery. 
ASA responsiveness was assessed both by urine 11-dhTXB2, which measures non-renal systemic 
TXA2 generation, and by AA-induced platelet aggregation, which specifically assesses platelet 
TXA2 generation. Similar to other studies32-35, the RIGOR study found that ASA suppressed 
platelet TXA2 generation in 95% and 99% of subjects 3 days and 6 months after CABG surgery, 
respectively. However, urine 11-dhTXB2 was also elevated in 70% and 33% of subjects at these 
same time points, often to high levels (Figure 2A). While no association was found between the 
failure of ASA to suppress platelet TXA2 generation and VG thrombosis, 11-dhTXB2 ≥450 pg/mg 
creatinine measured 6 months after surgery was independently associated with VG occlusion (OR 
2.6, P<0.02; Figure 2B) compared to values <450 pg/mg creatinine.3 In an analysis of long-term 
outcome, 11-dhTXB2 ≥450 pg/mg creatinine in 288 RIGOR subjects with suppressed platelet 
TXA2 generation independently predicted 5-year MACE (adjusted HR 1.79, P=0.02) and death 
(adjusted HR 2.90, P =0.01) compared to lower values (Figure 3).1 These data provide compelling 
evidence that, in patients with CVD, unlike healthy individuals, there is substantial TXA2 
Cell Type 
Physiologic Effect 
Platelet 
↑Shape change, 
↑Aggregation 
SMC 
↑Contraction, ↑Hypertrophy, 
↑Proliferation 
Myocyte 
↑Apoptosis, ↑Arrhythmia 
EC 
↓NO, ↑ROS, ↑Adhesion 
Molecules, ↑Tissue Factor 
Leukocyte 
↑Activation, ↑Adhesion 
Molecules, ↑Tissue Factor 
Lymphocyte 
↑Cytokine release, ↓Acquired 
Immunity 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 14 
 
 
generation from non-platelet sources that is not suppressed by standard ASA therapy and is a 
potent risk factor for atherothrombosis and death. 
 
 
To determine if TXA2 generation predicts 
outcome in a large unselected population, 
TXB2-M was recently measured 3042 
subjects 
participating 
in 
Exam 
8 
Offspring and Exam 3 Omni cohorts of 
the Framingham Heart Study (FHS). The 
median 
urine 
TXB2-M 
in 
the 
1361subjects on ASA and the 1681 
subjects not on ASA were 1145 and 4179 
pg/mg 
creatinine, 
respectively. 
Regardless of ASA use, urine TXB2-M 
above the respective median values was 
associated with more than a two-fold risk 
of long-term mortality (Figure 4; Rade, 
unpublished data). 
 
These data indicate that systemic TXA2 
generation is a potent risk factor for 
mortality, not only in patients with established CVD, but also in a general population and is 
independent of ASA use. 
 
1.4 
Factors Associated with Non-platelet TXA2 Generation 
Multivariable modelling of RIGOR subjects with documented ASA-mediated suppression of 
platelet TXA2 generation identified independent risk factors for non-platelet TXA2 generation 
(Table 2).2 Oxidative stress, as measured by urine 8-isoPGF2α, was identified as the strongest risk 
factor for non-platelet TXA2 generation, accounting for nearly half of the modelled effect (Figure 
Figure 2. A. Urine 11-dhTXB2 in RIGOR subjects 3 
days and 6 months after CABG surgery.3 Red line 
denotes 400 pg/mg creatinine. B. Percentage of 
occluded SVG 6 months after surgery in 229 subjects 
stratified by quartile of 11-dhTXB2. 3 
Figure 3. 5-year survival of 288 RIGOR 
subjects stratified by urine 11-dhTXB2 <450 
pg/mg creatinine (red line) or above (blue 
Figure 4. Long-term survival in 2727 FHS subjects 
stratified by mean urine TXB2-M. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 15 
 
 
5). In addition to being a marker for 
oxidative stress, 8-iso-PGF2α is also a 
stable circulating eicosanoid capable of 
activating TPr to cause vasoconstriction 
and potentiate agonist-induced platelet 
activation.36 Other independent risk 
factors are shown in Table 2. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.5 
Oxidative Stress Stimulates TXA2 Generation in Endothelial Cells 
While the predominant eicosanoid produced in healthy EC is PGI2, TXA2, is induced in 
dysfunctional EC where it counteracts the vasodilator effects of PGI2 and nitric oxide (NO), thus 
impairing endothelial-dependent vasodilatation.37, 38 Oxidative stress, a major mediator of EC 
dysfunction39, stimulates TXA2 and isoprostane generation in human EC (Figure 6A)2. Similar to 
porcine cerebral and retinal microvessels 40, 41, macrovascular EC generates substantial amounts 
of TXA2 when directly stimulated with 8-iso-PGF2α (Figure 6B), an effect that is inhibited by TPr 
blockade (Figure 6C). Furthermore, 8-isoPGF2α may not be the most physiologic relevant 
isoprostane species to mediate this effect. For example, oxidative stress also results in formation 
of isothromboxanes, which would be expected to bind TPr more avidly than 8-isoPGF2α.42 These 
data suggest that EC under oxidative stress are a potentially major source of non-platelet TXA2 
generation and that autocrine/paracrine stimulation of the TP receptor by TXA2 and/or isoprostanes 
amplify this process. Furthermore, they identify TP receptor blockade as a promising strategy to 
not only block the effects of TXA2 but also inhibit its generation. 
Urine 8-iso 
PGF2α (ln pg/mg 
creatinine) 
Age (years) 
Female gender 
White race 
(versus non- 
white) 
Statin therapy 
(mg/dL) 
LVEF (%) 
ASA dose (81mg 
vs. >81 mg) 
*Normalized using natural log transform 
Weight 
p-value 
Coefficient 
Table 2. Independent risk factors for UTXB2* 
identified by multivariable regression analysis. 
Figure 5. Linear regression of urine 11- 
dhTXB2 to 8-isoPGF2α in 228 subjects 
with suppressed platelet TXA2 
generation.2 
0.442 
<0.001 
0.472 
0.239 
<0.001 
0.102 
0.129 
0.015 
0.093 
-0.172 
0.009 
0.085 
-0.161 
0.004 
0.077 
-0.152 
0.002 
0.072 
-0.113 
0.032 
0.048 
-0.145 
0.004 
0.052 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 16 
 
 
 
 
 
1.6 
Non-platelet Thromboxane Generation and Atherothrombosis 
Little is known about how non-platelet TXA2 generation increases cardiovascular risk. While 
ASA-inhibited platelets could potentially still respond to non-platelet-derived TXA2, in the 
RIGOR cohort 11-dhTXB2 and 8-isoPGF2α were independent of any tested parameter of platelet 
function3 and in the CHARISMA trial the addition of clopidogrel did not reduce adverse events in 
subjects with elevated 11-dhTXB2.29 These data argue against non-platelet-derived TXA2 or 
circulating 8-iso-PGF2α mediating thrombosis in vivo by “priming” or potentiating platelet 
reactivity. 
 
Stronger evidence exists for non-platelet TXA2 and 8-iso-PGF2α generation promoting 
atherothrombosis and impairing EC thromboresistance. In apo E-deficient mice, for example, 
atherogenesis is inhibited by the administration of a TP receptor antagonist but not by ASA.43 
Preliminary data reveal that TPr stimulation with isoprostane induces expression of multiple 
adhesion molecules in EC (Figure 7A; Rade unpublished data). TPr activation has been shown by 
others to potentiate cytokine-mediated TF expression in endothelial cells and monocytes19, 44 and 
TF is also directly upregulated by oxidative stress (Figure 7B; Rade unpublished data). Additional 
evidence indicates that TPr inhibition blunts cytokine-mediated TXA2 generation (Figure 7C) and 
downregulation of thrombomodulin (TM), the key component of the protein C anticoagulant 
pathway that is critical for maintaining thromboresistance (Figure 7D; Rade unpublished data).45- 
Figure 6. A. Oxidative stress induced by hydrogen peroxide (H2O2) increases generation of 8-isoPGF2α, 11- 
dhTXB2 and TXB2 measured by LC-MS/MS in the supernatants of human umbilical vein endothelial cells 
(HUVEC).2 B. Direct exposure of HUVEC to 8-isoPGF2α increases generation of TXB2 measured by LC- 
MS/MS. C. TPr antagonism with SQ 29,548 (10-6M) inhibits HUVEC TXB2 generation in response to 8- 
isoPGF2α (10-6M) as measured by ELISA. 
Figure 7. A. Isoprostanes stimulate adhesion molecule expression by HUVEC. B. Oxidative stress induced by 
1-hour incubation with 200 μM H2O2 stimulates tissue factor expression in HUVEC. C. Cytokine-induced 
TXA2 generation in HUVEC is inhibited by TP receptor antagonism with SQ 29548. D. TP receptor 
antagonism with SQ 29548 blunts cytokine-mediated TM downregulation in HUVEC. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 17 
 
 
48 These data suggest that autocrine/paracrine TPr activation by TXA2 and/or isoprostane alter EC 
thromboresistance and provide the rationale using a TPr antagonist to reduce the risk of 
atherothrombosis. 
 
1.7 
Renal Effects of TXA2 and TPr Inhibition 
Prostaglandins play an important role in regulating renal blood flow and kidney function. The TPr 
is expressed in multiple places within the kidney (glomerulus, renal arterioles, thick ascending 
loop of Henle, collecting ducts and convoluted tubules) and renal TXA2 generation is thought to 
play a role in regulating renal blood flow and glomerular filtration.49, 50 TPr antagonism has been 
shown to inhibit renal vasoconstriction and prevent kidney injury in endotoxemic mice.51 In 
diabetic apolipoprotein E-deficient mice, TPr antagonism was shown to attenuate oxidative stress 
and proteinuria.52 In patients with cyclosporin-induced renal toxicity, infusion of a TXAS inhibitor 
reduced renal TXA2 generation and improved glomerular filtration.53 These data suggest TPr 
inhibition could potentially improve renal function in patients with CVD and increase non-platelet 
TXA2 generation, especially those with heart failure. 
 
1.8 
Summary of Mechanistic Hypothesis Tested in this Study 
Clinical and in vitro experimental data point to oxidative stress as a potent stimulus for endothelial 
TXA2 generation. TXA2 generation by vascular EC is hypothesized to be both a manifestation as 
well as a mediator of endothelial dysfunction. This provides the rationale for TPr antagonism as a 
means of improving vascular function. Figure 8 depicts a mechanistic model encompassing these 
concepts. Key features of the model include: 
• Oxidative stress stimulates TXA2 generation and inhibits PGI2 generation by; 1) enhancing 
COX-1 activity and altering the relative activities of TXAS to prostacyclin synthase (PGIS), 
and; 2) non-enzymatic formation of isoprostanes. 
• Autocrine/paracrine activation of 
the TPr by TXA2 and/or 
isoprostanes; 1) potentiates 
TXA2 generation via a positive 
feedback loop; 2) induction of 
adhesion molecules and other 
inflammatory mediators, and; 3) 
impairs thromboresistance by 
stimulating TF expression and 
inhibiting the TM-APC 
anticoagulant pathway. 
Figure 8. Proposed model for the effects of oxidative stress and 
mechanical stretch on EC/EEC eicosanoid generation and change 
in cellular phenotype. IsoP= isoprostane; ROS= reactive oxygen 
species; AA= arachidonic acid; TM= thrombomodulin; TF= 
tissue factor; APC= activated protein C. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 18 
 
 
2 
Ifetroban. 
2.1 
Pharmacology 
Ifetroban is a small potent and selective antagonist of the TPr with a molecular weight of 462.5 
kD (Figure 9). Though readily absorbed after oral dosing, absorption is delayed after a meal and 
thus should be given to patients 30 minutes or more prior to a meal. Ifetroban has a plasma half- 
life of approximately 22 hours. Elimination is primarily by metabolism and biliary excretion. 
 
Each capsule contains 250 mg of ifetroban. Other ingredients 
include mannitol, microcrystalline cellulose, crospovidone, 
magnesium oxide, colloidal silicon dioxide, and magnesium 
stearate. Capsules are filled into high density polyethylene 
bottles and sealed with screw-cap closures. Placebo for 
ifetroban capsules are formulated as a dry powder blend filled 
into capsules with similar matrix. Available data suggest the 
capsules will be stable for at least 48 months. A complete 
description of the pharmacology and toxicology of ifetroban is 
provided in the Investigator’s Brochure. 
2.2 
Pre-Clinical and Human Studies 
Ifetroban was initially developed for cardiovascular indications involving atherothrombosis and 
ischemia.54 It has been the subject of extensive pre-clinical and clinical testing including eight 
randomized, placebo-controlled clinical trials in various cardiovascular indications, several of 
which included daily ifetroban oral doses of 250 mg for more than two weeks in subjects taking 
ASA. Ifetroban was well-tolerated in clinical studies with a cumulative enrollment of nearly 1,300 
ifetroban-exposed subjects. Doses as high as 1000 mg were administered to healthy volunteers 
with no serious adverse events. 
 
Ifetroban is currently under study for the treatment of several non-cardiac conditions including 
systemic sclerosis with and without pulmonary hypertension, ASA exacerbated respiratory disease, 
hepatorenal syndrome, portal hypertension and prevention of tumor metastases. By virtue of its 
direct TPr blocking effect and its potential ability to reduce TXA2 generation by inhibiting a 
positive feedback loop involving the TPr, ifetroban is an ideal candidate to mitigate the effects of 
as well as potentially reduce non-platelet TXA2 generation. A complete description of the pre- 
clinical and clinical experience with ifetroban, including extensive safety data is provided in the 
Investigator’s Brochure. 
2.3 
Bleeding Risks 
Ifetroban, by virtue of its ability to block the effects of TXA2, has similar effects on platelet 
function and hemostasis as ASA, which exerts its effects by inhibiting TXA2 generation. In 
subjects already on ASA therapy, the addition of ifetroban does not further impair platelet or 
hemostatic function and does not increase the risk of bleeding 
 
TXA2 is only one of several platelet agonists that play an important role in hemostasis. Platelet 
function studies reveal that ifetroban blocks platelet activation by AA and TXA2 but not in response 
to ADP, thrombin or collagen. Thus, bleeding risk in subjects taking ifetroban plus dual antiplatelet 
Figure 9. Structural formula of 
ifetroban. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 19 
 
 
therapy (ASA + ADP receptor antagonist) would not be expected to be greater than in subjects 
taking dual antiplatelet therapy alone. 
 
In a study of 17 subjects on stable warfarin therapy, the addition of 250 mg/d ifetroban for 7 days 
did not alter the INR (BMS Internal Report). The available clinical data has not demonstrated an 
increase in hemorrhagic events, though the number of subjects taking ifetroban and warfarin are 
limited (see Investigator’s Brochure for details). The interaction of ifetroban and novel oral 
anticoagulants has not been studied. 
 
2.4 
Effect on Blood Pressure and Renal Function 
TXA2, through its activation of the TPr, is a potent vasoconstrictor. While ifetroban has been shown 
in pre-clinical animal models to reverse the hypertensive effects of infusions of arachidonic acid, 
it has not been shown to have significant hypotensive or adverse renal effects in humans: the 
incidence of hypotension and renal failure in all studies was <0.5% and not different with placebo 
treatment (see Investigator’s Brochure for details). 
2.5 
Heart Failure 
While ifetroban has not yet been studied specifically in a heart failure population, many of the 
subjects enrolled in the historic cardiovascular trials had recent myocardial infarctions and would 
have been expected to have had a diminished LVEF. In over 1500 subjects studied with 
cardiovascular disease, the incidence of new heart failure was low (≤1%) and no different between 
ifetroban and placebo treated subjects. 
 
3 
Study Objectives 
This study will test the hypothesis that administration of the TPr receptor antagonist ifetroban will 
improve endothelial function by blocking the effects and generation of TXA2 from non-platelet 
sources. 
 
The objectives of this study are: 
 
• To determine if thromboxane receptor antagonism improves endothelial function in 
subjects with non-platelet thromboxane generation. 
• To determine if thromboxane receptor antagonism reduces non-platelet thromboxane 
generation. 
• To determine if thromboxane receptor antagonism reduces oxidative stress, inflammation 
and thrombotic potential. 
• To determine if thromboxane receptor antagonism alters renal prostaglandin secretion 
and improves renal function. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 20 
 
 
4 
Study Design 
The TRAP study is a phase 2b, single center, prospectively randomized, double-blinded, placebo- 
controlled outpatient clinical trial conducted at the UMass Medical School/UMass Memorial 
Medical Center. A schedule of study events is outlined in Appendix A. 
4.1 
Screening Phase 
Patients with established cardiovascular disease on ≥81 mg daily ASA as part of their normal 
therapeutic regimen will be screened for non-platelet TXA2 generation by measuring urine TXB2 
metabolites using either the AspirinWorks 11-dehydro Thromboxane B2 Test Kit (Corgenix Inc., 
Bromfield, CO) or the essentially identical 11-dehydro Thromboxane B2 ELISA Kit (Cayman 
Chemical, Ann Arbor, MI). Those with normalized values ≥1145 pg/mg creatine and who meet all 
other inclusion/exclusion criteria will be eligible for participation. 
 
Urine TXB2-M measured as part of a related on-going observational study of this same patient 
population under separate consent may be used to determine eligibility for this randomized trial. 
4.2 
Randomization 
Eligible subjects will be randomized to receive either five 50 mg ifetroban oral capsules (250 mg 
total does) daily or matching placebo in a 1:1 ratio for 4 weeks. Double-blind randomization will 
occur at the first study visit using the UMass Flexible Randomization and Inventory Management 
System (FRIMS). 
 
4.3 
Study Interventional Phase 
4.3.1 
Study Visit 1 (Baseline Day 0, Outpatient) 
• If not already done prior to this visit, subjects will be consented for the study. 
• Compliance with uninterrupted daily aspirin use for the preceding 5 days will be verified. 
• Baseline physical exam will be performed, vital signs will be recorded. 
• Concomitant medications will be recorded. 
• Baseline urine and blood samples will be obtained. 
• Subjects will undergo baseline vascular testing. 
• Subjects will be randomized. 
• Subjects will be provided study drug enough for 35 days and instructed to take one pill 
daily. They will be instructed to continue all other medications, especially aspirin, as 
prescribed by their physician and to avoid unnecessary changes to their medical regimen. 
 
4.3.1.1 
Study Visit 2 (Interim Day 7 ± 3 Days, Phone Contact) 
• Subjects will be contacted approximately 1 week after starting study drug to monitor for 
compliance and possible adverse events (AE). 
 
4.3.1.2 
Study Visit 3 (Follow-up Day 28 ± 3 Days, Outpatient) 
• Subjects will take study drug on the day of the visit. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 21 
 
 
• Subjects will bring in unused study drug and pill counts will be performed. 
• Physical exam will be performed, vital signs will be recorded. 
• Concomitant medications will be recorded. 
• Subjects will be assessed for possible AE. 
• On-drug urine and blood samples will be obtained. 
• On-drug physical exam with vital signs will be performed. 
• On-drug vascular studies will be performed. 
 
4.3.1.3 
Study Visit 4 (Day 42 ± 3 Days, Phone Contact) 
• Subjects will be contacted 2 weeks after stopping study drug to monitor for possible AE. 
 
5 
Study Population and Eligibility Criteria 
5.1 
Study Population 
Patients suitable for this protocol are individuals with cardiovascular disease on standard aspirin 
therapy with elevated non-platelet thromboxane generation. 
 
5.2 
Inclusion Criteria 
1. Males and females 18-80 years of age with established cardiovascular disease, defined by 
either: 
a. Angiographic (invasive or non-invasive) evidence of coronary atherosclerosis. 
b. Prior myocardial infarction. 
c. Positive stress test with documented peripheral or cerebrovascular disease. 
2. Taking ≥81 mg daily ASA as part of their standard medical regimen. 
3. Urine TXB2-M >1145 pg/mg creatinine during screening. 
4. Able to provide written consent and comply with protocol-specific procedures. 
 
5.3 
Exclusion Criteria 
1. Chronic oral anticoagulation with a non-vitamin K antagonist. 
2. Anticipated change or interruption in aspirin therapy during the study period. 
3. STEMI within 30 days. 
4. Cardiac surgery within 30 days. 
5. Stage 4-5 kidney failure or on renal replacement therapy. 
6. An uncontrolled ongoing severe inflammatory condition. 
7. Pregnant, intending to become pregnant or breast feeding. (All women of child-bearing 
potential must have a negative pregnancy test within 24 hours of randomization.) 
8. Known ifetroban or aspirin sensitivity. 
9. Inability to perform vascular testing or comply with study protocol. 
10. Participation in another investigational drug trial within 30 days of randomization. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 22 
 
 
 
 
6 Recruitment and Screening Procedures 
6.1 
Common Recruitment Procedures 
Patients referred to the UMass Memorial Medical Center Heart and Vascular Interventional 
Laboratory for clinically-indicated cardiac catheterization who are participating in an ongoing 
observational clinical study (IRB #H00014594_3) will form the major screening population for 
this interventional trial. As part of this observational study, non-platelet thromboxane generation 
is assessed in each subject by measuring urine TXB2-M. Subjects not participating in this screening 
study but who otherwise meet all the inclusion/exclusion criteria will be eligible to participate in 
this interventional trial. 
 
6.2 
Estimated Enrollment Period 
This study will enroll a maximum of 57 subjects over a period of 30 months. 
 
6.3 
Informed Consent Procedures 
Designated study personnel will explain to eligible subjects the purpose of the study, interventions, 
risk and benefits of participation and will answer questions. Wherever possible, subjects will be 
provided a copy of the UMass IRB approved consent form to review in advance of meeting with 
study personnel. All subjects who sign consent forms will be provide a full copy of the signed and 
executed document. 
 
6.4 
Confidentiality and HIPAA Requirements 
All information collected on study participants will be stored in a confidential manner. Only 
approved study personnel will have access to data collected as part of the study. Consented study 
participants will be identified by a participant ID number on all study documents. Data will be 
stored securely using procedures described under Data Management. 
 
6.5 
Protection of Human Subjects 
Protections for human subjects of research are required under Department of Health and Human 
Services regulations in 21 CFR parts 50, 56, and 312. 
 
6.6 
Summary of Risks and Benefits 
6.6.1 Risks 
6.6.1.1 Drug-related 
Given its established safety profile, especially in patients with high-risk cardiovascular 
disease, the risks associated with ifetroban administration are expected to be minimal (see 
above section 2 and Investigator’s Brochure). Given that both ifetroban and ASA inhibit 
TXA2-mediate platelet activation, they have similar antiplatelet effects and bleeding risk. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 23 
 
 
Administration of ifetroban to subjects already taking ASA has not been shown to increase 
the risk of bleeding as the TXA2 pathway in platelets is already maximally suppressed. 
6.6.1.2 Blood draws 
The risks of drawing blood include bleeding at the puncture site, bruising and pain. These 
would be expected to occur in a very small portion of the population. 
6.6.1.3 Vascular Studies 
The risks of vascular studies include discomfort in the arm during prolonged blood pressure 
cuff inflation. 
 
7 Study Drug Handling 
7.1 
Drug Dispensing 
50 mg ifetroban oral capsules and matching placebo will be provided by Cumberland 
Pharmaceuticals Inc. and packaged by the UMass Investigational Pharmacy into plastic bottles 
containing 175 pills. Bottle labels will contain instructions to take one pill daily in the morning, 
the name and contact information of the PI as well as a code that can be used in case of emergency 
unblinding. 
 
7.2 
Drug Storage, Accountability and Destruction 
Study product will be stored prior to dispensing at the UMass Investigational Pharmacy at room 
temperature. Unused study drug will be returned by subjects at Visit 3 and disposed of by the 
UMass Investigational Pharmacy, which will hold all destruction documentation. 
 
7.3 
Emergency Unblinding 
The UMass Investigational Pharmacy will maintain a log of study product codes that can be used 
in the unlikely event that emergency unblinding is required. 
 
 
8 Outcome Determinations 
8.1 
Primary Endpoint 
The primary trial endpoint will be change from baseline in on-treatment Reactive Hyperemia Index 
(RHI) measured by peripheral arterial tonometry (PAT) using the standard protocol described in 
Appendix C . 
 
8.2 
Secondary Endpoints 
Secondary trial endpoints will be the change from baseline in on-treatment: 
 
• Brachial % flow-mediated vasodilation (FMD) using the standard protocol described in 
Appendix B. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 24 
 
 
• Urine 11-dhTXB2 as measured by mass spectrometry or immunoassay and normalized to 
urine creatinine. 
 
8.3 
Exploratory Endpoints 
Exploratory trial endpoints will be the change from baseline in on-treatment: 
 
• Urine 8-iso-PFG2α as measured by immunoassay and normalized to urine creatinine. 
• Plasma tissue factor as measured by immunoassay. 
• Plasma ICAM-1 as measured by immunoassay. 
• Plasma activated protein C as measured by immunoassay. 
• hs-CRP as measured by immunoassay. 
• NT pro-BNP as measured by immunoassay. 
• Urine 2,3-dinor-6-ketoPGF1α and ratio of 11-dhTXB2 to 2,3-dinor-6-ketoPGF1α as 
measured by immunoassay and normalized to urine creatinine. 
• Urine TXB2, 6-ketoPGF1α and the ratio of TXB2 to 6-ketoPGF1α ketoPGF1α as measured 
by immunoassay and normalized to urine creatinine. 
• Glomerular filtration rate as calculated by the Cockroft-Gault formula. 
 
9 
Subject Safety 
9.1 
Institutional Review Board 
The Study Protocol, Investigator Brochure, consent form, Investigational New Drug application 
(IND) and other relevant documents will be submitted to the UMass IRB for approval prior to 
commencement of the trial. Any amendment other than minor administrative changes, must be 
reviewed and approved by the IRB before implementation. 
 
9.2 
Food and Drug Administration 
Use of ifetroban in this clinical trial is covered under an approved IND application to the US 
Food and Drug Administration (FDA). 
 
9.3 
Independent Clinical Event Adjudicator 
An independent Clinical Event Adjudicator, with expertise in the field of cardiology will be 
identified and trained on the study protocol and Investigator Brochure prior to the first subject 
enrollment in this study. The role of this CEA is to provide an independent, expert review of data 
on clinical events based on protocol specific definitions outlined below. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 25 
 
 
9.4 
Adverse Events 
9.4.1 Definitions 
9.4.1.1 Adverse Event 
An adverse event (AE) is any untoward medical occurrence associated with the use of a drug 
in a participant, whether or not considered drug or biologic related. An AE can therefore be 
any undesirable sign, symptom or medical condition occurring after starting study drug, even 
if the event is not considered to be related to the pharmaceutical product. Study drug includes 
the drug under evaluation, and any reference or placebo drug given during any phase of the 
trial. 
9.4.1.2 Suspected Adverse Reaction 
A suspected adverse reaction (SAR) is any adverse event for which there is a reasonable 
possibility that the drug caused the event. “Reasonable possibility” suggests there is a causal 
relationship between the drug and the adverse event. “Suspected adverse reaction” implies a 
lesser degree of certainty about causality than adverse reaction, which means any adverse 
event caused by a drug. 
9.4.1.3 Serious Adverse Events (SAE) 
An adverse event or suspected adverse reaction is considered serious if the investigator or 
sponsor believes any of the following outcomes occurred: 
• Death 
• Life-threatening AE: Places the participant at immediate risk of death at the time of 
the event as it occurred. It does not include an AE that, had it occurred in a more 
severe form, might have caused death. 
• Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 
• Inpatient hospitalization or prolongation of hospitalization. 
• Results in congenital anomaly or birth defect. 
• Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon 
appropriate medical judgment, they may jeopardize the participant and may require 
medical or surgical intervention to prevent one of the outcomes listed in this 
definition above. This determination is based on the opinion of either the investigator 
or sponsor (e.g., if either believes it is serious, it must be considered serious). 
 
9.4.1.4 Laboratory Test Abnormalities 
For laboratory test abnormalities that meet the definition of an SAE, that required the 
participant to have the investigational product discontinued or interrupted or required the 
participant to receive specific corrective therapy, the clinical diagnosis rather than the 
laboratory term will be used by the reporting investigator (e.g., anemia versus low 
hemoglobin value). 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 26 
 
 
9.4.1.5 Assessment of Causal Relationship 
A medically-qualified investigator must assess the relationship of any AE to the use of study 
drug, based on available information, using the following guidelines: 
• Not related: There is not a reasonable causal relationship to the investigational 
product and the adverse event. 
• Unlikely related: No temporal association or the cause of the event has been 
identified, or the drug or biologic cannot be implicated. 
• Possibly related: There is reasonable evidence to suggest a causal relationship 
between the drug and adverse event. 
• Related: There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. 
 
9.4.1.6 Assessment of Adverse Event Severity 
The determination of adverse event severity rests on medical judgment of a medically- 
qualified Investigator. The severity of AEs will be graded using the following definitions: 
• Mild: Awareness of sign, symptom, or event, but easily tolerated. 
• Moderate: Discomfort enough to cause interference with usual activity and may 
warrant intervention. 
• Severe: Incapacitating with inability to do usual activities or significantly affects 
clinical status and warrants intervention. 
 
9.4.1.7 Expectedness 
The expectedness of an AE or SAR shall be determined according to the most current 
Investigator’s Brochure. Any AE that is not identified in nature, severity, or specificity in the 
current investigator’s brochure is considered unexpected. Events that are mentioned in the 
Investigator's Brochure as occurring with a class of drugs or as anticipated from the 
pharmacological properties of the drug, but not specifically mentioned as occurring with the 
particular drug under investigation are considered unexpected. 
 
9.4.2 Recording and Reporting of Adverse Events 
The principal investigator, CEA and study team are responsible for monitoring the safety of 
participants enrolled into the trial. Events significant enough to necessitate discontinuation of 
study drug dosing will be captured on the AE electronic case report forms (eCRF). 
 
All AEs/SAEs, except for those anticipated AEs, occurring from signed informed consent to 
14 (+/- 3 days) after visit 3 will be captured on the AE/SAE eCRF. Unless exempted, all SAEs, 
whether or not deemed drug-related or expected, must be reported by the investigator or 
qualified designee within 24 hours of first becoming aware of the event. For this study, all 
deaths will be reported on the SAE eCRF, as well as the Death eCRF page. The investigator 
or qualified designee will enter the required information regarding the AE/SAE into the 
appropriate module of the eCRF, which will be shared with the CEA. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 27 
 
 
When additional relevant information becomes available, the Investigator will record follow- 
up information according to the same process used for reporting the initial event as described 
above. The Investigator will follow all reportable events until resolution, stabilization or the 
event is otherwise explained. The investigators are also responsible for promptly reporting 
SAEs to the IRB per our institutional policy.. 
 
9.4.3 Suspected Unexpected Serious Adverse Reaction 
AEs that meet the criteria of serious, related to study drug, and unexpected per the 
Investigator’s Brochure, qualify for expedited reporting to the regulatory authorities. The PI 
will notify the FDA and all participating investigators in a written IND safety report of an SAR 
that is serious and is unexpected, based on the opinion of the Adjudicator as soon as possible, 
but not later than 15 calendar days after the event is confirmed to be a serious, unexpected SAR 
and qualifies for expedited reporting. 
 
 
10 Statistical Considerations 
10.1 
Sample Size Calculations 
10.1.1 Primary Endpoint 
Data from several studies was used to determine the power to detect a difference in RHI with 
drug treatment. In one study of PAT reproducibility in cardiovascular patients, the intra-patient 
standard deviation was 0.31.55 In a meta-analysis of >1500 subjects with cardiovascular disease 
the mean RHI was 1.52.56 Based on these data, a sample size of 48 total subjects (24 
randomized each to ifetroban or placebo) is required to detect a 20% increase in RHI with over 
85% power with a two-sided alpha of =0.05. Adjusting for 10% loss to follow up, we will need 
52 randomized subjects. 
10.1.2 Secondary Endpoints 
Data from a preliminary study conducted at UMass (Keaney, unpublished data) in which FMD 
was measured in patients after non-STEMI ACS was used for primary endpoint power 
calculations. In that pilot study, mean FMD was 9.6 ± 2.0% in normal controls (n=16) and 4.6 
± 2.8% in ACS patients (n=22). Based on these data, a sample size of 48 total subjects (24 each 
randomized to ifetroban or placebo) is required to detect a 50% increase in FMD with 80% 
power for an analysis of covariance of each treatment separately with a two-sided alpha=0.05. 
Data from the RIGOR study was used to determine the power to detect a difference in non- 
platelet TXA2 generation with drug treatment.3 With a median 11-dhTXB2 of 330 pg/mg 
creatinine, a 33% reduction would be to 220 pg/mg creatinine (reduction from 5.8 to 5.4 in the 
natural log scale) with a s.d. of 0.55 in the log scale. A sample size of 48 total subjects (24 each 
randomized to ifetroban or placebo) will have the ability to detect treatment group differences 
described above with 80% power for an analysis of covariance of each treatment separately 
with a two-sided alpha=0.05, assuming an R2 = 0.50 with the baseline level and treatment 
included in the model. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 28 
 
 
10.2 
Statistical Analysis Plan 
Descriptive statistics of patient characteristics will be generated using conventional methods. 
The main analysis of the outcome data will be a standard ANCOVA approach (SAS PROC 
GLM). The full model will be fit for change in RHI(or other measure of interest) as the outcome 
for patient i based on treatment group (as two dummy (0,1) variables:x1, x2), a vector of patient 
characteristics x3,I, and an error term ei: ln(11-dhTXB2)i=b0 + b1x1i + b2x2i + b3x3i +ei. We will test 
the main effects (treatment as well as patient characteristics) included in the model with the 
treatment main effects always included. We will investigate interactions between patient 
characteristics and treatments to provide insight into the effect of these treatments on the 
outcomes overall and the effect in larger patient subgroups. 
 
11 Data Management 
Study data will be recorded in a Research Electronic Data Capture (REDCap) database in the 
secure regulated environment at the UMass Medical School. REDCap is a secure, web-based 
application designed to support data capture for research studies, providing 1) formatted data entry 
screens; 2) audit trails; 3) automated export procedures for seamless data downloads to statistical 
packages; 4) procedures for importing data from external sources; and 5) an Application 
Programming Interface that enables it to communicate with our web-based randomization system. 
Data will be exported from REDCap into SAS (SAS Institute) for all analysis. 
 
12 Regulatory Issues 
12.1 
Ethics and Good Clinical Practice 
This study will be carried out in compliance with the protocol. These procedures are designed 
to ensure adherence to Good Clinical Practice, as described in: 1) ICH Harmonized Tripartite 
Guidelines for Good Clinical Practice 1996, and; 2) US 21 Code of Federal Regulations dealing 
with clinical studies (including parts 50 and 56 concerning informed consent and IRB regulations). 
All investigators and study personnel agree to adhere to the instructions and procedures described 
in it and hereby to adhere to the principles of Good Clinical Practice to which it conforms. 
 
12.2 
Institutional Review Board 
Before implementing this study, all required documents including the protocol, proposed informed 
consent form and Investigator’s Brochure must be reviewed and approved by the UMMS IRB. 
Any amendments to the protocol, other than administrative ones, must be approved by this 
committee. 
 
12.3 Informed Consent 
The investigator or designee must explain to each participant the nature of the study, its purpose, 
the procedures involved, the expected duration, the potential risks and benefits involved and any 
discomfort it may entail. Each participant must be informed that participation in the study is 
voluntary and that he/she may withdraw from the study at any time and that withdrawal of consent 
will not affect his/her subsequent medical treatment or relationship with the treating physician. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 29 
 
 
The informed consent form(s)and any amendments must be submitted by the investigator for IRB 
approval. 
 
12.4 
Quality Control and Assurance 
All CRFs and corresponding office and clinical laboratory records for each subject will be 
periodically independently audited by personnel from the UMass Office of Clinical Research. 
These audits will help to verify the accuracy and completeness of CRFs, to resolve any 
inconsistencies in the study records, and to assure that all protocol requirements, applicable FDA 
regulations, other requirements, and investigator’s obligations are being fulfilled. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 30 
 
 
 
 
13 References 
 
1. Kakouros N, Gluckman TJ, Conte J.V. et al. Differential impact of serial measurment of 
non-platelet thromboxane generation on long-term outcome after cardiac surgery. J Am 
Heart Assoc 2017;6(11):e007486. 
 
2. Kakouros N, Nazarian SM, Stadler PB, Kickler TS, Rade JJ. Risk factors for non-platelet 
thromboxane generation after coronary artery bypass graft surgery. J Am Heart Assoc 
2016;5(3):e002615. 
 
3. Gluckman TJ, McLean RC, Schulman SP et al. Effects of aspirin responsiveness and 
platelet reactivity on early vein graft thrombosis after coronary artery bypass graft 
surgery. J Am Coll Cardiol 2011;57(9):1069-1077. 
 
4. Patrono C, Ciabattoni G, Davi G. Thromboxane biosynthesis in cardiovascular diseases. 
Stroke 1990;21(12 Suppl):IV130-IV133. 
 
5. Capra V, Back M, Angiolillo DJ, Cattaneo M, Sakariassen KS. Impact of vascular 
thromboxane prostanoid receptor activation on hemostasis, thrombosis, oxidative stress, 
and inflammation. J Thromb Haemost 2014;12(2):126-137. 
 
6. Roberts LJ, Sweetman BJ, Oates JA. Metabolism of thromboxane B2 in man. 
Identification of twenty urinary metabolites. J Biol Chem 1981;256(16):8384-8393. 
 
7. Fritsma GA, Ens GE, Alvord MA, Carroll AA, Jensen R. Monitoring the antiplatelet 
action of aspirin. JAAPA 2001;14(5):57-62. 
 
8. Catella F, Nowak J, FitzGerald GA. Measurement of renal and non-renal eicosanoid 
synthesis. Am J Med 1986;81(2B):23-29. 
 
9. Huang JS, Ramamurthy SK, Lin X, Le Breton GC. Cell signalling through thromboxane 
A2 receptors. Cell Signal 2004;16(5):521-533. 
 
10. 
Nakahata N, Takano H, Ohizumi Y. Thromboxane A2 receptor-mediated tonic contraction 
is attributed to an activation of phosphatidylcholine-specific phospholipase C in rabbit 
aortic smooth muscles. Life Sci 2000;66(5):L-6. 
 
11. 
Santilli F, Mucci L, Davi G. TP receptor activation and inhibition in atherothrombosis: 
the paradigm of diabetes mellitus. Intern Emerg Med 2011;6(3):203-212. 
 
12. 
Feletou M, Vanhoutte PM, Verbeuren TJ. The thromboxane/endoperoxide receptor (TP): 
the common villain. J Cardiovasc Pharmacol 2010;55(4):317-332. 
 
13. 
Sasaki M, Miyosawa K, Ohkubo S, Nakahata N. Physiological significance of 
thromboxane A(2) receptor dimerization. J Pharmacol Sci 2006;100(4):263-270. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 31 
 
 
14. 
Bauer J, Ripperger A, Frantz S, Ergun S, Schwedhelm E, Benndorf RA. Pathophysiology 
of isoprostanes in the cardiovascular system: implications of isoprostane-mediated 
thromboxane A2 receptor activation. Br J Pharmacol 2014;171(13):3115-3131. 
 
15. 
Liu CQ, Leung FP, Wong SL et al. Thromboxane prostanoid receptor activation impairs 
endothelial nitric oxide-dependent vasorelaxations: the role of Rho kinase. Biochem 
Pharmacol 2009;78(4):374-381. 
 
16. 
Moss NG, Vogel PA, Kopple TE, Arendshorst WJ. Thromboxane-induced renal 
vasoconstriction is mediated by the ADP-ribosyl cyclase CD38 and superoxide anion. Am 
J Physiol Renal Physiol 2013;305(6):F830-F838. 
 
17. 
Ball SK, Field MC, Tippins JR. Regulation of thromboxane receptor signaling at multiple 
levels by oxidative stress-induced stabilization, relocation and enhanced responsiveness. 
PLoS One 2010;5(9):e12798. 
 
18. 
Valentin F, Field MC, Tippins JR. The mechanism of oxidative stress stabilization of the 
thromboxane receptor in COS-7 cells. J Biol Chem 2004;279(9):8316-8324. 
 
19. 
Del TS, Basta G, Lazzerini G, Chancharme L, Lerond L, De CR. Involvement of the TP 
receptor in TNF-alpha-induced endothelial tissue factor expression. Vascul Pharmacol 
2014;62(2):49-56. 
 
20. 
Nakahata N. Thromboxane A2: physiology/pathophysiology, cellular signal transduction 
and pharmacology. Pharmacol Ther 2008;118(1):18-35. 
 
21. 
Bode M, Mackman N. Regulation of tissue factor gene expression in monocytes and 
endothelial cells: Thromboxane A2 as a new player. Vascul Pharmacol 2014;62(2):57-62. 
 
22. 
Ueharaguchi Y, Honda T, Kusuba N et al. Thromboxane A2 facilitates IL-17A production 
from Vgamma4(+) gammadelta T cells and promotes psoriatic dermatitis in mice. J 
Allergy Clin Immunol 2018;142(2):680-683. 
 
23. 
Touchberry CD, Silswal N, Tchikrizov V et al. Cardiac thromboxane A2 receptor 
activation does not directly induce cardiomyocyte hypertrophy but does cause cell death 
that is prevented with gentamicin and 2-APB. BMC Pharmacol Toxicol 2014;15:73. 
 
24. 
Dorn GW, Becker MW. Thromboxane A2 stimulated signal transduction in vascular 
smooth muscle. J Pharmacol Exp Ther 1993;265(1):447-456. 
 
25. 
Ali S, Davis MG, Becker MW, Dorn GW. Thromboxane A2 stimulates vascular smooth 
muscle hypertrophy by up-regulating the synthesis and release of endogenous basic 
fibroblast growth factor. J Biol Chem 1993;268(23):17397-17403. 
 
26. 
Wacker MJ, Kosloski LM, Gilbert WJ et al. Inhibition of thromboxane A2-induced 
arrhythmias and intracellular calcium changes in cardiac myocytes by blockade of the 
inositol trisphosphate pathway. J Pharmacol Exp Ther 2009;331(3):917-924. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 32 
 
 
27. 
Eikelboom JW, Hirsh J, Weitz JI, Johnston M, Yi Q, Yusuf S. Aspirin-resistant 
thromboxane biosynthesis and the risk of myocardial infarction, stroke, or cardiovascular 
death in patients at high risk for cardiovascular events. Circulation 2002;105(14):1650- 
1655. 
 
28. 
Ames PR, Batuca JR, Muncy IJ et al. Aspirin insensitive thromboxane generation is 
associated with oxidative stress in type 2 diabetes mellitus. Thromb Res 
2012;130(3):350-354. 
 
29. 
Eikelboom JW, Hankey GJ, Thom J et al. Incomplete Inhibition of Thromboxane 
Biosynthesis by Acetylsalicylic Acid. Determinants and Effect on Cardiovascular Risk. 
Circulation 2008;118(1689):1690. 
 
30. 
McCullough PA, Vasudevan A, Sathyamoorthy M et al. Urinary 11-Dehydro- 
Thromboxane B2 and Mortality in Patients With Stable Coronary Artery Disease. Am J 
Cardiol 2017;119(7):972-977. 
 
31. 
Antiplatelet Trialists' Collaboration. Collaborative overview of randomised trials of 
antiplatelet therapy-II:Maintenance of vascular graft or arterial patency by antiplatelet 
therapy. BMJ 1994;308(159):168. 
 
32. 
Faraday N, Becker DM, Yanek LR et al. Relation between atherosclerosis risk factors and 
aspirin resistance in a primary prevention population. Am J Cardiol 2006;98(6):774-779. 
 
33. 
Tantry US, Bliden KP, Gurbel PA. Overestimation of platelet aspirin resistance detection 
by thrombelastograph platelet mapping and validation by conventional aggregometry 
using arachidonic acid stimulation. J Am Coll Cardiol 2005;46(9):1705-1709. 
 
34. 
Homorodi N, Kovacs EG, Lee S et al. The lack of aspirin resistance in patients with 
coronary artery disease. J Transl Med 2016;14:74. 
 
35. 
Kovacs EG, Katona E, Bereczky Z et al. New direct and indirect methods for the 
detection of cyclooxygenase 1 acetylation by aspirin; the lack of aspirin resistance among 
healthy individuals. Thromb Res 2013;131(4):320-324. 
 
36. 
Milne GL, Yin H, Hardy KD, Davies SS, Roberts LJ. Isoprostane generation and 
function. Chem Rev 2011;111(10):5973-5996. 
 
37. 
Schror K. Prostaglandins, other eicosanoids and endothelial cells. Basic Res Cardiol 
1985;80(5):502-514. 
 
38. 
Feletou M, Huang Y, Vanhoutte PM. Vasoconstrictor prostanoids. Pflugers Arch 
2010;459(6):941-950. 
 
39. 
Schulz E, Anter E, Keaney JF, Jr. Oxidative stress, antioxidants, and endothelial function. 
Curr Med Chem 2004;11(9):1093-1104. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 33 
 
 
40. 
Hou X, Gobeil F, Jr., Peri K et al. Augmented vasoconstriction and thromboxane 
formation by 15-F(2t)-isoprostane (8-iso-prostaglandin F(2alpha)) in immature pig 
periventricular brain microvessels. Stroke 2000;31(2):516-524. 
 
41. 
Lahaie I, Hardy P, Hou X et al. A novel mechanism for vasoconstrictor action of 8- 
isoprostaglandin F2 alpha on retinal vessels. Am J Physiol 1998;274(5 Pt 2):R1406- 
R1416. 
 
42. 
Morrow JD, Awad JA, Wu A, Zackert WE, Daniel VC, Roberts LJ. Nonenzymatic free 
radical-catalyzed generation of thromboxane-like compounds (isothromboxanes) in vivo. 
J Biol Chem 1996;271(38):23185-23190. 
 
43. 
Cayatte AJ, Du Y, Oliver-Krasinski J, Lavielle G, Verbeuren TJ, Cohen RA. The 
thromboxane receptor antagonist S18886 but not aspirin inhibits atherogenesis in apo E- 
deficient mice: evidence that eicosanoids other than thromboxane contribute to 
atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20(7):1724-1728. 
 
44. 
Bode M, Mackman N. Regulation of tissue factor gene expression in monocytes and 
endothelial cells: Thromboxane A2 as a new player. Vascul Pharmacol 2014;62(2):57-62. 
 
45. 
Kapur NK, Deming CB, Kapur S et al. Hemodynamic modulation of endocardial 
thromboresistance. Circulation 2007;115(1):67-75. 
 
46. 
Kapur NK, Bian C, Lin E et al. Inhibition of transforming growth factor-beta restores 
endothelial thromboresistance in vein grafts. J Vasc Surg 2011;54(4):1117-1123. 
 
47. 
Sohn RH, Deming CB, Johns DC et al. Regulation of endothelial thrombomodulin 
expression by inflammatory cytokines is mediated via activation of nuclear factor-kappa 
B. Blood 2005;105:3910-3917. 
 
48. 
Hiroi T, Deming CB, Zhao H et al. Proteasome Inhibitors Enhance Endothelial 
Thrombomodulin Expression via Induction of Kruppel-Like Transcription Factors. 
Arterioscler Thromb Vasc Biol 2009;29(10):1587-1593. 
 
49. 
Harding SD, Sharman JL, Faccenda E et al. The IUPHAR/BPS Guide to 
PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to 
IMMUNOPHARMACOLOGY. Nucleic Acids Res 2018;46(D1):D1091-D1106. 
 
50. 
Sakariassen KS, Alberts P, Fontana P, Mann J, Bounameaux H, Sorensen AS. Effect of 
pharmaceutical interventions targeting thromboxane receptors and thromboxane synthase 
in cardiovascular and renal diseases. Future Cardiol 2009;5(5):479-493. 
 
51. 
Boffa JJ, Just A, Coffman TM, Arendshorst WJ. Thromboxane receptor mediates renal 
vasoconstriction and contributes to acute renal failure in endotoxemic mice. J Am Soc 
Nephrol 2004;15(9):2358-2365. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 34 
 
 
52. 
Xu S, Jiang B, Maitland KA et al. The thromboxane receptor antagonist S18886 
attenuates renal oxidant stress and proteinuria in diabetic apolipoprotein E-deficient mice. 
Diabetes 2006;55(1):110-119. 
 
53. 
Smith SR, Creech EA, Schaffer AV et al. Effects of thromboxane synthase inhibition with 
CGS 13080 in human cyclosporine nephrotoxicity. Kidney Int 1992;41(1):199-205. 
 
54. 
Rosenfeld L, Grover GJ, Stier CT, Jr. Ifetroban sodium: an effective TxA2/PGH2 
receptor antagonist. Cardiovasc Drug Rev 2001;19(2):97-115. 
 
55. 
Nil M, Schafer D, Radtke T, Saner H, Wilhelm M, Eser P. Reproducibility of peripheral 
arterial tonometry measurements in male cardiovascular patients. Eur J Clin Invest 
2014;44(11):1065-1071. 
 
56. 
Matsuzawa Y, Kwon TG, Lennon RJ, Lerman LO, Lerman A. Prognostic Value of Flow- 
Mediated Vasodilation in Brachial Artery and Fingertip Artery for Cardiovascular Events: 
A Systematic Review and Meta-Analysis. J Am Heart Assoc 2015;4(11). 
 
57. 
Widlansky ME, Gokce N, Keaney JF, Jr., Vita JA. The clinical implications of endothelial 
dysfunction. J Am Coll Cardiol 2003;42(7):1149-1160. 
 
58. 
Flammer AJ, Anderson T, Celermajer DS et al. The assessment of endothelial function: 
from research into clinical practice. Circulation 2012;126(6):753-767. 
 
59. 
Corretti MC, Anderson TJ, Benjamin EJ et al. Guidelines for the ultrasound assessment 
of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the 
International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002;39(2):257- 
265. 
 
60. 
Hamburg NM, Benjamin EJ. Assessment of endothelial function using digital pulse 
amplitude tonometry. Trends Cardiovasc Med 2009;19(1):6-11. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 35 
 
 
14 Appendix A: Table of Study Events 
 
 
 
Event 
Screening 
Visit 1 
Day 0 
Visit 2 
Day 7±3 
Visit 3 
Day 28±3 
Visit 4 
Day 42±3 
Informed Consent 
X 
Medical History 
X 
Physical Exam 
X 
X 
Vital Signs 
X 
X 
Concomitant Medications 
X 
X 
X 
Study Drug Pill Counts 
X 
X 
AE Assessment 
X 
X 
X 
X 
Vascular Studies 
FMD 
X 
Xa 
PAT 
X 
Xa 
Laboratory Studies 
UrineTXB2-M 
X 
Urine Prostanoids (8-iso- 
PGF2α; 6-ketoPGF1α; 2,3- 
dinor-6-ketoPGF1α) 
X 
X 
Urine Creatinine 
X 
X 
X 
Plasma ICAM-1; APC; TF 
X 
X 
Serum creatinine; hs-CRP; 
NT pro-BNP 
X 
X 
aPerformed while still on study drug. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 36 
 
 
15 Appendix B: Brachial Reactivity 
15.1 Introduction 
Healthy blood vessels dilate in response to an increase in blood flow, a phenomenon known as 
flow-mediated dilation (FMD).57 FMD is predominantly a function of endothelial nitric oxide 
(NO) release that relaxes underlying vascular smooth muscle cells. Endothelial release of PGI2 
also plays a role in mediating FMD. TXA2, in addition to being a potent platelet activator, is 
also a potent vasoconstrictor that antagonizes the effects of NO and PGI2. 
 
Vascular dysfunction is associated with the development and presence of atherosclerosis and 
characterized by an impaired vasodilatory response to blood flow. FMD is traditionally 
measured non-invasively by ultrasound in the brachial artery following a period of blood flow 
cessation caused by an inflated blood pressure cuff.58 Typically, the cuff is inflated to at least 
50 mm Hg above systolic pressure to occlude arterial inflow for 5 minutes. This 
causes ischemia and consequent dilation of downstream resistance vessels via autoregulatory 
mechanisms. Subsequent cuff deflation induces a brief high-flow state through the brachial 
artery (reactive hyperemia) to accommodate the dilated resistance vessels. The resulting 
increase in shear stress causes the brachial artery to dilate. The ratio of the brachial artery 
diameter after the occlusive period to that at baseline is recorded as the percent FMD. Impaired 
FMD is associated with cardiovascular risk factors and the development of clinical 
cardiovascular disease. The protocol that will be used is based on guidelines published by the 
American College of Cardiology.59 
15.2 Protocol 
15.2.1 Subject preparation 
1. 
Prior to the study, ensure the patient has fasted for at least 4 hours, and has refrained 
for at least 8 hours from caffeine, tobacco, vitamins or medications that might affect 
vascular tone. The patient may wish to use the restroom prior to the study. 
2. 
The brachial reactivity study will be conducted in a quiet, dimly lit, temperature- 
controlled exam room to reduce fluctuations in vascular tone. 
3. 
Cell phones or paging devices should be silenced, and restrictive clothing that could 
interfere with blood flow to the arms should be removed. The patient should also 
remove watches, rings, or other jewelry on the hands or fingers. 
4. 
The patient should be supine and comfortable for 15 minutes so as to attain a 
cardiovascular steady-state. Place the two arm supporters along each of the patient's 
sides. 
5. 
Place a blood pressure cuff on the arm to be occluded during the brachial reactivity 
study. Apply the cuff snugly, but without excess pressure. Do not inflate the cuff at 
this time. 
6. 
The brachial artery is imaged above the antecubital fossa in the longitudinal plane 
(Fig. 1) using a linear array transducer with a minimum frequency of 7 MHz, attached 
to a high-quality mainframe ultrasound system. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 37 
 
 
7. 
A segment with clear anterior and posterior intimal interfaces between the lumen 
and vessel wall is selected for continuous 2D grayscale imaging. During image 
acquisition, anatomic landmarks such as veins and fascial planes are noted to help 
maintain the same image of the artery throughout the study. A stereotactic probe- 
holding device can be helpful. 
 
 
 
Figure 1. Ultrasound image of the brachial artery (longitudinally) at 8× 
magnification, 11-MHz transducer frequency annotated for anatomic 
landmarks. 
 
15.2.2 
Conduct of the Brachial Reactivity Study 
 
1. Tell the patient that you are going to inflate the cuff for the occlusion phase and that he or 
she should stay relaxed and not move the fingers. 
 
2. Rapidly inflate the blood pressure cuff to a supra-systolic pressure of 60mmHg above the 
patient's systolic pressure or 200mmHg, whichever is higher. A mid-artery pulsed 
Doppler signal is obtained to confirm cessation of blood flow. 
 
3. After 5 minutes, rapidly deflate the blood pressure cuff. A midartery pulsed Doppler 
signal is obtained upon immediate cuff release and no later than 15 s after cuff deflation 
to assess hyperemic velocity. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 38 
 
 
 
 
15.3 Representative Results 
4. 
 
Figure 2. Schematic drawing of ultrasound imaging of the brachial artery and representative 
normal and abnormal results. 
 
16 Appendix C: Peripheral Arterial Tonometry 
16.1 
Introduction 
Assessment of digital vasodilator responses using a commercially-available fingertip peripheral 
arterial tonometry (PAT) device (EndoPAT; Itamar Medical LTD, Caesarea, Israel) is a novel 
measure of vascular function that provides different but complimentary information compared 
with FMD.60With this device, a beat-to-beat plethysmographic record of the finger arterial 
pulse wave amplitude is captured using pneumatic probes placed on the index finger of each 
hand (Figure 1). The same reactive hyperemia technique that is used to measure FMD is used 
for Endo-PAT and the two techniques can be performed simultaneously. The baseline digital 
pulse amplitude is a measure of local arterial tone in the fingertip. The PAT ratio records post- 
ischemic arterial responsiveness to reactive hyperemia produced by a cuff occlusion in the 
forearm. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 39 
 
 
 
 
 
 
Figure 1. The Endo-PAT device. 
 
16.2 Protocol 
16.2.1 Subject Preparation 
1. 
Prior to the study, ensure the patient has fasted for at least 4 hours, and has refrained 
for at least 8 hours from caffeine, tobacco, vitamins or medications that might affect 
vascular tone. The patient may wish to use the restroom prior to the study. 
2. 
The Endo-PAT study will be conducted in a quiet, dimly lit, temperature-controlled 
exam room to reduce fluctuations in vascular tone. 
3. 
Cell phones or paging devices should be silenced, and restrictive clothing that could 
interfere with blood flow to the arms should be removed. The patient should also 
remove watches, rings, or other jewelry on the hands or fingers. 
4. 
Inspect the patient's fingers for any deformities or injuries that could affect the study. 
Do not place the probes on a finger that is cut or injured. Fingernails should not 
extend more than 5mm or 1/5 of an inch beyond the tip of the finger tissue. Trim or 
file fingernails if necessary to avoid damaging the internal membranes of the PAT 
probes and displacing the finger from the sensing region of the probe. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 40 
 
 
5. 
The index finger is recommended for the study; however, if this finger is unsuitable, a 
different digit (except the thumb) may be used, as long as the same finger is used on 
both hands. 
6. 
The patient should be supine and comfortable for 15 minutes so as to attain a 
cardiovascular steady-state. Place the two arm supporters along each of the patient's 
sides. 
7. 
Measure the blood pressure using the control arm (the arm that is not occluded during 
the Endo-PAT study. 
8. 
Place a blood pressure cuff on the arm to be occluded during the Endo-PAT study. 
Apply the cuff snugly, but without excess pressure. Do not inflate the cuff at this time 
16.2.2 
Prepare the Endo-PAT System for Study 
1. 
Launch the Endo-PAT 2000 software and click the "Patient Information" icon on the 
tool bar to create a new patient file. 
2. 
Complete the Patient Information dialog box, including patient ID, name (optional), 
age, gender, height, weight, systolic and diastolic blood pressures. Optional fields 
allow for free text comments. Select your name from the pre-defined list in the 
Patographer name field. If your name has not been inserted add it the list and select. 
3. 
Select two new PAT probes and connect to the pneumo-electrical tubing. To connect 
the probes, insert the connector tab into the probe slit and gently press the connector 
onto the probe until it clicks into place. 
4. 
Place the connected probes into the sockets of the arm-supports and press the 
"Deflate" button on the top of the Endo-PAT 2000 device. 
16.2.3 
Conduct of the Endo-PAT Study 
1. 
Place the patient’s index fingers completely into the probes, confirm with the patient 
that he or she can feel the very end of the probes, and press the "Inflate" button on the 
top of the Endo-PAT 2000 device. 
2. 
Place a foam anchor ring at the base of the adjacent middle finger. Ensure that the 
foam ring and the PAT sensor do not touch. Otherwise the ring may mechanically 
interfere with the sensor. 
3. 
Create an approximately 7-10cm loop with the pneumo-electrical tubing. The loop 
should extend from the PAT sensor and return to the foam ring on the adjacent finger 
while the rest of the tubing that connects to the EndoPAT device is pointing out 
tubing to the tip of the finger. 
4. 
Position the patient's arms so the forearms are supported on the arm supports and the 
fingers dangle freely off the edge of the support. Make sure the probes are not in 
contact with any object, including the arm support, foam ring, tubing, the mattress or 
another finger. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 41 
 
 
5. 
Ask the patient to refrain from moving the fingers, as this will create mechanical 
artifacts. It is important for the patient to be relaxed throughout the study. Explain to 
the patient that during the test you will inflate the arm cuff, and during that time they 
may feel some discomfort, numbness, or tingling. 
6. 
Click the "Standby" icon on the Endo-PAT's computer interface. Adjust the time base 
to 1 minute and adjust the signal gain on the screen to maximize signal clarity. 
Inspect the tracings of the PAT signals from the two probes to confirm that they are 
free of artifactual signals. If artifactual signals are present, veriify that the probes are 
not touching anything and that the patient is not moving the fingers. 
7. 
To begin the study, click the "Go" icon on the computer interface. Start the 
stopwatch, by clicking the "Start/Stop Timer" icon. This will initiate a five minute 
count down for the baseline recording period. After five minutes, stop the stopwatch 
by clicking the "Start/Stop Timer" icon. 
8. 
Tell the patient that you are going to inflate the cuff for the occlusion phase and that 
he or she should stay relaxed and not move the fingers. 
9. 
Rapidly inflate the blood pressure cuff to a supra-systolic pressure of 60mmHg above 
the patient's systolic pressure or 200mmHg, whichever is higher and start the 
stopwatch again. Complete cessation of blood flow to the hand is verified by the 
absence of a PAT signal from the occluded arm. To confirm occlusion increase the 
gain on the screen of the channel of the occluded side to 20,000 while keeping the 
gain of the contra-lateral side constant. Decrease the time base of both channels to 30 
seconds. Verify that you do not observe any signals at a periodicity that matches the 
signal from the control arm as this indicates an incomplete occlusion. If this is the 
case then further inflate the cuff until no signals are seen. The cuff may be inflated to 
a maximum of 300mmHg. 
10. 
This will initiate a five minute count down for the arterial occlusion recording period. 
Toward the end of the occlusion period tell the patient you are going to release the 
cuff and that they should continue to refrain from moving their fingers. After exactly 
five minutes, deflate the cuff abruptly as quickly as possible and stop the stopwatch 
by clicking the "Start/Stop Timer" icon. 
11. 
Click the "Start/Stop Timer" icon again to initiate a five-minute post occlusion 
recording period. Stop the timer after five minutes and click the "Test Stop" icon to 
complete the study. The probes will automatically deflate. 
12. 
Remove the probes, tape, and foam rings from the patient's fingers and disconnect the 
PAT probes from the pneumo-electrical tubing. Discard the used probes. 
 
16.2.4 
Review and Analysis 
1. 
Load the study file to the screen using the load icon. To run the automatic analysis, 
click the "magician stick" icon. The occlusion period will be highlighted in blue and 
the test result will be displayed, including the Reactive Hyperemia Index (RHI) and 
Heart Rate (HR), in the right-hand column of the screen. 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 42 
 
 
2. 
To review additional data, including study parameters, calculated variables, patient 
information, and measures of signal quality, click the "Open Results of Last 
Calculation" icon. This will open a spread sheet with study parameters and results for 
all analyses performed to date, with the last line in the table containing data from the 
most recent analysis. 
16.3 Representative Results 
A representative Endo-PAT screen of a study performed on an individual with normal 
endothelial vasodilator function is shown in Figure 2 and a representative screen performed on 
an individual with endothelial vasodilator dysfunction is shown in Figure 3. 
 
Figure 2: Normal Endothelial Vasodilator Function. Representative 
recording of an individual with normal endothelial vasodilator function, 
characterized by an increase in the signal amplitude after cuff release relative 
to baseline. 
 
 

Protocol H00017901 
Version 5, August 2022  
CONFIDENTIAL 
Page 43 
 
 
 
Figure 3: Abnormal Endothelial Vasodilator Function. Representative 
recording of an individual with abnormal endothelial vasodilator function, 
characterized by lack of increase in the signal amplitude after cuff release 
relative to baseline.