Platelet Activity in Vascular Surgery and Cardiovascular Events
Conditions: Cardiovascular Disease, Peripheral Artery Disease, Critical Limb Ischemia
Sponsor: NYU Langone Health

 
 
Official Title: 
Platelet Activity in Vascular Surgery and Cardiovascular Events 
NCT Number: 
NCT02106429 
Study Number: 
s14-00531 
Document Type:  
Study Protocol and Statistical Analysis Plan 
Date of the 
Document: 
• 
February 11, 2022 
 
 

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Platelet Activity in Vascular Surgery and Cardiovascular Events 
 
 
 
 
 
Protocol 
 
 
 
 
 
 
Study Principal Investigator 
Jeffrey S. Berger, MD, MS, FAHA, FACC 
Assistant Professor of Medicine (Cardiology and Hematology) 
Assistant Professor of Surgery (Vascular Surgery) 
Director of Cardiovascular Thrombosis 
New York University School of Medicine 
530 First Avenue, Skirball 9R 
New York, NY 10016 
Tel. 212-263-4004 
Fax. 212-263-3988 
E-mail. jeffrey.berger@nyumc.org 
 
 
  

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Table of Contents 
1. Synopsis……………………………………………………………………………………………………..……… 3 
2. Background and Significance……………………………………………………………………………..   3 
3. Specific Aim ……………………………………………………………………………………………………… 3 
4. Research Design...……………………………………………………………………….…………………… 4 
 
a. Recruitment………………………………………………………………………………………………… 
4 
 
b. Informed Consent………………………………………………………………………………………… 4 
 
c. Methods……………………………………………………………………………………………………… 
5 
 
d. Genetic Profiling……….……………………………………………………………………………….…    6 
 
e. Future Sample Storage……………………………………………………………………………….…   7 
 
f. After Surgery and Follow Up…………………………………………………………………….……   7  
 
g. Inclusion Criteria…………………………………………………………………………………………..  8 
 
h. Exclusion Criteria…………………………………………………………………………………………… 8 
i. Endpoint Assessments………………………………………………………………………………….. 8 
5. Potential Risks and Benefits……………………………………………………………………………………… 8 
 
5.1 Benefits……………………………………………………………………………………………………..     9 
6. Cost………………………………………………………………………………………………………………………..…    9  
7. Screening Log……………………………………….………………………………………………………………..…    9  
8. Statistical Analysis…………………………………………………………………………………………………….. 10 
 
8.1 Data Analysis…………………………………………………………………………………………….… 10 
8.2 Data Safety Monitoring Plan……………………………………………………………………….. 11 
9. Confidentiality……………………………………………………………………………………………………………  11 
10. Appendix A: Schedule of Study Related Procedure………………………………………………….   12 
11. References…………………………………………………………………………..…………………………….......  14 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Synopsis 
Peripheral artery disease (PAD) is a highly prevalent condition in the United States, and a significant 
cause of cardiovascular morbidity and all-cause mortality.  Although vascular surgical procedures are 
commonly performed to improve patient survival and quality of life and to prevent vascular disease 
related complications, these procedures carry heightened risk of adverse perioperative cardiovascular 
events.  
2. Background and Significance 
Pathological and clinical studies have consistently demonstrated that abnormalities in thrombosis and 
hemostasis play a major role in the pathogenesis of atherosclerosis and atherothrombosis.  Screening 
for abnormalities in thrombosis and hemostasis by measuring platelet activity, thrombin generation, 
markers of coagulation, and inflammatory biomarkers has been proposed to identify individuals at high-
risk for cardiovascular events, however, it remains a research tool not ready for implementation in 
standard care.  While many abnormalities in thrombosis and hemostasis have been widely studied in 
cardiac disease, there is a paucity of data in the setting of vascular surgery.  It is unknown how vascular 
surgery affects different measurements of platelet activity, thrombin, coagulation and inflammatory 
markers. It is also unknown what the association of these markers is with perioperative and long-term 
cardiovascular and bleeding events. 
 
Factors responsible for platelet activity are poorly understood.  Recent advances in genomic and 
transcriptomic profiling technologies may offer the possibility of identifying novel biomarkers and 
pathways involved in platelet activity.  Anucleate platelets lack genomic DNA, yet are known to retain a 
small amount messenger-RNA that can regulate protein expression, and have a diverse array of 
microRNA (miRNA).  Comparison of platelet RNA and miRNA could potentially provide vital information 
on platelet function and disease states where platelet activity is affected, such as atherosclerosis.  
Combining descriptive analysis of platelet activity with novel RNA and DNA profiling may offer the 
unique opportunity to develop biomarkers for the diagnostic, prognostic, and predictive management of 
subjects undergoing vascular surgery. 
 
The proposed study will add to the growing understanding of platelet activity and markers of 
coagulation in cardiovascular disease; examine a comprehensive battery of platelet activity markers, 
thrombin generation, markers of coagulation, and inflammatory biomarkers in subjects undergoing 
vascular surgery; and will provide important data on the mechanism of increased platelet activity using 
micro RNA, RNA and DNA expression profiling.  The study design is prospective and the main outcome 
measures are platelet activity, coagulation markers and incident cardiovascular and bleeding events.  
 
Previous research suggests that the incidence of dementia and Alzheimer’s disease (AD) is higher in 
those with prevalent cardiovascular disease (CVD), and is also associated with increased platelet activity. 
Whether the platelet abnormalities are related to a common atherogenic process or rather represent 
different inflammatory pathways is unclear and needs to be examined. In addition to their role in CVD, 

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platelets have been implicated in the pathology of AD at several levels. First, abnormal platelet 
activation markers are frequently observed in AD patients when compared with age-matched 
cognitively normal controls. Additionally, results in experimental animal models and postmortem human 
brains show that platelet markers accumulate in perivascular lesions. Since platelets contain Aβ and tau, 
which are the components of amyloid plaques and neurofibrillary tangles found in AD lesions, it is 
possible that platelets are contributors to lesion formation at earlier stages of the disease.  
 
We hypothesize that the platelet phenotypes (already measured) in patients with CVD are associated 
with cognitive decline and our preliminary studies suggest that this is the case. Using data from the 
Framingham Heart Study, we recently demonstrated that platelet aggregation in cognitively unimpaired 
middle age subjects is independently associated to the risk of incident dementia even after adjusting for 
potential confounders suggesting that the detection of platelet phenotypes in patients with CVD could 
be used for the detection of risk of cognitive decline in this population.  
 
We designed the PACE clinical study initially to investigate platelet activity in PAD patients and the 
incidence of major adverse cardiac and limb events after lower extremity revascularization. PACE was 
set up to investigate the relationship between platelet activity and cardiovascular risk. Many studies 
suggest the overlapping nature of cardiovascular risk and dementia. Thus, we would like to extend 
findings that have established the link between heart disease and dementia to platelet activity and 
dementia. The comprehensive platelet characterization already performed would facilitate our study 
investigating the association between platelets and cognitive decline in this cohort of patients with 
established CVD. We will attempt to call all living participants and obtain telephone consent for the 
additional measure, assess vital status, and perform a validated survey of subclinical cognitive 
impairment in the PACE cohort with 2 previously validated neuropsychiatric tests that are frequently 
used in aging studies: TICS and Montreal Cognitive Assessment, MoCA. Both tests can be easily done 
remotely by trained personnel and are particularly good at detecting early subclinical impairment.  
 
 
3. Specific Aims 
 
Aim 1. To determine whether preoperative platelet activity measurements are independently 
associated with short-term cardiovascular events in PAD patients undergoing non-emergent lower 
extremity revascularization. We will characterize the platelet phenotype in 350 PAD patients before 
vascular surgery and use Cox proportional hazard models to determine the independent association of 
the platelet phenotype with risk of cardiovascular events in the first 30 days after surgery.  
 
Aim 2. To determine whether postoperative platelet activity measurements are independently 
associated with long-term cardiovascular events in patients with established PAD. We will characterize 
the postoperative platelet phenotype following surgery and use Cox proportional hazards models to 
determine the independent association of the platelet phenotype with risk of long-term composite of 
myocardial infarction, stroke, or all-cause mortality with a mean follow-up of 2-years following vascular 
surgery.   

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Aim 3. To investigate mRNA-microRNA co-expression profiles in patients with and without elevated 
platelet activity measurements. We will establish the relationship between differentially expressed 
microRNAs and their target mRNAs related to platelet activity and thus identify new diagnostic markers 
and potential therapeutic targets of increased platelet activity.  
 
Aim 4. To investigate whether the platelet phenotype of patients with PAD associates with measures 
of cognitive impairment. We will contact participants enrolled in PACE to conduct a screening test for 
cognitive function via telephone and/or video-call using the MoCA and/or TICS tests. We will correlate 
these measures with already collected platelet activity measures. 
 
4. Research Design 
 
a. 
Recruitment 
 
350 non pregnant adults older than 21 years with peripheral artery disease undergoing planned lower 
extremity revascularization for lower extremity peripheral artery disease will be recruited from the 
inpatient setting at NYU Medical Center, Bellevue Hospital, the Manhattan VA, as well as the outpatient 
setting in the Faculty Group Practice.  For the inpatients, health care providers, generally vascular 
surgeons and research coordinators will attempt to identify patients who present to one of the 
participating hospitals (mentioned above) and are scheduled to undergo vascular surgery.  Once these 
patients are identified, one of the health care providers or a member of the research staff will make 
certain the patient fulfills the inclusion and exclusion criteria and will inform assigned health care 
provider who will obtain verbal consent for further contact by a member of the research staff prior to 
the surgery.  For the outpatients, patients fulfilling inclusion and exclusion criteria will be identified by 
their health care professional, who will obtain verbal consent for contact by a member of the research 
staff. Vascular surgeons will not be involved in the consenting process due to their limited time but once 
consent for contact is obtained, research staff will approach subjects for consent of study participation. 
Subjects are not consented over the phone. Patients may be approached ahead of time to eliminate 
added stress of surgery and/or for ease of enrollment.  The patient’s vascular surgeon will contact 
patients by phone or in person prior to the surgery (e.g. pre-surgical testing or at the group practice), to 
obtain consent for contact by research staff and then the research staff would consent subjects in 
person. Only subjects that have the capacity to consent will be asked to consent. Subject’s capacity is 
assessed by the research staff member performing the consent. The staff member will ask the subject if 
he/she understands the study procedures and consent form as well as answer subject questions to 
gauge capacity. 
 
 
 
b. 
Informed Consent 
 
 
If a subject wishes to participate, a member of the research team will review the informed consent 
documents, answer any questions, and obtain written consent for participation in the study and obtain 

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written consent for future analysis, contact, and sample storage. Stored samples will not undergo 
further genetic testing. Consenting will take place in the hospital (in the preadmission testing or in the 
pre-operative holding area) or in a private office (in the vascular surgery or medical practice).  Subjects 
will be given the opportunity to be contacted for future studies that they may qualify for as well. All 
subjects will be given a copy of the consent.  A member of the research team will conduct the baseline 
interview with the subject and review the clinical history, demographic information, diet questionnaire, 
lifestyle assessment, medication use and adherence, with the individual.  A case report form is attached 
as an appendix.  All information including the consent will be kept under double lock and key at the PI’s 
office in NYU Skirball 9th floor  
 
 
c. 
Methods 
 
Following the interview and signed informed consent, subjects will have their blood drawn, and will be 
asked to provide a urine sample prior to surgery. Blood will be obtained from the patient using standard 
aseptic phlebotomy techniques.  Blood will be collected from an arterial line placed for surgery (if 
available) or at the time of the intravenous line being placed for surgery or using a standard technique 
of blood drawing.  A brief description of the standard technique follows: after cleansing of the 
venipuncture site with an alcohol wipe and removal of excess alcohol with sterile gauze, a tourniquet 
will be applied to the patient’s bicep region.  A 19G or 21G butterfly needle will be inserted into the 
antecubital vein, the tourniquet will be removed, and free-flowing blood will be collected with minimal 
trauma and stasis.  The needle will be removed when blood collection is complete and sufficient 
pressure will be applied using sterile gauze at the puncture site until cessation of bleeding.  A sterile 
band-aid will be applied to cover the venipuncture site.  For each patient, no more than 60 ml (≈4 table 
spoons) of blood will be collected in gold (serum separator tube) top, lavender (EDTA anticoagulant) top, 
green (heparin), blue (sodium citrate anticoagulant) top and Pax gene tubes.  Approximately 40 cc of 
blood will be used for the different measurements of platelet function, inflammation, coagulation and 
other biomarkers of cardiovascular risk and approximately 20 cc of blood will be used to purify platelet 
RNA, whole blood RNA and DNA and then stored in -80C for the RNA, microRNA, and DNA profiling.  The 
samples will be placed in the PI’s lab at NYU New Science Building- Cardiology 7th floor without any 
identifying information other than a code number.  The code number will not be based on any 
information that could be used to identify the subject (for example, social security number, initials, birth 
date, etc).  The master list linking names to code numbers will be kept in a locked file cabinet, separate 
from all research information in the PI’s office at NYU Skirball 9th floor and will be accessed by the PI.  
The urine specimen will be collected to measure abnormalities in thrombosis, hemostasis and 
inflammation as well.  At the end of the patient recruitment and measurement of platelet function 
(probably within 4 years time), based on ones level of platelet activity, thrombin generation, coagulation 
and inflammation we will measure RNA, microRNA, and DNA in patients with different phenotypes of 
vascular disease, markers of thrombosis/hemostasis, and events from the stored specimens. 
 
There will be a follow-up component of this study that obtains blood and urine following surgery and at 
their first return visit following surgery.  This will allow us to assess the change in platelet activity, 

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thrombin, coagulation and inflammatory markers during and following the perioperative period.  This 
additional component would consist of a blood collection on postoperative day 2 (+/- 1 day).  No more 
than 60 ml (≈4 table spoons) of blood will be collected at this time point.  In addition, a blood collection 
will be obtained at the subjects’ return visit following surgery to the vascular surgeon.   No more than 60 
ml (≈4 table spoons) of blood will be collected at this time point.  The total blood drawn for the follow-
up component would be no more than 120ml (8 tablespoons).    This blood will be used for the different 
measurements of platelet activity, thrombin, coagulation and inflammatory markers.  Data gathered 
from this component of the study will help understand the dynamic profile of biomarkers during and 
following the perioperative period.  
 
Total amount of blood withdrawn will be no more than 180 ml (approximately 12 tablespoons or 40% of 
a standard blood donation).  We believe this amount of blood is small and spread out over a month and 
therefore patient is at minimal risk.  Blood collection at three different time points (before surgery, 
following surgery while still in the hospital, and at the subjects’ first return visit to the vascular surgeon 
following surgery) will allow us to assess the dynamic change in platelet activity, coagulation and 
inflammation during the perioperative period.  We believe that markers of clotting and bleeding will 
change during the course of surgery, and that some of these markers may be used to help predict the 
likelihood of developing a clotting or bleeding event following surgery.  The long-term goal is to develop 
a clinically useful assessment of platelet activity, thrombin generation, coagulation and inflammation for 
risk stratification that may ultimately serve as a target for therapeutic intervention.  The platform 
proposed will serve as a basis for future research and intervention, and hopefully, will have a great 
impact on subjects undergoing vascular surgery.  
 
During surgery, clot and or part of the diseased vascular wall is sometimes removed. If available at the 
time of surgery, we will collect tissue samples that will be removed as a part of the surgery, which would 
otherwise be discarded. Preservation of this tissue does not involve additional risk to the subjects. A 
portion of this tissue will be preserved in formalin, and sectioned in order to study factors contributing 
to cardiovascular disease risk. Similar to the blood and urine collection, all samples will be stored under 
unique identifiers (as all samples taken in the study).  Samples will be stored for up to 20 years. If 
subjects request the withdrawal of data and samples from the study, samples and all identifiers will be 
destroyed.  
 
We may also implement an assessment of endothelial function prior to and following vascular surgery.  
Since measurement of peripheral vasodilator response as a measure for endothelial dysfunction is 
correlated with adverse cardiovascular events and measurements of this response using fingertip pulse 
amplitude tonometry (peripheral arterial tonometry-PAT) is a useful method for non-invasive 
assessment of vascular health.  Peripheral arterial tonometry signals will be obtained using the EndoPAT 
2000 device (Itamar Medical Inc., Caesarea, Israel), which has been validated and used previously to 
assess peripheral arterial tone in high-risk populations. This measurement would be done prior to and 
following surgery (same time points that blood is collected).  
 

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d. 
Genetic Profiling 
The blood specimen will be analyzed upon completion of the study for mRNA, microRNA, and DNA 
profiling.  We will perform detailed molecular profile study, describing genetic arrays with different 
phenotypes associated with platelet activity.  We will be looking at specific transcripts associated with 
thrombotic and/or bleeding outcomes and different cardiovascular diseases. Platelet-rich plasma (PRP) 
will be isolated for platelet purification and subsequent genetic analysis will be performed.  We will be 
analyzing those with high and low levels of platelet activity and other cardiovascular biomarkers as well 
as those with clotting/no clotting or bleeding/no bleeding. Biomarkers of cardiovascular disease will be 
shared as per NIH. The samples will be stored in the PI’s lab, NYU New Science Building- Cardiology 7th 
floor, without any identifying information other than a unique code number.  The code number will not 
be based on any information that could be used to identify the subject (for example, social security 
number, initials, birth date, etc.).  The master list linking names to code numbers will be kept in a locked 
file cabinet, separate from all research information.  Biomarker testing is separate from genetic testing. 
Genetic information will not be disclosed to anyone, including the participants and their physicians.  
Positive test result can be an indication that the individual may be predisposed to high platelet activity 
associated with peripheral vascular disease.  There is no need for independent testing since patients are 
already diagnosed with this condition.  No tests other than authorized shall be performed on the 
biological sample.  Samples shall be destroyed at the end of the testing process.   
 
 
e.  
Future Sample Storage 
All patients will be offered an option to have their blood stored for future processing.  If a patient does 
not agree to store his/her blood for future tests not related to this study, then his/her blood will be 
destroyed after all planned laboratory tests have been performed.   
Blood samples will be assigned a unique code number and stored in the PI’s lab without any identifying 
information other than a code number. The unique code number will not be based on any information 
that could be used to identify the subject (for example, social security number, initials, birth date, etc).  
The master list linking names to code numbers will be kept in a locked file cabinet, separate from all 
research information placed under double lock and key at the PI’s office at NYU Skirball 9th floor. All 
confidential data will be stored with a unique code as an identifier and will be protected by a double 
electronic lock.  All physical data will be kept under double physical lock.  Access to the data will be given 
to the study personnel only.   
Samples will be stored for no more than 10 years following completions of the study.  Samples will be 
used for testing other potential biomarkers in patient plasma and/or serum related to other endpoints 
not specified in the protocol (e.g. cancer, metabolic disease, etc.).  Genetic testing will not be conducted 
on stored samples. Results of the future research will not be shared with the subjects or their study 
doctor, and will not become part of the medical records.  
Subjects can request for withdrawal of samples at any time by contacting PI Dr Berger in writing.  His 
mailing address is Faculty Practice Tower 9 R, 530 First Ave NEW YORK 10016.  Withdrawing 

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Authorization only affects uses and sharing of information after written request has been received, and 
subject may not withdraw his/her Authorization for uses or disclosures that have previously been made 
or must continue to complete analyses or report data from the research.  
 
 
 
f. 
After Surgery and Follow Up 
 
Starting on the day of surgery and continuing throughout hospitalization, a physician monitors the 
patient’s course for clinically evident cardiovascular events (MI, stroke, TIA, major bleeding, kidney 
disease, or any surgical complication.  Blood collections and/or ECG’s that are performed as part of routine 
care and/or for study purposes will be recorded and may be used to help ascertain whether a 
cardiovascular and/or bleeding event has occurred. After hospital discharge, patients will be contacted by 
telephone (or during their routine clinical follow-up) at 30 days and every 6-month thereafter out to 5-
years.  At the patients’ follow-ups they will be asked questions about their health, lifestyle, and medication 
use.  
To ascertain vital status, we may use the social security index, medical record chart review, or contact the 
participant directly. If we learn that the participant is deceased, we may perform a chart review to check 
for any prior clinical diagnosis of cognitive impairment. 
We will seek remote verbal re-consent for sub-clinical cognitive function testing (via TICS and/or MoCA 
test) from all living study participants. We will document in the study record whether a participant 
consents or declines to participate in the cognitive function testing. For participants who provide verbal 
consent, testing will be administered remotely by trained study personnel and will not require any in-
person visits. If we identify sub-clinical signs of cognitive impairment based on testing, we will inform the 
patient and will offer them the possibility of writing a letter to their primary healthcare provider.  
Participants with existing mild cognitive impairment: Given the advanced age of study participants, we 
will first ask participants if they have ever been diagnosed with dementia (including Alzheimer’s) by their 
healthcare provider and record their response in the study record. In collaboration with the PI, our 
neuroscience experts, and our senior research coordinator, we will assess decision-making capacity while 
talking to the participant. If the participant has sufficient capacity to consent, we will proceed with seeking 
verbal consent for cognitive function testing. If the participant declines cognitive function testing but gives 
permission for us to record their history of cognitive impairment, we will record this information in the 
study record. If the participant appears unable to provide verbal consent, they will not undergo cognitive 
function testing.  
 
g.  
Inclusion Criteria 
1. Subjects undergoing non emergent lower extremity revascularization* 
2. Age > 21 years of age 

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3. Able and willing to provide written informed consent for the study 
*Subjects who participated in this trial may be re-enrolled if they undergo another lower extremity revascularization >90 days 
after the original procedure. This modification is being done to increase enrollment and because the major aim is to look at peri-
procedural events and thus would not interfere with the overall goal of the study.  
 
h. 
Exclusion Criteria 
1. Use of any NSAID (ibuprofen, naproxen, etc.) within 72 hours   
2. Thrombocytopenia (platelet count<100) or Thrombocytosis (platelet count>500) 
3. Anemia (hemoglobin<8) 
4. Any known hemorrhagic diathesis 
 
 
Subjects will be re-enrolled and contacted the same way any subject is enrolled in the study.  If the 
subject is undergoing a lower extremity revascularization procedure and they are referred to the study 
by a health care professional taking care of the subject, they will be approached by a member of the 
research team.    This does not give a benefit to the patient other than to help answer an important 
scientific question. This poses no additional risk to the patient. 
i. 
Endpoint Assessments 
 
At every visit after randomization, the study coordinator will ask participants if they have had any 
symptoms or a report from a healthcare provider consistent with an endpoint event since the last study 
visit.  If at any time the subject refuses to continue with follow-up, every attempt will be made to 
continue contact by written communication, email, by proxy contact with family, friends, or allied health 
care providers, or record review to determine if outcome events have occurred, unless the subject 
specifically refuses such follow-up.  An independent clinical event adjudication committee (CEC) will 
review and adjudicate primary and selected secondary endpoint events in a blinded fashion based on 
study definitions.   
 
5. Potential Risks and Benefits 
 
The potential risks of performed phlebotomy are pain, bruising, fainting, or small infection at puncture 
site.  The use of the blood for future sample storage and genetic testing raises special issues of 
confidentiality.  Genetic testing can generate information about a subjects’ personal health risks and can 
cause or increase anxiety, damage family relationships, and/or compromise insurability, employability 
and can even lead to discrimination.  All confidential data will be stored with a unique code as an 
identifier and will be protected by a double electronic lock.  All physical data will be kept under double 
physical lock.  Access to the data will be given to the study personnel only thereby greatly reducing the 
possibility of psychological or social risks that could arise from knowledge of this genetic information, 
such as risk for employability or insurability or the risk of discrimination. 

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Additionally, there is a potential risk for a breach of confidentiality for all confidential data.  All data will 
be stored with a unique code that does not identify the subject.  Furthermore, data will be saved using a 
double electronic lock.  All physical data will also be kept under double physical lock.  Access to the data 
will be given to the study personnel only.  
 
There are no known risks associated with endothelial function assessment. The blood pressure cuff on 
the forearm may rarely cause pain or bruising. If a subject experiences pain, the cuff will be deflated. 
 
5.1 Benefits 
 
The proposed study will examine a panel of markers related to platelet function, inflammation, and 
coagulation and assess their ability to predict cardiovascular events following vascular surgery. The long-
term goal is to develop a clinically useful assessment of platelet activity for risk stratification that may 
ultimately serve as a target for therapeutic intervention.  The platform proposed will serve as a basis for 
future research and intervention, and hopefully, will have a great impact on subjects undergoing 
vascular surgery.  There might be no direct benefit to enrolled subjects. The cognitive function follow-up 
screening will act as another clinically valuable measure of risk predicted by platelet activity. We aim to 
use the information as future clinical research goal and will not provide direct benefit to patients. 
 
6. Cost 
 
There is no cost or compensation to the study participants.  There is also no direct benefit expected 
from the participation in this study.  This study will provide potentially valuable information with 
minimal risk to study subjects.  It is hoped that knowledge gained will be of benefit to others in the 
future.   
 
7. Screening Log 
 
During the study enrollment period, sites may collect de-identified data on all patients undergoing lower 
extremity revascularization. The goal of this effort will be to describe the characteristics of patients who 
are screened but not enrolled and to document the major reasons for exclusion.  Examples of data 
elements to be collected, when available, include: (age [recorded for patients <90 years of age, recorded 
as 90 if >90 years of age], sex, race/ethnicity, basic medical history from medical chart (if available), in-
hospital events (if available from the chart) and, if excluded from the study, reason(s) for exclusion will 
be recorded).  Importantly, only de-identified health information will be recorded and stored in the PI’s 
office at NYU Skirball 9th floor, under double physical lock and key. 
 
 
8. Statistical Analysis 
 

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Power and sample size analysis was performed to investigate appropriate sample size for evaluating 
platelet activity markers in subjects with cardiovascular disease CVD versus age- and sex matched 
controls.  The study is powered based on the analysis of association between platelet activity and 
cardiovascular events.  Based on published and preliminary data, we expect the 30-day event rate to be 
22%.  The relationship between platelet activity and short- term cardiovascular events will be assessed 
using 2 co-primary measures: LTA and MPA.  Based on preliminary data, LTA levels have skewed 
distribution and MPA levels are normally distributed.  Consider a transformation (e.g. log- or Box-Cox) 
on LTA measures that achieves Normality.  With 200 participants, two-sided Wilcoxon test will have 80% 
power at the 0.025 significance level to detect an association between platelet reactivity and short-term 
events corresponding to a 0.5*standard deviation difference in platelet 
 
8.1 
Data Analysis  
All eligible subjects will be assigned a unique identification number upon enrollment in the study.  Initial 
study data will be recorded on a printed form, which will be later converted to an IRB approved 
electronic database, REDCap. Only study personnel will have access to this database. All potential 
identifiers will be kept separately in a safe location at the PI’s office in NYU Skirball 9th floor, where it will 
be placed under double lock and key and the PI will have access.  After verification of entered data at 
completion of the study all potential identifiers will be stripped from the database. 
Descriptive statistics, including frequency tables, measures of center and variability, and graphical displays 
will present baseline subject characteristics. Similar analyses will present values of platelet activity 
measures.  
 
Primary Platelet Activity Measures: LTA and MPA To test the hypothesis that platelet activity 
measurements are independently associated with 30-day (Aim 1) and long-term (Aim 2) cardiovascular 
events in patients undergoing lower extremity revascularization we will employ the following analysis. For 
Aim 1, platelet activity will be measured preoperatively and the main outcome will be 30-day 
cardiovascular events. For Aim 2, platelet activity will be measured 48 hours postoperatively and the main 
outcome will be long-term (mean follow-up ~ 2 years) cardiovascular events. We will perform univariate 
analysis to investigate the association between LTA and MPA platelet activity measures and cardiovascular 
events. Next, we will examine differences in sociodemographic/clinical factors between participants with 
and without a cardiovascular event using two-sided tests of proportions for categorical variables (chi-
square, Fisher’s exact) and t-tests or non-parametric alternatives (e.g., Wilcoxon test) for continuous 
measures. 
 
The analysis plan outlined above for the primary platelet activity measures will be applied to the set of 
secondary measures including thrombin. In addition, the analysis plan outlined above for the primary 
outcome measures will be applied to the secondary outcome measures including major bleeding. 
 
To identify novel mRNA-miRNA target pairs involved in platelet activation, we will computationally 
inversely correlate miRNA expression with expression of gene targets using established prediction 
databases 
 
8.2 
Data Safety Monitoring Plan 

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There are no primary or secondary endpoints. Patients undergoing vascular surgery as part of their 
routine clinical care will have a blood collection before and after surgery to help identify future markers 
in their blood related to clotting and bleeding episodes. The proposal is deemed to be low risk for 
subjects.  Safety information will be collected via telephone at 30 days after the surgery, case report 
form, and/or scheduled follow-up visits as part of standard of care.   
 
PI will conduct interim monitoring of accumulated data from research activities to assure the continuing 
safety of participants, relevance of the study question, appropriateness of the study, protocol 
compliance and integrity of the accumulating data.  The PI in conjunction with NYU IRB will be 
responsible for monitoring the data and conducting safety reviews at a minimum of every 12 months 
when annual re-approval is sought.  Any unanticipated serious adverse event will be reported to IRB 
within 72 hours.  During the review process, the PI and NYU IRB will evaluate whether the study should 
continue unchanged, require modification/amendment, or close to enrollment. 
 
 
      9. Confidentiality: 
 
 
Study is for research purposes only.  Individual result will not be given back to study participant.  This 
will include information from final results of the study, interim results of the study and incidental 
findings.  Research result will never occur in medical records.  Confidentiality will be preserved to the 
fullest extent by the research team.  All data will be stored with a unique code that does not identify the 
subject.  Furthermore, data will be saved using a double electronic lock.  All physical data will also be 
kept under double physical lock.  Access to the data will be given to the study personnel only. 
 
 
10. Appendix A: Schedule of study related procedure 
 
Pre-op 
Period 
POD 2 
(+/- 1 day) 
Discharge 
Post-op 
Appointment  
follow-up  
Long-term 
follow-up  
Eligibility 
X 
 
 
 
 
Informed consent 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical history/status 
X 
X 
X 
X 
X 
Medication use 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
 
 
Standard lab results 
X 
X 
X 
X 
X 
Troponin 
X 
X 
 
 
 

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ECG 
X 
X 
 
 
 
Blood draw 
X 
X 
 
X 
 
Urine sample 
X 
 
 
X 
 
Endothelial dysfunction 
assessment 
X 
X 
 
X 
 
QOL assessment 
X 
 
 
X 
X 
Diet questionnaire 
X 
 
 
 
 
Lifestyle assessment 
X 
 
 
X 
X 
Medication adherence 
X 
 
 
X 
X 
Hospitalization assessment 
 
 
X 
X 
X 
Endpoint assessment 
 
X 
X 
X 
X 
Cognitive Function Test 
 
 
 
 
X 
 
1. Pre-operative period 
1.1. Determine eligibility 
1.2. Obtain informed consent 
1.3. baseline interview with the subject and review the clinical history, demographic information, 
lifestyle assessment, QOL assessment, medication use and adherence  
1.4. Vital signs 
1.5. Obtain ECG 
1.6. Measure troponin 
1.7. Urine sample  
1.8. Blood sample  
1.9. Endothelial function assessment 
 
2. Post-operative day # 2 (+/- 1 day): 
2.1. review medical status 
2.2. Medication use 
2.3. Vital signs 
2.4. Review standard lab tests 
2.5. Obtain ECG 
2.6. Measure troponin 
2.7. Blood sample  
2.8. Endothelial function assessment 
2.9. Endpoint assessment 
 
3. Day of discharge 
3.1. review medical status 

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3.2. Medication use 
3.3. Vital signs 
3.4. Review standard lab tests 
3.5. Hospitalization assessment 
3.6. Endpoint assessment 
 
4. Post-op Appointment follow-up): 
4.1. review medical status 
4.2. Medication use and adherence 
4.3. Review standard lab tests 
4.4. Blood sample  
4.5. Urine sample 
4.6. Endothelial function assessment 
4.7. Lifestyle and QOL assessment 
4.8. Hospitalization and assessment 
4.9.  Endpoint assessment 
 
5. Long term follow up (q 6months [+/- 2 months]): 
5.1. review medical status 
5.2. Medication use and adherence 
5.3. Review standard lab tests 
5.4. Lifestyle and QOL assessment 
5.5. Hospitalization and assessment 
5.6.  Endpoint assessment 
5.7. Cognitive function test (will be assessed once) 
5.8. Question regarding history of dementia (will be assessed once) 
5.9. Vita status assessment 
 
 
 
 
 
 
 
 
 
 
 
 
 

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11. 
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