Influence of Rivaroxaban 2.5 mg Two Times a Day for Intermittent Claudication and Exercise Tolerance in Patients With Symptomatic Peripheral Arterial Disease (PAD) - a Randomised Controlled Trial
Conditions: Peripheral Arterial Disease
Sponsor: Poznan University of Medical Sciences

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Title:  
Influence of Rivaroxaban for Intermittent Claudication and Exercise Tolerance in 
Patients With Symptomatic PAD 
 
NCT number: 
NCT04305028 
 
Document date 
02/13/2020  
 
 

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Tables of contents 
SUMMARY  
EXISTING KNOWLEDGE AND MAIN ASSUMPTIONS OF THE PROJECT................................ 3 
1.New possibilities of pharmacotherapy for patients with symptomatic peripheral artery disease 4 
2. Pleiotropic effects of rivaroxaban on arteriosclerosis ................................................................. 4 
3. The objective ............................................................................................................................... 6 
4. Type of study ............................................................................................................................... 6 
5. Description of the study group (age, gender, health status, size of the group) ........................... 6 
5.1 Size of the study group .............................................................................................................. 7 
5.2 Characteristics of the study group and collected material ......................................................... 7 
5.3 Inclusion and exclusion criteria ................................................................................................. 7 
5.4 Pharmacotherapy ....................................................................................................................... 8 
6. Methods ....................................................................................................................................... 8 
6.1 Clinical characteristics of the studied population ................................................................... 10 
6.2. Treadmill test .......................................................................................................................... 15 
6.2.1. Qualification for the treadmill test ...................................................................................... 15 
6.2.2 The evaluation of the claudication distance ......................................................................... 15 
6.2.3. Evaluation of exercise tolerance. ........................................................................................ 16 
6.2.4. Post-test examination .......................................................................................................... 17 
6.3. Length of observation ............................................................................................................. 17 
6.4. Endpoints of the study ............................................................................................................ 17 
6.5. Biological samples ................................................................................................................. 18 
6.5.1. Biological material collection - blood samples ................................................................... 18 
6.5.2.  Preparation and storage of blood samples .......................................................................... 19 
6.5.3. Material and methods for laboratory testing of patient blood ............................................. 20 
6.5.4. Biochemical blood tests ...................................................................................................... 20 
6.5.5. Material and methods for testing peripheral blood biomarkers .......................................... 20 
6.5.6. DNA testing ......................................................................................................................... 21 
6.7. Equipment .............................................................................................................................. 21 
7. Use of results ............................................................................................................................. 21 
8. Statements ................................................................................................................................. 21 
 
 
 
 

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SUMMARY 
This study aims to assess the efficacy of rivaroxaban (trade name of the medicinal product 
Xarelto) administered together with acetylsalicylic acid (ASA) in comparison with the efficacy of ASA 
itself with respect to peripheral artery disease (PAD) severity and cardiological capacity in patients with 
PAD. Current COMPASS results (Cardiovascular Outcomes for People Using Anticoagulation 
Strategies) have shown that rivaroxaban at a vascular dose (2.5 mg 2x/day) combined with ASA (75-
100 mg 1x/day) provides more effective cardiovascular protection (defined collectively as 
cardiovascular death, myocardial infarction, and stroke) compared to ASA alone. Basic and clinical 
studies indicate that rivaroxaban not only has a positive effect on thrombotic and embolism processes, 
but also probably causes pleiotropic effects, which are associated with the reduction of vascular damage 
resulting from cardiovascular risk factors, and has the potential to inhibit the development of 
atherosclerotic changes. 
However, so far, no clinical trials have been conducted on the protective effects of rivaroxaban in 
relation to the progress of peripheral atherosclerosis and exercise tolerance in patients with PAD in 
short-term observation.  
The studies will be based on the prospective assessment of the clinical condition of PAD patients 
hospitalized in the Clinical Hospital of Transfiguration, Department of Vascular, Endovascular Surgery, 
Angiology and Phlebology of the Poznan University of Medical Sciences or monitored in the adjacent 
vascular Outpatient Clinic where anticoagulants are used in thrombotic events prevention. The expected 
observation period is three months. The proposed pharmacotherapy does not exceed the standard of 
medical care in patients with PAD. The assessment of intermittent claudication (IC) distance will be 
performed on the basis of a treadmill test, according to Gardner protocol. The assessment of exercise 
tolerance will be performed on the basis of exercise duration, questionnaire, and biochemical exponents. 
The individual oxygen ceiling value (VO2max) and energy expenditure expressed (metabolic 
equivalent MET-s) will be also estimated. Fatigue levels change will be independently assessed using 
the Borg subjective scale. At rest and after exercise, the intensity of metabolic exercise acidosis will be 
assessed (a measurement of lactic acid in blood before and after exercise). 
An additional effect of the research will be the collection of biological material for further genetic 
research and research on vascular biomarkers. It is assumed that the genetic characterization of patients 
with respect to functional polymorphisms of genes affecting the function of blood vessels (e.g., 
MTHFR, ApoE, PON1, HIF1A, VEGF) will be performed.  
The obtained results will be the basis for applying for external funding for research (NCN, 
NCBiR) and further multi-center research.. 
 
EXISTING KNOWLEDGE AND MAIN ASSUMPTIONS OF THE PROJECT 
Atherosclerosis is the most common cause of peripheral artery disease (PAD), the first 
symptom of which is intermittent claudication (IC). IC is characterized by the occurrence of pain, 
cramps, numbness, and discomfort in the lower limb muscles. They gradually intensify while 
walking, forcing the patient to stop. The ailments are caused by continuous, specific physical effort 
and quickly disappear when it is stopped. The limitation of the possibility of walking leads to a 
decrease in the quality of life of patients. It adds to other dangerous consequences of 

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atherosclerosis, increased risk of cardiovascular incidents (myocardial infarction, stroke, acute 
lower limb ischemia) (Micker M et al. 2006). 
Treatment of patients with IC is aimed at modifying cardiovascular risk factors and improving 
the quality of life by relieving pain and prolonging the IC distance. (Goszcz et al., 2009) 
1. New possibilities of pharmacotherapy for patients with symptomatic 
peripheral artery disease 
According to the adopted TASC (The Trans-Atlantic Inter-Society Consensus), and its 
TASC II supplement concerning aspects of management and treatment of patients with chronic 
limb ischemia, drugs used in PAD patients were divided into several groups because of their 
justified and documented therapeutic efficacy in multicenter clinical trials. These include: drugs 
with documented clinical effect used in intermittent claudication (cilostazol and naphthydrofuryl), 
drugs with probable clinical effect used in intermittent claudication (carnitine and propyl-L-
carnitine), lipid-lowering drugs (statins), drugs with insufficiently proven clinical effects 
(penthoxyphilin), anticoagulants, L-arginine, 5-hydroxytrypatamine (ketanserin) antagonism, 
buflomedil, and defibrotide.  
Antiplatelet drugs play an essential role in the therapy of PAD patients. They are used to 
prevent thrombotic complications in the course of myocardial infarction, stroke and thrombi in 
peripheral arteries. Acetylsalicylic acid (ASA, aspirin) at doses of 75-325 mg per day, ticlopidine 
at doses of 250 mg 2x/day and clopidogrel at doses of 75 mg 1x/day (CAPRIE, 1996) are proven 
to be effective in PAD patients. Recently there have been reports of beneficial effects of 
policosanol in patients with peripheral circulation disorders. This drug lowers cholesterol levels 
but also has an antiplatelet effect, demonstrating in clinical trials a significant prolongation of the 
claudication distance after ten weeks of treatment (10 mg 1x/day), while the results after ASA (100 
mg daily) were inconclusive (Illnait et al., 2008). 
The group of new antiplatelet drugs, used in PAD patients, also includes rivaroxaban (trade 
name of the medicinal product Xarelto). Currently, the results of the COMPASS study have shown 
that rivaroxaban in a vascular dose (2.5 mg 2 x/day) combined with ASA (75-100 mg 1 x/day) 
provides more effective cardiovascular protection (defined together as cardiovascular death, 
myocardial infarction and stroke) compared to ASA alone (Anand SS et al. 2018; Olinic DM et al. 
2018; Connolly SJ, 2018). Other studies with the acronyms MACE and MALE, conducted in the 
PAD patients of nearly 5,000 cases in total, confirm that the use of rivaroxaban provides 
comprehensive vascular protection in this group of patients. The former showed a 28% reduction 
in the risk of cardiovascular death/myocardial infarction/brain stroke, the latter a 44% reduction 
in the risk of acute lower limb ischemia and a 46% reduction in the risk of acute/chronic limb 
ischemia and a 70% reduction in the risk of large amputations. This is the only anticoagulant that, 
in combination with ASA, reduces mortality in patients with coronary artery disease (CAD) and 
PAD. At the same time, Xarelto, in a vascular dose in combination with ASA, does not cause a 
significant increase in the risk of major bleedings compared to ASA itself. So far, this drug has 
not been tested in the context of prolongation of the claudication distance 
 
2. Pleiotropic effects of rivaroxaban on arteriosclerosis  
Rivaroxaban inhibits the formation of thrombin and clots by directly inhibiting factor Xa 
(activated factor X), thus preventing the transformation of prothrombin into thrombin and 
interrupting the final common path of the blood clotting cascade. This drug competitively inhibits 

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factor Xa with 10,000 times the selectivity of related serine proteases such as thrombin and factors 
VIIa, IXa, and XIa. 
Numerous experimental data suggest the role of clotting system components in regulating 
atherosclerosis progression (Borensztajn K et al., 2008; Borissoff et al., 2009). This is 
particularly expressed in the case of platelets, which not only play a role in the process of 
hemostasis, but their action is also important in proinflammatory states, such as atherosclerosis. 
Moreover, many clotting proteins are involved in atherosclerotic processes such as endothelial 
damage, oxidative stress, recruitment of leukocytes, inflammation, migration, and proliferation of 
vascular smooth muscle cells, activation of the immune system, platelet apoptosis and 
angiogenesis (Platek and Szymański, 2019). These activities are mediated by various factors of 
the coagulation cascade, and the broadest spectrum of action concerns the effect of the 
complex of factor Xa and thrombin precisely. 
 After activation, factor Xa initiates intracellular signaling in different types of cardiovascular 
cells, preferentially through type 2 protease-activated receptors (PAR-2); or both PAR receptors 
(-1 and -2). 
Figure 1. Pleiotropic mechanism of rywaroxaban in relation to atherosclerosis. Representation of 
cellular locations of PAR1 and PAR2 and the potential effects of thrombin- or factor Xa-mediated 
PAR activation on the arterial vessel wall and the heart, and the resulting contribution to 
atherosclerosis and AF (according to Spronk H.M. i wsp. Cardiovasc. Res. 2014; 101: 344–351) . 
 
PAR-1 and/or -2 receptors are present in large numbers on endothelial cells, leukocytes, 
smooth muscle cells, fibroblasts, and dendritic cells. The signal transmitted through PAR 
contributes to the production of proinflammatory cytokines (e.g., IL-6) and expression of cellular 
adhesion molecules (E-selectin, ICAM-1, VCAM-1), together with tissue factor growth regulation, 
smooth muscle cell proliferation and release of growth factors. All these factors may contribute to 
atherosclerotic plaque progression, including inflammation, leukocyte transmigration, restenosis, 
and angiogenesis. It should also be noted that vascular wall remodeling and the formation of new 
atherosclerotic plaque can be reduced by administering non-specific Xa factor inhibitors (low 

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molecular weight heparin and low molecular weight heparin) conjugated to the anti-fibrin antibody 
(Borissoff JI et al., 2009). 
Scientific research confirms the pleiotropic effects of rivaroxaban on the cardiovascular 
system. However, most of the currently available observations come from basic research whose 
results need to be confirmed in clinical trials. The results of in vitro studies using human cells 
indicate that rivaroxaban reduces the expression of proinflammatory cytokines in the vascular 
endothelium and may contribute to the stabilization of atherosclerotic plaques. On the other 
hand, it has been shown in animal models that selective inhibition of factor Xa ensures a reduction 
of restenosis after balloon angioplasty of the arteries, and this drug is used to weaken 
atherosclerotic plaque progression and its destabilization in mice with ApoE gene excluded.  
Rivaroxaban positively influences neovascularization and angiogenesis, especially in 
hyperglycemic conditions, increasing the concentration of endothelial nitric oxide synthesis and 
expression of vascular endothelial growth factor (VEGF). The beneficial effects of rivaroxaban 
under hypoxia-reperfusion and periodic hypoxia conditions were also observed. 
Among the planned clinical trials that may establish pleiotropic rivaroxaban actions, the 
PRE ¬FER-AF study (PREvention oF thromboembolic events - European Registry in Atrial 
Fibrillation) is mentioned as the most important. This study aims to determine the efficacy of oral 
anticoagulants other than vitamin K antagonists (NOAC) in preventing endothelial dysfunction 
and atherosclerosis progression in patients with atrial fibrillation. Patients with no previous 
clinically apparent history of CAD, PAD, or stroke will be randomly assigned to the NOAC 
(dabigatran or rivaroxaban) or placebo-taking group. It is planned to determine vascular 
endothelial function in long-term therapy after 12 and 24 months. The thickness of the intima-
media complex in the carotid arteries will also be measured. This study will assess whether long-
term treatment with rivaroxaban brings measurable benefits in relation to the first stages of 
atherogenesis and atherosclerosis development. However, it will not provide information on the 
effect of the drug on the progress of PAD or cardiological capacity of patients. 
 
3. The objective 
The study aims to assess the protective efficacy of rivaroxaban (trade name of the medicinal 
product Xarelto), administered together with acetylsalicylic acid (ASA), in comparison with the 
effectiveness of ASA alone, with respect to intermittend claudication and cardiological capacity 
in patients with PAD over a 3-month period. 
 
4. Type of study 
Prospective, observational, unblinded (open), randomized, clinical-control, single-centre.  
5. Description of the study group (age, gender, health status, size of the group) 
The study will include patients with symptomatic peripheral artery disease (PAD), hospitalized in 
the The Lord's Transfiguration Clinical Hospital of PUMS, the Department of Vascular, 
Endovascular, Angiology and Phlebology, monitored in the adjacent Vascular Outpatient Clinic, 
or referred by cooperating physicians who will use a different protocol of anticoagulation 

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pharmacotherapy to prevent thrombotic events. The recommended pharmacotherapy does not 
exceed the standard of treatment in patients with PAD. 
5.1 Size of the study group  
At least 60 patients with symptomatic PAD, i.e., those with symptoms of intermittent 
claudication, will be included in the study. In the case of outflow of patients from the study due to 
an unexplained course (lack of information about the reason for not attending the follow-up visit 
after three months) or lack of statistically significant effects, the possibility of increasing the 
recruitment of patients for the study is assumed. The maximum group size will be increased to 100 
patients. The collection and observation of the study group will take place until the scientific 
goal of the study is achieved. 
5.2 Characteristics of the study group and collected material  
Researchers will perform a clinical evaluation of patients with PAD. A clinical history of the 
inclusion/exclusion criteria, medical history, and cardiovascular risk factors will be performed. 
Patients will be subjected to the objective assessment of exercise tolerance, assessment of 
claudication distance, implementation of anticoagulation pharmacotherapy protocol, and 
prospective study protocol.  
The examined persons will be taken venous blood samples in the volume of 9-18 ml to 
perform necessary diagnostic tests (including blood morphology, lipid profile, glucose level, 
biochemistry) and molecular (genetic and biomarkers in the blood). Capillary and venous blood 
will also be collected to assess the intensity of metabolic exercise acidosis and other biochemical 
indicators 
The costs of blood collection and diagnostic tests will be covered from the budget of the 
Poznan University of Medical Sciences from the pool of funds allocated for scientific activities. 
The costs of biochemical and genetic research will be covered by the Poznan University of 
Physical Education and Institute of Human Genetics, Polish Academy of Sciences, respectively. 
5.3 Inclusion and exclusion criteria  
The primary selection criteria for all the subjects are the presence of PAD, Caucasian origin, 
inhabitants of the Great Poland region. Patients who do not have clinical contraindications to 
performing the exercise test will be included in the study. Detailed inclusion criteria are given in 
Table 1. In Table 2, exclusion criteria are collected.  
In order to qualify for inclusion in the study, patients with PAD of Fountain grade IIa or IIb 
will be included (with intermittent claudication). The disease will be confirmed by the ankle-
brachial pressure index (ABI), with a score of less than 0.90. In all subjects, the ABI will be 
measured at the start of the study; the result will be calculated from the ratio of the highest systolic 
limb blood pressure to the highest systolic arm blood pressure. 
Table 1. Inclusion criteria 
Parameter 
Study 
Age 50-70 years old 
This study 
Sex: women and men 
This study 
PAD with advancement on the Fountain IIa or IIb scale 
This study 

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Table 2. Exclusion criteria 
 
LP 
Parameter 
Study 
1 
Severe heart failure with known ejection fraction <30% or NYHA class III 
or IV symptoms 
COMPASS  
 
2 
High risk of bleeding 
COMPASS  
3 
Stroke with 1 month or any history of hemorrhaging or lacunar stroke 
COMPASS  
4 
Estimated glomerular filtration rate <15 ml/ml 
COMPASS  
5 
Need for dual antiplatelet therapy, other non-aspirin 
antiplatelet therapy/, oral anticoagulant therapy/ 
COMPASS  
6 
Known non-cardiovascular disease that is associated with poor prognosis 
(i.n, metastatic cancer) 
COMPASS  
7 
History of hypersensitivity or known contraindication for 
rivaroxaban, aspirin 
COMPASS  
8 
Systemic treatment with strong inhibitors of CYP 3A4 as well as p-
glycoprotein or strong inducers of CYP 3M 
COMPASS  
9 
Any known hepatic disease associated with coagulopathy 
COMPASS  
10 
[Subjects who are pregnant, breastfeeding, or are of childbearing potential, 
and sexually active] 
[COMPASS- 
not used in this 
study] 
11 
Concomitant participation in another study with investigational drug 
COMPASS 
12 
Known contraindication to any study-related procedures* 
Concerns: Absolute contraindications to perform an exercise test 
COMPASS 
13 
Respiratory failure 
This study 
14 
BMI  ≥ 40 
This study 
15 
Musculoskeletal dysfunction preventing walking (e.g. amputations) 
This study 
*- myocardial infarction within 2 days, unstable coronary artery disease, unmanaged by pharmacological 
treatment; symptomatic arrhythmia, severe, symptomatic aortic valve stenosis; unmanaged heart failure; 
fresh pulmonary embolism or myocardial infarction; acute pericarditis or myocardial infarction; acute 
aortic dilation. 
 
5.4 Pharmacotherapy 
Patients will be selected according to inclusion and exclusion criteria and randomly allocated to 
two groups with the different anticoagulation treatment protocol. The following treatment profile 
is planned: 
1. ASA 
2. ASA + rivaroxaban  
Vascular doses of drugs will be used, including in group 1: 1 x/day ASA 75-100 mg and in group 
2: 1 x/day ASA 75-100 mg + 2 x/day 2.5 mg Xarelto. Treatment costs will not be reimbursed 
by the researchers, patients pay for the drugs themselves. 
6. Methods  
The main plan of the research is shown in Figure 2. 

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Figure 2. The research plan. 
 
 
 
 
 

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6.1 Clinical characteristics of the studied population 
 
The following clinical data will be collected using existing clinical documentation, and/or 
commissioned laboratory tests (performed under contract with the hospital), and based on data 
obtained from the patient (see Figure 3 for a clinical history): 
 
1. Results of aortic and lower limb arteries imaging (angiography, computed tomography, 
ultrasounds; which will assess the severity and location of atherosclerosis in lower limb 
arteries) 
2. Results of heart function tests (echocardiography, electrocardiography- ECG) 
3. Assessment of the occurrence and severity of atherosclerotic lesions in blood vessels 
(including ankle-brachial index -ABI: at rest and after exercise, the degree of thickening of 
the intima-media complex; IMT-ultrasound-Doppler examination; blood supply and 
oxygenation of tissues by percutaneous oximetry ( Precise 8008 device for measurement 
of TcPO2) 
4. Results of fasting venous blood laboratory tests (Table 3) glucose metabolism: glucose 
(GLU), glycated hemoglobin (HbA1C), diabetes mellitus, lipid metabolism (TC, HDL, 
LDL, TG), uric acid concentration (URCA), CRP, renal function: glomerular filtration rate 
(eGFR); creatinine (CREA); urea (UREA), liver function: aspartate aminotransferase 
(AST), arginine aminotransferase (ALT), homocysteine (Hcy), folic acid (FA), peripheral 
blood morphology, blood coagulation parameters: fibrinogen, prothrombin time (PT), 
international normalized ratio (INR)  
5. Blood pressure values and the presence of hypertension. 
6. Results of virological and bacteriological tests 
7. Anthropometric data (weight, height, BMI, waist and hip circumference, WHR) 
(8) Interview on smoking (estimation of the "peak years" index),  
9.  Pharmacotherapy (drugs: antiplatelet, lipid-lowering, anti-hypertensive: angiotensin-
converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, proton pump 
inhibitor) 
10. Coexisting diseases 
11. Severity of pain  
12. Family history of cardiovascular disease and cancer 
 
 

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Tabela 3. Laboratory blood tests 
 
No. Parameter 
1 
Lipid profile (TC, HDL, LDL, TG) 
2 
Glucose (GLU) 
3 
Glycated hemoglobin (HbA1C) 
4 
Uric acid (URCA) 
5 
Glomerular filtration rate (eGFR) 
6 
Creatinine (CREA) 
7 
Urea (UREA) 
8 
Aspartate aminotransferase (AST) 
9 
Arginine aminotransferase (ALT) 
10 
Peripheral blood morphology 
11 
Fibrinogen  
12 
Prothrombin time (PT) 
13 
International Normalized Ratio (INR) 
14 
Homocysteine (Hcy) 
15 
Folic acid (FA) 
 
 
 

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Figure 3. Sample questionnaire for a clinical interview 
 
 
 

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6.2. Treadmill test  
The test will be carried out in the Laboratory of Functional Research of the Department of 
Physiology and Biochemistry of the University of Physical Education in Poznań; in the absence 
of contraindications. The subjects will be tested at each of the test dates to assess the claudication 
distance on the Katana S30p medical treadmill (Lode B.V., Netherlands). Before performing the 
test, patients will undergo an initial, minimum 6-minute implementation of the principles of 
research, primarily to familiarize them with the way of moving on the treadmill (Mika et al. 2009). 
Additionally, they will be instructed not to support themselves on treadmill railings during the 
march. The total time of each test will not exceed 16 minutes 40 seconds, and the regeneration 
time will be at least 5 minutes. 
 
6.2.1. Qualification for the treadmill test  
Patients will be qualified for the treadmill test based on the result of an electrocardiographic 
examination (ECG), and the test on a treadmill will be carried out by authorized investigators 
under the supervision of the doctor. 
 
6.2.2 The evaluation of the claudication distance  
The treadmill test will be carried out according to Gardner protocol (Gardner et al., 1991). 
Patients will march on a treadmill at a constant speed of 3.2 km/h. The initial angle of inclination 
will be 0% and will be raised by 2% every 2 minutes (Table 4). When the patient reports pain (i.e., 
grade 2 on a subjective pain assessment scale), the time of occurrence of the claudication will be 
recorded, from which the claudication distance will be calculated (DGCH).  The test will be 
interrupted when very severe pain occurs that prevents further walking (step 5 corresponding to 
maximum walking time), and the maximum walking time will then be recorded. The maximum 
walking time will be used to estimate the maximum walking distance (DGMAX). 
 
Table 4. Parameters of the treadmill test according to Gardner protocol 
Time 
Level 
Speed 
[mph] 
Speed 
[kph] 
Distance 
[m] 
Slope 
 [%] 
00:00 
1 
2,0 
3,2  
0 
0,0 
02:00 
2 
2,0 
3,2 
107 
2,0 
04:00 
3 
2,0 
3,2 
213 
4,0 
06:00 
4 
2,0 
3,2 
320 
6,0 
08:00 
5 
2,0 
3,2 
427 
8,0 
10:00 
6 
2,0 
3,2 
533 
10,0 
12:00 
7 
2,0 
3,2 
640 
12,0 
14:00 
8 
2,0 
3,2 
747 
14,0 
16:00 
9 
2,0 
3,2 
853 
16,0 
 
Abbreviations: mph- miles/hour; kph-km/hour 

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6.2.3. Evaluation of exercise tolerance. 
Evaluation of exercise tolerance will be carried out based on effort duration and a survey 
and biochemical exponent. 
Based on the duration of the effort, the individual oxygen ceiling value (VO2max) and the 
energy expenditure expressed as metabolic equivalent MET-s will be estimated. Table 5 gives the 
MET-s values calculated from the parameters for the cardiological treadmill test, according to 
Ramped Bruce Protocol; (Ramped Bruce Protocol; Bires AM et al., 2013).  
Table 5. Results of the treadmill test depending on the severity of ischemia in the Fountaine 
scale. Energy expenditure expressed as metabolic equivalent of MET-s is given. The values of 
MET-s were estimated based on parameters of cardiological treadmill test according to Bruce's 
ramp protocol. 
Time Level Speed [mph] Speed [kph] Distance 
[m] 
Slope 
[%] 
MET-s Fountaine scale 
00:00 
1 
2 
3,2 
0 
0,00 
2,1 
- 
00:20 
 
 
 
18 
 
 
IIb 
00:40 
 
 
 
36 
 
 
IIb 
01:00 
 
 
 
54 
 
 
IIb 
01:20 
 
 
 
71 
 
 
IIb 
01:40 
 
 
 
89 
 
 
IIb 
02:00 
2 
2 
3,2 
107 
2,00 
3,2 
IIb 
02:20 
 
 
 
125 
 
 
IIb 
02:40 
 
 
 
143 
 
 
IIb 
03:00 
 
 
 
161 
 
 
IIb 
03:20 
 
 
 
178 
 
 
IIb 
03:40 
 
 
 
196 
 
 
IIb 
04:00 
3 
2 
3,2 
213 
4,00 
4,3 
IIa 
04:20 
 
 
 
231 
 
 
IIa 
04:40 
 
 
 
249 
 
 
IIa 
05:00 
 
 
 
267 
 
 
IIa 
05:20 
 
 
 
284 
 
 
IIa 
05:40 
 
 
 
302 
 
 
IIa 
06:00 
4 
2 
3,2 
320 
6,00 
5,3 
IIa 
06:20 
 
 
 
338 
 
 
IIa 
06:40 
 
 
 
356 
 
 
IIa 
07:00 
 
 
 
374 
 
 
IIa 
07:20 
 
 
 
391 
 
 
IIa 
07:40 
 
 
 
409 
 
 
IIa 
08:00 
5 
2 
3,2 
427 
8,00 
6,4 
IIa 
08:20 
 
 
 
445 
 
 
IIa 
08:40 
 
 
 
463 
 
 
IIa 
09:00 
 
 
 
481 
 
 
IIa 
09:20 
 
 
 
498 
 
 
IIa 
09:40 
 
 
 
516 
 
 
IIa 

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10:00 
6 
2 
3,2 
533 
10,00 
7,5 
IIa 
10:20 
 
 
 
551 
 
 
IIa 
10:40 
 
 
 
569 
 
 
IIa 
11:00 
 
 
 
587 
 
 
IIa 
11:20 
 
 
 
604 
 
 
IIa 
11:40 
 
 
 
622 
 
 
IIa 
12:00 
7 
2 
3,2 
640 
12,00 
8,5 
IIa 
12:20 
 
 
 
658 
 
 
IIa 
12:40 
 
 
 
676 
 
 
IIa 
13:00 
 
 
 
694 
 
 
IIa 
13:20 
 
 
 
711 
 
 
IIa 
13:40 
 
 
 
729 
 
 
IIa 
14:00 
8 
2 
3,2 
747 
14,00 
8,5 
IIa 
14:20 
 
 
 
765 
 
 
IIa 
14:40 
 
 
 
783 
 
 
IIa 
15:00 
 
 
 
801 
 
 
IIa 
15:20 
 
 
 
818 
 
 
IIa 
15:40 
 
 
 
836 
 
 
IIa 
16:00 
9 
2 
3,2 
853 
16,00 
9,6 
IIa 
Abbreviations: mph- miles/hour; kph-km/hour; MET-s - metabolic equivalent. 
 
The degree of intensity of fatigue changes after the completed effort will also be assessed 
using a subjective scale to evaluate the degree of fatigue (Borg scale; Smarż et al. 2015). At rest 
and 3 minutes after the exercise, the intensity of metabolic exercise acidosis will be assessed based 
on lactate concentration in capillary blood taken from the fingertip. At the same time, venous blood 
will also be collected for biochemical and genetic tests. 
6.2.4. Post-treadmill test examination 
After completing the treadmill tests, patients will undergo a control ECG examination to 
exclude ischemic changes in the myocardium and cardiac arrhythmias.. 
6.3. Length of prospective observation 
The effects of the treatment will be assessed within three months. It is assumed that the 
observation period can be extended to a maximum of 12 months. 
6.4. Endpoints of the study 
The endpoints of this study and the complications were developed in accordance with the 
results of the COMPASS study. Parameters related to the results of the treadmill test were added. 
The study endpoints are summarized in Table 6. The complications related to possible bleeding 
risk for PAD patients are presented in Table 7. 
 
 

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Table 6. Endpoints of the study 
Parametr 
Study 
Primary outcomes 
Rate of stroke  
COMPASS 
Rate of myocardial infarction 
COMPASS 
Rate of cardiovascular death   
COMPASS 
Rate of death  
COMPASS 
Prespecified limb outcomes  
Rate of acute limb ischaemia 
COMPASS 
Rate of major amputation 
COMPASS 
Rate of all vascular amputations 
COMPASS 
Tredmill tests outcomes 
Change in the distance of intermittent claudication in the test according to 
Gardner's protocol (Δ Dch; Δ Dmax).  
This study 
Change in the value of oxygen uptake (Δ VO2max) 
This study 
Change in energy expenditure expressed as the metabolic equivalent of MET-s  
(Δ MET-s) 
This study 
 
Table 7. Safety outcomes for patients with PAD 
Parametr 
Study 
Rate of major bleeding 
COMPASS 
Rate of fatal bleeding 
COMPASS 
Rate of non-fatal symptomatic intracranial haemorrhage 
COMPASS 
Rate of non-fatal, non-intracranial haemorrhage symptomatic bleeding 
into a critical organ 
COMPASS 
Rate of other major bleeding (surgical site bleeding requiring 
reoperation or bleeding leading to hospitalization) 
COMPASS 
Rate of fatal or symptomatic bleeding into a critical organ 
COMPASS 
[Rate of fatal or symptomatic bleeding into a critical organ or surgical site 
bleeding leading to re-operation] 
[COMPASS- not 
used in this study] 
Rate of ISTH major bleeding 
COMPASS 
Sites of bleeding (frequency of occurrence) 
COMPASS 
Gastrointestinal 
COMPASS 
Intracranial 
COMPASS 
Genitourinary 
COMPASS 
Ocular 
COMPASS 
Skin 
COMPASS 
Respiratory 
COMPASS 
Other 
COMPASS 
Minor bleeding 
COMPASS 
 
6.5. Biological samples 
6.5.1. Biological material collection - blood samples 
For standard diagnostic and molecular tests, fasting venous blood samples will be taken from 
the ulnar vein in the volume of 9-27 ml, from a single puncture. Samples will be collected after 
the patient is qualified for tests (1 collection during the study).  

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Subsequent samples of venous blood from the ulnar vein (10 ml) and capillary blood from the 
fingertip (1 ml) will be collected during treadmill tests before and after the exercise to determine 
objective metabolic markers associated with the exercise (a total of 2 venous and 2 capillary 
blood collections during the tests). 
Blood collection from the ulnar vein is a standard invasive test that causes about 1 minute of 
discomfort and pain, and the risk of bruising, redness, or skin infection is very low. Whenever 
possible, to reduce the discomfort, additional blood samples will be taken when other routine 
laboratory diagnostic tests are carried out.  
Blood samples may be taken: at the wards of the Department of Vascular and Endovascular  
Surgery, Angiology and Phelbology of the PUMS at the blood collection point of the The Lord's 
Transfiguration Clinical Hospital of PUMS or the Department of Physiology and Biochemistry of 
the University of Physical Education by researchers or collaborators. As compensation, the 
examined persons will receive free of charge the results of additional laboratory tests conducted 
by the investigators. If it is not possible to collect blood from the ulnar vein, the blood will be 
collected from another location (wrist, hand) with the consent of the examined person. The 
capillary blood from the pad also causes short (30 s) pain and discomfort to the patient, and the 
risk of bruising, redness, or skin infection is very low in this case. 
 
6.5.2.  Preparation and storage of blood samples 
Blood samples will be used to perform laboratory tests (9 ml, The Lord's Transfiguration Clinical 
Hospital Central Laboratory) and to obtain material for further biochemical and molecular 
analyses: plasma/serum and genomic DNA (residual blood; Table 8). 
Table 8. Use of biological material 
LP Biological material 
 
Analyses 
1 
Plasma / peripheral fasting blood serum 
 
Assessment of blood biomarker 
concentrations 
2 
Morphotic elements of peripheral blood 
 
Isolation of genomic DNA, genetic 
analysis 
3 
Plasma / peripheral blood serum before 
and after exercise 
 
Biochemical analysis - effort 
markers 
4. 
Krew kapilarna przed i po wysiłku 
 
Capillary blood before and after 
exercise 
 
Serum samples (fasted venous blood) will be collected in serum-separating tubes containing 
a serum accelerator (SST; proposed: S-Monovette Serum Z, manufacturer of SARSTEDT), left to 
clot for 15 minutes at room temperature or overnight at 4°C and then centrifuged at 1000 x g for 
15 minutes. The serum will be divided into 500 µl samples, placed in 1.5 ml Eppendorf tubes and 
stored at -80°C. 
Blood samples for plasma collection (venous blood collected on an empty stomach) will be 
collected using the sodium version, EDTA, as an anticoagulant (S-Monovette EDTA, 

Unique Protocol ID: 
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20 
 
manufacturer of SARSTEDT), centrifuged for 15 minutes at 1,000 x g at 2-8°C within 30 minutes 
of collection. Part of the plasma will then be centrifuged for 15 minutes at 4000 x g (for blood 
micro RNA levels). The plasma will be divided into 500 µl samples, placed in 1.5 ml Eppendorf 
tubes and stored at -80°C. The remainder of the blood sample taken on EDTA, which contains all 
cellular elements, will be used for DNA extraction. 
Twice during each test, i.e., at rest and after exercise, approximately 10 ml of blood will be 
taken from the ulnar vein using an S-Monovette syringe without anticoagulant (made by 
SARSTEDT, Germany), then centrifuged (1500 g, 4°C, 4 minutes) to separate the serum and 
plasma. The samples will be frozen at -80°C pending biochemical analyses. Similarly, twice during 
each test 
Capillary blood samples will be analyzed immediately after collection. 
All samples (blood, fractions obtained) will be stored in the Department of Vascular Surgery, 
Endovascular Surgery, Phlebology and Angiology of the Medical University of Poland, 
Department of Physiology and Biochemistry of the University of Physical Education and the 
Institute of Human Genetics of the Polish Academy of Sciences in Poznań. 
6.5.3. Material and methods for laboratory testing of patient blood 
The results of laboratory tests will be performed in the Lord's Transfiguration Clinical 
Hospital Central Laboratory. These results will come from available clinical documentation or will 
be performed in the above mentioned Laboratory based on a separate agreement with the Hospital. 
It is also acceptable for the patient to pay for the tests themselves. The analyses will be carried out 
using standard laboratory techniques used in the Lord's Transfiguration Clinical Hospital Central 
Laboratory. 
 
6.5.4. Biochemical blood tests 
The concentration of lactate in capillary blood before and after exercise (assessment of the 
metabolic severity of exercise acidosis) will be tested using EDGE Blood Lactate Test Strip, 
ApexBio, (Taiwan) based on the analysis of blood taken from the fingertip. Similarly, venous 
blood will be collected before and after exercise for biochemical tests, including gasometry. 
 
6.5.5. Material and methods for testing peripheral blood biomarkers 
The level of selected proteins in the blood will be assessed by the immunoenzymatic method 
(ELISA). The assays of selected circulating miRNAs will be performed using TaqMan (Life 
Technologies) using the 7900HT Fast Real-Time PCR System (Life Technologies). These tests 
will be performed at Institute of Human Genetics PAS.   
Further analysis of biomarkers present in the blood, to obtain funds for testing, assumes the 
possibility of using high-pass transcriptomic (use of expressive microarrays) and proteomic (using 
mass spectrometry) methods.   

Unique Protocol ID: 
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21 
 
6.5.6. DNA analysis 
DNA extraction will be performed chemically using guanidine isothiocyanate, phenol, and 
chloroform to remove proteins. 
Analysis of genotypes for selected SNPs, including genes with proven role in cardiovascular 
susceptibility (related to: response to hypoxia, angiogenesis, antioxidant barrier,  lipid metabolism, 
homocysteine and hypertension). Among others, functional polymorphisms of genes MTHFR, 
ApoE, HIF1A, VEGF, PON1) will be assessed using allele discrimination tests (TaqMan SNP 
Genotyping Assays, Life Technologies) and 7900HT Fast Real-Time PCR System (Life 
Technologies) in at Institute of Human Genetics PAS.  
Material is to be collected for mutation evaluation, whole exome/genome sequencing and 
epigenome studies. These studies will be carried out based on the DNA sequencing method using 
the apparatus available in Poznan University of Medical Sciences / Institute of Human Genetics 
PAS or external services. 
 
6.7. Equipment 
In the course of the research project, the equipment of the Poznan University of Medical 
Sciences, the University of Physical Education in Poznań, the Institute of Human Genetics of the 
Polish Academy of Sciences in Poznań and cooperating units will be used.  
7. Use of results 
The results will be published in scientific journals and presented at conventions and scientific 
conferences. 
8. Statements 
The research is carried out in compliance with personal data protection. The prospective 
assessment will be carried out during the Clinic's inspection visits. Additionally, to conduct 
prospective research, contact with the patient will be made by phone and e-mail based on the 
submitted statement. 
 

Unique Protocol ID: 
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22 
 
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Websites 
http://leki.urpl.gov.pl/files/Xarelto.pdf