Enhanced Platelet Toll-like Receptor 2 and 4 Expression in Acute Coronary Syndrome and Stable Angina Pectoris

Accepted Manuscript Enhanced Platelet Toll- like Receptor- 2 and 4 Expression in Acute Coronary Syndrome and Stable Angina Pectoris Kadri Murat Gurses, MD, Duygu Kocyigit, MD, Muhammed Ulvi Yalcin, MD, Hande Canpinar, MD, Mehmet Ali Oto, MD, Necla Ozer, MD, Lale Tokgozoglu, MD, Dicle Guc, MD, Kudret Aytemir, MD PII:

S0002-9149(15)01934-7

DOI:

10.1016/j.amjcard.2015.08.048

Reference:

AJC 21409

To appear in:

The American Journal of Cardiology

Received Date: 19 June 2015 Revised Date:

22 August 2015

Accepted Date: 25 August 2015

Please cite this article as: Gurses KM, Kocyigit D, Yalcin MU, Canpinar H, Oto MA, Ozer N, Tokgozoglu L, Guc D, Aytemir K, Enhanced Platelet Toll- like Receptor- 2 and 4 Expression in Acute Coronary Syndrome and Stable Angina Pectoris, The American Journal of Cardiology (2015), doi: 10.1016/ j.amjcard.2015.08.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Enhanced Platelet Toll- like Receptor- 2 and 4 Expression in Acute Coronary Syndrome and Stable Angina Pectoris Kadri Murat Gursesa, MD; Duygu Kocyigitb, MD; Muhammed Ulvi Yalcinc, MD; Hande

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Canpinard, MD; Mehmet Ali Otob, MD; Necla Ozerb, MD; Lale Tokgozoglub, MD; Dicle Gucd, MD; Kudret Aytemirb, MD.

Department of Cardiology, Konya Training and Research Hospital, Konya Turkey.

b

Department of Cardiology, Hacettepe University Faculty of Medicine, 06100, Sihhiye Ankara,

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a

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Turkey. c

Department of Cardiology, Hakkari State Hospital, Hakkari Turkey.

d

Hacettepe University Cancer Institute Basic Oncology Department, 06100 Sihhiye Ankara,

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Turkey.

Corresponding author: Duygu Kocyigit, MD. [email protected] Department of Cardiology, Hacettepe University Faculty of Medicine, 06100, Ankara Turkey. Tel: +90312-

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3051780 Fax: +90312- 3114058

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ABSTRACT Evidence is accumulating that Toll-like receptors (TLR) are involved in the initiation and

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progression of cardiovascular disease. Enhanced expression of these receptors on monocytes has been shown in patients with acute coronary syndrome (ACS). However, expression on platelets in this group of patients has not been evaluated yet. We aim to demonstrate the possible association between platelet TLR- 2 and 4 expression on stable coronary artery disease and ACS

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pathogenesis. In this observational case- control study, 40 patients diagnosed with ACS (non-ST

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segment elevation and ST-segment elevation ACS), 40 patients diagnosed with stable coronary artery disease (CAD) and 40 age and gender- matched subjects with normal coronary arteries were involved. Platelet TLR- 2 and 4 expression were evaluated by flow cytometry in peripheral venous blood samples obtained prior to coronary angiography. 120 patients (60.7± 12.3 years, 50% male) were included. Median platelet TLR- 2 and TLR- 4 expression was higher in ACS

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patients when compared to patients with normal coronary arteries and stable angina pectoris (3.0 vs. 10.5 vs. 29.5%; p<0.001 and 3.0 vs. 11.5 vs. 40.5%, p<0.001, respectively). Median platelet TLR-2 and TLR- 4 expression was also greater in patients with stable angina pectoris when

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compared to those with normal coronary arteries (p< 0.05). In conclusion, this is the first study

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demonstrating enhanced TLR- 2 and 4 expression on platelets in ACS patients. These findings may suggest that platelet TLR expression as a novel potential prophylactic and therapeutic target in ACS.

KEYWORDS Toll- like receptor; platelets; acute coronary syndrome; stable coronary artery disease.

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INTRODUCTION Besides their primary role in hemostasis, platelets have been shown to secrete several adhesion molecules that make them essential in the interplay between endothelial cells and other immune

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cells1. Platelets have also been shown to interact and modulate leukocyte function, through the release of mediators such as P- selectin and CD40- L2,3 or formation of platelet- leukocyte

aggregates4. Toll- like receptor (TLR) expression on platelets5-7 may be the link between platelets

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and immune response that deserves attention. Prior studies have primarily focused on the role of

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TLR expression on platelets mostly concerning the pathogenesis of thrombocytopenia, sepsis, hemorrhagic shock or severe thrombotic complications (such as stroke or myocardial infarction) on the basis of infections8. However, TLRs have been reported to sense not only microbial molecules, but also molecules of host origin9. This has been suggestive of the possible role of TLRs on atherosclerosis, which is regarded as an inflammatory disease. In this study, we aimed

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to demonstrate the possible association between platelet TLR- 2 and 4 expression with stable coronary artery disease (CAD) and acute coronary syndrome (ACS).

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METHODS

40 patients diagnosed with acute coronary syndrome (ACS) [unstable angina pectoris

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(USAP), non-ST- segment elevation myocardial infarction (NSTEMI), ST- segment elevation myocardial infarction (STEMI)] and 40 patients with stable angina pectoris were included as the patient group, where 40 age and gender- matched patients with normal coronary arteries were included as the control group. Patients were recruited between November 2013 and June 2014. In our study, ACS group was defined as the group of patients who were diagnosed with ACS in accordance with the most recent guidelines10,11. Other patients were recruited from the

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outpatient clinics of our department. These patients had presented with stable angina pectoris and were scheduled for invasive coronary angiography following clinical evaluation and assessment of ischemia in accordance with the recommendations of guidelines on the management of stable

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angina pectoris12,13. Patients with normal epicardial coronary arteries in the coronary angiography were classified as the “normal coronary arteries” group. Patients who had significant coronary artery disease in the coronary angiography were classified as “stable coronary artery disease”

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group.

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Detailed medical history was recorded from each patient to identify co- morbidities, cardiovascular risk factors and drug use. All patients underwent detailed physical examination. Transthoracic echocardiographic examination was performed to evaluate left ventricular systolic functions.

Patients with a history of coronary artery disease, renal dysfunction (glomerular filtration

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rate< 60 mL/min/1.73 m2 calculated with MDRD formula), malignancy or prior surgery within last 3 months were not included in the study. None of the patients had any evidence of systemic

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inflammatory disease (eg. infections, autoimmune diseases). In addition, none had received antiplatelet or anticoagulant drugs before. The study was performed in accordance with the

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Declaration of Helsinki and was approved by the local Research Ethics Committee. All participants provided written informed consent. Peripheral venous blood samples were collected from all patients at the first encounter (ie.

the emergency room for ACS patients and outpatient clinic visits for stable angina pectoris and control groups). None of the patients had received antiplatelet or anticoagulant drugs prior to blood drawal. Complete blood cell count (CBC), serum biochemistry tests including fasting

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blood glucose, renal function tests were performed besides creatine kinase- myocardial band (CK- MB) and troponin- T measurement and flow cytometry.

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Flow cytometry was performed within 15 minutes of blood sample obtainment. Whole blood (5 ml) collected in citrate was centrifuged for 15 minutes at 210 g, and the Platelet Rich Plasma Fraction was collected. Platelets were washed twice with buffer (17.5 mM Na2HPO4, 8.9 mM Na2EDTA, 154 mM NaCl, pH 6.9, containing 0.1% bovine serum albumin) by centrifuging

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them 5 minutes at 2310 g. Platelets were resuspended in HEPES medium (132 mM NaCl, 6 mM

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KCl, 1 mM MgSO4, 1.2 mM KH2PO4, 20 mM HEPES, pH 7.4, containing 5 mM glucose). TLR expression on platelets was measured on a Flow cytometry (Beckman Coulter XLMCL, USA). Briefly, 106 platelets were incubated with 10 µl TLR 2 PE (CD282, cloneT2.5, Biolegend) and 10 µl TLR 4 FITC (CD284, HTA 125, Biolegend) antibodies and isotopic controls (MsIgG1 FITC, Biolegend and MsIgG1PE, Biolegend) for 30 minutes at 4°C in the dark and were washed once

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with PBS; then 10.000 events were acquired through a live gate drawn on forward light scatter (FSC) and side light scatter (SSC).

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Normally distributed continuous parameters are presented as mean± standard deviation and skewed continuous parameters are expressed as median (interquartile range defined as the

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difference between 25th and 75th centiles). Categorical data are presented as frequencies and percentages. Comparisons of continuous data among the 3 patient groups with normal distribution were performed using ANOVA followed by the Bonferroni correction. Skewed data from 3 patient groups were compared with Kruskal- Wallis H test followed by the adjusted Mann- Whitney U test. Comparison of two groups were made with Mann- Whitney U test and student’s t- test for parameters with and without normal distribution, respectively. Multinominal logistic regression analysis was performed to determine whether platelet TLR expression was 5

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independently associated with ACS occurrence. Spearman correlation analysis was done to investigate factors related with levels of platelet TLR- 2 and TLR- 4 expression. Statistical analyses are performed using SPSS statistical software (version 20.0; SPSS Inc., Chicago,

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Illinois, USA). A two-tailed p < 0.05 is considered statistically significant. RESULTS

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A total of 120 patients (60.7± 12.3 years; 64.2% male) were included in the study.

Patients were evaluated in separate groups, each containing age and gender- matched 40 patients,

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depending on their clinical presentation. Baseline characteristics of the patients regarding their clinical presentation is shown in Table 1. Among cardiovascular risk factors, prevalence of hypertension (p= 0.015), diabetes mellitus (p= 0.013), smoking (p= 0.023) and family history of CAD (p= 0.006) were higher in patients with ACS when compared to patients with stable angina pectoris and normal coronary arteries. Patients with stable angina pectoris and ACS had higher

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prevalence of hyperlipidemia when compared to patients with normal coronary arteries (p= 0.013). On the other hand, BMI did not differ between three groups (p= 0.595). Patients with

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stable angina pectoris more frequently received beta blockers (p=0.001), statins (p<0.001), where it was the group of patients with ACS who more frequently received angiotensin- converting

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enzyme inhibitor/ angiotensin receptor blockers (ACEi/ ARBs) (p=0.001) (Table 1). White blood cell (WBC) count was significantly greater in patients with ACS (p= 0.002).

Fasting blood glucose levels were higher in patients with ACS when compared to others (p= 0.001). High- density lipoprotein (HDL)- cholesterol levels were significantly lower in patients with ACS (p< 0.001); where low- density lipoprotein (LDL)- cholesterol (p=0.078) and serum creatinine (p= 0.689) levels did not significantly differ between three groups (Table 1).

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Median platelet TLR- 2 and TLR- 4 expression was higher in ACS patients when compared to patients with normal coronary arteries and stable angina pectoris (3.0 vs. 10.5 vs. 29.5%; p<0.001 and 3.0 vs. 11.5 vs. 40.5%, p<0.001, respectively). Median platelet TLR-2 and

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TLR- 4 expression was also greater in patients with stable angina pectoris when compared to those with normal coronary arteries (p< 0.05) (Figure 1). Platelet TLR- 2 (p= 0.799) or TLR-4 (p= 0.715) expression on platelets in patients with unstable angina pectoris, NSTEMI and STEMI

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did not differ among groups (Table 2).

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Median platelet TLR- 2 and TLR- 4 expressions were found to be higher in patients with hypertension (p=0.021 and p=0.012), diabetes mellitus (p=0.009 and p=0.002), hyperlipidemia (p=0.020 and p=0.047), smoking (p=0.014 and p=0.048) and family history of CAD (p=0.028 and p=0.020) (Table 3).

Results of correlation analysis between platelet TLR- 2 and TLR- 4 expression and

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baseline characteristics are shown in Table 4. Fasting blood glucose, WBC count, peak troponinT were positively and HDL- cholesterol, left ventricular ejection fraction (LVEF) were inversely

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correlated with platelet TLR- 2 expression. Fasting blood glucose, LDL- cholesterol, WBC count, peak CK- MB, baseline troponin- T, peak troponin- T were positively and HDL- cholesterol,

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LVEF were inversely correlated with platelet TLR- 4 expression (Table 4). Results of univariate multinominal regression analysis for identifying predictors of ACS

are shown in Table 5. When traditional cardiovascular risk factors such as hypertension, diabetes mellitus, smoking, family history of CAD and statin use were included, platelet TLR- 2 and 4 expression were found to be independently associated with ACS occurrence in relative to normal coronary arteries. Platelet TLR- 2 expression was also found to be independently associated with ACS occurrence in relative to stable angina pectoris, when the same model was applied. When 7

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platelet TLR- 4 expression was replaced with TLR- 2 expression in the same model, none of the variables independently predicted ACS in relative to stable angina pectoris.

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DISCUSSION This is the first study demonstrating the enhanced TLR- 2 and 4 expression on platelets in ACS patients when compared to patients with stable coronary artery disease and normal coronary

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arteries. Platelet TLR- 2 and TLR- 4 expression were found to be higher in patients with

conventional cardiovascular risk factors such as hypertension, diabetes mellitus, hyperlipidemia,

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smoking and family history of CAD. In addition, platelet TLR- 2 and TLR- 4 expression were the only independent predictors of ACS risk in relative to normal coronary arteries when they were included in the regression model separately.

Atherosclerosis is characterized by vascular endothelial damage, adhesion and migration

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of monocytes, lipid accumulation in macrophages and the formation of foam cells14,15. In the TLR family, especially TLR- 4 has been reported to be an important player in the initiation and progression of atherosclerotic diseases16. Association of TLRs in linking pathological signals in

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the circulation with pro- thrombotic and pro- inflammatory signals in ACS has previously been investigated on peripheral blood mononuclear cells (PBMC). Expression of TLR- 2 and TLR- 4

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on monocyte subsets, leukocytes, PBMC and also CD14-labelled monocytes localized in coronary thrombi were found to be increased in ACS patients 17-20. Several studies have identified a relationship between platelet activation and enhanced

platelet TLR expression. Cognasse et al.21 have demonstrated that surface TLR expression was enhanced following platelet activation. In their study, TLR- 2 and TLR- 9 expression on the platelet membrane were increased and TLR- 4 expression was decreased on activated platelets.

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On the other hand, intracellular expression of TLR- 2, -4 and -9 were increased in activated platelets21. Stimulation of platelet TLR- 2 has been shown to increase P-selectin surface expression, activation of integrin αIIbβ3, generation of reactive oxygen species, and, in human

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whole blood, formation of platelet–neutrophil heterotypic aggregates4. However, there is a lack of data regarding altered self- ligands, other than pathogen- associated ligands for TLR- 2 and TLR4 that have been shown to induce a thrombo- inflammatory response in platelets4,22, that could be

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recognized by platelet TLRs and translated into pathological responses.

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In our study, platelet TLR- 2 and TLR- 4 expression were enhanced in patients with ACS when compared to patients with stable angina pectoris and normal coronary arteries. Therefore, it may be possible to suggest that via TLRs altered self- ligands may cause activation of platelets and this may result in a pro- thrombotic state reflected with acute coronary syndrome. It may also be speculated that platelet TLR expression in ACS, especially in STEMI, may play a role in

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the non- self- limiting cascade leading to the thrombosis, owing to the significantly elevated expression. This is compatible with the findings of Semeraro et al.23, who have demonstrated that extracellular histones released from dying cells promote thrombin generation through platelet

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TLR- 2 and TLR- 4. However, we have not found a particular difference in platelet TLR

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expression regarding the presentation of ACS (ie. unstable angina pectoris, NSTEMI, STEMI). This may be explained with the relatively small study population. On the other hand, platelet TLR- 2 and TLR- 4 expression were found to be significantly correlated with peak troponin- T levels in ACS patients, showing that TLR expression was also associated with severity of MI. Other than ACS, TLR- 4 mRNA and protein expression on mononuclear cells were also found to be significantly increased in patients with stable CAD and expression levels were associated with the severity of CAD reflected with the number of vessels with critical stenosis24. 9

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Kuwahata et al.25 have suggested that high TLR- 2 expression level on monocytes may be an independent risk factor for atherogenesis. In our study, platelet TLR- 2 and TLR- 4 expression were greater in patients with stable angina pectoris when compared to patients with normal

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coronary arteries, suggesting that platelet TLR expression may have a role in atherogenesis, other than thrombosis leading to ACS.

Blair et al.4 have reported that incubation of isolated platelets with Pam3CSK4, a

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synthetic TLR2/TLR1 agonist, directly induce platelet aggregation and adhesion to collagen. They have also shown that these responses are inhibited in TLR-2- deficient mice and, in human

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platelets, by pre- treatment with TLR2-blocking antibody4. This data is of particular importance, since platelet TLRs may then also constitute a therapeutic target in the treatment of ACS. Further studies are required to demonstrate the exact role of TLRs on platelets on ACS pathogenesis. Altered self- ligands of TLR-2 and TLR-4, or TLRs themselves may constitute therapeutic

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targets26 in ACS when pathophysiology is better understood.

There are some limitations of the study. First, this is an observational study. Second,

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surface TLR expression is not an adequate measure of platelet TLR production and we have not evaluated intracellular TLR expression. mRNA measurement could have provided more accurate

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information.

CONFLICT OF INTEREST None. All authors have approved the final article.

FUNDING None.

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FIGURE LEGENDS Figure 1. Figure comparing platelet TLR- 2 (a) and TLR- 4 (b) expression in patient groups with

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normal coronary arteries, stable angina pectoris and acute coronary syndrome.

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Table 1. Baseline characteristics of the study population (n= 120). Total population (n= 120)

Normal coronary arteries (n= 40)

Age (years) 60.7± 12.3 60.4± 12.1 Men 60 (50.0%) 19 (47.5%) Conventional risk factors for coronary artery disease

Stable coronary artery disease (n= 40) 60.5± 12.2 20 (50.0%)

Acute coronary syndrome (n= 40) 61.0± 12.6 21 (52.5%)

p value

0.712 0.789

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Variable

15 (37.5%) 10 (25.0%)

21 (52.5%) 15 (37.5%)

28 (70.0%) 23 (57.5%)

0.015* 0.013*

20 (50.0%) 2 (5.0%) 5 (12.5%)

31 (77.5%) 4 (10.0%) 5 (12.5%)

31 (77.5%) 10 (25.0%) 15 (37.5%)

0.013* 0.023* 0.006*

28.1± 3.2

29.5± 3.3

Statin 75 (62.5%) Beta blocker 68 (56.7%) Angiotensin65 (54.2%) converting enzyme inhibitor/ Angiotensin receptor blocker Laboratory parameters

13 (32.5%) 13 (32.5%) 15 (37.5%)

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28.3± 3.1

0.595

26 (65.0%) 27 (67.5%) 29 (72.5%)

<0.001* 0.001* 0.001*

0.8± 0.2

0.9± 0.2

0.9± 0.3

0.689

91 (25.5)

99 (33.0)

119 (47.0)ǂɸ

0.001*

143.7± 41.5

126.2± 32.3

124.0± 36.6

0.078

50.5± 14.8

48.3± 13.2

35.1± 7.9ǂɸ

<0.001*

13.8± 1.5

13.5± 1.6

13.7± 1.3

14.1± 1.6

0.246

8.2± 3.1

7.1± 0.2

7.9± 2.9

9.5± 3.9ǂɸ

0.002*

242.6± 65.6

253.2± 68.7

233.1± 64.0

241.8± 64.4

0.417

-

-

-

2.4 (2.9) 6.9 (18.2)

-

0.9± 0.2 101.5 (37.0)

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130.7± 37.4

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36 (90.0%) 28 (70.0%) 21 (52.5%)

43.2± 13.5

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Serum creatinine (mg/ dL) Fasting blood glucose (mg/dL) Low- density lipoprotein cholesterol (mg/dL) High- density lipoprotein cholesterol (mg/dL) Hemoglobin (g/dL) White blood cell count (x103/µL) Platelet count (x103/µL) Creatine kinasemyocardial band (ng/mL) - Baseline - Peak Troponin- T

SC

Hypertension 64 (53.3%) Diabetes 48 (40.0%) mellitus Hyperlipidemia 82 (68.3%) Smoking 16 (13.3%) Family history 25 (20.8%) of coronary artery disease Body mass index 28.5± 3.4 (kg/ m2) Medications on admission

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(ng/mL) - Baseline - Peak Echocardiographic parameters 56.9± 10.5

-

0.03 (0.11) 0.41 (2.3)

-

60.0± 10.4

59.1± 9.1

52.3± 10.5ǂɸ

0.003*

RI PT

Left ventricular ejection fraction (%)

-

* p< 0.05

denotes the statistical significant difference when compared to the group of patients with normal coronary arteries.

TE D

M AN U

SC

denotes the statistical significant difference when compared to the group of patients with stable angina pectoris.

EP

ɸ

AC C

ǂ

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Table 2. Comparison of platelet Toll- like receptor - 2 and 4 expression in subgroups of acute coronary syndrome (n=40).

Unstable angina

Non-ST- segment

ST- segment

receptor expression (%)

pectoris (n=12)

elevation myocardial

elevation myocardial

infarction (n=16)

infarction (n=12)

p value

RI PT

Platelet Toll- like

26.0 (47.0)

29.5 (44.0)

32.0 (74.0)

0.799

Toll- like receptor- 4

29.5 (37.0)

45.5 (74.0)

51.5 (70.0)

0.715

AC C

EP

TE D

M AN U

SC

Toll- like receptor- 2

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Table 3. Comparison of platelet Toll- like receptor- 2 and 4 expression regarding presence of conventional cardiovascular risk factors and medications.

Hyperlipidemia Smoking Family history of coronary artery disease Beta blockers Statins Angiotensin- converting enzyme inhibitor/ angiotensin receptor blockers

12.0 (19.0)

AC C

EP

TE D

* p<0.05

0.021* 0.009* 0.020* 0.014* 0.028* 0.011*

Platelet Toll- like receptor- 4 expression 5.5 (16.3) 19.0 (29.8) 5.5 (17.0) 20.0 (36.3) 4.0 (21.0) 13.5 (24.5) 10.5 (18.3) 20.0 (31.8) 8.0 (17.5) 24.0 (39.0) 4.0 (20.5) 15 (28.0) 4.0 (22.8) 15.0 (21.0) 4.0 (21.0)

0.009* 0.008*

p value

RI PT

Diabetes mellitus

+ + + + + + + +

p value

SC

Hypertension

Platelet Toll- like receptor- 2 expression 6.5 (9.5) 13 (19.8) 6.0 (9.0) 13.5 (22.5) 5 (11.0) 12 (20.0) 8.0 (10.0) 16.5 (36.3) 8.0 (10.0) 14.0 (43.0) 5 (11.0) 12 (12.0) 4.0 (14.8) 12.0 (12.0) 5.0 (11.0)

M AN U

Variables

17.0 (29.0)

0.012* 0.002*

0.047*

0.048*

0.020* 0.006* 0.027* 0.005*

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Table 4. Correlation between baseline characteristics and platelet Toll- like receptor -2 and 4 expression.

0.125

0.353

0.001*

0.386

<0.001*

-0.135

0.190

-0.265

-0.496

<0.001*

-0.556

-0.329

0.001*

-0.379

<0.001*

0.126

0.179

0.128

0.174

0.272

0.003*

0.304

0.001*

-0.073

0.443

-0.076

0.423

0.081

0.554

0.112

0.413

0.168

0.215

0.328

0.014*

0.186

0.170

0.318

0.017*

0.452

<0.001*

0.282

RI PT

-0.469

AC C

* p<0.05

0.121

0.035*

0.175

<0.001*

SC

M AN U

White blood cell count (x103/µL) Platelet count (x103/µL) Baseline creatine kinase- myocardial band (ng/mL) Peak creatine kinase- myocardial band (ng/mL) Baseline troponin- T (ng/mL) Peak troponin- T (ng/Ml)

Platelet Toll- like receptor - 4 expression Spearman’s rho p value

-0.300

TE D

Body mass index (kg/m2) Fasting blood glucose (mg/ dL) Low- density lipoprotein cholesterol (mg/dL) High- density lipoprotein cholesterol (mg/dL) Left ventricular ejection fraction (%) Hemoglobin (g/dL)

Platelet Toll- like receptor - 2 expression Spearman’s rho p value

EP

Variables

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Table 5. Univariate multinominal regression analysis for identifying predictors of acute coronary syndrome. Model 1. Acute coronary syndrome vs. normal coronary arteries

95% confidence interval

lower

upper

p value

Hypertension

2.13

0.84

5.26

0.111

Diabetes mellitus

2.27

0.92

5.56

0.075

Hyperlipidemia

1.0

0.35

2.86

Smoking

3.03

0.85

11.11

Family history of coronary artery disease

4.57

1.46

14.28

Statins

4.85

1.43

16.42

Beta blockers Angiotensin- converting enzyme inhibitor/ angiotensin receptor blockers

1.12

0.44

2.89

1.31

0.47

Toll- like receptor - 2

11.96

Toll- like receptor - 4

9.02

EP AC C

lower

upper

p value

2.11

0.84

5.29

0.111

2.25

0.92

5.52

0.075

1.00

0.35

2.86

1.000

0.086

3.02

0.86

10.63

0.086

0.009

4.57

1.46

14.28

0.009*

0.011*

0.21

0.06

0.70

0.011*

0.809

0.89

0.35

2.29

0.809

3.61

0.606

0.77

0.28

2.11

0.606

4.10

34.87

<0.001*

2.55

1.35

4.83

0.004*

2.88

28.25

<0.001*

1.92

1.27

2.91

0.002*

M AN U

1.000

TE D

* p< 0.05

Odds ratio

SC

Odds ratio

RI PT

95% confidence interval

Variables

Model 2. Acute coronary syndrome vs. stable angina pectoris

AC C

EP

TE D

M AN U

SC

RI PT

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