J. of Cardiovasc. Trans. Res. (2014) 7:47–52 DOI 10.1007/s12265-013-9530-8
Impact of Cigarette Smoking on P2Y12 Receptor Binding Activity Before and After Clopidogrel Therapy in Patients with Coronary Artery Disease Jung Rae Cho & Bhaloo Desai & Michael J. Haas & Fabiana Rollini & Francesco Franchi & Ana Muñiz-Lozano & Antonio Tello-Montoliu & Elisabetta Ferrante & Luis A. Guzman & Theodore A. Bass & Dominick J. Angiolillo
Received: 9 November 2013 / Accepted: 9 December 2013 / Published online: 20 December 2013 # Springer Science+Business Media New York 2013
Abstract Smoking enhances the P2Y12 receptor inhibitory effects of clopidogrel. Nicotine increases P2Y12 receptor expression in platelet lysates from healthy volunteers. However, the impact of cigarette smoking on platelet P2Y12 receptor binding in clopidogrel-treated patients with coronary artery disease (CAD) is unknown. Clopidogrel-naïve patients with stable CAD (n =20) were enrolled and stratified according to smoking status. P2Y12 receptor binding activity was determined by radioligand receptor binding prior and 24 h after a 600-mg loading dose of clopidogrel. Baseline P2Y12 receptor binding was 1.8-fold higher in smokers compared with nonsmokers. After a 600-mg loading dose of clopidogrel, smokers showed a 6.4-fold reduction in P2Y12 receptor binding indicative of marked clopidogrel-mediated blockade, while there were minimal changes among nonsmokers. Among patients with stable CAD, smokers have more P2Y12 receptor binding than nonsmokers and have a higher degree of clopidogrelmediated platelet inhibition.
Keywords Smoking . Platelet P2Y12 receptors . Clopidogrel . Coronary artery disease
Associate Editor Emanuele Barbato oversaw the review of this article J. R. Cho : B. Desai : M. J. Haas : F. Rollini : F. Franchi : A. Muñiz-Lozano : A. Tello-Montoliu : E. Ferrante : L. A. Guzman : T. A. Bass : D. J. Angiolillo (*) University of Florida College of Medicine-Jacksonville, 655 West 8th Street, Jacksonville, FL 32209, USA e-mail: [email protected]
Introduction Clopidogrel is the most widely investigated platelet P2Y12 receptor inhibitor and in conjunction with aspirin has represented the cornerstone of treatment for prevention of recurrent ischemic events in patients with acute coronary syndromes and undergoing percutaneous coronary interventions [1]. Clopidogrel is an inactive prodrug which needs to be metabolized by the cytochrome (CYP) P450 system to give origin to an active metabolite, which in turn irreversibly inhibits the platelet P2Y12 receptor [2]. However, the pharmacodynamic (PD) effects of clopidogrel vary broadly which can explain its differential clinical benefit [2–4]. Several factors may contribute these findings [2–4]. Most recently, smoking status has been implied in this process. In particular, although smokers have higher event rates compared with nonsmokers, clinical studies have shown that clopidogrel therapy is associated with a greater relative benefit among smokers also known as the “smoker's paradox” [5–13]. Investigations have shown that the relative clinical benefit of clopidogrel among smokers may be attributed to the enhanced PD effects compared with nonsmokers [14–18]. The underlying mechanism appears to be its more favorable metabolism among smokers. In fact, smoking is an inducer of CYP1A2 and CYP2B6, key isoenzymes contributing to clopidogrel metabolism [19, 20]. Therefore, smoking can enhance generation of clopidogrel's active metabolite leading to greater PD effects [14–18]. In addition, the enhanced PD effects can also be explained at the cellular level given that, among healthy volunteers, smoking status is associated with increased P2Y12 binding on the platelet surface [21]. However, to date, there are no studies which have assessed the impact
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of smoking on P2Y12 receptor binding among patients with coronary artery disease (CAD) and how this impacts the inhibitory effects of clopidogrel, which represented the aim of this pilot investigation.
Methods Study Population and Research Design Patients were screened at the outpatient clinic of the Division of Cardiology of the University of Florida College Of Medicine-UF Health Jacksonville. A total of 20 patients (n = 10 smokers; n =10 nonsmokers) with stable CAD meeting study entry criteria were prospectively enrolled. Blood sampling for assessment of P2Y12 receptor enumeration was collected at two time points: before and 24 h after a 600-mg loading dose of clopidogrel. Patients needed to meet all of the following inclusion criteria: (1) established CAD (defined as a prior history of percutaneous or surgical coronary revascularization), (2) clinically stable, (3) on treatment with aspirin (81 mg/day) ≥7 days, (4) clopidogrel (or any P2Y12 receptor inhibitor) naïve for at least 30 days, and (5) age between 18 and 80 years. Smoking status was defined through patient interview. The cutoff of >10 cigarettes/day was used to define smokers. This cutoff was used in line with prior investigations showing an impact of smoking on the PD effects of clopidogrel [15, 17, 18]. Nonsmokers reported not to be actively smoking for at least 6 months. Individuals not meeting these criteria were not eligible for inclusion in the study. Other specific exclusion criteria included: (1) known allergies to aspirin or clopidogrel, (2) blood dyscrasia, (3) serum creatinine level >2.5 mg/dL, (4) any active bleeding or bleeding diathesis, (5) clinical indication to be on a P2Y12 receptor inhibitor such as an acute coronary syndrome or percutaneous coronary intervention in the last 12 months, (6) hemodynamic instability, (7) cerebrovascular accident within 3 months, (8) any active malignancy, (9) use of any antiplatelet agent other than aspirin (e.g., ticlopidine, clopidogrel, prasugrel, ticagrelor, glycoprotein IIb/IIIa antagonist, cilostazol, or dipyridamole) or oral anticoagulation (vitamin K antagonist, dabigatran, rivaroxaban, and apixaban) therapy within 30 days, (10) use of drugs interfering with clopidogrel effects (ketoconazole, itraconazole, diltiazem, erythromycin, clarithromycin, and omeprazole), (11) platelet count 2 mg/dL), (13) any major bleeding within the last 6 months, and (14) pregnant or lactating females. The study complied with the Declaration of Helsinki, was approved by the Institutional Review Board of the University of Florida College of Medicine-Jacksonville, and all patients gave their written informed consent.
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P2Y12 Receptor Binding Assay Peripheral venous blood samples were drawn with a loose tourniquet to avoid artifacts through a short venous catheter inserted into a forearm vein. The first 2–4 mL of blood was discarded to avoid spontaneous platelet activation. A total of 80 mL of blood was collected in a citrated tube before and 24 h after a 600-mg load of clopidogrel. Samples were processed for P2Y12 receptor binding assay within 2 h after blood drawing. Determination of P2Y12 receptor numbers was performed by radioligand binding assay according to standard protocols [22]. Platelet-rich plasma (PRP) was prepared by centrifuging at 1,000 rpm for 10 min and platelet count was also determined. PRP containing 2×108 platelets was incubated with a range of concentrations (0.1–100 nM) of radiolabeled [33P]2MeSADP (2-methylthioadenosine 5′-diphosphate from Perkin Elmer LAS Inc., Waltham, MA) for 15 min at room temperature. Nonspecific binding was determined at each ligand concentration by also including a maximal concentration (10 μM) of cold 2MeSADP (Sigma-Aldrich, Saint Louis, MO). In a separate set of experiments, a saturating amount of P2Y 1 antagonist, 100 μM MRS2179 (N6-methyl-2′deoxyadenosine-3′,5′-bisphosphate, Sigma-Aldrich) was used for the determination of P2Y12 specific binding. Specific P2Y12 binding and receptor number (B max) were calculated using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Statistical Analysis and Sample Size Calculations Continuous baseline variables are expressed as mean ± standard deviation, while categorical baseline variables are expressed as frequencies and percentages. P2Y12 receptor binding activity is expressed as mean ± standard error. Given the lack of prior investigations in the field, an arbitrary sample of ten patients per group (total of 20 patients) was chosen to provide pilot information. Such sample size selection is in line with recommendations for pilot studies [23]. Also, in line with recommendations for pilot studies with a limited sample size, no formal statistical comparisons were performed.
Results A total of 20 patients with stable CAD meeting study entry criteria were prospectively enrolled. There were no significant differences in baseline demographics between smokers (n = 10) and nonsmokers (n =10) (Table 1). At baseline, P2Y12 receptor binding was 1.8-fold higher in smokers compared with nonsmokers (Fig. 1). After a 600-mg loading dose of clopidogrel, P2Y12 receptor binding was 3.4-fold lower among smokers compared with nonsmokers (Fig. 1). Overall,
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Table 1 Baseline characteristics
ACEI/ARB angiotensin converting enzyme inhibitors/angiotensin receptor blockers, BMI body mass index, CABG coronary artery bypass graft surgery, CCB calcium channel blockers, LVEF left ventricular ejection fraction, MI myocardial infarction, PCI percutaneous coronary intervention, PPI proton pump inhibitor
Variable
Nonsmokers (n =10)
Smokers (n =10)
P value
Age, years Male BMI, kg/m2 Race Caucasian
61.2±8.5 9 (90 %) 33.5±6.7
56.9±7.1 8 (80 %) 32.3±7.0
0.233 0.531 0.686 0.639
7 (70 %)
6 (60 %)
3 (30 9 (90 9 (90 5 (50 6 (60
4 (40 %) 10 (100 %) 10 (100 %) 5 (50 %) 5 (50 %)
0.305 0.305 1.000 0.653
8 (80 %) 6 (60 %) 7 (70 %) 0 (0 %) 4 (40 %) 6 (60 %) 0.9±0.2 42.6±3.1
8 (80 %) 5 (50 %) 6 (60 %) 3 (30 %) 7 (70 %) 6 (60 %) 1.0±0.4 41.6±5.6
1.000 0.463 0.232 0.090 0.260 1.000 0.471 0.537
217.9±36.6
237.9±61.5
0.414
African-American Hypertension Dyslipidemia Diabetes mellitus Prior MI Medications Beta-blockers ACEI/ARB Statins CCB Nitrates LVEF >50 % Creatinine, g/dL Hematocrit, % Platelet count, 1,000/mm3
smokers had a 6.4-fold reduction in P2Y12 receptor binding activity compared with baseline (Fig. 1). There were minimal changes in P2Y12 receptor binding activity among nonsmokers (Fig. 1).
Discussion Recent investigations have consistently shown a greater relative clinical benefit of clopidogrel among smokers compared with nonsmokers [5–13]. These clinical findings have been attributed to the greater magnitude of PD effects achieved with clopidogrel among smoking subjects [14–18]. Enhanced clopidogrel metabolism secondary to upregulation of key CYP enzymes, in particular CYP1A2 and CYP2B6, among smoking subjects has been ascribed as a contributing cause [19, 20]. A dose-dependent effect of cigarette smoking on CYP activity has also been shown, with return baseline levels after smoking cessation [19]. Accordingly, a dose-dependent effect on PD response has been observed, with lower levels of platelet reactivity and rates of poor clopidogrel responders among heavy smokers [16]. A causal relationship between smoking and degree of clopidogrel-induced antiplatelet effects is also supported by the observation that the enhanced PD effect among smokers diminishes with smoking discontinuation [24]. Our investigation expands on prior mechanistic studies. First, our study shows that among patients with CAD, smokers have increased binding of P2Y12 receptor on the
%) %) %) %) %)
platelet surface compared with nonsmokers. Second, after clopidogrel loading dose, a greater number of P2Y12 receptor are inhibited by clopidogrel among smokers. Overall, these findings provide insights on differences in clinical outcomes observed among smokers compared with nonsmokers (smoker's paradox). Among patients with established cardiovascular disease, smoking is a strong independent risk factor for cardiovascular mortality and adverse cardiovascular outcomes [25]. These consequences may be attributed to the fact that smoking causes endothelial dysfunction, dyslipidemia, and increased platelet activation, which all lead to a prothrombotic state [26]. Increased P2Y12 receptor binding among smokers as observed in our study may contribute to their higher risk for recurrent events. In fact, the P2Y12 receptor signaling pathway is one of the key platelet activating pathways mediating arterial thrombosis as supported by the large number of clinical trials demonstrating that its blockade is associated with a reduction in atherothrombotic recurrences [1, 2, 27, 28]. Moreover, the magnitude of such effect is proportional to levels of P2Y12 inhibition, as shown by trials with more potent inhibitors (prasugrel and ticagrelor) having greater benefit over clopidogrel [29, 30]. Our findings therefore can explain why in PD studies conducted among aspirin treated patients, in the absence of a P2Y12 receptor inhibitor, platelet reactivity is higher among smokers compared with nonsmokers [31–34]. Smoking in fact has been associated with less biological efficacy of aspirin, also coined as aspirin resistance, as shown
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J. of Cardiovasc. Trans. Res. (2014) 7:47–52
Fig. 1 Radioligand receptor binding for P2Y12 receptor enumeration in smokers vs. nonsmokers before and after a 600-mg clopidogrel loading dose. Values indicate total number of P2Y12 receptors per 2×108 platelets and are expressed as mean ± standard error
by increased platelet reactivity and incomplete inhibition of thromboxane biosynthesis compared with nonsmokers [31–34]. Therefore, it may be hypothesized that the benefit of clopidogrel over aspirin, as observed in the only head to head comparison between these two agents in the Clopidogrel Versus Aspirin in Patients at Risk of Ischemic Events (CAPRIE) trial, may be due to the fact that smokers have higher platelet surface P2Y12 density, contributing to their higher risk for recurrent events, which can be suppressed by clopidogrel but not by aspirin [12]. The first observation on the impact of nicotine on P2Y12 receptor expression was from Shanker et al. who demonstrated a higher number of P2Y12 receptors in platelet lysate in healthy smokers compared to nonsmokers [21]. In this investigation, various human cell lines were used, including human megakaryoblastic cells and MEG-01. After nicotine treatment, the expression of P2Y12 receptor on MEG-01 increased significantly. The upregulation of P2Y12 by nicotine was mediated by nicotinic acetylcholine receptors (nAChR). Accordingly, long-term exposure to nicotine upregulates P2Y12 receptors first in megakaryocytes and subsequently in their platelet products. In addition, nicotine upregulates the expression of nAChR subtype α7, suggesting the possibility of an amplified positive feedback loop leading to enhanced P2Y12 expression [21]. Our study findings are in line with these prior observations in non-medicated healthy volunteers and expand upon these as it is the first to assess the impact of smoking status on P2Y12 receptor binding activity in patients with CAD as well as seeing how P2Y12 receptor binding can be modulated by clopidogrel exposure. The number of P2Y12 receptors on the platelet surface varies and has been suggested to be regulated by an intracellular pool [35]. There are several reports on intraplatelet
signaling (for example, P2Y12 receptor internalization and recycling) after prolonged stimulation with agonists to prevent inappropriate thrombosis in normal physiological condition [36, 37]. To this extent, megakaryocyte and derived platelets from smoking subjects may have deranged intraplatelet signaling due to increase systemic exposure to platelet stimulants. This in turn would lead to increased P2Y12 receptor expression on the platelet surface enabling greater exposure and thus inhibition by P2Y12 receptor antagonists. The greater density of P2Y12 receptors may be one of the mechanisms involved in the observed benefit of prasugrel over clopidogrel among smokers in the Targeted Platelet Inhibition to Clarify the Optimal Strategy to Medically Manage Acute Coronary Syndromes trial (TRILOGY-ACS) [38, 39]. In fact, although the active metabolite of prasugrel and clopidogrel are equipotent, the main differences between the two drugs is that prasugrel generates more efficiently its active metabolite compared with clopidogrel which can be of potential benefit in smokers who have higher P2Y12 receptor density. Study Limitations The criteria of smoker vs. nonsmoker were based on patient interview. Indeed, a more objective assessment, such as cotinine assessment, could discriminate more accurately the degree of smoking exposure of each individual patient. Further, this study only performed P2Y 12 binding assays. Indeed, performing additional pharmacodynamic tests could have been useful to corroborate our study findings. Moreover, experiments conducted with potent platelet stimulants could have allowed to determine the pool of internalized P2Y12 receptors [35–37]. However, the abovementioned additional experiments would have implied much larger blood sample
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volumes which would not be feasible for ethical reasons. Also, in this study, we did not assess individual response profiles to aspirin, which are known to be affected by smoking status [31–34]. Indeed, this could have provided additional insights on our study findings, including explaining some of the variability in P2Y12 receptor binding activity. Ultimately, this study should be considered as pilot in nature with a limited sample size, further limiting the formal statistical comparison, which would be subject to type II errors. However, this approach is in line with recommendations for pilot investigations [23].
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Conclusions
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Among patients with stable CAD, smokers have greater P2Y12 receptor binding activity than nonsmokers and have a higher degree of inhibition following a 600-mg loading dose of clopidogrel. Increased P2Y12 receptor binding among smokers may contribute to their higher risk for recurrent events, which can be suppressed to a greater extent by clopidogrel explaining their greater relative ischemic benefit (smoker's paradox) compared with nonsmokers.
8.
Source of Funding The present study was funded by an Institutional Grant of the University of Florida College of Medicine-Jacksonville and by a grant provided by The James and Esther King Biomedical Research Program (1KN01-33989). Conflict of Interest Jung Rae Cho, Bhaloo Desai, Michael J. Haas, Fabiana Rollini, Francesco Franchi, Ana Muñiz-Lozano, Antonio TelloMontoliu, Elisabetta Ferrante, Luis A. Guzman, and Theodore A. Bass have no conflict of interest to report. Dominick J. Angiolillo received payment as an individual for (a) consulting fee or honorarium from Bristol Myers Squibb, Sanofi-Aventis, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Merck, Evolva, Abbott Vascular, and PLx Pharma and (b) participation in review activities from Johnson & Johnson, St. Jude, and Sunovion. Institutional payments for grants from Bristol Myers Squibb, Sanofi-Aventis, Glaxo Smith Kline, Otsuka, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Evolva, Gilead and has other financial relationships with Esther and King Biomedical Research Grant.
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Impact of Cigarette Smoking on P2Y12 Receptor Binding Activity Before and After Clopidogrel Therapy in Patients with Coronary Artery Disease Jung Rae Cho & Bhaloo Desai & Michael J. Haas & Fabiana Rollini & Francesco Franchi & Ana Muñiz-Lozano & Antonio Tello-Montoliu & Elisabetta Ferrante & Luis A. Guzman & Theodore A. Bass & Dominick J. Angiolillo
Received: 9 November 2013 / Accepted: 9 December 2013 / Published online: 20 December 2013 # Springer Science+Business Media New York 2013
Abstract Smoking enhances the P2Y12 receptor inhibitory effects of clopidogrel. Nicotine increases P2Y12 receptor expression in platelet lysates from healthy volunteers. However, the impact of cigarette smoking on platelet P2Y12 receptor binding in clopidogrel-treated patients with coronary artery disease (CAD) is unknown. Clopidogrel-naïve patients with stable CAD (n =20) were enrolled and stratified according to smoking status. P2Y12 receptor binding activity was determined by radioligand receptor binding prior and 24 h after a 600-mg loading dose of clopidogrel. Baseline P2Y12 receptor binding was 1.8-fold higher in smokers compared with nonsmokers. After a 600-mg loading dose of clopidogrel, smokers showed a 6.4-fold reduction in P2Y12 receptor binding indicative of marked clopidogrel-mediated blockade, while there were minimal changes among nonsmokers. Among patients with stable CAD, smokers have more P2Y12 receptor binding than nonsmokers and have a higher degree of clopidogrelmediated platelet inhibition.
Keywords Smoking . Platelet P2Y12 receptors . Clopidogrel . Coronary artery disease
Associate Editor Emanuele Barbato oversaw the review of this article J. R. Cho : B. Desai : M. J. Haas : F. Rollini : F. Franchi : A. Muñiz-Lozano : A. Tello-Montoliu : E. Ferrante : L. A. Guzman : T. A. Bass : D. J. Angiolillo (*) University of Florida College of Medicine-Jacksonville, 655 West 8th Street, Jacksonville, FL 32209, USA e-mail: [email protected]
Introduction Clopidogrel is the most widely investigated platelet P2Y12 receptor inhibitor and in conjunction with aspirin has represented the cornerstone of treatment for prevention of recurrent ischemic events in patients with acute coronary syndromes and undergoing percutaneous coronary interventions [1]. Clopidogrel is an inactive prodrug which needs to be metabolized by the cytochrome (CYP) P450 system to give origin to an active metabolite, which in turn irreversibly inhibits the platelet P2Y12 receptor [2]. However, the pharmacodynamic (PD) effects of clopidogrel vary broadly which can explain its differential clinical benefit [2–4]. Several factors may contribute these findings [2–4]. Most recently, smoking status has been implied in this process. In particular, although smokers have higher event rates compared with nonsmokers, clinical studies have shown that clopidogrel therapy is associated with a greater relative benefit among smokers also known as the “smoker's paradox” [5–13]. Investigations have shown that the relative clinical benefit of clopidogrel among smokers may be attributed to the enhanced PD effects compared with nonsmokers [14–18]. The underlying mechanism appears to be its more favorable metabolism among smokers. In fact, smoking is an inducer of CYP1A2 and CYP2B6, key isoenzymes contributing to clopidogrel metabolism [19, 20]. Therefore, smoking can enhance generation of clopidogrel's active metabolite leading to greater PD effects [14–18]. In addition, the enhanced PD effects can also be explained at the cellular level given that, among healthy volunteers, smoking status is associated with increased P2Y12 binding on the platelet surface [21]. However, to date, there are no studies which have assessed the impact
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of smoking on P2Y12 receptor binding among patients with coronary artery disease (CAD) and how this impacts the inhibitory effects of clopidogrel, which represented the aim of this pilot investigation.
Methods Study Population and Research Design Patients were screened at the outpatient clinic of the Division of Cardiology of the University of Florida College Of Medicine-UF Health Jacksonville. A total of 20 patients (n = 10 smokers; n =10 nonsmokers) with stable CAD meeting study entry criteria were prospectively enrolled. Blood sampling for assessment of P2Y12 receptor enumeration was collected at two time points: before and 24 h after a 600-mg loading dose of clopidogrel. Patients needed to meet all of the following inclusion criteria: (1) established CAD (defined as a prior history of percutaneous or surgical coronary revascularization), (2) clinically stable, (3) on treatment with aspirin (81 mg/day) ≥7 days, (4) clopidogrel (or any P2Y12 receptor inhibitor) naïve for at least 30 days, and (5) age between 18 and 80 years. Smoking status was defined through patient interview. The cutoff of >10 cigarettes/day was used to define smokers. This cutoff was used in line with prior investigations showing an impact of smoking on the PD effects of clopidogrel [15, 17, 18]. Nonsmokers reported not to be actively smoking for at least 6 months. Individuals not meeting these criteria were not eligible for inclusion in the study. Other specific exclusion criteria included: (1) known allergies to aspirin or clopidogrel, (2) blood dyscrasia, (3) serum creatinine level >2.5 mg/dL, (4) any active bleeding or bleeding diathesis, (5) clinical indication to be on a P2Y12 receptor inhibitor such as an acute coronary syndrome or percutaneous coronary intervention in the last 12 months, (6) hemodynamic instability, (7) cerebrovascular accident within 3 months, (8) any active malignancy, (9) use of any antiplatelet agent other than aspirin (e.g., ticlopidine, clopidogrel, prasugrel, ticagrelor, glycoprotein IIb/IIIa antagonist, cilostazol, or dipyridamole) or oral anticoagulation (vitamin K antagonist, dabigatran, rivaroxaban, and apixaban) therapy within 30 days, (10) use of drugs interfering with clopidogrel effects (ketoconazole, itraconazole, diltiazem, erythromycin, clarithromycin, and omeprazole), (11) platelet count 2 mg/dL), (13) any major bleeding within the last 6 months, and (14) pregnant or lactating females. The study complied with the Declaration of Helsinki, was approved by the Institutional Review Board of the University of Florida College of Medicine-Jacksonville, and all patients gave their written informed consent.
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P2Y12 Receptor Binding Assay Peripheral venous blood samples were drawn with a loose tourniquet to avoid artifacts through a short venous catheter inserted into a forearm vein. The first 2–4 mL of blood was discarded to avoid spontaneous platelet activation. A total of 80 mL of blood was collected in a citrated tube before and 24 h after a 600-mg load of clopidogrel. Samples were processed for P2Y12 receptor binding assay within 2 h after blood drawing. Determination of P2Y12 receptor numbers was performed by radioligand binding assay according to standard protocols [22]. Platelet-rich plasma (PRP) was prepared by centrifuging at 1,000 rpm for 10 min and platelet count was also determined. PRP containing 2×108 platelets was incubated with a range of concentrations (0.1–100 nM) of radiolabeled [33P]2MeSADP (2-methylthioadenosine 5′-diphosphate from Perkin Elmer LAS Inc., Waltham, MA) for 15 min at room temperature. Nonspecific binding was determined at each ligand concentration by also including a maximal concentration (10 μM) of cold 2MeSADP (Sigma-Aldrich, Saint Louis, MO). In a separate set of experiments, a saturating amount of P2Y 1 antagonist, 100 μM MRS2179 (N6-methyl-2′deoxyadenosine-3′,5′-bisphosphate, Sigma-Aldrich) was used for the determination of P2Y12 specific binding. Specific P2Y12 binding and receptor number (B max) were calculated using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Statistical Analysis and Sample Size Calculations Continuous baseline variables are expressed as mean ± standard deviation, while categorical baseline variables are expressed as frequencies and percentages. P2Y12 receptor binding activity is expressed as mean ± standard error. Given the lack of prior investigations in the field, an arbitrary sample of ten patients per group (total of 20 patients) was chosen to provide pilot information. Such sample size selection is in line with recommendations for pilot studies [23]. Also, in line with recommendations for pilot studies with a limited sample size, no formal statistical comparisons were performed.
Results A total of 20 patients with stable CAD meeting study entry criteria were prospectively enrolled. There were no significant differences in baseline demographics between smokers (n = 10) and nonsmokers (n =10) (Table 1). At baseline, P2Y12 receptor binding was 1.8-fold higher in smokers compared with nonsmokers (Fig. 1). After a 600-mg loading dose of clopidogrel, P2Y12 receptor binding was 3.4-fold lower among smokers compared with nonsmokers (Fig. 1). Overall,
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Table 1 Baseline characteristics
ACEI/ARB angiotensin converting enzyme inhibitors/angiotensin receptor blockers, BMI body mass index, CABG coronary artery bypass graft surgery, CCB calcium channel blockers, LVEF left ventricular ejection fraction, MI myocardial infarction, PCI percutaneous coronary intervention, PPI proton pump inhibitor
Variable
Nonsmokers (n =10)
Smokers (n =10)
P value
Age, years Male BMI, kg/m2 Race Caucasian
61.2±8.5 9 (90 %) 33.5±6.7
56.9±7.1 8 (80 %) 32.3±7.0
0.233 0.531 0.686 0.639
7 (70 %)
6 (60 %)
3 (30 9 (90 9 (90 5 (50 6 (60
4 (40 %) 10 (100 %) 10 (100 %) 5 (50 %) 5 (50 %)
0.305 0.305 1.000 0.653
8 (80 %) 6 (60 %) 7 (70 %) 0 (0 %) 4 (40 %) 6 (60 %) 0.9±0.2 42.6±3.1
8 (80 %) 5 (50 %) 6 (60 %) 3 (30 %) 7 (70 %) 6 (60 %) 1.0±0.4 41.6±5.6
1.000 0.463 0.232 0.090 0.260 1.000 0.471 0.537
217.9±36.6
237.9±61.5
0.414
African-American Hypertension Dyslipidemia Diabetes mellitus Prior MI Medications Beta-blockers ACEI/ARB Statins CCB Nitrates LVEF >50 % Creatinine, g/dL Hematocrit, % Platelet count, 1,000/mm3
smokers had a 6.4-fold reduction in P2Y12 receptor binding activity compared with baseline (Fig. 1). There were minimal changes in P2Y12 receptor binding activity among nonsmokers (Fig. 1).
Discussion Recent investigations have consistently shown a greater relative clinical benefit of clopidogrel among smokers compared with nonsmokers [5–13]. These clinical findings have been attributed to the greater magnitude of PD effects achieved with clopidogrel among smoking subjects [14–18]. Enhanced clopidogrel metabolism secondary to upregulation of key CYP enzymes, in particular CYP1A2 and CYP2B6, among smoking subjects has been ascribed as a contributing cause [19, 20]. A dose-dependent effect of cigarette smoking on CYP activity has also been shown, with return baseline levels after smoking cessation [19]. Accordingly, a dose-dependent effect on PD response has been observed, with lower levels of platelet reactivity and rates of poor clopidogrel responders among heavy smokers [16]. A causal relationship between smoking and degree of clopidogrel-induced antiplatelet effects is also supported by the observation that the enhanced PD effect among smokers diminishes with smoking discontinuation [24]. Our investigation expands on prior mechanistic studies. First, our study shows that among patients with CAD, smokers have increased binding of P2Y12 receptor on the
%) %) %) %) %)
platelet surface compared with nonsmokers. Second, after clopidogrel loading dose, a greater number of P2Y12 receptor are inhibited by clopidogrel among smokers. Overall, these findings provide insights on differences in clinical outcomes observed among smokers compared with nonsmokers (smoker's paradox). Among patients with established cardiovascular disease, smoking is a strong independent risk factor for cardiovascular mortality and adverse cardiovascular outcomes [25]. These consequences may be attributed to the fact that smoking causes endothelial dysfunction, dyslipidemia, and increased platelet activation, which all lead to a prothrombotic state [26]. Increased P2Y12 receptor binding among smokers as observed in our study may contribute to their higher risk for recurrent events. In fact, the P2Y12 receptor signaling pathway is one of the key platelet activating pathways mediating arterial thrombosis as supported by the large number of clinical trials demonstrating that its blockade is associated with a reduction in atherothrombotic recurrences [1, 2, 27, 28]. Moreover, the magnitude of such effect is proportional to levels of P2Y12 inhibition, as shown by trials with more potent inhibitors (prasugrel and ticagrelor) having greater benefit over clopidogrel [29, 30]. Our findings therefore can explain why in PD studies conducted among aspirin treated patients, in the absence of a P2Y12 receptor inhibitor, platelet reactivity is higher among smokers compared with nonsmokers [31–34]. Smoking in fact has been associated with less biological efficacy of aspirin, also coined as aspirin resistance, as shown
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Fig. 1 Radioligand receptor binding for P2Y12 receptor enumeration in smokers vs. nonsmokers before and after a 600-mg clopidogrel loading dose. Values indicate total number of P2Y12 receptors per 2×108 platelets and are expressed as mean ± standard error
by increased platelet reactivity and incomplete inhibition of thromboxane biosynthesis compared with nonsmokers [31–34]. Therefore, it may be hypothesized that the benefit of clopidogrel over aspirin, as observed in the only head to head comparison between these two agents in the Clopidogrel Versus Aspirin in Patients at Risk of Ischemic Events (CAPRIE) trial, may be due to the fact that smokers have higher platelet surface P2Y12 density, contributing to their higher risk for recurrent events, which can be suppressed by clopidogrel but not by aspirin [12]. The first observation on the impact of nicotine on P2Y12 receptor expression was from Shanker et al. who demonstrated a higher number of P2Y12 receptors in platelet lysate in healthy smokers compared to nonsmokers [21]. In this investigation, various human cell lines were used, including human megakaryoblastic cells and MEG-01. After nicotine treatment, the expression of P2Y12 receptor on MEG-01 increased significantly. The upregulation of P2Y12 by nicotine was mediated by nicotinic acetylcholine receptors (nAChR). Accordingly, long-term exposure to nicotine upregulates P2Y12 receptors first in megakaryocytes and subsequently in their platelet products. In addition, nicotine upregulates the expression of nAChR subtype α7, suggesting the possibility of an amplified positive feedback loop leading to enhanced P2Y12 expression [21]. Our study findings are in line with these prior observations in non-medicated healthy volunteers and expand upon these as it is the first to assess the impact of smoking status on P2Y12 receptor binding activity in patients with CAD as well as seeing how P2Y12 receptor binding can be modulated by clopidogrel exposure. The number of P2Y12 receptors on the platelet surface varies and has been suggested to be regulated by an intracellular pool [35]. There are several reports on intraplatelet
signaling (for example, P2Y12 receptor internalization and recycling) after prolonged stimulation with agonists to prevent inappropriate thrombosis in normal physiological condition [36, 37]. To this extent, megakaryocyte and derived platelets from smoking subjects may have deranged intraplatelet signaling due to increase systemic exposure to platelet stimulants. This in turn would lead to increased P2Y12 receptor expression on the platelet surface enabling greater exposure and thus inhibition by P2Y12 receptor antagonists. The greater density of P2Y12 receptors may be one of the mechanisms involved in the observed benefit of prasugrel over clopidogrel among smokers in the Targeted Platelet Inhibition to Clarify the Optimal Strategy to Medically Manage Acute Coronary Syndromes trial (TRILOGY-ACS) [38, 39]. In fact, although the active metabolite of prasugrel and clopidogrel are equipotent, the main differences between the two drugs is that prasugrel generates more efficiently its active metabolite compared with clopidogrel which can be of potential benefit in smokers who have higher P2Y12 receptor density. Study Limitations The criteria of smoker vs. nonsmoker were based on patient interview. Indeed, a more objective assessment, such as cotinine assessment, could discriminate more accurately the degree of smoking exposure of each individual patient. Further, this study only performed P2Y 12 binding assays. Indeed, performing additional pharmacodynamic tests could have been useful to corroborate our study findings. Moreover, experiments conducted with potent platelet stimulants could have allowed to determine the pool of internalized P2Y12 receptors [35–37]. However, the abovementioned additional experiments would have implied much larger blood sample
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volumes which would not be feasible for ethical reasons. Also, in this study, we did not assess individual response profiles to aspirin, which are known to be affected by smoking status [31–34]. Indeed, this could have provided additional insights on our study findings, including explaining some of the variability in P2Y12 receptor binding activity. Ultimately, this study should be considered as pilot in nature with a limited sample size, further limiting the formal statistical comparison, which would be subject to type II errors. However, this approach is in line with recommendations for pilot investigations [23].
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Conclusions
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Among patients with stable CAD, smokers have greater P2Y12 receptor binding activity than nonsmokers and have a higher degree of inhibition following a 600-mg loading dose of clopidogrel. Increased P2Y12 receptor binding among smokers may contribute to their higher risk for recurrent events, which can be suppressed to a greater extent by clopidogrel explaining their greater relative ischemic benefit (smoker's paradox) compared with nonsmokers.
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Source of Funding The present study was funded by an Institutional Grant of the University of Florida College of Medicine-Jacksonville and by a grant provided by The James and Esther King Biomedical Research Program (1KN01-33989). Conflict of Interest Jung Rae Cho, Bhaloo Desai, Michael J. Haas, Fabiana Rollini, Francesco Franchi, Ana Muñiz-Lozano, Antonio TelloMontoliu, Elisabetta Ferrante, Luis A. Guzman, and Theodore A. Bass have no conflict of interest to report. Dominick J. Angiolillo received payment as an individual for (a) consulting fee or honorarium from Bristol Myers Squibb, Sanofi-Aventis, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Merck, Evolva, Abbott Vascular, and PLx Pharma and (b) participation in review activities from Johnson & Johnson, St. Jude, and Sunovion. Institutional payments for grants from Bristol Myers Squibb, Sanofi-Aventis, Glaxo Smith Kline, Otsuka, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Evolva, Gilead and has other financial relationships with Esther and King Biomedical Research Grant.
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