Introduction

2. Introduction • Clopidogrel is metabolized by P450 (CYP)-isoenzymes: CYP 3A4/5, 1A2, 2B6, 2C9, and 2C19 1 • Wide response variability and nonresponsiveness to clopidogrel may be related to functional and genetic variability in CYP isoenzymes.2 • Cigarette smoking induces CYP1A2. 3 • Smoking has been associated with an increased benefit of dual antiplatelet therapy on long-term clinical outcomes after PCI.4 • However, there are no laboratory data to support a relation between increased platelet inhibition by clopidogrel in current smokers (CS) as compared to non- smokers (NS). • 1. Brandt JT et al. J Thromb Haemost 2007;5:2426-36. 2. Gurbel PA et al. Thromb Res. 2007;120:311-321. • 3. Zevin S et al. Clin Pharmacokinet. 1999;36:425-38. 4. Saraff KY, et al. J Am Coll Cardiol 2006;36B:Abstract 2920-122.

3. Objectives • The primary objective was to investigate the effect of cigarette smoking on the platelet response to clopidogrel as measured by ADP-induced aggregation using conventional turbidometric aggregometry in platelet rich plasma in patients treated with elective PCI. • The secondary objectives were to analyze the relation between clopidogrel response and smoking by using flow cytometry measurements of ADP-induced expression of activation dependent receptors. • We hypothesized that 5M ADP-induced aggregation would be lower in CS following loading and maintenance doses of clopidogrel compared to NS.1,2 1. Matetzky S, et al. Circulation 2004;109:3171-5, 2. Gurbel PA et al. Circulation 2005;111:1153-9

4. Methods • Patients • Platelet function data in 259 current cigarette smokers or non-smokers undergoing elective PCI treated with clopidogrel consecutively enrolled in clinical trials was analyzed.1,2 • 104 current smokers (smoking within 2 weeks of PCI) • 155 nonsmokers (no smoking within 1 year of PCI). • Previous smokers were excluded since the influence of smoking on CYP1A2 activity is highly variable upon smoking cessation and the interval between the last smoking event and enrollment in the study was inconsistent. • Patients on chronic clopidogrel therapy prior to PCI(n=120, 75mg/day) were not loaded; 139 clopidogrel naïve patients were loaded with 600 mg immediately after PCI. • No patients had been treated with immunosuppressants, antibiotics, or anti-HIV agents that may influence CYP activity within 2 weeks of study. 1. Gurbel PA et al. J Am Coll Cardiol. 2005;46:1820-6. 2. Gurbel PA et al. Circulation 2005;111:1153-9

5. Methods Platelet Function Analysis • Conventional aggregometry • ADP-stimulated total and active GPIIb/IIIa expression by flow cytometry • Prior to PCI in all patients • In patients loaded with clopidogrel analyses done at 18-24 hours post-PCI in patients not treated with eptifibatide (n=63) , and at 5 days post-PCI in patients treated with eptifibatide (n=76). Statistical Analysis • Categorical variables compared by Fischer exact test • Continuous variables compared by Student's t-test • Logistic regression analysis - evaluate the impact of smoking status, age, body mass index, diabetes, prior MI, family history of CAD, hyperlipidemia, HBP, and statin and other concomitant medications on low post-treatment PA. (MedCalc software (Mariakerke, Belgium)).

6. Methods Definitions • Current smokers smoked 3 cigarettes/day within 2 weeks of PCI Non-smokers: no history of cigarette smoking within 1 year of PCI • Low platelet aggregation = lowest quartile of 5μM ADP- induced platelet aggregation measured by light transmittance aggregometry (29%). • Relative inhibition of platelet aggregation (IPA) = [(MPApredose – MPApostdose )/MPApredose ]x 100% • MPA = maximum platelet aggregation

7. Results • Patient demographics and procedural characteristics were similar between the groups. • Current smokers: greater prevalence of prior MI (46% vs. 23%, p = 0.005) treatment of bifurcation lesions (12% vs. 3%, p = 0.005) • Non-smokers: More diabetes (33% vs. 48%, p=0.01) • Current smoking was the only variable significantly associated with low post treatment platelet aggregation (odds ratio 2.63, p<0.007)

8. Results

9. Results 100 90 100 100 * * 80 90 90 * 70 80 80 60 * * * 70 70 50 60 60 20 uM ADP-Induced Aggregation (%) 5 uM ADP-Induced Aggregation (%) 40 50 50 30 40 40 20 30 30 10 20 20 CS CS CS CS NS NS (< ½ pack) (< ½ pack) (> ½ pack) (> ½ pack) 10 10 (n=72) (n=72) (n=11) (n=37) (n=11) (n=37) ADP-Induced Platelet Aggregation * p<0.05 for mean post-treatment platelet aggregation in CS smoking 1/2 pack/day compared to NS and CS smoking <1/2 pack/day.

10. Conclusions • Cigarette smoking influences platelet responsiveness to clopidogrel. • The mechanism of the effect deserves further study and may further elucidate the causes of response variability to clopidogrel and the clinical efficacy of clopidogrel in specific patient groups.