Author + information
- Received May 1, 2008
- Revision received August 14, 2008
- Accepted August 18, 2008
- Published online December 2, 2008.
- Jacqueline Saw, MD⁎,⁎ (, )
- Esben Hjorth Madsen, MD‡,
- Sammy Chan, MD‡ and
- Elisabeth Maurer-Spurej, MD, PhD†,‡,§
- ↵⁎Reprint requests and correspondence:
Dr. Jacqueline Saw, Vancouver General Hospital, 2775 Laurel Street, 9th Floor, Vancouver, British Columbia, V5Z 1M9, Canada
Objectives This study was designed to evaluate the effects of long-term clopidogrel and aspirin administration on platelet aggregation, activation, and inflammation.
Background Clopidogrel resistance was described in 15% to 30% of patients with short-term therapy, but its antiplatelet effects with long-term therapy is unknown.
Methods We performed a prospective study of patients undergoing coronary stenting who were on aspirin for ≥5 days but not previously on clopidogrel. Clopidogrel 600 mg was given before stenting. Clopidogrel 75 mg/day and aspirin 325 mg/day were continued for 1 year. Light-transmittance aggregometry with 5-μmol/l adenosine diphosphate and 1-mmol/l arachidonic acid stimulation; VerifyNow clopidogrel and aspirin assays; platelet activation receptor expression of CD40L, CD62P, and PAC-1 (antibody against activated glycoprotein IIb/IIIa); and inflammatory markers of soluble CD40L and P-selectin, high-sensitivity C-reactive protein, interleukin-10, and interleukin-18 were measured at baseline; 1 day; and 1, 6, and 12 months. Our primary analysis compared light-transmittance aggregometry aggregation at 1 versus 12 months.
Results We enrolled 26 patients who completed a 1-year follow-up. Maximal platelet adenosine diphosphate-stimulated aggregation was 61.8 ± 25.9% at baseline, 22.1 ± 18.3% at 1 day, 30.6 ± 16.8% at 1 month, 29.0 ± 13.3% at 6 months, and 26.7 ± 13.6% at 12 months (p = 0.099 for 12 months vs. 1 month). VerifyNow clopidogrel platelet inhibition was similar at 12 months versus 1 month (38.9 ± 19.7% vs. 45.6 ± 26.7%, p = 0.578). Likewise, there was no difference in aspirin's effects on platelet aggregation at 12 months versus 1 month. In contrast, platelet activation receptor expression of CD40L, CD62P, and PAC-1 were higher at 12 months versus 1 month.
Conclusions Our pilot study showed no attenuation of clopidogrel's effects on platelet aggregation with long-term administration. However, platelet activation receptor expression increased with time and should be further evaluated.
Aspirin and thienopyridines are indispensable pharmacologic arsenals in modern cardiovascular practices with several indications for long-term therapy: percutaneous coronary intervention (PCI) with drug-eluting stents, presentation with acute coronary syndrome, and patients with established myocardial infarction (MI), ischemic stroke, or symptomatic peripheral arterial disease (1). Thus, it is essential that clinicians comprehend the platelet response to long-term dual antiplatelet therapy.
Resistance to either aspirin or clopidogrel at baseline or development of resistance with prolonged administration could jeopardize their clinical efficacy. Although it is widely recognized that 15% to 30% of patients may be resistant to clopidogrel 75 mg/day, this is derived from short-term (≤30 days) studies (2,3), with no data pertaining to resistance prevalence with longer-term administration. Controversial results were reported on aspirin resistance (4–6), and no studies have addressed the development of resistance to both these agents with long-term administration.
We, therefore, designed a prospective pilot study to evaluate the effects of long-term clopidogrel and aspirin on platelet aggregation, activation, and inflammation.
We included patients ≥18 years of age undergoing PCI at Vancouver General Hospital, who received aspirin 81 mg/day for ≥5 days, but had not had clopidogrel within 2 weeks. Exclusion criteria were known allergy or intolerance to clopidogrel; platelet count <100,000/μl or >500,000/μl; history of chronic inflammatory disease; clopidogrel, ticlopidine, dipyridamole, abciximab, steroidal and nonsteroidal anti-inflammatory drugs use within 2 weeks; eptifibatide or tirofiban within 48 h; MI within 1 week; active bleeding (excluding menses) or significant gastrointestinal bleed within 2 months; major surgery within 2 weeks; and pregnancy. Our study was approved by the Vancouver General Hospital and University of British Columbia Institutional Review Board, and written informed consent was obtained from all patients.
Study drugs and protocol
Clopidogrel 600 mg was given before PCI. Clopidogrel 75 mg/day and aspirin 325 mg/day were continued for 1 year. Procedural anticoagulant choice was at the discretion of the interventionalists. Eptifibatide or tirofiban was permitted, but had to be discontinued ≥10 h before the next day's blood work. Abciximab was not permitted due to its long effective half-life. The choice of bare-metal or drug-eluting stents was at the discretion of the interventionalists. The study protocol is depicted in Figure 1.
Blood samples were drawn at baseline (before clopidogrel); at 16 to 24 h (1 day) after PCI; and at 1, 6, and 12 months. Patients were fasting for ≥4 h prior to blood withdrawal. The first 3 ml of blood drawn were discarded. Baseline samples were collected from the arterial sheath into ethylenediaminetetraacetic acid, sodium citrate (3.2%), heparin, and serum-separator vacutainer tubes. Post-PCI samples were drawn from peripheral venipunctures. The following were assessed with each collection: 1) light-transmittance aggregometry (LTA) with 5-μmol/l adenosine diphosphate (ADP) and 1-mmol/l arachidonic acid (AA) stimulation; 2) VerifyNow aspirin and clopidogrel assays; 3) flow cytometry platelet-bound CD40L, CD62P, and PAC-1 (antibody against activated glycoprotein IIb/IIIa); 4) soluble CD40L and P-selectin; 5) high-sensitivity C-reactive protein, interleukin (IL)-10, and IL-18; and 6) platelet count, hemoglobin, and hematocrit.
Platelet-rich plasma and platelet-poor plasma were prepared at 24°C, and LTA was performed as previously described (7). The platelet-rich plasma platelet count was 316 ± 66 × 109/l. Using a lumi-aggregometer (Chronolog Corporation, Havertown, Pennsylvania), platelet-rich plasma was stimulated with final concentrations of AA 1.0 mmol/l or ADP 5.0 μmol/l. The LTA was performed <2 h after blood withdrawal in duplicates (means reported as percent aggregation), and relative light transmission values were recorded at maximum and 6 min after agonist addition.
Flow cytometry preparation
Binding of phycoerythrin-labeled anti-CD40L (Beckman Coulter, Mississauga, Ontario), fluorescein isothiocyanate-labeled PAC-1 (BD Biosciences, Mississauga, Ontario), and anti–CD62P-phycoerythrin to platelets stimulated by 5.0-μmol/l thrombin receptor activating peptide (Peninsula Laboratories Europe, Merseyside, England) was quantified by flow cytometry as previously described (7). Results were expressed as percent positive cells of the platelet population. Instrument stability was monitored by daily measurement of calibration beads (Flow-Check fluorospheres, Beckman Coulter). The mean antibody binding to platelets from 6 unmedicated normal controls were 30.9 ± 8.7 (CD40L), 83.9 ± 7.5 (CD62), and 93.7 ± 5.4 (PAC-1).
Enzyme-linked immunosorbent assay tests
Soluble CD40L, soluble P-selectin, IL-10, and IL-18 were measured using commercial enzyme-linked immunosorbent assay kits according to the manufacturer's instructions. Plasma CD40L and P-selectin were measured with kits from Bender MedSystems (Burlingame, California). Plasma IL-10 and -18 were measured with kits by BioSource (Invitrogen Canada, Burlington, Ontario).
The VerifyNow (Accumetrics, San Diego, California) aspirin and clopidogrel (P2Y12) assay cartridges contain fibrinogen-coated beads and platelet agonists (aspirin assay contains AA, and clopidogrel assay contains ADP/prostaglandin E1 and iso-thrombin receptor activating peptide). Activated platelets in the whole blood samples bind and aggregate the fibrinogen-coated beads in proportion to the number of expressed activated glycoprotein IIb/IIIa receptors, with resulting increase in light transmittance. This change in optical signal is reported as the aspirin reaction unit (ARU) and P2Y12 reaction unit (PRU). The P2Y12 assay also reports “percent inhibition” (percent change from baseline aggregation calculated from the PRU and the baseline thrombin receptor activating peptide channel value). Patients with ARU ≥550 were labeled as aspirin-resistant.
Clopidogrel and aspirin resistance definitions
Clopidogrel resistance was defined as an absolute difference between baseline and post-clopidogrel platelet aggregation <10% with LTA using 5.0-μmol/l ADP (2). Aspirin resistance was defined as mean aggregation >20% with LTA using 1.0-mmol/l AA (8).
End points evaluated
Our primary analysis compared LTA platelet aggregation, for the 75-mg/day clopidogrel dosage, at 12 months versus 1 month. We chose 1 month for the comparison in order to achieve a steady state with the maintenance dose. Secondary assessments include the prevalence of clopidogrel and aspirin resistance, and the change in platelet activation and inflammatory markers. Death, stroke, MI, and target vessel revascularization at 12 months were also assessed. MI was defined as chest pain with creatine kinase (CK) or CK-myocardial band elevation >2× the upper limit of normal or any troponin elevation. Peri-procedural MI was defined as CK or CK-myocardial band elevation >3× the upper limit of normal. Stroke was defined as new focal neurologic deficits lasting >24 h.
Hypothesis tests were done using 2-sided tests at the 5% significance level. Comparisons of binary variables were performed with the McNemar test, and continuous variables with the Wilcoxon signed rank test for paired samples. We hypothesized that LTA platelet aggregation would increase by an absolute 15% after 12 months of clopidogrel compared with 1 month of clopidogrel. Assuming a mean platelet aggregation of 30% at 1 month, we needed to enroll 21 patients to detect platelet aggregation increase to 45% at 12 months, with 90% power and 2-sided alpha = 0.05 (estimated SD for mean change 20%). We anticipated 35% attrition, thus we planned to enroll 33 patients. Statistical analyses were performed with SPSS (version 13, SPSS Inc., Chicago, Illinois).
We enrolled 33 patients from September 2005 through August 2006, and 26 patients completed the 1-year follow-up after successful PCI. Table 1 shows the baseline demographics. Seven patients withdrew prematurely: 2 had contraindications to antiplatelet therapy (nosebleed and colon cancer surgery), and 5 withdrew on their own accord without medical contraindications.
The baseline maximal platelet aggregation measured by LTA 5.0-μmol/l ADP was 61.8 ± 25.9%. This was significantly reduced after the administration of clopidogrel at 1 day and at 1, 6, and 12 months (Table 2,Fig. 2). Our primary comparison showed no difference in platelet aggregation at 12 months versus 1 month (26.7% vs. 30.6%, p = 0.099). Likewise, no difference was shown in our comparison of platelet aggregation at 12 months versus 1 day after PCI (26.7% vs. 22.1%, p = 0.093). Similar results were obtained with 6-min aggregation.
With administration of aspirin at 325 mg/day, platelet aggregation measured with LTA 1.0-mmol/l AA was significantly reduced at each follow-up time point compared with platelet aggregation at baseline (aspirin 81 mg/day at baseline). However, we did not observe any difference in maximal platelet aggregation at 12 months versus 1 month (p = 0.940), or at 12 months versus 1 day after PCI (p = 0.183) (Table 3,Fig. 2). Similar results were obtained with 6-min aggregation.
Baseline PRU was 292.4 ± 58.8 and platelet inhibition 4.9 ± 8.1%. After clopidogrel administration, repeat PRU measurements were significantly reduced and platelet inhibitions were significantly increased at each follow-up. However, there were no significant differences in PRU (p = 0.520) or platelet inhibitions (p = 0.578) at 12 months versus 1 month (Table 2, Fig. 2). Likewise, there were no differences in PRU (p = 0.551) or platelet inhibitions (p = 0.446) at 12 months versus 1 day after PCI. Similarly, with the aspirin assays, there was no difference in ARU measurements at 12 months versus 1 month (p = 0.212) or at 12 months versus 1 day after PCI (p = 0.687) (Fig. 3).
Clopidogrel and aspirin resistance
Using our LTA definition, the proportion of patients who were clopidogrel-resistant was 16.0% at 1-day, 12.0% at 1-month, 8.0% at 6-month, and 12.0% at 12-month follow-ups (p = 1.0 for 12 months vs. 1 month). Among the 3 who were clopidogrel-resistant at 12 months, all were previously labeled clopidogrel-resistant at 1 day or 1 month. Using LTA with 1.0-mmol/l AA, no patient was identified as aspirin-resistant at any time point. However, with the VerifyNow aspirin assay, 4% of patients were aspirin-resistant at baseline, 0% at 1-day, 0% at 1-month, 0% at 6-month, and 15% at 12-month follow-ups (p = 0.625 for 12 months vs. baseline; p = 0.250 for 12 months vs. 1 month).
Platelet activation markers
Platelet expression of PAC-1 was significantly lower after starting clopidogrel at 1 day and 1 month after PCI. In contrast, CD40L and CD62P expressions appeared lower at 1 day and 1 month, but were not statistically significant compared with baseline expressions. Nevertheless, platelet expressions of all 3 markers were significantly higher at 12 months compared with expressions 1 month or 1 day after clopidogrel administration (Table 2, Fig. 2).
Soluble P-selectin remained unchanged at 1 day and 1 month following clopidogrel administration, but became significantly higher at 6 and 12 months. Conversely, soluble CD40L levels were stable at 1 day and 1 month, but became progressively and significantly lower at 6 and 12 months. Serum IL-18 levels were stable at 1 day and 1 month, but became significantly higher at 6 and 12 months compared with baseline levels. However, the 12-month IL-18 level was not significantly higher compared with the 1-month level. Serum IL-10 levels were unchanged from baseline to all subsequent visits. High-sensitivity C-reactive protein was significantly elevated 1 day after PCI, but returned to baseline values at all subsequent visits (Table 2, Fig. 3).
Three non–ST-segment elevation MIs occurred during the 1-year follow-up (1 had procedural-related MI requiring target vessel revascularization). None of these patients were resistant to aspirin or clopidogrel. There were no death or stroke events.
Our prospective study showed no increase in platelet aggregation or prevalence of resistance with long-term clopidogrel and aspirin administration. However, the inhibitory effects of clopidogrel on platelet expression of CD40L, CD62P, and PAC-1 diminished over the course of long-term treatment.
Our study is the first to evaluate the long-term effects of clopidogrel on platelet aggregation. With rising concerns of clopidogrel resistance and the associated risks of atherothrombotic complications, it is prudent to assess the longer-term platelet response, even among patients who were initially clopidogrel-responsive. In our primary analysis, we found no increase in platelet aggregation with 1-year clopidogrel administration. This should alleviate some lingering concerns among clinicians who manage patients with drug-eluting stents, because the risk of stent thrombosis is in part related to heightened platelet reactivity (4).
Likewise, our study showed no increase in platelet aggregation or resistance prevalence with 1-year aspirin administration. These results differed from prior studies (4–6), which may be explained by the use of different agonists and doses, lack of clopidogrel use, and different patient populations.
The expression of platelet activation markers CD40L, CD62P, and PAC-1 were significantly higher at 12 months compared with expression at 1 month after PCI. Expressions of these receptors are known to promote binding and mediate interactions among platelets, the endothelium, and leukocytes, which may play important roles in inducing inflammation, atherosclerosis, and thrombosis (9). However, in our study, this increase in platelet receptor expression did not correspond to an increase in platelet aggregation. This apparent discrepancy is baffling, and our study is too small to assess for potential clinical events that may correspond to these contradictory effects.
The inflammatory marker findings in our study were atypical and discordant with the different markers assessed. Although the high-sensitivity C-reactive protein, IL-10, and IL-18 levels remained unchanged at 12 months versus 1 month, suggesting that the overall systemic inflammatory status remained stable in our patients, we could not explain the contradicting increase in soluble P-selectin and decrease in soluble CD40L levels at the 1-year follow-up. These findings are perplexing and should be further explored in larger studies.
Our study has several limitations. Although prospective, our study population is small, and we could not adequately assess clinical events corresponding to platelet function tests. However, we rigorously followed our patients with laboratory and clinical assessments longitudinally and exceeded our power calculation with 26 patients completing the 1-year follow-up. The definition that we used for clopidogrel resistance, although not universally accepted, had been widely used (2,3).
Our prospective pilot study showed that long-term therapy with clopidogrel and aspirin did not attenuate the initial inhibitory effects on platelet aggregation. In contrast, platelet activation receptor expression increased with long-term therapy and should be further evaluated in large-scale prospective studies corresponding laboratory findings with clinical end points.
The authors thank the Vancouver General Hospital Interventional Cardiology Research coordinators (Rebecca Fox, Andrew Starovoytov, Naomi Uchida, and Sandy MacLeod) for screening, enrolling patients, and data entry. They also thank Cheryl Pittendreigh, Luba Cermakova, and Willie Yu for specimen processing and analysis.
Funded by a research grant from the Vancouver General Hospital and University of British Columbia Hospital Foundation and by the Danish Heart Association.
- Abbreviations and Acronyms
- arachidonic acid
- adenosine diphosphate
- aspirin reaction unit
- creatine kinase
- light-transmittance aggregometry
- myocardial infarction
- antibody against activated glycoprotein IIb/IIIa
- percutaneous coronary intervention
- P2Y12 reaction unit
- Received May 1, 2008.
- Revision received August 14, 2008.
- Accepted August 18, 2008.
- 2008 American College of Cardiology Foundation
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