The effect of clopidogrel is not uniform in all patients, and decreased platelet inhibition by clopidogrel is seen in about 20% of patients taking clopidogrel and is associated with an 8-fold increased risk of major adverse cardiac events (1- 4). Clopidogrel is metabolized to an active thiol metabolite by the CYP3A4 enzyme (5- 6). The variability in the response to clopidogrel has been, at least in part, linked to its metabolism by cytochrome P450 enzymes (2). For example, some lipophilic statins may inhibit the CYP3A4 enzyme, and therefore decrease the formation of the active metabolite of clopidogrel (7). Calcium-channel blockers (CCBs), another frequently used class of cardiovascular drugs, also inhibit CYP3A4 (8- 10).

Therefore, we hypothesized that intake of CCBs may be associated with decreased responsiveness to clopidogrel. We performed the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay and platelet aggregometry. The VASP assay is a fairly new platelet function assay (11- 13). It is specific for clopidogrel and other P2Y12 antagonists in the absence of cilostazol (14- 15), and it can be used to quantify platelet inhibition by clopidogrel (16). It has been shown that adjusting the clopidogrel loading dose according to the platelet reactivity index (PRI) in the VASP assay improves the clinical outcome in patients with decreased platelet inhibition by clopidogrel (17- 18).

This prospective study aimed to compare the responsiveness to clopidogrel in 200 coronary artery disease (CAD) patients with and without concomitant treatment with CCBs.

This was a prospective observational study. Patients were followed up for 6 months. The study protocol was approved by the ethics committee of the Medical University of Vienna in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants before study entry. Two hundred patients with CAD undergoing percutaneous coronary intervention (PCI) were enrolled. Patients had been on a regimen of clopidogrel (75 mg/day) therapy for 3 months on average (since at least 1 week before study entry) or had received a 600 mg loading dose of clopidogrel (at least 2 h before study entry). The primary end point was a composite of death from cardiovascular causes, nonfatal myocardial infarction, stent thrombosis, and revascularization (PCI or coronary artery bypass graft [CABG] surgery). During the study, samples of 20 healthy volunteers with a normal platelet function were run in parallel to exclude any potential shifts in the readouts of the platelet function tests.

Blood samples from patients were obtained from the arterial sheath (6-F) in the catheterization laboratory. Blood samples from volunteers were obtained from an antecubital vein, using a 21-gauge needle.

To determine the VASP phosphorylation state of whole blood, we used a standardized flow cytometric assay (Platelet VASP, BioCytex, Marseille, France), which is an adaptation of the method of Schwarz et al. (19). Blood samples collected in 3.8% sodium citrate (Vacutainer, Becton Dickinson Biosciences, Vienna, Austria) were incubated in vitro with ADP and/or prostaglandin E1 (PGE1) before fixation. After 10 min, platelets were permeabilized, labeled with a primary monoclonal antibody against serine 239-phosphorylated VASP (clone 16C2) or its isotype, followed by a secondary fluorescein isothiocyanate-conjugated polyclonal goat-antimouse antibody. All procedures were performed at room temperature. Platelet geometric mean fluorescence intensity (MFI) was determined using a flow cytometer (FACSCalibur System, Becton Dickinson Biosciences). The platelet population was identified by its forward and side scatter distribution, and 10,000 platelet events were gated and analyzed for MFI. Platelet reactivity was expressed as the PRI, calculated as: PRI% = [(MFI (PGE1) – MFI (PGE1 + ADP)/MFI (PGE1)] × 100. The ratio is expressed as mean percentage platelet reactivity, which inversely correlates with the clopidogrel effect. The VASP assay was performed within 24 h after blood sampling. Using the test results from 20 healthy volunteers without clopidogrel therapy, the reference value for the assay 69% to 100% was calculated by using a nonparametric percentile method (95%). The coefficient of variation of the assay was <5% for duplicates and for testing the same samples on 2 different days.

In 10 additional patients, who were receiving clopidogrel but not CCBs, PRI and aggregometry were measured before and after incubation of blood (10 min, 37°C) with 4 different CCBs: verapamil (Isoptin [90 ng/ml], Abbott, Vienna, Austria), diltiazem (Dilzem [200 ng/ml], Elan Pharma International, Ltd., Athlone, Ireland), amlodipine (40 ng/ml, Sigma Aldrich, Vienna, Austria), and nimodipine (Nimotop [10 ng/ml], Bayer, Vienna, Austria). The concentrations of CCBs are the maximum concentrations in plasma (Cmax) described (20- 23).

Whole blood aggregation was determined using an impedance aggregometer (Multiple Platelet Function Analyzer/Multiplate Analyzer, Dynabyte Medical, Munich, Germany). The system detects the electrical impedance change due to the adhesion and aggregation of platelets on 2 independent electrode-set surfaces in the test cuvette (24- 25). A 1:2 mixture of 0.9% NaCl and whole blood anticoagulated with hirudin (200 U/ml, Dynabyte, Munich, Germany) were stirred at 37°C for 3 min in the test cuvettes; adenosine diphosphate (ADP [6.4 μM, Dynabyte Medical, Munich, Germany]) was added, and the increase in electrical impedance was recorded continuously for 5 min. The mean values of the 2 independent determinations are expressed as the area under the curve of the aggregation tracing (24). The reference values for the test are 29 to 118 U (26). The results measured by the Multiplate Analyzer are reproducible with a <6% variability (24).

A sample size calculation was based on the observed mean ± SD (61 ± 17) of the PRI under clopidogrel treatment (27). We calculated that we needed to include 200 patients to be able to detect a 15% relative difference in PRI with a power of 95% and a 2-sided alpha value of 0.05. Normal distribution was tested with the Kolmogorov-Smirnov test. Data are expressed as mean and SEM, SD, or 95% confidence intervals (CIs). Statistical comparisons were performed with the t test, the Mann-Whitney U test, and the chi-square test. Stepwise multivariable logistic regression analysis was used to estimate possible associations between PRI, platelet aggregation, and use of CCBs. The logistic model included age, serum creatinine levels, diabetes mellitus, arterial hypertension, hypercholesterolemia, previous myocardial infarction, smoking, and use of beta-blockers, statins (lipophilic vs. hydrophilic), antidiabetic agents, and angiotensin-converting enzyme inhibitors. Two-year cumulative incidence rates of composite clinical outcomes were estimated by the Kaplan-Meier method. Stepwise multivariable Cox proportional hazards regression modeling was used to estimate the independent effect of concomitant CCB treatment on clinical outcome. The Cox regression model included age, serum creatinine levels, diabetes mellitus, arterial hypertension, hypercholesterolemia, previous myocardial infarction, smoking, and use of beta-blockers, statins (lipophilic vs. hydrophilic), antidiabetic agents, and angiotensin-converting enzyme inhibitors. A 2-tailed p value of <0.05 was considered significant for the primary outcome variable (PRI in the VASP assay). All statistical calculations were performed using commercially available statistical software (version 14.0, SPSS, Inc., Chicago, Illinois).

Patient demographics are shown in (Table 1). Patients receiving CCBs suffered more frequently from high blood pressure (91% vs. 78%; p = 0.026) and diabetes mellitus (49% vs. 29%; p = 0.007) as compared with patients not taking CCBs. These risk factors and higher age were associated with increased serum creatinine values in patients taking CCBs (p < 0.05).

Using the test results of volunteers without clopidogrel therapy, the 95% reference range for the PRI was 69% to 100%. Similarly, the cut-off value for the fourth quartile of PRI in CAD patients was 69%, indicating that 25% of patients on clopidogrel therapy had decreased platelet inhibition by clopidogrel (Figure 1). The PRI in patients taking clopidogrel and in volunteers not taking clopidogrel exhibited a normal distribution (Figure 1). The mean PRI in patients was 52% (SD ±21%).

A total of 93% of patients treated with CCBs were taking dihydropyridines (Table 2). The PRI was 21% higher in patients receiving CCBs (61% PRI) as compared with patients not taking CCBs (48% PRI; absolute difference 13%; 95% CI: 6% to 20%; p = 0.001) (Figure 2A). This significant difference was also seen in the largest subgroup of patients treated with amlodipine (p = 0.001) (Table 2). The distribution of PRI values among patients treated with CCBs was shifted to the right, compared with patients not treated with CCBs (Figure 3). A decreased platelet inhibition by clopidogrel (PRI >69%) was seen in 40% and 20% of patients with and without concomitant treatment CCB treatment, respectively (chi-square test p = 0.008). The significant increase in PRI in patients receiving CCBs persisted after adjustment for CAD risk factors and other cardiac medication (p = 0.002). There was no apparent difference in PRI values between different groups of CCBs (Table 2). Moreover, differences in baseline characteristics between patients with or without CCB treatment did not affect the PRI: the PRI was not altered by high blood pressure, diabetes mellitus, age, increased serum creatinine values, or use of beta-blockers, as tested by univariate analysis (U test and Spearman rank-sum correlation test). Similarly, there was no difference in PRI values among patients presenting with stable angina, unstable angina, or myocardial infarction; and there was no difference in PRI values for other medications such as beta-blockers, angiotensin-converting enzyme blockers, antidiabetic agents, and statins. Platelet inhibition by clopidogrel was not significantly altered by intake of lipophilic statins (50% PRI) as compared with patients without intake of statins (52% PRI). Up to 40 mg, there was no dose-dependent increase in PRI by intake of lipophilic statins such as atorvastatin. The 80-mg dose of atorvastatin was associated with higher PRI values (60%; p > 0.05), although the small sample size in this subgroup (n = 13) does not allow us to draw any conclusions.

ADP-induced platelet aggregation was 30% higher in patients being treated with both clopidogrel and CCBs (52 U) as compared with patients treated with clopidogrel but not CCBs (40 U; 95% CI: −0.3 to −20; p = 0.046) (Figure 2B). This difference, however, lost significance when it was adjusted for diabetes and beta-blocker intake. Differences in baseline characteristics between patients with or without CCB therapy did not affect the ADP-induced platelet aggregation: the ADP-induced platelet aggregation was not altered by high blood pressure, diabetes mellitus, age, increased serum creatinine values, and use of beta-blockers, as tested by univariate analysis (U test and Spearman rank-sum correlation test). Similarly, there was no difference in the ADP-induced platelet aggregation between patients presenting with stable angina, unstable angina, or myocardial infarction; and there was no difference in ADP-induced platelet aggregation for other medications such as beta-blockers, angiotensin-converting enzyme blockers, antidiabetic agents, and statins (lipophilic vs. hydrophilic; data not shown).

In vitro incubation of blood from patients administered clopidogrel with CCBs (nimodipine, verapamil, amlodipine, and diltiazem) slightly but insignificantly decreased the average PRI as compared with the control group (average absolute difference: −6% to −3%). Similarly, incubation with CCBs did not alter the ADP-induced platelet aggregation (absolute difference: −3.5 to 3 U; data not shown). In summary, CCBs did not directly affect platelet function as measured by either test system.

The composite end point occurred more frequently among patients with concomitant CCB treatment (25%) than among patients without concomitant CCB treatment (8%; crude hazard ratio [HR]: 3.9, p = 0.001, 95% CI: 1.7 to 8.5; adjusted HR: 3.5, p = 0.005, 95% CI: 1.4 to 8.6) (Figure 4). The composite end point was driven by the higher rate of revascularization procedures.

The most important finding is that intake of CCBs is associated with a relative increase in platelet reactivity (index) by 21% (Figure 2A). To our knowledge, no trial has investigated the drug-drug interaction between clopidogrel and CCBs in the VASP assay. The VASP assay is the most specific test for clopidogrel action. It measures the inhibition of clopidogrel's biochemical target, the P2Y12 receptor and its intracellular signalling: the PGE1-stimulated, cyclic adenosine monophosphate-dependent phosphorylation of VASP (2,28). The VASP assay has been validated (29) and found to be predictive for stent thrombosis and cardiac adverse events after coronary artery stenting (16,28,30). Aggregometry consistently showed that intake of CCBs was associated with reduced efficacy of clopidogrel to inhibit platelet aggregation (Figure 2B).

The cut-off value for the VASP assay indicating decreased platelet inhibition by clopidogrel was based on the lower limit of the normal range (69% PRI) (Figure 1). This is consistent with another study in 39 patients untreated with clopidogrel, and confirms the external validity of the test (31). It indicates that 25% of patients had a decreased response to clopidogrel. When comparing patients with and without CCB therapy, the prevalence of decreased platelet inhibition by clopidogrel was 40% and 20%, respectively. A higher rate of decreased platelet inhibition by clopidogrel in the CCB group corresponded to a 3.9 higher risk for the occurrence of the composite end point as compared with patients not having CCB treatment.

However, our cut-off value (<69% PRI) may still underestimate the PRI value necessary for true efficacy. Receiver-operator characteristics curve analysis has previously shown that a PRI value of 48% was most sensitive to predict adverse cardiovascular events (28). Applying such a cut-off value of 48% PRI to our patients would result in 64% of patients with decreased platelet inhibition by clopidogrel. Consistent with our results, the prevalence of decreased platelet inhibition by clopidogrel would then be higher among patients treated with CCBs (71%) as compared with patients not treated with CCBs (50%; p < 0.05).

There is only 1 small study, involving 6 patients with peripheral arterial obstructive disease, which investigated platelet inhibition by clopidogrel when added to ongoing nifedipine therapy (32). Clopidogrel inhibited platelet aggregation by 30%. However, the study design, including the very small sample size, does not allow any conclusion to be made about the drug–drug interaction between clopidogrel and nifedipine.

Our in vitro experiments indicate that CCBs do not directly affect platelet inhibition by clopidogrel as measured by the VASP assay or aggregometry. This finding supports the concept that CCBs conceivably alter the in vivo biotransformation of clopidogrel at the level of the CYP3A4 cytochrome. Clopidogrel is a pro-drug, which requires hepatic bioactivation by the cytochrome P450 isoform 3A4 to generate the active metabolites (2). Also, diltiazem and dihydropyridines are highly metabolized (60% to 90%) in the liver by the cytochrome P450 isoform 3A4 to inactive metabolites (10,33- 34). Clopidogrel is hydrolyzed in vivo to an inactive metabolite, which represents more than 85% of the circulating drug-related compounds in plasma (35). The intrahepatocyte level of clopidogrel, which is available for metabolism, is 10-fold lower than the plasma level of the inactive carboxylic acid metabolite of clopidogrel (7). The plasma concentration of the main circulating active metabolite of clopidogrel is very low, and generally below detection limits (0.25 ng/ml) (36). For comparison, the plasma concentrations of CCBs are much higher (amlodipine, 41 ng/ml; diltiazem, 200 ng/ml; nisoldipine, 100 ng/ml) (20,22,37). The degree of competitive inhibition between 2 substrates depends on the relative affinity of the substrates for the binding site of CYP3A4 and their relative concentrations (7). As CCBs inhibit CYP3A4 (8- 10), they may inhibit clopidogrel's metabolism. Our results call for formal clinical trials that study the effects of CCBs on the pharmacokinetics of clopidogrel to substantiate these seminal observations. Moreover, it will be interesting to investigate a possible effect of CCBs on prasugrel.

A drug–drug interaction study showed that the potent CYP3A4 inhibitor ketoconazole significantly reduced the level of clopidogrel's active metabolite (35). Moreover, concomitant treatment with ketoconazole decreased platelet inhibition in response to clopidogrel as measured by aggregometry. Another study showed that the CYP3A4 inhibitor itraconazole significantly decreased the ability of clopidogrel to inhibit platelet aggregation (38). Finally, a negative effect of statins on clopidogrel has been reported on the level of CYP3A4. Some studies suggested that lipophilic statins (atorvastatin, lovastatin, and simvastatin) may competitively inhibit CYP3A4 and decrease the generation of clopidogrel's active metabolite, reducing the antiplatelet effect of clopidogrel (7,39- 40). On the contrary, other reports were not able to confirm this findings (41- 46). Similarly, we did not observe any drug–drug interaction between clopidogrel and lipophilic statins as measured with both assays in the current study.

In our study, the largest subgroup of CCBs was amlodipine. Further studies are needed to confirm a similar effect for other CCBs and to examine whether this effect is a class effect. Additional limitations are that the duration of CCB therapy as well as the true baseline for platelet reactivity in both groups was unknown.

Coadministration of CCBs and clopidogrel is associated with a decreased response to clopidogrel.

The authors are grateful to Knut Prillinger and Katrin Haberl for their help in data acquisition, and to Alexander Spiel, MD, for his help for the settings of the VASP assay.