Calcium-Channel Blockers Attenuate the Antiplatelet Effect of Clopidogrel

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1 ORIGINAL RESEARCH ARTICLE Calcium-Channel Blockers Attenuate the Antiplatelet Effect of Clopidogrel Thomas Gremmel, 1 Markus Durstberger, 1 Beate Eichelberger, 2 Renate Koppensteiner 1 & Simon Panzer 2 1 Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria 2 Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria Keywords Calcium-channel blockers; Clopidogrel; Flow cytometry. Correspondence Prof. Dr. Thomas Gremmel, M.D., Department of Internal Medicine II, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Tel.: ; Fax: ; thomas.gremmel@meduniwien.ac.at doi: / SUMMARY Aims: Dihydropyridine calcium-channel blockers (CCBs) inhibit cytochrome 3A4 and could therefore interfere with the conversion of clopidogrel to its active form. The impact of CCBs on the antiplatelet effect of clopidogrel has not been studied with assays directly capturing platelet activation to adenosine diphosphate (ADP), so far. We therefore sought to investigate platelet activation in response to ADP by flow cytometry in clopidogrel-treated patients without and with CCBs. Methods: Platelet surface P-selectin expression and activated glycoprotein (GP) IIb/IIIa in response to ADP were determined by flow cytometry in 302 patients on dual antiplatelet therapy with aspirin and clopidogrel after successful angioplasty with stent implantation. Results: Ninety-two patients (30.5%) received CCBs. Patients with concomitant CCB therapy showed significantly higher platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP than patients without CCBs (both P 0.03). Moreover, the fold increase of P-selectin and activated GPIIb/IIIa in response to ADP was significantly more pronounced in patients taking CCBs (both P 0.03). The associations of ADP-inducible activated GPIIb/IIIa and fold increase of activated GPIIb/IIIa after the addition of ADP with CCB therapy remained significant after adjustment for differences in patient characteristics and factors that were previously associated with clopidogrel response by multivariate regression analyses (both P < 0.05). High levels of ADP-inducible P-selectin and activated GPIIb/IIIa were seen significantly more frequent in patients with CCBs than in patients without CCB therapy (both P 0.01). Conclusion: Dihydropyridine CCBs attenuate the effect of clopidogrel on ADP-inducible platelet activation in patients undergoing angioplasty and stenting for cardiovascular disease. Introduction Despite the emergence of the new P2Y 12 inhibitors prasugrel and ticagrelor, clopidogrel remains the most frequently prescribed adenosine diphosphate (ADP) receptor antagonist in patients with atherosclerotic cardiovascular disease. The addition of clopidogrel to aspirin reduces adverse ischemic events in patients with acute coronary syndromes by 20% [1]. However, clopidogrel is a prodrug and requires biotransformation to its active metabolite by the hepatic cytochrome P450 enzyme system to finally exert its antiplatelet effect [2]. Previous studies have shown that exogenous and endogenous factors impairing the hepatic metabolism of clopidogrel reduce its antiplatelet effect. [3] In detail, loss-of-function polymorphisms of cytochrome 2C9 and 2C19 [4 7], demographical patient characteristics [8], various comorbidities [9 12], and comedication [13 15] have been associated with increased on-treatment residual platelet reactivity to ADP during clopidogrel therapy. Furthermore, high on-treatment residual platelet reactivity (HRPR) to ADP was linked to the occurrence of adverse ischemic events in patients undergoing percutaneous coronary angioplasty and stenting [16]. One of the most intensely discussed influencing factors for clopidogrel-mediated platelet inhibition is the potential interaction of clopidogrel metabolism with calcium-channel blockers (CCBs). Dihydropyridine CCBs inhibit cytochrome 3A4 and could therefore interfere with the conversion of clopidogrel to its active form [17,18]. Indeed, several studies reported increased on-treatment residual platelet reactivity to ADP by different platelet aggregation tests in clopidogrel-treated patients, who concomitantly received CCBs [14,19,20]. In contrast to these observations, a large study found no significant association between CCB therapy and clopidogrel-mediated platelet inhibition as assessed by multiple electrode platelet aggregometry [21]. Moreover, the impact of CCBs on the antiplatelet effect of clopidogrel has not been studied with assays directly capturing platelet activation to ADP, so far. We therefore sought to investigate platelet activation in response to ADP by whole-blood flow cytometry in clopidogrel-treated patients without and with CCB therapy following angioplasty and stenting for cardiovascular disease. 264 Cardiovascular Therapeutics 33 (2015) ª 2015 John Wiley & Sons Ltd

2 T. Gremmel et al. Calcium-Channel Blockers and Clopidogrel Methods Study Population The study population consisted of 302 patients on dual antiplatelet therapy after percutaneous intervention with endovascular stent implantation. All patients received daily aspirin (100 mg/day) and clopidogrel therapy (75 mg/day). Exclusion criteria were a known aspirin or clopidogrel intolerance (allergic reactions, gastrointestinal bleeding), a therapy with vitamin K antagonists (warfarin, phenprocoumon, acenocoumarol) or novel oral anticoagulants (rivaroxaban, apixaban, dabigatran, edoxaban), treatment with ticlopidine, dipyridamole, or nonsteroidal anti-inflammatory drugs, a family or personal history of bleeding disorders, malignant paraproteinemias, myeloproliferative disorders or heparin-induced thrombocytopenia, severe hepatic failure, known qualitative defects in thrombocyte function, a major surgical procedure within 1 week before enrollment, a platelet count <100,000 or >450,000/lL, and a hematocrit <30%. The study protocol was approved by the Ethics Committee of the Medical University of Vienna in accordance with the Declaration of Helsinki, and written informed consent was obtained from all study participants. Blood Sampling Blood was drawn by aseptic venipuncture from an antecubital vein using a 21-gauge butterfly needle ( mm; Greiner Bio-One, Kremsm unster, Austria) 1 day after the percutaneous intervention. To avoid procedural deviations, all blood samples were taken by the same physician applying a light tourniquet, which was immediately released, and the samples were mixed adequately by gently inverting the tubes. After the initial 3 ml of blood had been discarded to reduce procedurally induced platelet activation, blood was drawn into 3.8% sodium citrate Vacuette tubes (Greiner Bio-One; 9 parts of whole blood, 1 part of sodium citrate M/L) for evaluations by flow cytometry. Determination of P-Selectin Expression and Glycoprotein (GP) IIb/IIIa Activation The expression of P-selectin and the binding of the monoclonal antibody PAC-1 to activated glycoprotein IIb/IIIa (GPIIb/IIIa) were determined in citrate-anticoagulated blood, as previously published [22,23]. In brief, whole blood was diluted in phosphatebuffered saline to obtain 20x10 3 /ll platelets in 20 ll, and incubated for 10 min without agonists, and after in vitro exposure to suboptimal concentrations of ADP (final concentration 1 lm; Dynabyte, Munich, Germany). The platelet population was identified by staining with anti-cd42b (5 ll of clone HIP1, allophycocyanin labelled, final dilution 1:9; Becton Dickinson (BD), San Jose, CA, USA), and the expression of P-selectin and activated GPIIb/IIIa were determined by the binding of the monoclonal antibodies PAC-1 fluorescein (5 ll, final dilution 1:9; BD) and anti-cd62p-phycoerythrin (5 ll of clone CLB-Thromb6, final dilution 1:9; Immunotech, Beckman Coulter, Fullerton, CA, USA), respectively. Isotype-matched control antibodies were used in separate vials for the determination of nonspecific binding. After 15 min of incubation in the dark, the reaction was stopped by adding 500 ll PBS and samples were acquired immediately on a FACSCalibur flow cytometer (BD) with excitation by an argon laser at 488 nm and a red diode laser at 635 nm at a rate of events per second. FITC- and PE-labelled beads were used to compensate manually for the FITC signal into the PE channel and vice versa. Platelets were gated in a side scatter vs. FL4 dot plot. A total of 10,000 events were acquired within this gate. Positive analysis regions for P-selectin and activated GPIIb/IIIa, respectively, were set with appropriate nonspecific controls. The gated events were further analyzed in histograms for FL-1 and FL-2 for PAC-1 and P-selectin, respectively, using the CellQuest Pro software (BD). Standard BD Calibrite beads were used for daily calibration of the cytometer. The results are presented as mean fluorescence intensities (MFI) of ADP-inducible P-selectin expression and activated GPIIb/IIIa, and as fold increase of P-selectin expression and activated GPIIb/IIIa after the addition of ADP. Statistical Analysis A sample size calculation was based on the observed mean standard deviation of ADP-inducible activated GPIIb/IIIa ( MFI) in 100 patients on dual antiplatelet therapy after angioplasty with stent implantation. We calculated that we needed to include 290 patients to be able to detect a 25% relative difference of ADP-inducible activated GPIIb/IIIa between patients without and with CCBs with a power of 90% (using a two-sided alpha level of 0.05). To compensate for potential technical problems, we included 12 additional patients. Statistical analysis was performed using the Statistical Package for Social Sciences (IBM SPSS version 22, Armonk, New York, USA). Median and interquartile range of continuous variables are shown. Categorical variables are given as number (%). We performed Mann Whitney U-tests to detect differences in continuous variables. The chi-square test and the Fisher s exact test were used to assess differences in categorical variables, respectively. Multivariate linear regression analyses were used to adjust for patient characteristics that were significantly different between patients without and with CCBs, and for factors that were previously associated with response to clopidogrel therapy. The Kolmogorov Smirnov test was used to test for normal distribution, and variables with skewed distribution were log-transformed for multivariate regression analyses. Two-sided P-values <0.05 were considered statistically significant. Results Clinical, laboratory, and procedural characteristics of the overall study population and of patients without and with CCBs are given in Table 1. Ninety-two patients (30.5%) were concomitantly treated with CCBs. Among these, 90 patients (97.8%) received dihydropyridine CCBs (78.9% amlodipine, 5.6% nifedipine, 5.6% nisoldipine, 4.4% lercanidipine, 4.4% nitrendipine, 1.1% felodipine), and only 2 patients (2.2%) received a phenylalkylamine CCB (verapamil). As expected, patients with CCBs were older and had hypertension more often than patients without CCBs. Moreover, serum creatinine levels and the frequency of diabetes were higher in patients receiving CCBs (Table 1). ª 2015 John Wiley & Sons Ltd Cardiovascular Therapeutics 33 (2015)

3 Calcium-Channel Blockers and Clopidogrel T. Gremmel et al. Table 1 Clinical, laboratory, and procedural characteristics of the overall study population and of patients without and with calcium-channel blockers (CCBs) Characteristics Overall (n = 302) No CCBs (n = 210) CCBs (n = 92) P Demographics Age, years 66 (58 74) 63 (56 72) 69 (62 77) Male sex 199 (65.9) 134 (63.8) 65 (70.7) 0.3 Body mass index, kg/m ( ) 26.7 ( ) 27.3 ( ) 0.06 Medical history Hypertension 270 (89.4) 178 (84.8) 92 (100) <0.001 Hypercholesterolemia 282 (93.4) 196 (93.3) 86 (93.5) 1 Diabetes 99 (32.8) 60 (28.6) 39 (42.4) 0.02 Active smoking 126 (41.7) 89 (42.4) 37 (40.2) 0.7 Laboratory data Hematocrit, % 39 ( ) 39.2 ( ) 38.3 ( ) 0.08 White blood cell count, G/L 8.4 ( ) 8.4 ( ) 8.4 ( ) 0.1 Platelet count, G/L 208 ( ) 208 ( ) 204 ( ) 0.7 Serum creatinine, mg/dl 1 ( ) 1 ( ) 1.1 (1 1.4) <0.001 C-reactive protein, mg/dl 1 ( ) 1 ( ) 0.8 ( ) 0.7 Procedure Stent implantation 302 (100) 210 (100) 92 (100) 1 Number of stents/patient 1 (1 2) 1 (1 2) 1 (1 2) 0.3 Medication pre-intervention Clopidogrel 302 (100) 210 (100) 92 (100) 1 Aspirin 302 (100) 210 (100) 92 (100) 1 Statins 288 (95.4) 203 (96.7) 85 (92.4) 0.1 ACE inhibitors/arb 260 (86.1) 179 (85.2) 81 (88) 0.5 Beta blockers 206 (68.2) 150 (71.4) 56 (60.9) 0.07 Proton pump inhibitors 157 (52) 110 (52.4) 47 (51.1) 0.8 Continuous data are shown as median (interquartile range). Dichotomous data are shown as n (%). ACE inhibitors, angiotensin converting enzyme inhibitors; ARB, angiotensin receptor blockers. Genotyping for loss-of-function polymorphisms of cytochrome 2C9 (wt/*3, *2/*2, *3/*3) and cytochrome 2C19 (*2/*2, *2 8*/ wt, *2/*17) was available for 275 patients (91.1%). Cytochrome 2C9 and cytochrome 2C19 loss-of-function polymorphisms were found in 41 (14.9%) and 83 patients (30.2%), respectively. The frequency of loss-of-function polymorphisms of cytochrome 2C9 and cytochrome 2C19 was similar in patients without and with CCBs (both P > 0.5). The platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP were significantly higher in patients concomitantly treated with CCBs than in patients without CCB therapy (Figure 1; P-selectin expression: 13.6 MFI [ MFI] vs MFI [ MFI], P = 0.03; activated GPIIb/IIIa: 12.4 MFI [ MFI] vs. 9.8 MFI [ MFI], P = 0.001). Similarly, the fold increase of platelet surface P-selectin expression and activated GPIIb/IIIa was significantly more pronounced in patients receiving CCBs than in patients without CCBs (Figure 2; P-selectin expression: 4.1 [ ] vs. 3.4 [ ], P = 0.03; activated GPIIb/IIIa: 4.1 [ ] vs. 3.4 [2.4 5], P = 0.001). The associations of ADP-inducible activated GPIIb/IIIa and fold increase of activated GPIIb/IIIa after the addition of ADP with CCB therapy remained statistically significant after adjustment for age, hypertension, diabetes, serum creatinine, smoking status, cytochrome 2C9 and 2C19 loss-of-function polymorphisms, and use of proton pump inhibitors and statins by multivariate regression analyses (both P < 0.05). In a second step, ADP-inducible platelet surface P-selectin expression and activated GPIIb/IIIa in the fourth quartile were defined as high P-selectin and high GPIIb/IIIa, respectively. The resulting cutoff values were a MFI >19.6 for high P-selectin and a MFI >16.4 for high activated GPIIb/IIIa. With use of these thresholds, high P-selectin and high activated GPIIb/IIIa were significantly more frequent in patients with CCBs compared to patients without CCB therapy (high P-selectin: 34.8% vs. 21%, P = 0.01; high activated GPIIb/IIIa: 35.9% v. 21.4%, P = 0.008). Discussion To the best of our knowledge, this is the first study investigating the impact of CCBs on the antiplatelet effect of clopidogrel by flow cytometric parameters of platelet activation. We found significantly higher surface expressions and a significantly higher increase of surface expressions of P-selectin and activated GPIIb/ IIIa in response to ADP in patients concomitantly treated with CCBs compared to patients without CCBs. Moreover, high P-selectin and high activated GPIIb/IIIa were more frequent in patients with CCB therapy than in patients without CCBs. While the new ADP receptor inhibitors prasugrel and ticagrelor are only approved for patients with acute coronary syndromes (ACS) so far, clopidogrel can also be prescribed in stable coronary artery disease, peripheral arterial disease (PAD), and cerebrovascular disease. Moreover, especially in older patients with ACS and 266 Cardiovascular Therapeutics 33 (2015) ª 2015 John Wiley & Sons Ltd

4 T. Gremmel et al. Calcium-Channel Blockers and Clopidogrel (A) (A) (B) (B) Figure 1 (A) Platelet surface P-selectin expression in response to adenosine diphosphate (ADP) in patients without and with calciumchannel blockers (CCBs). (B) Platelet surface expression of activated glycoprotein (GP) IIb/IIIa in response to ADP in patients without and with CCBs. MFI, mean fluorescence intensity. The boundaries of the box show the lower and upper quartile of data, and the line inside the box represents the median. Whiskers are drawn from the edge of the box to the highest and lowest values that are outside the box but within 1.5 times the box length. Figure 2 (A) Fold increase of platelet surface P-selectin expression in response to adenosine diphosphate (ADP) in patients without and with calcium-channel blockers (CCBs). (B) Fold increase of platelet surface expression of activated glycoprotein (GP) IIb/IIIa in response to ADP in patients without and with CCBs. The boundaries of the box show the lower and upper quartile of data, and the line inside the box represents the median. Whiskers are drawn from the edge of the box to the highest and lowest values that are outside the box but within 1.5 times the box length. in those, who previously experienced an ischemic stroke, clopidogrel is often used in conjunction with aspirin to prevent detrimental platelet activation. Hypertension is a main risk factor for atherosclerosis, and dihydropyridine CCBs are frequently prescribed to lower blood pressure in patients with all manifestations of cardiovascular disease. Consequently, the interaction between dihydropyridine CCBs and the antiplatelet effect of clopidogrel would still affect a large number of patients. In contrast to previous studies from others and us [14,19 21], we decided to directly assess platelet activation by flow cytometry in the current study instead of measuring platelet aggregation as the final step of platelet response to agonist stimulation. Flow cytometry allows capturing fine regulations of platelet activation in response to ADP and is therefore suitable to further investigate the potential interaction between CCBs and clopidogrel. In detail, the expressions of P-selectin and activated GPIIb/IIIa on platelet surface are considered sensitive markers of platelet activation [24,25]. Platelet surface P-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes and thereby allows the attachment of leukocytes to activated platelets [22]. The resulting heterotypic leukocyte platelet aggregates are elevated in different manifestations of cardiovascular disease including myocardial infarction and stable coronary artery disease [26 28]. Activated GPIIb/IIIa mediates the interaction of platelets with coagulation factors and other platelets [29]. Recently, we associated platelet surface P-selectin expression and activated GPIIb/IIIa with the occurrence of adverse ischemic outcomes in patients undergoing angioplasty and stenting for peripheral arterial disease [30]. In the last years, others and we have shown that therapy with CCBs is associated with higher on-treatment residual platelet reactivity to ADP by different platelet aggregation and reactivity tests [14,19,20]. We found significantly higher levels of on-treatment residual ADP-inducible platelet reactivity by light transmission aggregometry (LTA) and the VerifyNow P2Y 12 assay in patients taking CCBs after angioplasty and stenting [14]. In line with our study, Harmsze et al. [20] observed a worse response to clopidogrel as assessed by LTA in clopidogrel-treated patients, who were coadministered the CCB amlodipine. Another study reported increased on-treatment residual platelet reactivity to ADP by the vasodilator-stimulated phosphoprotein phosphorylation assay in patients receiving both clopidogrel and CCBs as compared to patients receiving only clopidogrel [19]. In contrast, Sarafoff et al. [21] found no significant association between CCB therapy and on-treatment residual platelet reactivity to ADP by multiple electrode aggregometry. This discrepancy may be explained by the fact that the various test systems use different approaches to estimate the extent of platelet aggregation and consequently capture different aspects of platelet activation. Indeed, we have previously shown that the influencing factors for clopidogrel-mediated platelet inhibition vary between different test systems and can be con- ª 2015 John Wiley & Sons Ltd Cardiovascular Therapeutics 33 (2015)

5 Calcium-Channel Blockers and Clopidogrel T. Gremmel et al. sidered as assay dependent to some extent [31]. Therefore, we directly determined platelet activation in response to ADP by flow cytometry in the current study. Our findings of higher ADPinducible platelet activation and a stronger increase of P-selectin expression and activated GPIIb/IIIa in response to ADP in patients taking CCBs strengthen the hypothesis that dihydropyridine CCBs interact with clopidogrel metabolism and thereby attenuate its antiplatelet potency. Besides the inhibition of cytochrome 3A4 by dihydropyridine CCBs [17,18], the inhibition of the drug transporter P-glycoprotein by several CCBs (verapamil, nifedipine, diltiazem, barnidipine) may attenuate the antiplatelet effect of clopidogrel by decreasing its intestinal absorption. As most of our patients received amlodipine, which does not act as P-glycoprotein inhibitor, our study was not designed to assess the effects of P-glycoprotein inhibiting CCBs on clopidogrel-mediated platelet inhibition. However, Harmsze et al. [20] recently reported that only amlodipine and not the use of P-glycoprotein inhibiting CCBs was associated with poor response to clopidogrel in 623 consecutive patients undergoing elective percutaneous coronary intervention. These findings suggest that the inhibition of cytochrome 3A4 is the main mechanism for the attenuation of clopidogrel-mediated platelet inhibition by CCBs. The significance of the interaction between CCBs and clopidogrel for clinical outcomes of patients with cardiovascular disease has only been addressed by observational studies and retrospective analyses, so far. While some of these reported increased rates of adverse ischemic events in clopidogrel-treated patients who also received CCBs [19,32], others found no such association [21,33,34]. Consequently, large prospective randomized clinical trials are needed to confirm or rule out a clinically relevant interaction between clopidogrel and CCBs. A limitation of our study is the lack of clinical outcome data. In conclusion, dihydropyridine CCBs attenuate the effect of clopidogrel on ADP-inducible platelet activation in patients undergoing angioplasty and stenting for cardiovascular disease. Prospective randomized clinical trials are warranted to clarify whether this interaction affects the long-term outcomes of clopidogrel-treated patients with different manifestations of cardiovascular disease. Conflict of Interest The authors declare no conflict of interest. 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