Clin Res Cardiol 95:212216 (2006) DOI 10.

1007/s00392-006-0363-1

ORIGINAL PAPER

D. Skowasch A. Viktor M. Schneider-Schmitt B. Lderitz G. Nickenig G. Bauriedel

Differential antiplatelet effects of angiotensin converting enzyme inhibitors

Comparison of ex vivo platelet aggregation in cardiovascular patients with ramipril, captopril and enalapril

Received: 3 May 2005 Accepted: 22 December 2005 Published online: 17 February 2006

Dr. Dirk Skowasch ()) Achim Viktor Melanie Schneider-Schmitt Prof. Dr. Dr. h.c. Berndt Lderitz Prof. Dr. Georg Nickenig Prof. Dr. Gerhard Bauriedel Department of Internal Medicine II Cardiology Heart Center University of Bonn Sigmund-Freud-Str. 25 53105 Bonn, Germany E-Mail: Dirk.Skowasch@ukb.uni-bonn.de

n Summary Background Increasing evidence suggests that angiotensin converting enzyme (ACE) inhibitors exert antithrombotic effects. Based on the assumption of differential effects of various ACE inhibitors on coagulation, the aim of the present study was to evaluate the coagulative activities of cardiovascular (CV) patients treated with either ramipril, captopril, and enalapril, and to compare these with patients treated with established antithrombotics such as aspirin (ASA) and clopidogrel or none of these medication. Methods Blood samples of 320 CV patients with coronary artery disease and/or arterial hypertension were analyzed by wholeblood aggregometry. Platelet aggregation was determined by measuring the increase in impedance across paired electrodes in response to the aggregatory agents collagen and adenosine diphosphate (ADP), respectively. These data were correlated with medical treatment. Results Platelet aggre-

gation was attenuated ex vivo by ramipril and captopril as well as by ASA and clopidogrel. While collagen-induced platelet aggregation was significantly reduced by ramipril (35%; P < 0.01) and captopril (27%; P = 0.01), no change was seen with enalapril. After induction with ADP, platelet aggregation was reduced in the presence of captopril therapy by 46% (P < 0.05). There was a trend of inhibition with ramipril (32%, P = n.s.), whereas no antithrombotic effect was seen with enalapril. Conclusion Our findings demonstrate that ACE inhibitors decrease platelet aggregation ex vivo. The differential antiaggregatory profile may explain at least in part different effects of ACE inhibitors on cardiovascular endpoints as observed in large clinical trials. n Key words ACE inhibitors atherosclerosis arterial hypertension platelet aggregation

Introduction
Angiotensin-converting enzyme (ACE) inhibitors are widely used in patients with various cardiovascular diseases [4, 14]. ACE inhibitor therapy has been shown to reduce the incidence of acute myocardial infarction (MI) in patients with left ventricular dysfunction [11, 19]. In addition, mortality was lower

in subgroups of these patients post myocardial infarction [68, 16]. Likewise, the Heart Outcomes Prevention Evaluation (HOPE) study showed a 22% reduction in cardiovascular death, stroke, and MI with ramipril in patients with high cardiovascular risk in the absence of heart failure or left ventricular dysfunction [17]. The EURopean trial On reduction of cardiac events with Perindopril in stable coronary

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Artery disease (EUROPA) demonstrated a 20% relative reduction in the combined primary endpoint cardiovascular mortality, non-fatal myocardial infarction, and resuscitated cardiac arrest [3]. Recently, we and others demonstrated attenuated platelet aggregation with ACE inhibitor therapy, which might at least partly explain the pleiotropic beneficial effects of this drug class on cardiovascular events [1, 13]. Since individual ACE inhibitors differ substantially in their physiochemical properties, enzymebinding kinetics, pharmacokinetic profile, organ specific affinity and selectivity, the purpose of the present study was to evaluate different ACE inhibitors versus aspirin and clopidogrel as classical antithrombotics in their effect on platelet aggregation, by use of whole-blood aggregometry in patients with cardiovascular disease.

Methods
We prospectively enrolled 320 stable cardiovascular patients, between February 2000 and February 2003. These patients were recruited from consecutive patients who presented at our Heart Center. All participants gave informed consent before enrollment. Inclusion criteria were coronary artery disease, arterial hypertension, or both [1]. Treatment with glycoprotein II b/IIIa receptor blockers, aspirin as analgesic therapy within the last four weeks, angiotensin II type 1 receptor antagonists or combined antithrombotic/ACE inhibitor medication led to study exclusion. At the baseline study visit, detailed demographic data and a background medication profile were collected (Table 1). Patients were classified into five groups of study participants: group A, patients with cardiovascular disease who were not taking ACE inhibitors or antithrombotic medication (control group); group B, patients taking aspirin (100 to 300 mg/d) and/or clopidogrel (75 mg/d); group C, patients taking ramipril; group D, patients taking captopril; group E, patients taking enalapril. Of the patients with ACE inhibitor medication, 21 were treated with ramipril (2.510 mg/d, mean 5.5 3.0 mg/d), 33 were treated with captopril (6.25-150 mg/d; mean 45.3 23.4 mg/d) and 25 with enalapril (2.5-20 mg/d; mean 9.6 6.2 mg/d); patients received ACE inhibitors for a minimum of 2 weeks. After recruitment of the patients, venous blood samples were collected in plastic tubes containing a 1 : 10 volume of acid citrate anticoagulant (2 parts of 0.1 mol/l citic acid to 3 parts of 0.1 mol/l trisodium citrate) and subsequently (between 30 min and 5 h) processed, as recently described [1]. Platelet aggregation in whole blood samples were examined by an

impedance aggregometer (Model 560, Chrono-Log, Havertown, PA, USA). Measurements were performed at 37 8C and a stirring speed of 900 rpm. According to the recommendations of the manufacturer, citrate blood (500 ll) was diluted 1-to-1 with 0.9% NaCl and prewarmed for 5 min at 37 8C. After the electrode was placed, aggregation was induced by the stimulatory agents collagen (final concentration 2 lg/ml) and adenosine diphosphate (ADP; final concentration 5 lmol/ml). Collagen and ADP were purchased from Nobis (Endingen, Germany). Platelet aggregation was monitored continually for 6 min, and responses were recorded as electrical impedance across paired electrodes (in Ohm). Inhibition of aggregation was evaluated as the percentage comparing the extent of aggregation in the presence (groups B-E) or absence (group A) of the antiaggregatory regimens studied. Baseline demographics, use of medication, and data of whole-blood aggregometry were compared. Group comparisons were performed by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. P values < 0.05 were considered to be statistically significant. Data are given as mean SD.

Results
Patient characteristics such as age, gender, cardiovascular risk factors and medication did not differ between groups and are presented in Table 1. Platelet aggregation was significantly attenuated by aspirin/ clopidogrel as well as by ramipril and captopril. Specifically, patients receiving aspirin/clopidogrel (group B) showed a 28% decrease in collagen-induced platelet aggregation (P < 0.001), indicated by a lower impedance increase compared with that of the untreated patients of group A (Fig. 1 a). With ramipril therapy (group C), the increase in impedance was reduced by 35% (P = 0.005), with captopril (group D) by 27% (P = 0.014), whereas no effect was seen with enalapril (group E, P = 1.0). Likewise, ADP-induced platelet aggregation was found to be significantly attenuated with aspirin/clopidogrel (group B) by 35% (P = 0.032; Fig. 1 b). The decrease in platelet aggregation averaged 46% with captopril (group D, P = 0.047). With ramipril therapy (group C), there was a trend of inhibition (32%; P = 0.96). No change in impedance was seen with enalapril (group E, P = 1.0). A subgroup analysis of group B was performed to evaluate specific inhibition pathways of the established antithrombotics. As expected, the increase in impedance after collagen-induction was reduced by 27% by aspirin monotherapy (n = 67; P = 0.01) and

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Table 1 Baseline characteristics of patients Group No. Age (yr) Male sex CAD Previous MI Hypertension Systolic BP (mmHg) Diastolic BP (mmHg) Other risk factors Diabetes Hyperlipidemia Current smoker Familial disposition Obesity Medication Aspirin/clopidogrel ACE inhibitors Nitrates b-blockers Calcium channel bl. Statins A 151 61 16 70 (46) 71 (47) 21 (24) 90 (60) 139 16 80 10 17 73 50 38 31 (11) (48) (33) (25) (21) B 90 63 12 45 (50) 58 (64) 19 (21) 58 (64) 138 15 79 11 21 55 38 25 17 (23) (61) (42) (28) (21) C 21 67 10 8 (38) 11 (52) 5 (24) 14 (67) 143 19 83 9 7 (33) 12 (57) 6 (29) 5 (24) 5 (24) 0 21 (100) 9 (43) 11 (52) 4 (19) 10 (48) D 33 65 9 12 (36) 15 (44) 7 (21) 19 (58) 141 17 81 12 5 (15) 16 (48) 10 (30) 13 (39) 4 (12) 0 33 (100) 10 (30) 14 (42) 6 (18) 11 (33) E 25 68 11 14 (56) 18 (72) 7 (21) 18 (72) 141 18 81 10 8 (32) 16 (60) 11 (44) 5 (20) 5 (20) 0 25 (100) 10 (40) 14 (56) 4 (16) 10 (40)

0 0 40 (26) 62 (41) 22 (15) 42 (28)

90 (100) 0 27 (30) 46 (51) 15 (17) 41 (46)

Baseline characteristics [number of patients (%) or mean SD]. A cardiovascular patients without ACE inhibitors or antithrombotic medication; B patients with aspirin/clopidogrel; C patients with ramipril; D patients with captopril; E patients with enalapril; ACE angiotensin converting enzyme; CAD coronary artery disease; MI myocardial infarction

a
Fig. 1 Influence of established antithrombotics and angiotensin-converting enzyme (ACE) inhibitors on platelet aggregation. Impedance changes in Ohm after induction with collagen (a) or with adenosine diphosphate (b) in pa-

b
tients not receiving antithrombocyte or ACE inhibitor medication (group A), in patients receiving aspirin/clopidogrel (group B), and in participants receiving ramipril (C), captopril (D) or enalapril (E). n.s. not significant

by 47% by clopidogrel monotherapy (n = 12; P = 0.19). After ADP induction, platelet aggregation was reduced by 22% with aspirin (P = 0.01) and by 72% with clopidogrel (P = 0.02).

Discussion
Beyond confirming the antiaggregatory effects of the established antithrombotics aspirin and clopidogrel, the present study shows that ACE inhibitors attenu-

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ate platelet aggregation and thereby demonstrates a differential antiaggregatory profile of different ACE inhibitors. While ramipril and captopril therapy reduced ex vivo platelet aggregation, no antithrombotic effect was seen with enalapril. The underlying mechanisms remain unclear so far. It has been hypothesized that tissue affinity might be responsible for some of the beneficial cardiovascular properties of ACE inhibitors. However, the present findings suggest that tissue ACE inhibitor affinity does not affect the antithrombotic effects of ACE inhibitors, since ramipril is a tissue-ACE inhibitor, and captopril and enalapril are non-tissue-ACE inhibitors. Likewise, others have not found a correlation between tissue-ACE affinity and risk of myocardial infarction in patients with arterial hypertension [12]. Possible mechanisms for reduced platelet aggregation with ACE inhibitor therapy are decreased tissue factor expression with ACE inhibition [20], lowered concentrations of circulating tissue factor [10], and reduced vascular [5] and systemic inflammation [9]. At first glance, parallel implications may result from the present ex vivo study and recent data from HOPE [17] and other trials [68, 11, 15, 16, 19]. The HOPE trial tested ramipril in patients with atherosclerosis in the absence of heart failure or left-ventricular dysfunction, and showed a significant decrease in the combined end point of cardiovascular death, myocardial infarction, and stroke, but not in unstable angina with electrocardiographic changes or in hospitalization rate [17]. One may conclude that clinical end points caused by total thrombotic vascular occlusion, but not those by incomplete coronary obliteration are significantly influenced. Beyond normalization of endothelial dysfunction and plaque stabilization, which were both discussed as being responsible for the clinical benefit of ramipril [17], our results support the concept of an additional antithrombotic effect of ramipril. Although it is unclear whether the benefits of ramipril can be extrapolated to other ACE inhibitors, data from unselected patients of the prospective multicenter registry MITRA PLUS suggested that ramipril has more beneficial effects on cardiovascular events than treatment with other ACE inhibitors [18]. The cardioprotective findings seen in HOPE and EUROPA do not necessarily presume a cardiovascular class effect of ACE inhibitors. The Prevention of Events with An-

giotensin Converting Enyzme Inhibition (PEACE) trial failed to provide any further benefit in terms of death from cardiovascular causes, myocardial infarcion, or coronary revascularization among a large cohort of patients with stable coronary disease treated with the ACE inhibitor trandolapril [2]. To date, several well-controlled clinical trials involving post-MI patients with left ventricular dysfunction have been performed with the different ACE inhibitors studied in the present work. The reduction in all-cause mortality observed in shortterm trials (CCS-1 [8] and ISIS-4 [7] with captopril) has been very modest, and in the CONSENSUS II trial there was even an increase in mortality with enalapril [15]. In long-term studies (SAVE [11] with captopril, AIRE [16] and AIREX [6] with ramipril), post-MI patients with left ventricular dysfunction and/or clinical signs and symptoms of heart failure were included and followed up for a longer period. The reduction in all-cause mortality observed in these studies was even greater compared to that in the short-term trials. One may speculate that antithrombotic effects of ramipril and captopril, as observed in the present study, were, at least in part, responsible for these beneficial effects, whereas no antithrombotic effect was seen with enalapril in the present study, analogous to an increase in mortality in the CONSENSUS II trial [15]. The concept of our present study was to screen the impact on platelet aggregation brought in by basic drug prescription in cardiovascular patients as seen in daily practice. Of course, there are several limitations of the study. No information can be drawn regarding the clinical relevance of ACE inhibitor reduced platelet aggregation. In remains open how long and in what dosage ACE inhibitors should be taken to achieve antithrombotic effects. In this study, medication use was based on answers to questionnaires; drug levels or pill counts were not performed. In summary, the present study demonstrates a differential antiaggregatory profile among different ACE inhibitors, which may explain at least in part different effects on cardiovascular events as observed in large clinical trials. Further head-to-head investigations with equivalent dosages of different ACE inhibitors may be warranted in large-scale studies to fully elucidate clinical differences and similarities among ACE inhibitors.

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