Limited early antiplatelet effect of 300 mg clopidogrel in patients with aspirin therapy undergoing percutaneous coronary interventions

Aino Lepäntaloa, Kari S Virtanenb, Juhani Heikkiläb, Ulla Wartiovaarac and Riitta Lassilaa,d,*

a Wihuri Research Institute, Kalliolinnantie 4, Fin-00140 Helsinki, Finland
b Division of Cardiology, Department of Internal Medicine, Helsinki University Central Hospital, PO Box 340, Fin-00029 HUCH, Helsinki, Finland
c Laboratory Department, Helsinki University Central Hospital, PO Box 340, Fin-00029 HUCH, Helsinki, Finland
d Division of Haematology, Department of Internal Medicine, Helsinki University Central Hospital, PO Box 340, Huch, Helsinki, Finland

* Corresponding author. Tel.: +358-(9)-681411; fax: +358-(9)-637476
E-mail address: riitta.lassila{at}hus.fi

See page 454 for the editorial comment on this article1


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Aim Our aim was to evaluate the early efficacy and variability of the platelet inhibition exerted by 300 mg clopidogrel for the purpose of acute percutaneous coronary interventions using platelet function tests.

Methods and results Elective percutaneous coronary intervention was used as a timely model in which clopidogrel was added to ongoing acetylsalicylic acid (aspirin) (100 mg/day) at 2.5 h prior to procedure. Blood samples were collected before administration of clopidogrel and immediately before the intervention from 50 patients. Platelet functions were assessed with traditional aggregation and PFA-100®.

At baseline, 14 (28%) patients were poor responders to aspirin according to PFA and 9 (18%) continued to show arachidonic acid-induced aggregation. After clopidogrel ADP-triggered aggregation was only modestly inhibited in 40% of the patients. Eight percent of the study population was left without any measurable antiplatelet effect. The patients with modest response to clopidogrel had higher levels of c-peptide (1.5 nmol/L) than the ones responding well (0.9 nmol/L, ).

Conclusion Neither ongoing aspirin treatment nor added clopidogrel did reach an expected extent of platelet inhibition. This study shows that aspirin-treated patients undergoing PCI gain highly variable levels of platelet inhibition with short-term clopidogrel 300 mg. At 2 h after adding clopidogrel it failed to enhance platelet inhibition in 40% of the patients. In future, targeted platelet function tests may be helpful to individually select an effective antiplatelet medication for these patients. This study suggests that for acute PTCA clopidogrel does not reach the optimal antithrombotic efficacy in all patients.

Key Words: Angioplasty • Aspirin • Clopidogrel • Platelet • Thrombosis


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The effects of acetylsalicylic acid (aspirin) and ADP (P2Y12-receptor) – antagonist clopidogrel as well as their combination over aspirin alone have proven beneficial in patients with cardiovascular disease.1–5 The reduction of acute vessel closure in patients who have undergone percutaneous coronary intervention (PCI) by combination of these antiplatelet agents was detected both in the CLASSICS and the CURE studies.6–8

Theoretically, administration of clopidogrel only hours before the endoluminal procedure should be efficacious, as 70% inhibition of platelet aggregation is reported to be gained within 2 h in healthy volunteers.9 Previously, in a smaller study of 20 patients with stable arterial disease and ongoing aspirin medication, ADP-induced platelet aggregation was observed to be reduced by 17% at 2 h after clopidogrel loading dose of 300 mg.10 In CREDO study, comparing aspirin with combination of aspirin and clopidogrel, it was found that when 300 mg clopidogrel was administered to patients at least 6 h before PCI their risk of death, myocardial infarction or urgent revascularisation at 28 days was reduced by 39%. Yet, still 7% suffered from subacute thrombotic complications in the combination treatment group.11 A large interindividual variability in platelet responses to combined aspirin and postprocedural clopidogrel was detected in patients with coronary artery disease after PCI by Gurbel et al.12 For an acute PCI setting it will be of interest to further investigate how these patients will respond to short-term clopidogrel. There may be individual differences in the P2Y12 receptor activity13 and also drugs affecting liver metabolism may slow down the generation of the active metabolite of clopidogrel.14

Our aim was to assess the antiplatelet effect: (1) of long-term use of aspirin in patients undergoing elective PCI and (2) of an additional loading dose (300 mg) of clopidogrel administered 2.5 h prior to the intervention. The antiplatelet efficacy of this medication was assessed with traditional platelet aggregation tests and simultaneous PFA-100, which has been suggested to be useful in evaluating the platelet effects of aspirin.15–18 Our specific interest was directed to the individual variability of the measurable antiplatelet effects.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients
Fifty patients who were to undergo elective PCI participated in this study. Patients treated with either insulin, warfarin, heparins or antiplatelet agents other than aspirin were excluded from the study. The procedures of the study followed the institutional guidelines. The study was approved by the Ethics Committee of Helsinki University Central Hospital, complied with the Declaration of Helsinki and informed written consent was obtained from all patients.

Study medication and specimen collection
The patients were treated with a daily dose of 100 mg of acetylsalicylic acid (aspirin), already when admitted to the outpatients' clinic. An additional platelet inhibitor, 300 mg of clopidogrel (methyl-(S)-2-(2-chlorophenyl)-2-(4,5,6,7-tetrahydrothieno(3,2-c)pyridin-5-yl)acetate, hydrogen sulphate) was orally administered prior to the PCI. All blood samples were obtained before the actual procedure; (1) before the administration of 300 mg clopidogrel, to study the effect of aspirin alone and (2) 2.5±0.5 h after the administration of clopidogrel to study the combined effect of aspirin and clopidogrel. The samples were collected by venipuncture through a 20-gauge needle into evacuated tubes. The samples for platelet function tests were anticoagulated with: (1) 130 mmol/L of trisodium citrate for PFA-100 and aggregations and (2) with 100 U/mL of hirudin for collagen monomer-induced aggregations, as monomer-induced aggregation depends on Mg2+, which is chelated by citrate.19

Platelet function analysis PFA-100
PFA-100® (Dade-Behring AG, Düdingen, Switzerland) is a platelet function analyser that measures primary hemostasis in whole blood, under capillary flow conditions, with a high shear rate (4000 1/s). Platelets interact with a collagen mesh spiked with either ADP (CADP) or epinephrine (CEPI), resulting in occlusion of an aperture in the respective cartridge. The results are expressed as closure times. The PFA method has been suggested to be useful in the assessment of the antiplatelet effects of aspirin and thus the closure time of epinephrine-stimulated cartridge should be prolonged in aspirin-treated patients.15–18 The manufacturer of the PFA-100 refers to 170 s as the limit of aspirin efficacy with a sensitivity of 95%. In our laboratory the intraindividual method- and donor-dependent variability of PFA in CADP and CEPI was found to be 7% and 11%, respectively (three repeated samples from eight healthy donors during 3 weeks).

Aggregations
Blood was prepared for aggregation experiments by centrifuging (180g, 12 min) platelet-rich plasma from citrate- and hirudin-anticoagulated blood. The platelet count of platelet-rich plasma was adjusted to 300x106/mL with platelet-poor plasma, which had been centrifuged (1500g, 10 min) from platelet-rich plasma. Aggregation was induced with: (1) soluble collagen monomers (5 or 10 µg/mL) (bovine type I)18 in hirudin-anticoagulated platelet-rich plasma and with (2) ADP (2 or 5 µmol/L) (Sigma Chemical Co.), (3) arachidonic acid (1.5 mmol/L) (Sigma) and (4) epinephrine (2 or 5 µmol/L) (Sigma) in citrate-anticoagulated platelet-rich plasma. Aggregation was traced with a traditional, turbidometric, PPACKS-4 aggregometer (Helena Laboratories, Beaumont, USA). Rate (%/min) and maximal aggregation (% change of light transmission) at 5 min were recorded.

Definition of responders and poor responders to aspirin and clopidogrel
Poor responders to aspirin were defined in two alternative manners. First, we used PFA-100. Thus, if the patients had a closure time shorter than 170 s in collagen and epinephrine-induced PFA-experiments, they were defined as poor responders to aspirin according to PFA. These criteria have been previously reported to detect aspirin resistance.15–18 Second, we used arachidonic acid (AA)-induced aggregation. Thus, if the patients had an aggregation slope 10% during the first minute of the test, they were defined as poor responders to aspirin according to AA-induced aggregation.

Poor response to clopidogrel was defined by inhibition of 5 µmol/L ADP-induced platelet aggregation. Aggregations were performed before and after clopidogrel administration and if the inhibition of aggregation was less than 10% (post value was subtracted from the pre value), these patients were defined as poor responders to clopidogrel.

Laboratory analyses
The following blood and plasma analyses were performed at baseline prior to the administration of clopidogrel. Standard techniques were used: blood cell counts, levels of von Willebrand factor (vWf) (ELISA, Asserachrom®vWf, Diagnostica Stago, France), fibrinogen (method of Clauss), glycosylated haemoglobin (Hb A1c) (immunochemical, photometric method), c-peptide (RIA), C reactive protein (CRP) (sensitive immunoturbidimetric method), blood glucose levels (enzymatic, photometric method), activated partial thromboplastin time (APTT) (automated coagulometry), epinephrine and norepinephrine (HPLC-EC).20

Statistical methods
The efficacy of antiplatelet medication was studied by using previously determined parameters of poor ASA response and by comparison between intraindividual samples before and after clopidogrel treatment. The data are presented as means±SD. We used SPSS for Windows, version 9.0. Spearman's non-parametric test was used to assess the correlations between variables. The differences between groups were assessed with Mann–Whitney U-test and inhibition exerted by clopidogrel was assessed with Wilcoxon test for paired samples. The level of significance used was 0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients
Fifty patients, 41 males and 9 females of a mean age of 60 (range 37–80), were included. Their mean body mass index was 28 (range 19–47). Four patients had type II diabetes mellitus. Ten were current smokers. Sixty-two percent () of patients were treated with statins, 70% () with nitrates, 92% () with Beta-blockers, 22% () with ACE-inhibitors and 18% () with Ca-channel blockers. Of the 31 patients with statin treatment 3 (9%) were treated with water-soluble statins (pravastatin or fluvastatin) and 28 (91%) with lipid-soluble statins (atorvastatin , other ). Twenty (40%) patients had previously suffered acute myocardial infarction. Thirteen (26%) of the patients had undergone previous PCI and six (12%) coronary artery bypass grafting. In this study the initial success rate of the PCI procedure was 46/50.

Routine laboratory variables
Routine laboratory samples for assessing blood coagulation, inflammation, catecholamines and glycemic control were collected at baseline prior to the administration of clopidogrel. In the whole study population they were within normal reference limits (data not shown) with the exception of elevated c-peptide levels (mean 1.2±0.8, reference values 0.3–0.9 nmol/L). Higher levels of vWf correlated with shortened PFA closure times in CEPI cartridge (, ).

Baseline PFA-100 and platelet aggregation – the effect of aspirin
Patients were studied with two methods to assess their response to aspirin: (1) with the PFA closure times in the epinephrine-spiked collagen cartridge and (2) with the extent of arachidonic acid-induced platelet aggregation.

PFA-100
Fourteen patients (28%) showed closure times in CEPI cartridges shorter than 170 s (Table 1). These patients were classified as poor responders to aspirin. Rest of the patients () were considered responders to aspirin by PFA. In patients with impaired aspirin response the aggregation responses were stronger (Table 1). Shortened closure times in CEPI associated with enhanced ADP-induced aggregations with both ADP concentrations (at 2 µmol/L, , and at 5 µmol/L, , ). The PFA method seemed also to detect the patients who had previously suffered acute myocardial infarction (). These patients had shorter closure times in the CEPI cartridge (214±84 s) than those who did not have previous history (259±68 s) ().


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Table 1 Shear-induced platelet interaction with collagen (PFA) and traditional platelet aggregation (maximal %) in patients with either normal or poor response to aspirin

 
Platelet aggregation
Nine patients continued to show response (slope 7–94) to arachidonic acid (1.5 mmol/L) despite aspirin treatment and were classified as poor aspirin responders according to more strict criteria on the basis of arachidonic acid-induced aggregation (Table 2). The patients with poor response to aspirin according to AA-induced aggregation had shorter APTT at baseline (29±2 s, ) than the aspirin responders (34±9 s, ). Number of responders to aspirin by AA-aggregation was 40. Three of these patients responded with a slope steeper than 88 %/min (Fig. 1). Furthermore, also the secondary wave of aggregation in response to other agonists was enhanced in poor responders (Table 2).


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Table 2 Arachidonic acid-induced platelet aggregation in patients with either normal or poor response to aspirin

 


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Fig. 1 Aggregation curves of the three least aspirin-responding patients who had sustained arachidonic acid-induced aggregation at the baseline (a) and after clopidogrel (b). A control curve, representative of the 40 aspirin responders, is also displayed.

 
PFA-100 and platelet aggregation after administration of clopidogrel
Inhibition induced by clopidogrel was measured with both PFA and aggregations. The ADP(5 µmol/L) -induced aggregation was used to define limited clopidogrel response.

PFA-100
Clopidogrel did not markedly prolong closure times in PFA-100 (neither CEPI nor CADP cartridges) in the entire study population. The higher the level of catecholamines and vWf the less was the prolonging effect of clopidogrel in closure times of CADP (, ) and (, ), respectively.

Platelet aggregation
The platelet inhibition exerted by added clopidogrel was evident in aggregations induced by ADP, collagen and arachidonic acid (Table 3). In contrast, epinephrine-induced aggregation was much less affected.


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Table 3 Inhibitory effect of clopidogrel assessed with platelet aggregations

 
The reduced inhibition of clopidogrel measured with CADP associated also with the number of drugs used by the patient (, ). There was no association with the antiplatelet efficacy of clopidogrel and any statin used (). The mean inhibition of ADP (2 and 5µmol/L )-induced aggregation was 21±12% and 15±8% in patients with atorvastatin, 4±13% and 6±12% with water-soluble statins, 19±16% and 19±16% with lipid-soluble statins other than atorvastatin and 17±10% and 15±11% in patients with no statin treatment.

Limited response to clopidogrel
In a subgroup of patients clopidogrel treatment did not inhibit platelet aggregation. There were two patients (measured with 5 µmol/L) and four patients (measured with 2 µmol/L) in whom ADP-induced aggregation did not attenuate at all after administrating clopidogrel. Furthermore, we identified 20 (assessed with ADP 5 mol/L) patients and 14 (assessed with ADP 2 µmol/L) patients in whom percent of maximal aggregation was decreased by less than 10%. Due to that, 40% () of the study population was defined as poor responders to clopidogrel according to the limited inhibition of ADP(5 µmol/L)-induced aggregation. The average inhibition of ADP-induced aggregation exerted by clopidogrel in poor responders (10%) was 5±4% and in responders 23±11% () (Fig. 2). When compared with the responders the poor responders showed also a limited reaction to clopidogrel in PFA (-8±27, range–63-50 vs. +36±71, range–44-225 s, respectively, ). The effect of clopidogrel did not associate with the time between drug and blood sample collection (). The time interval in responders to clopidogrel was 143±25 min and in poor responders 139±21 min.



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Fig. 2 Aggregations at baseline and after 300 mg clopidogrel administration in the responders () and poor responders () to clopidogrel. The response to clopidogrel was defined by limited inhibition of ADP (5 mol/L)-induced aggregations. Additionally aggregations were induced by (a) ADP (2 and 5µmol/L), (b) collagen (5 and 10 µg/ml) or (c) epinephrine (2 and 5 µmol/L). Data are expressed as means±SD. * and **.

 
Efficacy of clopidogrel and insulin resistance
Attenuated response to clopidogrel correlated with insulin resistance. Patients with poor long-term glucose balance measured with glycosylated haemoglobin (Hb A1c) and elevated insulin levels measured with c-peptide did not benefit from clopidogrel as much as the normoglycemic patients. Clopidogrel-induced inhibition of aggregation triggered by ADP (5 µmol/L) was less in patients with high levels of glycosylated haemoglobin (Hb A1c) or c-peptide (, , , ). The patients with limited response (change in aggregation 10%) to clopidogrel had higher serum c-peptide levels than patients who responded well to clopidogrel. As expected, increased c-peptide levels correlated with increased body mass index (, ).

Distribution of poor responders and responders to aspirin and clopidogrel
Of the poor responders to aspirin (), measured with arachidonic acid-induced aggregations, 4 patients benefited from clopidogrel, whereas five patients did not. These five patients, 10% of the entire patient group, did not achieve any measurable antiplatelet effect from either aspirin (arachidonic acid 1.5 mmol/L) or clopidogrel (measured with ADP 5 µmol/L) (Fig. 3).



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Fig. 3 Number of responders (+) and poor responders (–) to aspirin (ASA), clopidogrel (CLOPI) or both. Responders to aspirin have been defined based on fully blocked (rate=0) arachidonic acid-induced aggregation and to clopidogrel based on a decrease of maximal ADP-induced (5 µmol/L) aggregation exceeding 10% of the baseline response.

 
In patients with poor response to aspirin according to PFA (), clopidogrel inhibited ADP-induced maximal platelet aggregation by more than 10% in 10 patients. Thus, four patients remained poor responders to both aspirin and clopidogrel. Clopidogrel prolonged closure times in CEPI ({Delta}94±79 s, , ), but not in CADP ({Delta}6±19 s, ) cartridges in patients with limited response to aspirin.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study involved 50 elective PTCA patients using permanent aspirin, in whom short-term clopidogrel was administered to mimic acute PTCA setting. As a patient group without any antiplatelet treatment is not feasible, the results must be evaluated accordingly. Ongoing aspirin (100 mg) failed to suppress platelet activity in nearly one third of the patients in comparison with the general standard, and added clopidogrel (300 mg) in 40% of the patients in comparison with their baseline value on aspirin. A study which doubled the loading dose to 600 mg has suggested an improved antiaggregatory effect.21 From the acquired conditions high levels of vWf and insulin resistance impaired the antiplatelet effect of aspirin and that of combined therapy, respectively.

Effect of aspirin
In spite of ongoing aspirin 28% patients did not meet the closure time criteria for aspirin responses (170 s) in collagen-epinephrine cartridge of PFA-100, and 18% continued to show arachidonic acid-induced aggregation. Limited response to aspirin in patients undergoing elective PCI could result from several factors. Daily dose of aspirin (100 mg) suppresses platelet activity in healthy subjects,22 but this may not apply uniformly to patients with a high thrombogenic burden. Stress reaction, elevated levels of epinephrine and vWf are known to activate platelets synergistically with other agonists,23–25 and may impair the drug responses. In parallel with the resisting role of vWf in aspirin responses, PFA-closure times associated with levels of vWf. After initial collagen-induced activation, GP Ib/IX and GP IIb/IIIa, an axis bridged by vWf, mediate platelet recruitment from flowing blood. This mechanism is promoted by epinephrine,23 which is also able to circumvent the antiplatelet effect of aspirin.25 Clinically, excretional urinary thromboxane and the activity of thromboxane/isoprostanoid-receptor have associated with cardiovascular events.26,27 Both functional and genetic differences of isoprostanoid, cyclooxygenase or other platelet receptors may also affect the efficacy of aspirin.28

Effect of short-term clopidogrel
The aim of this study was to assess the short-term effects of 300 mg clopidogrel for acute PCI. In healthy subjects the rapid antiplatelet onset (at 2 h) of clopidogrel (375 mg) reached 70% inhibition of ADP (5 µmol/L)-induced platelet aggregation.9 However, in the present study 40% of the patients showed persisting aggregation to ADP. Therefore, we conclude that under acute PCI the inhibition exerted by the loading dose (300 mg) of clopidogrel will not be complete in all patients. CREDO study suggests that time interval shorter than 6 h does not offer clinical benefit in contrast to prolonged (6 h) clopidogrel exposure. Alike, at 2.5 h time point we noted only partial functional benefit. However, only a study with significant patient numbers will be able to address the clinical impact of the poor response in laboratory tests.

In addition to ADP large individual variability exists in collagen-induced platelet activation.29,30 It remains to be studied whether genetic or acquired variability in responses to ADP and its feedback actions by other agonists plays a role in the efficacy of antiplatelet agents. The overexpression or activity of P2Y1-receptors may explain poor responses to clopidogrel. 31 In all, differences in genotypes of ADP-, collagen- and GPIIb/IIIa-receptors on platelets may explain the individual functional differences, both short- and long-term.32

Poor response to clopidogrel may also associate with the simultaneous ingestion of other drugs that may prolong the appearance of the active metabolite of clopidogrel. However, unlike Lau et al.,14 but in accordance with Müller et al.,33 we did not find impaired clopidogrel responses to associate with any statin.

Efficacy of clopidogrel and insulin resistance
We found a correlation between a decreased response to clopidogrel and increased levels of body mass index and c-peptide. This is compatible with the physiological finding that insulin inhibits platelet aggregation and activation,34 an effect that is absent in insulin-resistant obese subjects.35 Furthermore, insulin resistance attenuates the efficacy of aspirin when compared with normal insulin response.36 Also diabetics benefit from clopidogrel,2 but in CAPRIE, CREDO and CURE the benefit tended to be less than in non-diabetics. However, only subgroup analyses have been reported on the efficacy of clopidogrel in diabetics without assessing insulin resistance.2,6,7,11,37

Conclusions
This study suggests that the patients undergoing elective PCI do not obtain the expected platelet inhibitory effect with acetylsalicylic acid combined with short-term 300 mg loading dose of clopidogrel and call upon more potent antiplatelet therapy acutely. It remains to be studied whether increasing the dose or prolonging the effective time for clopidogrel from hours to days will improve the efficacy in all or some patients. As a relevant clinical factor insulin resistance impaired the antiplatelet effect of the combination therapy.

The large individual variability in platelet activity should tailor antiplatelet medication to meet the individual needs both in acute and chronical settings. How these platelet responses in functional tests associate with patient outcome needs to be answered by future, larger studies.


    Acknowledgments
 
The authors are grateful to the cardiologists of Helsinki University Central Hospital for their help in recruiting and treating the patients in this study. We acknowledge the excellent technical assistance of Marja Lemponen and Tuula Järvenpää. This study was supported financially by the Helsinki University Central Hospital research funds and Aarne Koskelo Foundation.


    Footnotes
 
1 doi:10.1016/j.ehj.2004.02.005. Back


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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