Recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers

Abstract

Aims

To study the recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers.

Methods

Ten healthy volunteers were treated with clopidogrel (75 mg day⁻¹) for 7 days. CD62P expression and PAC-1 binding were measured by flow cytometry.

Results

Adenosine diphosphate (ADP, 30 µm)-induced platelet responses were almost completely inhibited by clopidogrel. After discontinuation of the drug, platelet function gradually increased and complete recovery was seen 7 days after the last clopidogrel dose. The mean difference (95% CI) for ADP-induced PAC-1 binding (fluorescence intensity) between baseline and 7 days after the last dose was 0.01 (0.61, −0.59). Single cell analysis provides direct evidence for an irreversible mode of action of clopidogrel.

Conclusions

This is the first report to directly demonstrate irreversibility of clopidogrel action in humans.

Introduction

The thienopyridine clopidogrel, an inhibitor of the P2Y₁₂ adenosine diphosphate (ADP) receptor [1], is at least as effective as aspirin in preventing cardiovascular events in patients with advanced atherosclerosis (reviewed in [2]). Although in vitro activity of this compound has been demonstrated [3], the mechanism did not involve the G_i-coupled ADP receptor, and desensitization of other ADP receptor subtypes (most likely P2Y1) might account for these effects [4]. Recently, the structure of the putative active metabolite of clopidogrel has been published and it was postulated that this metabolite binds irreversibly to a cysteine residue of the platelet P2Y₁₂ ADP receptor [5].

However, data on the recovery of platelet function after discontinuation of clopidogrel treatment are sparse. In the only published study, the standard dosing regimen for clopidogrel (75 mg day⁻¹ for 14 days) caused 46% inhibition of ADP-induced platelet aggregation 24 h after the last dose but no significant inhibition of platelet aggregation was observed 4 days after the last dose [6]. Paradoxically, in the same study, a long-lasting inhibition of platelet aggregation was seen, when a dose of 50 mg day⁻¹ clopidogrel was administered to the volunteers. Thus the duration of action of clopidogrel needs to be clarified.

Using flow cytometry [7, 8], the current study presents data on the recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers. In addition, single cell analysis provides direct evidence for an irreversible mode of action of clopidogrel.

Methods

Subjects and drug administration

This study was conducted according to the Helsinki Declaration and was approved by the Ethics Committee of the Heinrich-Heine-Universität Düsseldorf. Ten healthy male volunteers (age 28.7±2.1 years) gave informed written consent to participate in the study. The effects of clopidogrel (Plavix®, Sanofi Pharma Bristol-Myers Squibb SNC, Paris, France, 75 mg day⁻¹ for 7 days) on platelet function were studied. Platelet analyses were performed at baseline and at the end of the treatment period (1 h after the last clopidogrel dose). The recovery of platelet function was monitored 3, 5, and 7 days after the last clopidogrel dose.

Preparation of washed human platelets and flow cytometry

Fresh citrated blood (acidic citrate dextrose, ACD, 1:7 v/v) was obtained by venipuncture. Platelet-rich plasma (PRP) was prepared by centrifugation at 1000 × g for 45 s and acidified with ACD (1:5 v/v). Platelets were pelleted by centrifugation at 1700 × g for 30 s and resuspended in HEPES-buffered Tyrode buffer (134 mm NaCl, 12 mm NaHCO₃, 2.9 mm KCl, 2 mm CaCl₂, 0.36 mm NaH₂PO₄, 1 mm MgCl₂, 5 mm HEPES, 5 mm glucose, 0.5 mg ml⁻¹ bovine serum albumin, pH 7.4). For flow cytometry analysis, washed platelets (30,000 µl⁻¹) were stimulated with 30 µm ADP (Sigma, Deisenhofen, Germany) for 5 min. Platelets (25 µl) were incubated with 5 µl anti-CD62P-FITC (Coulter-Immunotech, Marseille Cedex, France), or 5 µl PAC-1-FITC (Becton Dickinson, San Jose, CA, USA) for 30 min in the dark. Samples were diluted with 500 µl Isotone® and immediately analysed on a EPICS-XL cytometer (Beckman Coulter, Krefeld, Germany). The platelet population was identified on its forward and side scatter distribution which was validated by staining with anti-CD42b antibodies (Dako, Hamburg, Germany) (data not shown). Detectors were set to logarithmic amplification and the net geometrical mean fluorescence (Mn X) was measured in 10,000 platelets using the System II (3.0) software. Non-specific binding, as assessed using isotype-matched FITC-conjugated antibodies, was subtracted from the fluorescence measurements.

Statistics

Data presented are means±s.e. mean. Statistical analysis was performed using one-way anova followed by the Bonferroni test for multiple comparisons. P levels <0.05 were considered significant.

Results

ADP (30 µm)-induced CD62P expression and PAC-1 binding were almost completely inhibited by clopidogrel (Figure 1). After discontinuation of clopidogrel treatment, ADP-induced platelet responses increased in a time-dependent fashion and complete recovery of platelet function was seen 7 days after the last clopidogrel dose. For ADP-induced CD62P expression, the mean difference (95% CI) in fluorescence intensity between baseline and 7 days after the last dose was −0.30 (−1.26, 0.66). For ADP-induced PAC-1 binding, the mean difference (95% CI) in fluorescence intensity between baseline and 7 days after the last dose was 0.01 (0.61, −0.59).

Figure 1.

Effects of clopidogrel (75 mg day⁻¹ for 7 days) on adenosine diphosphate (ADP, 30 µm)-induced expression of CD62P (a) and PAC-1 binding (b) in washed human platelets. Platelet function was measured at baseline (pre-treatment), 1 h after the last dose (1 h post-treatment), 3 days post-treatment, 5 days post-treatment and 7 days post-treatment. Data are means±s.e. mean, n = 10 subjects, *P < 0.05 vs ADP-stimulated controls at baseline. Solid bars: control, shaded bars: ADP.

Representative flow cytometry tracings are shown in Figure 2. At baseline, an increase in fluorescence intensity of the respective activation marker was seen in the whole platelet population upon stimulation with ADP. Three days after the last clopidogrel dose, a platelet subpopulation occurred which was fully activated by ADP. However, the majority of platelets, did not change fluorescence levels. Platelet responsiveness subsequently increased and a uniform shift in fluorescence intensity in ADP-stimulated platelets was observed 7 days after the last clopidogrel dose. A similar pattern was obtained when platelets obtained 1 h after the last clopidogrel dose were mixed with platelets from an untreated volunteer. For example, when 30% untreated platelets were mixed with 70% clopidogrel-treated platelets, flow cytometry histograms were comparable with those from clopidogrel-treated volunteers 3 days after the last clopidogrel dose (data not shown).

Figure 2.

Representative flow cytometry histograms demonstrating the effects of clopidogrel (75 mg day⁻¹ for 7 days) on adenosine diphosphate (ADP, 30 µm)-induced expression of CD62P (a) and PAC-1 binding (b) in washed human platelets. Platelet function was measured at baseline (pre-treatment), 1 h after the last dose (1 h post-treatment), 3 days post-treatment, 5 days post-treatment and 7 days post-treatment.

Discussion

This short report presents data on the kinetics of the recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers. In addition, single cell analysis provides direct evidence for an irreversible mode of action of clopidogrel.

It is generally assumed that clopidogrel irreversibly inhibits ADP-mediated platelet responses (reviewed in [9]). However, the irreversibility of the effects of clopidogrel has never been directly demonstrated in humans and data on the duration of action of clopidogrel are conflicting [6].

Alternative mechanisms may be responsible for the long-lasting effect of clopidogrel on platelet function. For example, the long elimination half-life of clopidogrel (>300 h for the [¹⁴C]-labelled drug, which was found predominantly in the plasma and not in blood cells [10], may contribute to its long duration of action.

The present study demonstrated that after discontinuation of clopidogrel treatment, platelet function gradually recovers and that a complete restoration of ADP-induced platelet responses occurs 7 days after the last clopidogrel dose. In addition, single cell analysis demonstrates that, 3 days after the last clopidogrel dose, a platelet subpopulation fully responsive to ADP occurred while the majority of platelets did not respond to this stimulus. In coincubation experiments with 30% untreated and 70% clopidogrel-treated platelets, similar histograms were observed to those at 3 days after the last clopidogrel dose. About 10–15% of platelets are replaced each day [11]. Thus, our data are compatible with the view that the inhibitory effects of clopidogrel are terminated by de novo formed platelets.

Interestingly, there was some tendency for an increased expression of activation markers in ADP-stimulated platelets 3 and 5 days after the last clopidogrel dose (c.f. Figure 2), which is compatible with a transient hyperreactivity of the newly formed platelets. However, when the fluorescence intensity of the highly reactive platelets (arbitrarily defined as 10% of the population with the highest fluorescence levels) at baseline was compared with that at 3 days after the last clopidogrel dose, the difference did not reach statistical significance.

Taken together, the present study has directly demonstrated that clopidogrel causes irreversible inhibition of platelet function in humans. Seven days after treatment discontinuation, platelet function was fully restored.