Individual variations of platelet inhibition after loading doses of clopidogrel

Authors


  • This work was presented in part on the 4th International Congress on Coronary Artery Disease, Prague, October 21–24, 2001

 P. Järemo MD, Department of Cardiology, Linköping University Hospital, S-581 85 Linköping, Sweden (fax: +11 125 662; e-mail: petter.jaremo@beta.telenordia.se).

Abstract

Abstract. Järemo P, Lindahl TL, Fransson SG, Richter A (Linköping University Hospital, Linköping, Sweden). Individual variations of platelet inhibition after loading doses of clopidogrel. J Intern Med 2002; 252: 233–238.

Objective.  To investigate individual variations of platelet inhibition after clopidogrel-loading doses.

Setting.  Department of Cardiology, Linköping University Hospital, Linköping, Sweden.

Subjects.  Individuals with stable angina pectoris (n = 18) subject to percutaneous coronary interventions (PCI) and subsequent stenting were investigated.

Methods and experimental protocol.  A 300-mg clopidogrel loading dose was administrated immediately after stenting (day 1) followed by an additional 75 mg clopidogrel after 24 h (day 2). The ADP-evoked platelet fibrinogen binding was analysed to estimate platelet reactivity immediately before angiography and on day 2. A flow cytometry technique was used with two ADP solutions (final concentrations 0.6 and 1.7 μmol L−1) employed as platelet activating agents. Soluble P-selectin was used as a marker of platelet activity.

Results.  When using 1.7 μmol L−1 ADP to activate platelets four individuals had a strong inhibition (i.e. platelet reactivity <10% of the day 1-value day 2). In contrast, five patients demonstrated a weak inhibition (i.e. platelet reactivity >60% of the day 1-value day 2). Similar results were obtained when using 0.6 μmol L−1 ADP as a platelet-activating agent. Clopidogrel, however, fails to suppress platelet activity as estimated from soluble P-selectin.

Conclusions.  Clopidogrel evoked platelet inhibition exhibits a considerable individual heterogeneity. Some individuals only had weak responses whereas others displayed strong platelet inhibition. The present flow cytometry technique appears suitable for identifying patients with abnormal reactions after clopidogrel exposure.

Introduction

The well-established role of platelets in acute ischaemic coronary disease [1] has focused attention on platelet inhibitory drugs [2]. The recently developed drug clopidogrel irreversibly inhibits ADP induced platelet aggregation and the binding of fibrinogen to the GPIIb-IIIa receptor [3]. At present aspirin concomitantly given with clopidogrel is the standard antiplatelet therapy in conjunction with coronary stenting [4]. However, despite the medication the incidence of stent thrombosis within the first month after the procedure is about 2% [5]. The onset of antiaggregating effects of clopidogrel is rapid and the use of loading doses significantly reduces the time necessary to achieve maximal inhibition of platelet aggregation [6]. The objective of this investigation is to examine individual differences of platelet inhibition after clopidogrel loading doses in a cohort of individuals with stable angina pectoris subject to percutaneous coronary interventions (PCI) and subsequent stenting.

Materials and methods

Subjects and experimental protocol

Eighteen patients with stable angina pectoris participated in the trial (Table 1). Patients were considered eligible if they were below 75 years of age, if they were without myocardial infarctions for the preceding month and if they did not have diabetes mellitus or rheumatic disorders. Coronary angiography with subsequent PCI and stenting was performed using standard procedures. An ionic contrast medium (ioxoglate) was used [7]. A loading dose of clopidogrel (300 mg) was administered orally immediately after stent deployment followed by an additional 75 mg of the drug the next day. Blood samples were drawn immediately before (day 1) and on the day after stenting (day 2). The ADP evoked platelet fibrinogen binding was used as a measure of platelet reactivity. Platelet activity was estimated through analysing plasma concentrations of P-selectin.

Table 1.  Clinical and demographic characteristics for the study patients
Number of patients18
Age59 ± 8
Gender (male/female)16/2
Current smokers, n4
Previous myocardial infarction, n7
Hypertension requiring medical treatment, n6
Medication at admission
 Aspirin, n16
 Beta-blocker, n17
 ACE-inhibitor, n2
 Calcium uptake antagonist, n6
 Prophylactic nitro-glycerine, n10
 Diuretics, n4
 Lipid lowering medication, n13

Analytical procedures

A Cytoron Absolute Flow Cytometer (Ortho, Raritan, NJ, USA) was employed for flow cytometry studies. The protocol has been described previously [7]. The samples were run in duplicate. A phycoerytrin-conjugated antibody directed towards GPIb (Dako AS, Glostrup, Denmark) was used to identify platelets. Two ADP solutions (final concentrations 0.6 and 1.7 μmol L−1) were employed as activating agents. We used platelet bound fibrinogen as a marker for platelet activation. Fibrinogen was identified using a fluorescein isothiocyanate conjugated chicken antihuman fibrinogen antibody (Biopool AB, Umeå, Sweden). A negative control with identical preparation steps included 10 mmol L−1 ethylenediaminetetraacetic acid (EDTA) to prevent the platelet fibrinogen binding was run simultaneously. Platelets having a fluorescence intensity exceeding the negative control were identified as fibrinogen-positive cells. An immunoenzymoassay (R & D Systems, Minneapolis, MN, USA) was employed for soluble P-selectin. The local ethics committee approved the study protocol.

Results

The left part of Fig. 1 shows individual results for platelet reactivity when stimulating with 0.6 μmol L−1 ADP. Before clopidogrel the individual variability of activated platelets after stimulation reached from 1 to 58% (diagram 1, left side). As expected, clopidogrel caused a substantial decrease of platelet reactivity with day 2 fibrinogen positive cells varying from 0 to 29% (diagram 2, left side). Diagram 3 summarizes the data and shows that clopidogrel caused a strong platelet inhibition in seven individuals. On day 2, their ADP induced platelet reactivity was below 10% of the day 1-value. In contrast, one subject had a weak inhibition with a day 2 platelet reactivity exceeding 60% of the day 1-value. The remaining patients belonged to an intermediate group with platelet reactivity after clopidogrel within the range 10–60% of the day 1-value.

Figure 1.

Individual results for the 18 patients with respect to platelet reactivity when stimulating with 0.6 μmol L−1 ADP (left section) and 1.7 μmol L−1 ADP (right section). The first row shows platelet reactivity before clopidogrel. The diagrams in the second line display the corresponding reactivity after clopidogrel (day 2) (0.6 μmol L−1 ADP to the left and 1.7 μmol L−1 ADP to the right). The third row of diagrams presents day 2 platelet reactivity expressed as percentage of the day 1-value. The order of the patients is the same in each diagram.

Similar results were obtained when using 1.7 μmol L−1 ADP to activate platelets (Fig. 1, right side). Before clopidogrel the individual variability of activated cells reached from 6 to 80% of the total population (diagram 1, right side). After clopidogrel a platelet inhibition was observed and the corresponding day 2 figures were 1 and 50%, respectively (diagram 2, right side). The third diagram of the right section condenses the results by showing that four individuals displayed very low (<10% of day 1-value) platelet reactivity on day 2. In contrast, a platelet reactivity exceeding 60% of the day 1 figure on day 2 was found in five subjects. The remainder had a day 2 reactivity ranging from 10 to 60% of the day 1-value.

Figure 2.

Individual results for the 18 patients with respect to platelet activity as estimated from soluble P-selectin values. The first diagram shows platelet activity before clopidogrel. The second diagram displays the corresponding activity after clopidogrel (day 2). The third diagram presents soluble P-selectin concentrations on day 2 expressed as percentage of the day 1-value. The order of the subjects is the same as in Fig. 1.

Contrasting results were obtained when using soluble P-selectin to estimate the effects of clopidogrel (Fig. 2). Before angiography a substantial variation was found and the soluble P-selectin levels varied from 17 to 84 μg L−1. The corresponding figures on day 2 were 27–81 μg L−1, respectively. The third diagram summarizes the effects of clopidogrel on platelet activity. The drug caused in the majority (n = 13) of cases slightly reduced P-selectin. The remainder (n = 5), however, had higher circulating P-selectin after clopidogrel exposure (Fig. 2).

Discussion

The combined use of clopidogrel and aspirin is common to prevent thrombotic events in conjunction with coronary stenting [4]. Loading doses of clopidogrel reduce the time necessary to achieve impaired ADP induced platelet reactivity [6]. This study shows a considerable heterogeneity of clopidogrel induced platelet inhibition. Some subjects had strong platelet inhibition. It is possible that they are susceptible to bleeding. In contrast, others had inadequate responses upon clopidogrel. They appear to lack protection through medication.

Cardiovascular medication does not appear to cause pharmacodynamic interactions with clopidogrel [8]. Consequently, drug interference does not explain individual differences of platelet inhibition. Noncompliance is an obvious explanation for treatment failure. This trial was performed in a clinical setting with the medications given being documented by nurses. Therefore, most likely it is good compliance with intended medication. Many patients had their angiography early in the morning thus having clopidrogrel after an overnight fast. Other individuals had their loading doses after food intake. Administration of clopidogrel with food does not seem to alter the bioavailability significantly [9] but fasting status may contribute to the individual heterogeneity of platelet inhibition.

Some limitations regarding our data should be considered. The present laboratory study by necessity contains a small number of patients. Much larger clinical trials are needed to ascertain the clinical implications of individual clopidogrel evoked platelet suppression. The concentrations of ADP in the present ex vivo assays could be beyond the physiological range constituting an additional source of error. We do not know how ADP-induced fibrinogen binding translates to clinically effective platelet inhibition and more importantly prevention of thrombotic complications. Most but not all patients had aspirin at admission. Furthermore, 10 patients had prophylactic nitro-glycerine. Both drugs have the potential to affect platelets [2, 10] comprising a further source of error.

The degree of platelet fibrinogen binding after ADP challenge was used as a measure of platelet reactivity and determined with a flow cytometry technique. Soluble P-selectin was used as an estimate of platelet in vivo activity without the stimulus of a platelet-activating agent. The present study confirms a recent report in demonstrating that clopidogrel does not suppress platelet activity as estimated from soluble P-selectin [11]. A possible dissociation exists between platelet P-selectin expression and soluble P-selectin concentrations as the latter is also derived from endothelial cells. Thus, its use as a surrogate marker for platelet activation is open for criticism. The cytokine prevents neutrophil adhesion thus sustaining the nonadhesive property of neutrophils [12]. Clopidogrel may fail to suppress P-selectin deriving from the endothelial cells therefore maintaining the possible anti-inflammatory properties of circulating P-selectin.

Clopidogrel in combination with aspirin provides synergistic effect as compared with the drugs alone. Approximately 10% of the population fail to show platelet inhibition after aspirin exposure [13]. It is possible that an individual with aspirin resistance and impaired clopidogrel induced platelet inhibition has increased risk for adverse events in conjunction with coronary stenting.

In conclusion, the present study shows substantial individual variations after loading doses of clopidogrel. Some subjects had weak platelet responses to clopidogrel most likely having an increased risk for thrombotic events in conjunction with coronary stenting. Others had strong reactions suggesting an increased bleeding tendency. The present flow cytometry technique appears suitable for identifying patients with abnormal clopidogrel evoked platelet inhibition.

Acknowledgements

The County Council of Östergötland, Sweden supported the research.

Ancillary