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Multiple studies have demonstrated that the addition of clopidogrel to lifelong aspirin therapy is effective in reducing the composite endpoint of death from cardiovascular causes, non-fatal myocardial infarction, or stroke in patients undergoing percutaneous coronary intervention (PCI) [1,2]. However, the problem with the ‘one size fits all’ 75 mg/day clopidogrel maintenance therapy is the wide variability in individual responses to it [3]. Therefore, it has been demonstrated that an individual risk-stratification with platelet function tests may identify patients who are at a high risk for the occurrence of atherothrombotic events [4–7]. Light transmission aggregometry (LTA) is generally considered to be the ‘gold standard’ method of measurement of platelet function and it has been suggested that the focus of interpretation of the results from LTA in clopidogrel-treated patients should be the absolute level of late aggregation instead of ‘peak’ aggregation [8]. The rationale behind this theory is the following. Two platelet surface adenosine diphosphate (ADP) receptors have now been characterized: the Gq-protein coupled P2Y1 receptor, which is responsible for platelet shape change, phospholipase C activation and calcium release from internal stores, and the Gi-protein coupled P2Y12 receptor, which is responsible for the inhibition of adenylyl cyclise [9]. The active metabolite of clopidogrel selectively inhibits ADP binding to P2Y12 but not P2Y1. Platelets from patients with a P2Y12 deficiency and platelets from patients with a structural defective P2Y12 are still capable of changing shape and aggregating (although this aggregation is rapidly reversible with loosely bound platelets with few contact points), and fail to attenuate PGE1-induced cAMP production. Therefore, it has been hypothesized that P2Y1 is mainly responsible for the maximal amplitude of platelet aggregation whereas P2Y12 is responsible for the stabilization of aggregation [10–12]. The aim of the present study was to investigate whether a difference exist between ADP-induced peak aggregation and late aggregation in patients who have been treated with an aspirin and clopidogrel maintenance dose for a long period of time (> 5 days).

‘Classical’ LTA induced by four different concentrations (2, 5, 10 and 20 μmol L−1) of ADP was performed in 164 patients who were on a maintenance of 75 mg of clopidogrel for >five days. All patients were also on ≥80 mg of aspirin for at least seven days. The local Institutional Review Board approved the protocol and written informed consent was obtained before elective PCI.

LTA was quantified in non-adjusted platelet-rich plasma (PRP) at maximal (peak) aggregation and after 360 s (late aggregation) with an APACT 4004 four-channel light transmission aggregometer (LABiTec, Arensburg, Germany). Samples were centrifuged for 10 min at 150 × g to obtain native PRP and the platelet count was assessed on a routine cell-counter (LH 750, Beckman Coulter, Krefeld, Germany). Samples tubes containing 3.2% of citrate were used for the analysis of platelet function.

Both peak and late aggregation at 360 s were highly variable between subjects (mean ± SD were 27 ± 11% vs. 12 ± 9%, 46 ± 12% vs. 23 ± 16%, 55 ± 13% vs. 34 ± 21%, and 61 ± 12% vs. 44 ± 22% for 2, 5, 10, and 20 μmol L−1 of ADP, respectively). Furthermore, as illustrated in Fig. 1, peak aggregation was strongly correlated with late aggregation (r = 0.72, 0.76, 0.86, and 0.93, all P < 0.01, for 2, 5, 10, and 20 μmol L−1 of ADP, respectively).

image

Figure 1.  Correlation between adenosine diphosphate (ADP)-induced maximal aggregation (peak aggregation) and the absolute level of aggregation at 360 s (late aggregation) for different concentrations of ADP.

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Multiple studies have demonstrated that patients with an inadequate response to clopidogrel therapy have an increased risk of developing atherothrombotic events [4–6]. However, to date, there is no predefined cutoff level to segregate clopidogrel poor responders from clopidogrel responders. As a consequence, the multiple studies linking clopidogrel poor response with a higher incidence of atherothrombotic events have all used different definitions to allocate clopidogrel poor responsiveness. In addition, some of them use the absolute change from baseline (off-drug aggregation) whereas others use one single measurement after a certain (but adequate) clopidogrel pretreatment period. Importantly, several of these studies are biased because platelet function testing was performed 24 h after the PCI with its consequent additional platelet activation.

Recently, it has been suggested that the absolute level of platelet aggregation after a fixed period of time (e.g. 360 s after the addition of ADP) is a better representation of the inhibitory effects of clopidogrel treatment because P2Y12 is mainly responsible for the stabilization of aggregation and not initiation of aggregation [7]. We found a strong correlation between maximal aggregation and late aggregation as measured by ‘classical’ LTA. This observation again fuels the ongoing discussion about whether peak or late aggregation represents the best pharmacodynamic evaluation of the platelet response to clopidogrel.

In general, LTA remains poorly standardized and every laboratory needs to thoroughly evaluate its own method of performing LTA [13]. This must include batch and brand differences of ADP, concentration of ADP, time between blood drawing and LTA measurements, to adjust or not to adjust the platelet count to a certain standardized platelet count, stirring speed, and type of aggregometer. However, LTA remains eventually poorly reproducible despite the attempts of standardization and it remains difficult to compare and extrapolate the results of the multiple late-breaking studies with the local findings in hospitals. Therefore, it is of great importance that simple, rapid point-of-care platelet function assays are developed that are easier to use and more reproducible. This development will warrant inter-hospital comparisons.

Recently, it has been suggested that D-Phe-Pro-Arg-chloromethylketone (PPACK) is the preferred anticoagulant when testing for clopidogrel responsiveness as citrate decreases the plasma concentration of ionized calcium in the plasma thereby affecting platelet–receptor–agonist interactions and presumably overestimates the true inhibition of platelet function in vivo [8]. Whether the present observation that peak and late aggregation are almost interchangeable also counts when PPACK is used as an anticoagulant needs to be evaluated in future studies.

References

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  4. Disclosure of Conflict of Interests
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Acknowledgements

  1. Top of page
  2. References
  3. Acknowledgements
  4. Disclosure of Conflict of Interests

The authors wish to thank C. A. K. van der Stelt and T. Seesing for their work and assistance in performing platelet function testing.

Disclosure of Conflict of Interests

  1. Top of page
  2. References
  3. Acknowledgements
  4. Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.