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Therapeutic failure of antiplatelet drugs has elicited considerable interest to search for surrogate markers to predict therapeutic benefit from new pharmaceutical agents. The Platelet Function Analyzer (PFA-100) is a well-standardized high shear dependent device [1]. However, until recently there was little evidence that the PFA-100 could be used to detect the effect of clopidogrel on platelets. This concept was based on cross-sectional studies. In contrast, recent longitudinal studies showed that the PFA-100 is sensitive to clopidogrel in a subset of stroke patients [2], and that it even predicts adverse outcome in patients following percutaneous interventions of the lower limbs [3]. We hypothesized that there is a considerable time lag before relevant clopidogrel effects would set in, and thus aimed to characterize the time course of platelet inhibition by clopidogrel in stroke patients over a 4-week period. The trial was approved under a broad provision of the Henry Ford Hospital Institutional Review Board, and was a prospective study in 15 ischemic stroke patients > 18 years of age, with normal blood counts. The attending physicians initiated either a therapy according to his or her discretion with clopidogrel (75 mg day−1; Plavix; Sanofi, New York, NY, USA, n = 9) alone or in combination with aspirin (325 mg day−1, n = 6) within 1 week after the acute event. During the clopidogrel treatment no concomitant heparin or thrombolytic agents were administered.

Blood samples were taken with butterfly needles into 3.2% citrate containing Vacutainer tubes (Becton Dickinson, San Jose, CA, USA) at times indicated in Fig. 1 and platelet function was assessed with the Food and Drug Administration-approved PFA-100 device [1]. Patients with collagen adenosine diphosphate closure time (CADP-CT) values above the normal range of 107 s were considered as responders.

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Figure 1. Effects of clopidogrel monotherapy (75 mg day−1, solid symbols, n = 9) or in combination with aspirin (325 mg day−1, open symbols, n = 6) on closure times (CT) measured with the platelet function analyzer (PFA-100): collagen/epinephrine CT (CEPI-CT, top layer) and collagen/adenosine diphosphate CT (CADP-CT, bottom layer) in patients suffering from ischemic stroke. Clopidogrel with/without aspirin markedly increased CEPI-CT and CADP-CT (median of monotherapy depicted by solid line, and median of combination therapy by dotted line). Data points are the mean of two replicates. Every individual patient is presented by the same symbol throughout both layers, and symbols are slightly offset between groups for clarity of presentation. Notably, there was at least a 12-day time lag before the onset of the effect, and maximal values were not seen before 4 weeks of clopidogrel monotherapy.

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There was a considerable time lag (Fig. 1) before clopidogrel prolonged collagen epinephrine closure time (CEPI-CT) by a maximum of 104%[confidence interval (CI) 71, 137] and CADP-CT by a maximum of 205% (CI 143, 266; P = 0.008, Wilcoxon test). While this prolongation was seen in all patients, there was a clear interindividual variability (CV% 31%) in the maximal CADP-CT.

Combination therapy with aspirin and clopidogrel prolonged CEPI-CT by 144% (CI 96, 192) already after 5 days (P = 0.04, Fig. 1). CEPI-CT was maximally prolonged in all patients (> 300 s, corresponding to an average 195% increase) from 12 to 26 days under combined treatment. CADP-CT increased by a maximum of 173% (CI 63, 282, P = 0.04) after 26 days of treatment. Combination therapy more effectively prolonged CEPI-CT compared with clopidogrel monotherapy (P < 0.001 for Mann–Whitney U-test between groups), whereas no consistent difference was seen for CADP-CT between groups. The CADP-CT was slightly more prolonged in the clopidogrel-only group than in the combined treatment group from day 12 to 25. However, this observation may be due to the small number of subjects evaluated.

Our data confirm and extend previous results in patients with stroke [2] or peripheral arterial vascular disease [3]. The rate of responders appeared to be higher than in a previous stroke population [2], which could be due to differences between study populations. The CEPI-CT prolongation by clopidogrel indicates a potentiating effect of released ADP on epinephrine-induced platelet activation under high shear [4]. Most importantly, we found a time lag of almost 2–4 weeks before the effect of clopidogrel on CT-values was maximal (Fig. 1), when therapy was initiated with a maintenance dose of 75 mg. Notably, single doses of 100 mg clopidogrel inhibited ADP-induced aggregation, but 600 mg were needed to inhibit collagen-induced aggregation [5]. Steady-state inhibition of the former by 50–60% is reached after 4–7 days [6] because clopidogrel is used routinely at 1 × the dose necessary to inactivate the P2Y12 receptor fully upon repeated dosing [7]. Moreover, 75 mg is only about 1/5 of the dose necessary to achieve maximal inhibition of ADP-induced platelet aggregation after single dosing.

Recently a loading dose of 600 mg clopidogrel [before percutaneous coronary interventions (PCI)] was found to acutely prolong CADP-CT 2–3-fold within 24 h [8]. Similarly, a prolongation of CT values could be seen only after a loading dose of 300 mg clopidogrel, but not with the maintenance dose of 75 mg in such patients during the first 5 days of treatment [9]. Our data now extend these observations for the 75-mg dose to a stroke population with a longer duration of observation. While a loading dose is currently not recommended for the treatment of stroke patients, it is expected to increase the therapeutic efficacy in the acute setting.

Based on our data under high shear rates, starting clopidogrel monotherapy with a maintenance dose of 75 mg may not yield desirable antiplatelet effects during or shortly after the acute phase of stroke. Although CADP-CT has recently been shown to be of predictive value in a small population of patients with peripheral arterial occlusive disease [3], an association of CT values with stroke reoccurrence has been investigated in only one cross-sectional study [10]. Hence, stroke trials should investigate whether the variability in the PFA-100 response to clopidogrel correlates with clinical outcome.

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