Comparison between hirudin and citrate in monitoring the inhibitory effects of P2Y12 receptor antagonists with different platelet function tests

Dual treatment with aspirin and clopidogrel is currently the therapy of choice in the prevention of recurrent cardiovascular events following coronary stent implantation. However, not all patients benefit equally from this antiplatelet regimen, as inadequate suppression of platelet aggregation by clopidogrel and/or aspirin – also referred to as high on-treatment platelet reactivity or antiplatelet therapy resistance – is associatedwith worsened clinical outcome. The prevalence of this phenomenon varies widely, and is highly dependent on the type of platelet functiontestandthetypeofanticoagulantthat isbeingused[1,2]. Classic light transmission aggregometry (LTA), using ADP as agonist, is considered to be the reference standard platelet function test for determining the effectiveness of clopidogrel. Its use in daily practice is, however, not viable, owing to important limitations [3]. To overcome these limitations, various wholeblood platelet function assays designed to evaluate the efficacy of P2Y12 receptor antagonism have been introduced to the commercial market. Trisodium citrate dihydrate is the preferred anticoagulant agent for platelet function testing, as it has been in use for more than 40 years. However, measurements in citrate do not resemble physiologic conditions, as citrate inhibits coagulation by chelating calcium. This could result in an underestimation or overestimation of the true inhibitory effects of P2Y12 receptor antagonists in vivo. Other anticoagulants, such as hirudin and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK), prevent coagulation of blood by direct inhibition of thrombin, therebymaintaining physiologic calcium levels [4]. This approach might therefore mimic the in vivo situation of platelet function more precisely than anticoagulation with citrate. Previous studies on this issue have demonstrated a generally higher magnitude of ADP-induced platelet aggregation in blood anticoagulated with citrate than in blood anticoagulated with PPACK or hirudin when measured with classic LTA [5–7]. The manufacturers of the multiple-electrode aggregometry (MEA) assay strongly recommend the use of hirudin instead of citrate as an anticoagulant agent. Two recent publications reported a reduced area under theMEA curve, which indicates a lower magnitude of platelet aggregation, when platelet function was measured in citrated blood than when it was measured in hirudin-anticoagulated blood [8,9]. This finding is opposite to the results obtained with classic LTA when measurements are made in citrate vs. PPACK (or hirudin) [5,7]. For the whole-blood assays determining the efficacy of P2Y12 receptor antagonists, no information regarding the effects of anticoagulants other than citrate on the test performance is currently available. The aim of the present study was to compare the effects of the anticoagulants citrate and hirudin on the test performance of four different platelet function assays that are currently in use for the monitoring of platelet inhibition with P2Y12 receptor antagonists. Seventeen healthy volunteers, nine male and eight female, without coronary artery disease were enrolled after written informed consent was obtained. The mean ages were 28.9 ± 7.2 years and 35.9 ± 13.3 years for males and females, respectively. Subjects were ineligible if they had used any medication in the past 7 days known to influence platelet function. Other exclusion criteria were a known platelet dysfunction or bleeding disorder, a platelet count < 150 · 10 L, and age < 18 years. To avoid platelet activation, blood was drawn without a tourniquet from the antecubital vein into tubes containing either 3.2% trisodium citrate or lepirudin (25 lg mL, Refludan, hirudin blood collection tubes; Dynabyte, Munich, Germany), after the first 5 mLof blood had been discarded. Aliquots of both the citrateanticoagulated and the hirudin-anticoagulated blood samples were spiked with increasing concentrations of the direct P2Y12 Correspondence: JochemW. van Werkum, Department of Cardiology, St Antonius Hospital, 3435 CM Nieuwegein, the Netherlands. Tel. : +31 306099111; fax: +31 306034420. E-mail: w.van. werkum@antonius.net