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

2009; Elsevier BV; Volume: 7; Issue: 11 Linguagem: Inglês

10.1111/j.1538-7836.2009.03585.x

1538-7933

C.A.C.M. Pittens, Heleen Bouman, Jochem W. van Werkum, Jurriën M. ten Berg, C. M. Hackeng,

Atrial Fibrillation Management and Outcomes

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 associated with worsened clinical outcome. The prevalence of this phenomenon varies widely, and is highly dependent on the type of platelet function test and the type of anticoagulant that is being used [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 whole-blood 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, thereby maintaining 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 the MEA 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 × 109 L−1, 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 μg mL−1, Refludan, hirudin blood collection tubes; Dynabyte, Munich, Germany), after the first 5 mL of blood had been discarded. Aliquots of both the citrate-anticoagulated and the hirudin-anticoagulated blood samples were spiked with increasing concentrations of the direct P2Y12 receptor antagonist cangrelor (The Medicines Company, Parsippany, NJ, USA; in final subtherapeutic concentrations of 0.015 μmol L−1 and 0.15 μmol L−1) or 0.9% saline (for baseline measurements), and incubated for exactly 5 min. Platelet function testing was performed in parallel using LTA (using 20 μmol L−1 ADP induced peak and late aggregation) [10], the PFA-100 Collagen/ADP (CADP) cartridge and the novel PFA-100 cartridge INNOVANCE PFA P2Y* (product under development – not available for sale) (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany), the VerifyNow P2Y12 assay (VN; Accumetrics, San Diego, CA, USA), the Multiplate analyzer (also referred to as MEA) (Dynabite, Munich, Germany) ADP test (20 μmol L−1 ADP) and the high-sensitivity ADP test (ADP HS test, 20 μmol L−1 ADP + 9.4 nmol L−1 prostaglandin E1). Detailed descriptions of these tests have been published elsewhere [1, 3]. All tests were performed at 1 h after blood sampling and according to the manufacturers' recommendations. The membrane of the INNOVANCE PFA P2Y* is coated with ADP, prostaglandin E1, and calcium chloride. Differences between citrate-anticoagulated and hirudin-anticoagulated blood samples were determined using paired Student's t-test and Pearson correlation. For PFA-100 results, the non-parametric Mann–Whitney U-test and Spearman correlation were used. PFA-100 results reported as > 300 s were displayed as 300 s in the analyses. As shown in Fig. 1, absolute aggregation values obtained with LTA and VN revealed a significantly higher magnitude of platelet aggregation when measured in citrate than when measured in hirudin. In hirudin-anticoagulated and citrate-anticoagulated blood samples, respectively, peak platelet aggregation was 64% ± 19% and 84% ± 6% for baseline (P < 0.01), and 38% ± 13% vs. 60% ± 21% after in vitro addition of 0.015 μmol L−1 cangrelor (P = 0.01) and 6.4% ± 2.7% vs. 18% ± 8.8% after addition of 0.15 μmol L−1 cangrelor (P < 0.001). Similar results were obtained for late platelet aggregation values. Inhibition of platelets function by cangrelor as measured with the various platelet function tests. The effects of in vitro addition with cangrelor (in final concentrations of 0.015 μmol L−1 and 0.15 μmol L−1) when measured in either citrate or hirudin are shown. P2Y12 reaction units, obtained with VN, were 194 ± 52 vs. 257 ± 51 for hirudin and citrate, respectively, for baseline measurements (P < 0.01), 132 ± 55 vs. 184 ± 85 after in vitro addition of 0.015 μmol L−1 cangrelor (P = 0.01), and 17 ± 25 vs. 19 ± 17 after addition of 0.15 μmol L−1 cangrelor (P = not significant). In both tests, the absolute difference in the magnitude of ADP-induced aggregation between hirudin and citrate declined with increasing concentrations of cangrelor. Thrombin receptor-activating peptide (TRAP)-induced platelet aggregation (represented by the VN 'BASE' value) was also highly affected by the anticoagulant agent used (Fig. 1C). Remarkably, the absolute magnitude of TRAP-induced platelet aggregation in either hirudin or citrate was not affected by cangrelor. For the CADP cartridge, baseline closure times were 101 ± 19 s in hirudin vs. 97 ± 13 s in citrate (P = not significant). A notable prolongation of the closure times to 188 ± 76 s and 155 ± 62 s, for hirudin and citrate, respectively, was observed after incubation with 0.15 μmol L−1 cangrelor (P = not significants). Similar results were obtained with INNOVANCE PFA P2Y*, with closure times of 72 ± 15 s and 69 ± 9 s at baseline (P = not significant) and 272 ± 57 s and 290 ± 32 s after 0.15μmol L−1 cangrelor (P = not significant) for hirudin and citrate, respectively. However, the magnitude of closure time prolongation after in vitro cangrelor addition was much higher with INNOVANCE PFA P2Y* than with the CADP. In contrast to the results of the other platelet function assays, results obtained with MEA demonstrated an inverse trend towards a higher magnitude of platelet aggregation when measured in hirudin-anticoagulated blood than when measured in citrate-anticoagulated blood. The baseline area under the curve (AUC) of the MEA ADP test was 49.5 ± 23.6 for hirudin and 40.6 ± 15.7 for citrate. The AUC decreased after in vitro addition of increasing concentrations of cangrelor, but a trend towards a higher AUC in hirudin was still present (Fig. 1G). The results obtained with the MEA ADP HS test showed similar trends (Fig. 1H). Importantly, a strong intraindividual correlation was observed between hirudin-anticoagulated and citrate-anticoagulated blood samples (r = 0.84 for LTA peak aggregation, r = 0.79 for LTA late aggregation, r = 0.88 for VN, r = 0.72 for the MEA ADP test, and r = 0.63 for the MEA ADP HS test, with P < 0.0001 for all). Results of the PFA-100 collagen/ADP and INNOVANCE PFA P2Y* were, respectively, r = 0.57 and r = 0.61, with P < 0.0001 for both. The present study was designed to study the impact of different anticoagulants on the test performance of multiple platelet function assays. Clearly, our results extend the current knowledge that the nature of the anticoagulant used influences platelet aggregation when measured with various platelet function methods. Results obtained with both LTA and VN demonstrated a higher magnitude of platelet reactivity when measured in citrate than when measured in hirudin. These results are in agreement with previous findings of Labarthe et al. and a previous finding from our group [5, 7]. In contrast, results obtained with the PFA-100 CADP cartridge and the PFA-100 INNOVANCE PFA P2Y* cartridge were not significantly influenced by the anticoagulant used. Interestingly, the results obtained with MEA are opposite to those obtained with the other platelet aggregation tests, as a higher AUC was measured in hirudin than in citrate. Whether this effect might be due to the higher conductivity of the hirudinized blood samples than of the citrated blood samples or, indeed, to increased platelet functionality remains to be clarified [8, 9]. Another interesting finding is the fact that the novel PFA-100 INNOVANCE PFA P2Y* cartridge has an increased sensitivity to P2Y12 receptor inhibition than the CADP cartridge, as INNOVANCE PFA P2Y* revealed a more pronounced prolongation of the closure time, in particular after in vitro addition of relatively low concentrations of cangrelor. More studies are underway to define the sensitivity of the PFA-100 INNOVANCE PFA P2Y* cartridge to the effects of different P2Y12 inhibitors (e.g. clopidogrel, prasugrel, and ticagrelor). Three limitations of the present study need to be acknowledged. First, the study did not explore relationships between platelet function test findings and clinical outcomes, as platelet function was evaluated using healthy control samples, spiked with a P2Y12 inhibitor ex vivo. Second, platelet function analysis was performed in the absence of acetylsalicylic acid. Therefore, it remains to be determined whether the findings would be the same in aspirin-treated subjects. Third, although the present study contains a sufficient number of subjects to study the effects of different anticoagulants on the magnitude of platelet reactivity, it does not allow for an answer to be given to the question of which of these tests – if any – is the most predictive for the (re)occurrence of atherothrombotic events. In conclusion, the present study confirms that the results of platelet function assays are highly dependent on the type of ex vivo anticoagulation. Methods based solely on aggregation, such as 'classic' LTA and VN, revealed a higher magnitude of platelet aggregation when measured in citrate than when measured in hirudin. In contrast, MEA demonstrated a higher magnitude of aggregation when hirudin was used as anticoagulant than when citrate was used as anticoagulant. The authors state that they have no conflict of interest.