Ticagrelor is the first direct-acting and reversibly binding ADP P2Y12 receptor antagonist. It provides faster, greater and more consistent platelet inhibition compared with clopidogrel and has demonstrated superiority over clopidogrel in a large trial of patients with acute coronary syndrome (ACS).1,2 As a result, both European and American Societies of Cardiology recommend ticagrelor over clopidogrel, as first-line therapy for patients with ACS (ST elevation myocardial infarction or moderate to high-risk non-ST elevation ACS) in combination with aspirin.3,4
Like other antithrombotic agents, ticagrelor is associated with an increased risk of spontaneous and perioperative bleeding complications.5 In the rare cases of severe haemorrhage, management of bleeding remains challenging; no antidote is currently available, ticagrelor is not removed by dialysis and platelet transfusion, usually proposed to reverse antiplatelet drugs, has been suggested to be ineffective because circulating ticagrelor and its active metabolite are likely to inhibit the new platelets.6 The few ex-vivo studies performed on blood samples from patients on dual antiplatelet therapy combining aspirin and ticagrelor support this hypothesis.7–10 In addition, we recently reported a case in which platelet transfusion failed to restore platelet aggregation in a ticagrelor-treated patient requiring emergent surgery.11 There is, however, no comprehensive demonstration of the effectiveness of platelet transfusion for the reversal of ticagrelor. We designed an in-vitro study to assess the effects of platelet supplementation on ticagrelor-induced inhibition of platelet aggregation using aggregometry on whole blood and platelet-rich plasma. We also report a second clinical case of platelet transfusion in a ticagrelor-treated patient.
Material and methods
Blood samples were obtained from the French Blood Bank Institute (Etablissement Français du Sang, EFS) according to the agreement between the EFS and the Paris Descartes University (convention ref. C CPSL UNT n°12/EFS/038). The EFS delivered anonymised blood samples after blood donors gave written informed consent that specifies the exclusive research purpose and the respect of ethical guidelines. Blood samples were obtained from 24 healthy donors who had not taken antiplatelet or nonsteroidal anti-inflammatory drugs during the past 10 days. Blood samples were collected in 0.105 M sodium citrate tubes (BD Vacutainer, Franklin Lakes, New Jersey, USA) or acid citrate dextrose (ACD) tubes (BD Vacutainer, citric acid 5.7 nM, trisodium citrate 11.2 mM and dextrose 20 mM, final concentrations) to prepare washed platelet suspension.12 Whole blood platelet count was measured for each sample (MS9-3, Melet Scloesing, La Chaux de Fonds, Switzerland).
Ticagrelor was provided by AstraZeneca (Mölndal, Sweden) and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg ml−1 stock solution, which was diluted in Tris buffer saline (1/100). Serial dilutions in Tris buffer saline and 1% DMSO were performed to obtain the working solutions. The final DMSO concentration in all test samples was 0.026%, a value shown under our experimental conditions not to have any influence on platelet aggregation (data not shown). Blood samples were preincubated with ticagrelor for 15 min before experiments. The 3.25 μM final ticagrelor concentration was chosen after a dose-ranging study.
Aspirin was used as a positive control for the effect of platelet supplementation. Aspirin solution (Aspegic Injectable 500 mg; Sanofi-Aventis, Paris, France) was diluted in 0.9% saline to obtain a 25 μM aspirin solution. Blood samples were preincubated with aspirin for 60 min before the experiments. This concentration, also used by others, was selected after a dose-ranging study performed in preliminary experiments, as the lowest dose that inhibited at least 80% of platelet aggregation in all samples using both impedance aggregometry and light transmission aggregometry (LTA).13
Blood samples collected on ACD samples were mixed with wash buffer (citric acid 36 mM, glucose 5 mM, KCl 5 mM, CaCl2 2 mM, MgCl2 1 mM and NaCl 103 mM; pH = 6.5) containing 2.10−7 μM prostaglandin E1 and 0.06 IU ml−1 apyrase (Sigma Aldrich, St. Louis, Missouri, USA) to avoid unintended platelet activation during centrifugation. The first spin (216 G for 11 min at room temperature) produced platelet-rich plasma. Wash buffer was added before the second centrifugation (1200 G for 11 min) that yielded a platelet pellet and supernatant platelet-poor plasma. The platelet pellet was resuspended in suspension buffer (Hepes 10 mM, NaCl 140 mM, KCl 3 mM, NaHCO3 5 mM, MgCl2 0.5 mM, and glucose 10 mM; pH = 7.35) to a final concentration of 4000 ×109 l−1.
Platelet aggregation was performed in whole blood using impedance aggregometry on a Chrono-log aggregometer (Model 700; Chrono-log Corporation, Havertown, Pennsylvania, USA), which measures the increase in electrical resistance between two electrodes immersed in a diluted sample of whole blood caused by the platelet adhesion to the electrodes and the subsequent platelet aggregation. Warm 0.9% saline (37°C) was added to 475 μl whole blood (v/v 1/1) preincubated with ticagrelor, aspirin or vehicle. Aggregation was induced by 20 μM ADP or 1 mM arachidonic acid, for ticagrelor or aspirin-spiked samples, respectively. Results were expressed in maximum aggregation in ohms during a 6 min record.
Platelet aggregation was also performed in platelet-rich plasma using LTA on the Chrono-Log aggregometer. LTA measures the changes in transmission of a beam of light through a sample of platelet-rich plasma that occur when platelets aggregate upon stimulation. Whole blood incubated with ticagrelor, aspirin or vehicle was centrifuged to obtain, successively, platelet-rich (100 G, 11 min) and platelet-poor plasma (2630 G, 20 min). Platelet-rich plasma was adjusted to 300 ×109 l−1. Aggregation was induced in the platelet-rich plasma samples using 20 μM ADP or 1 mM arachidonic acid, for ticagrelor or aspirin-spiked samples, respectively. Results were expressed in percentage of final aggregation, after a 6 min record.
Dose-ranging study with ticagrelor (preliminary study)
A preliminary dose-ranging study was performed to determine the concentration to use in the main study. We defined it as the lowest concentration that inhibited at least 80% of platelet aggregation in all samples. Whole blood (2 ml) was spiked with 52 μl ticagrelor working solutions to obtain final concentrations of 0.2, 0.4, 0.81, 1.63, 3.25, and 6.5 μM, or vehicle and incubated for 15 min at room temperature. Impedance aggregometry was performed on six samples.
Effects of platelet supplementation (main study)
Platelet supplementation was performed by adding 75 μl fresh concentrated washed platelet suspension to whole blood for 15 min at room temperature. This volume was chosen to increase sample test platelet count by at least 60% and guarantee the presence of more than 100 ×109 l−1 fresh platelets. Impedance aggregometry (n = 6) and LTA (n = 6) were performed on whole blood and platelet-rich plasma in both ticagrelor and aspirin-spiked samples. We also used washed platelets as negative control pretreated in vitro with ticagrelor (3.25 μM for 15 min) or aspirin (25 μM for 60 min) before being added to ticagrelor or aspirin-spiked samples, respectively.
Data management was performed using Microsoft Excel 2008 (Microsoft Corporation, Redmond, Washington, USA) and statistical analysis using GraphPad PRISM (version 4 for Windows; GraphPad Software, San Diego, California, USA). Platelet count values are reported as mean ± standard deviation. Other quantitative data are reported as median (range) or box-and-whisker plots showing median, 25 and 75% percentiles, minimum and maximum values. Nonparametric tests were used for comparisons. Variables were compared using the Friedman test, followed by the Wilcoxon test when significant P values less than 0.05 in two-tailed tests were required to reject the null hypothesis.
In the dose-ranging study (n = 6), ticagrelor concentration dependently inhibited ADP-induced platelet aggregation in whole blood (Fig. 1); 3.25 μM ticagrelor was the lowest concentration that inhibited at least 80% of platelet aggregation in all samples (from 80.2 to 100%), thus this concentration was chosen for further experiments (main study). The main study confirmed that this condition was satisfied. Platelet aggregation was strongly decreased in ticagrelor-spiked samples compared with control in whole blood [2 (1.3 to 2.8) vs. 13 (10.8 to 13.8) Ω, P < 0.05, n = 6] and in platelet-rich plasma [15 (10 to 17) vs. 75 (72 to 81)%, P < 0.05, n = 6].
As expected, 25 μM aspirin clearly inhibited arachidonic acid-induced platelet aggregation in whole blood [1 (0.3 to 1.8) vs. 7.5 (6.3 to 9.5) Ω, P < 0.05, n = 6] and in platelet-rich plasma [5 (4.3 to 5.8) vs. 77.5 (66.5 to 78.8)%, P = 0.01, n = 6]. Platelet supplementation increased platelet count from 267 ± 50 to 431 ± 20 ×109 l−1 and 225 ± 20 to 385 ± 25 ×109 l−1 in whole blood and platelet-rich plasma, respectively, and normalised platelet aggregation both in whole blood [10 (8.5 to 10.8) vs. 1 (0.3 to 1.8) Ω, P = 0.008] and platelet-rich plasma [73 (63.8 to 76.3) vs. 5 (4.3 to 5.8)%, P = 0.01] (Fig. 2a and b). As a control, aspirin-pretreated platelet supplementation failed to restore aggregation using whole blood and platelet-rich plasma. We also verified that platelet supplementation did not modify control platelet aggregation.
Platelet supplementation increased the platelet count from 231 ± 26 to 427 ± 39 ×109 l−1 in whole blood and 218 ± 15 to 352 ± 15 ×109 l−1 in platelet-rich plasma. In 3.25 μM ticagrelor-spiked samples, platelet supplementation failed to correct ticagrelor-induced inhibition of ADP-triggered platelet aggregation as compared with control, both in whole blood [2 (0.5 to 2.8) vs. 2 (1.3 to 2.8) Ω, P > 0.05] and platelet-rich plasma [13.5 (12.5 to 15.5) vs. 15 (10 to 17)%, P > 0.05] (Fig. 3a and b). As expected, supplementation with ticagrelor-pretreated platelets had no corrective effect. We also verified that platelet supplementation did not modify control platelet aggregation.
Platelet transfusion failed to restore platelet aggregation in blood from a ticagrelor-treated patient: a case report
A 52-year-old man (weight 87 kg) with type II diabetes mellitus presented to a local hospital complaining of tearing chest pain. A diagnosis of non-ST segment elevation myocardial infarction was made and he received ticagrelor 180 mg, aspirin 250 mg and heparin 5000 IU. Coronary angiography revealed significant stenoses in three major coronary arteries requiring surgical revascularisation. The patient was transferred for surgical management. Coronary artery bypass grafting was performed using cardiopulmonary bypass (CPB), with standard heparin anticoagulation and 4 g tranexamic acid for routine bleeding prophylaxis. He received three units of red blood cells during the 80 min of CPB and nine units of platelets at the end of CPB in an attempt to reverse the effects of antiplatelet therapy. During the next hour, two units of fresh-frozen plasma were also transfused. Postoperative haemorrhagic drainage was 800 ml. The patient was discharged on postoperative day 7.
Platelet function was investigated with LTA on platelet-rich plasma using 20 μM ADP as an activator at three time points (before surgery and before and after platelet transfusion) corresponding to 14, 17.5 and 18 h, respectively, after ticagrelor administration (Fig. 4). Platelet transfusion increased platelet count from 95 to 140 ×109 l−1 but had no effect on inhibition of ADP-induced platelet aggregation.
This in-vitro study demonstrated that a large platelet supplementation failed to restore ADP-induced platelet aggregation in ticagrelor-spiked samples. These data support the hypothesis that platelet transfusion might be ineffective for the reversal of ticagrelor, because of the unique pharmacologic properties of this antiplatelet agent.6,11 First, unlike thienopyridines, ticagrelor is directly active, without the requirement for metabolic activation to exert its antiplatelet effect. It also has a circulating active metabolite, representing about one-third of the concentration of the parent compound, which is as potent as ticagrelor at blocking the P2Y12 receptor. Second, plasma concentrations of ticagrelor are high, with a maximum drug concentration of 931 ± 474 ng ml−1 after 180 mg loading dose (1.77 μM), whereas the maximum drug concentration of the clopidogrel active metabolite after 300 mg loading dose is 17.82 ± 20.38 ng ml−1.14,15 Third, ticagrelor and its active metabolite have long half-lives of 7 and 8.5 h, respectively, whereas the half-lives of the active metabolites of aspirin and clopidogrel are 20 and 30 min, respectively.16,17 Finally, ticagrelor reversibly binds to the platelet P2Y12 receptor, in a constant balance between plasma and receptors [inhibitory constant (Ki) 4 nM]. Thus, circulating ticagrelor and its active metabolite are likely to redistribute and inhibit newly introduced platelets in vitro as well as transfused platelets in clinical practice.
Our results are in line with the previously published data, despite apparently conflicting conclusions. Hobl et al.8 concluded that platelet supplementation ex vivo was able to reverse ticagrelor in healthy individuals after a 180 mg loading dose; the addition of increasing amounts of platelet-rich plasma in a dose-dependent manner improved ADP-induced platelet aggregation assessed with multiple electrode aggregometry. Nevertheless, even a massive supplementation obtained by the addition of platelet-rich plasma in a ratio of 1 : 3 failed to return platelet activity to baseline in 12 of 20 volunteers. Hansson et al.7 also concluded that platelet supplementation improved platelet aggregability in whole blood samples from patients treated with aspirin and ticagrelor using multiple electrode aggregometry. Although increasing the platelet concentration by 138 ×109 l−1 significantly improved ADP-induced aggregability, it only reached 37% of normal values and was markedly lower than a suggested cutoff level for an increased bleeding risk in cardiac surgery patients.18 Similarly, Scharbert et al.9 demonstrated ex vivo that whole blood or platelet-rich plasma from ticagrelor-treated patients completely abolished ADP-mediated platelet functions of blood from healthy volunteers, as measured by flow cytometry, LTA and impedance aggregometry. Recently, Bonhomme et al.10 reported that mixing increasing amounts of platelet-rich plasma from healthy volunteers with platelet-rich plasma from ACS patients treated with ticagrelor and aspirin failed to correct ADP and TRAP-induced platelet aggregation using LTA. Moreover, in a rat model, platelet transfusion showed no influence on ticagrelor-induced prolongation of tail bleeding time.19 Last, in a patient taking ticagrelor in combination with aspirin and requiring emergent surgery, we previously demonstrated that the transfusion of 17 units of platelets increased the platelet count but failed to restore platelet aggregation. This illustrated the ineffectiveness of platelet transfusion in reversing the effects of ticagrelor using VerifyNow (Accumetrics) and vasodilator-stimulated phosphoprotein assays.11
Our study complements previous ones in that we specifically focused on ticagrelor/platelet interactions in a mechanistic and translational approach.7–9,18 We evaluated platelet aggregation through two complementary methods, using both whole blood and platelet-rich plasma. Platelet-rich plasma aggregometry, which is poorly influenced by the platelet count when normal, is considered the gold standard for testing platelet function, whereas aggregation on whole blood, although strongly dependent on the platelet count, allows a more global analysis.20,21 In contrast to other studies, we used ticagrelor alone in order to avoid the synergic inhibitory effects of aspirin upon platelets. We also used an in-vitro model to explore the reversal of the ticagrelor parent drug and not its active metabolite produced from cytochrome P450-mediated metabolism. We carefully selected the 3.25 μM ticagrelor concentration by performing a dose-ranging study. This concentration is higher than those reported in pharmacological studies performed with therapeutic doses.14,16 The choice of the concentration, however, was driven by its effect on platelet aggregation rather than on the concentration itself. Indeed, in this in-vitro study, we selected the concentration that led to 80% inhibition of platelet aggregation, which is close to that observed in clinical practice.16,22
The case report adds clinical relevance to the in-vitro data. It showed similar results using the same assays; namely that perioperative platelet transfusion increased the platelet count substantially but did not correct ADP-induced aggregometry in a ticagrelor-treated patient. This case complements our earlier publication, reporting the ineffectiveness of platelet transfusion to reverse ticagrelor as assessed by two other platelet tests: VerifyNow, a point-of-care device that measures platelet aggregation in whole blood, and the vasodilator-stimulated phosphoprotein assay to specifically monitor P2Y12 receptor function.11
These results, suggesting that platelet transfusion is unlikely to reverse ticagrelor, may impact the clinical management of ticagrelor-induced bleeding and encourage physicians to consider other therapeutic options. A specific antidote is currently in development.23 This monoclonal antibody fragment binds to ticagrelor and its main circulating active metabolite to reverse their antiplatelet activity. Whilst approval for this antidote is awaited, nonspecific haemostatic agents may be proposed. The summary of the product characteristics suggests using recombinant activated factor VII or tranexamic acid to reduce ticagrelor-bleeding risk as these agents are likely to improve haemostasis.6 However, there are few data to support this recommendation.
Regarding aspirin reversal, efficacy of platelet supplementation is more substantiated. Ex vivo, Li et al.24 showed that 30 to 40% uninhibited platelets are required to restore LTA platelet aggregation in the presence of aspirin. Hansson et al.7 demonstrated that arachidonic acid-induced aggregation was restored and increased above baseline levels after platelet supplementation corresponding to the transfusion in vivo of two to five units of apheresis platelets. In vivo, Taylor et al.25 observed that a mean platelet transfusion dose of 0.13 IU kg−1 (0.10–0.15 IU kg−1) restored platelet function as assessed by VerifyNow assay in neurosurgical patients receiving aspirin. Finally, a double-blind, randomised controlled trial was performed to assess the effectiveness of platelet transfusion to reverse aspirin in patients requiring emergent craniotomy.24 This study showed a significant reduction in postoperative haemorrhage, disability and mortality with platelet transfusion compared with control in aspirin-sensitive patients. Regarding P2Y12 antagonists, platelet transfusion is also usually recommended despite a low-level of supporting evidence, even for clopidogrel.26
Our study has several limitations. First, it is an in-vitro study with inherent limitations, including the extent to which the results can be translated into the clinical setting. Second, although ticagrelor is usually prescribed in association with aspirin, we used it in isolation because it was more relevant for a mechanistic approach. This is also why we conducted experiments using the most widely used concentrations of ADP and arachidonic acid for ticagrelor and aspirin, respectively. Regarding platelet supplementation, we used fresh washed platelets instead of stored platelet concentrates that are more clinically relevant but are known to lose efficacy over time because of change in activation status and aggregability. Moreover, we added a massive amount of platelets; the increase in platelet count was calculated to correspond to the increase in platelet count achieved by in-vivo transfusion of five units of apheresis platelet concentrates. One unit of apheresis platelet concentrate provides approximately 50 ×109 l−1 functional platelets and this is usually sufficient to ensure haemostasis. However, even in these optimal conditions, platelet supplementation was unable to reverse the effects of ticagrelor.
In conclusion, in this in-vitro study, platelet supplementation failed to restore platelet aggregation in samples preincubated with ticagrelor. These results question the effectiveness of platelet transfusion for the reversal ticagrelor in patients and suggest that a specific antidote is required.
Acknowledgements relating to this article
Assistance with the study: the authors would like to thank Valérie Dias (Universite[Combining Acute Accent] Paris Descartes) for the linguistic support.
Financial support and sponsorship: Ticagrelor was kindly provided by AstraZeneca (Mölndal, Sweden). The study was otherwise funded by institutional sources, by grants from CSL Behring and the Conny Maeva Charitable Foundation. In 2014, ACM received a prize from the Société Française d’Anesthésie Réanimation for this work.
Conflicts of interest: CMS has been a lecturer for AstraZeneca. No conflicts of interest for remaining authors. This work was performed at Inserm UMR-S1140, Paris, France.
Comment from the editor: CMS is an Associate Editor of the European Journal of Anaesthesiology.
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