http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, 2015; 26(4): 324–330 ! 2015 Informa UK Ltd. DOI: 10.3109/09537104.2015.1035247

ORIGINAL ARTICLE (FAST TRACK)

Impact of non-inhibited platelet supplementation on platelet reactivity in patients treated with prasugrel or ticagrelor for an acute coronary syndrome: An ex vivo study Fanny Bonhomme1, Robert Bonvini2, Jean-Luc Reny3,4, Antoine Poncet5, & Pierre Fontana4,6 1

Division of Anesthesiology, Geneva University Hospitals, Geneva, Switzerland, 2Division of Cardiology, Geneva University Hospitals, Geneva, Switzerland, 3Division of Internal Medicine, and Rehabilitation, Trois-Cheˆne Hospital, Geneva University Hospitals, Geneva, Switzerland, 4Geneva Platelet Group, Faculty of Medicine, Geneva, Switzerland, 5Division of Clinical Epidemiology, Faculty of Medicine, Geneva University Hospitals, Switzerland, and 6Division of Angiology and Hemostasis, Geneva University Hospitals, Geneva, Switzerland

Abstract

Keywords

Managing bleeding in patients receiving P2Y12 inhibitors is challenging. Few data are available regarding the efficacy of platelet transfusion in patients treated with prasugrel or ticagrelor. The aim of this study was to evaluate the minimal amount of platelet supplementation (in terms of ratio of non-inhibited platelets to inhibited platelets) necessary to restore platelet reactivity in platelet-rich plasma (PRP) of patients treated with aspirin and a prasugrel or ticagrelor loading dose for an acute coronary syndrome. PRP samples from patients were mixed ex vivo with increasing proportions of pooled PRP from healthy volunteers. Platelet reactivity was challenged with adenosine diphosphate (ADP), arachidonic acid, collagen or thrombin receptor activating peptide using light transmission aggregometry. The primary endpoint was the proportion of patient samples recovering an ADP-induced maximal aggregation (ADP-Aggmax) value above 40%. In patients treated with prasugrel (n ¼ 32), ADP-Aggmax increased progressively with supplements of pooled PRP, with an average increase of 7.9% (95% CI [7.1; 8.8], p50.001) per each 20% increase in the ratio of non-inhibited platelets to inhibited platelets. A ratio of 60% was associated with 90% of patients reaching the primary endpoint. In patients treated with ticagrelor (n ¼ 15), ADP-Aggmax did not significantly increase with any level of supplements. In conclusions, ex vivo addition of non-inhibited platelets significantly improved ADP-Aggmax in patients treated with prasugrel with a dose-dependent effect. There was no evidence of such a reversal in patients treated with ticagrelor. These results suggest that platelet transfusion may be more effective in blunting bleeding in patients treated with prasugrel, than those treated with ticagrelor.

Platelet function tests, platelet transfusion, prasugrel, ticagrelor

Introduction Since 2011, prasugrel and ticagrelor have been front line therapies for patients presenting with acute coronary syndrome (ACS) [1, 2]. Prasugrel, a thienopyridine, is a prodrug requiring metabolization to an active compound that binds irreversibly to the platelet P2Y12 receptor. Rapidly, 30 min after a loading dose (LD), prasugrel’s active metabolite reaches peak concentration and is detectable up to 6 h after ingestion [3], providing fast inhibition of platelet aggregation. Ticagrelor is a non-thienopyridine, oral P2Y12 antagonist (cyclopentyl-triazolopyrimidine), that does not need bioactivation and binds reversibly to the P2Y12 receptor. Ticagrelor concentrations in plasma reach a peak within 1 h of oral intake, with significant levels between twice daily doses [4]. Both prasugrel and ticagrelor provide stronger platelet inhibition than clopidogrel, whether after a LD or during a

Correspondence: Fanny Bonhomme, Department of Anesthesiology, Pharmacology and Intensive Care, Geneva University Hospitals, 4 Rue Gabrielle-Perret-Gentil, CH-1211 Geneva 14, Switzerland. Tel: +41 22 372 74 03. Fax: +41 22 372 76 90. E-mail: [email protected]

History Received 1 February 2015 Revised 24 March 2015 Accepted 25 March 2015 Published online 22 April 2015

maintenance regimen [5, 6], and are therefore associated with an increased risk of bleeding. In the TRITON-TIMI 38 trial [7], prasugrel increased the rate of reoperation after coronary artery bypass grafting (CABG), major bleeding events, life-threatening bleeding and fatal bleeding, while in the PLATO trial [8], ticagrelor increased the rate of non-CABG-related bleeding and the rate of fatal intracranial bleeding. In elective invasive surgical procedures, current guidelines recommend that P2Y12 inhibitors should be discontinued five to seven days beforehand in order to limit the risks of perioperative bleeding [9]. However, the management of spontaneous hemorrhagic events or excessive bleeding during emergency surgery in patients receiving antiplatelet drugs remains challenging and no standardized reversal strategies are available. Platelet transfusions are deemed the most efficient therapy, yet few data are available regarding the efficacy of platelet transfusions in the management of bleeding in patients receiving prasugrel or ticagrelor. This ex vivo study aimed to evaluate the minimal proportion of non-inhibited platelets (functional platelets from healthy volunteers) that should be added to platelets inhibited by prasugrel or ticagrelor in order to significantly improve overall platelet reactivity in most patients.

Prasugrel/ticagrelor and platelet supplementation

DOI: 10.3109/09537104.2015.1035247

Methods Participants This was a single-center, observational, prospective ex vivo study. Patients admitted to the tertiary intensive care or coronary care units at Geneva University Hospitals, Switzerland, from October 2011 to December 2013, were eligible for inclusion if they had an ACS as defined by the European Society of Cardiology [1], and had received a prasugrel LD (60 mg) from 6 to 24 h prior to study inclusion. Following ticagrelor’s approval in Switzerland, a protocol amendment allowed the inclusion of patients who had received a ticagrelor LD (180 mg) between 6 and 24 h beforehand. The choice for selecting prasugrel or ticagrelor was left to the discretion of the cardiologist in charge of the patient, according to institutional guidelines: before ticagrelor’s approval, prasugrel was preferred over clopidogrel for percutaneous coronary intervention in all ACS patients, excluding those with prasugrel contraindications; after ticagrelor’s approval, internal guidelines have been modified and prasugrel was the first choice for ST elevation myocardial infarction (STEMI) patients and ticagrelor for those presenting with non-STEMI (NSTEMI) [1, 2]. Patients who had previously received a clopidogrel LD, or intravenous glycoprotein IIb/IIIa inhibitors within 10 days, were excluded. Others exclusion criteria were a known congenital thrombopathy and/or coagulation defect, and pregnancy. Venous blood was collected using a 21-gauge needle and no tourniquet, in tubes containing 3.2% sodium citrate (BD VacutainerÔ, Becton Dickinson, Meylan, France). The first 3 ml were discarded. Eight apparently healthy control subjects, with no known congenital thrombopathy and/or coagulation defect and not taking any medication, were recruited at the beginning of the study. At the inclusion of each new patient, three of the eight control subjects were contacted to obtain blood samples. All participants (patients and control subjects) provided written informed consent. The study was approved by Geneva University Hospitals’ Central Ethics Committee (No 11-117), and the trial was registered (clinicalTrials.gov identifier NCT01839968). Platelet function evaluation Platelet-rich plasma from both patients (P-PRP) and healthy control subjects (C-PRP) were obtained from whole blood by centrifugation at 150 g for 10 min at room temperature. C-PRP (source of the non-inhibited platelets) from three control subjects were pooled together. Several PRP samples were then prepared by mixing increasing amounts of pooled C-PRP to P-PRP according to their respective platelet counts, in order to obtain a final volume of 280 mL of mixed PRPs with different ratios of inhibited and non-inhibited platelets. The volume of C-PRP in the PRP mixture was calculated with the following formula: volume of C-PRP ¼ 280  [(280 x platelet count in C-PRP)/(F x platelet count in P-PRP + platelet count in C-PRP)], with F ¼ 0.20 for PRP-20%, F ¼ 0.40 for PRP-40%, F ¼ 0.60 for PRP-60%, and F ¼ 0.80 for PRP-80%. F represents the ratio of non-inhibited platelets (added potent platelets) to inhibited platelets (referred below as ‘‘ratio’’) in the mix. Platelet aggregation assays were performed on unadjusted PRP [10] within 3 h of blood collection, using an eight-channel aggregometer (TA-8V; SD Medical, Heillecourt, France) and in response to 10 mmol L1 ADP (Sigma-Aldrich, Buchs, Switzerland), 2.5 mg mL1 of Horm collagen (COL, Nycomed, Linz, Austria), 7.5 mmol L1 thrombin receptor activating peptide (TRAP-6, Bachem, Switzerland), and 1 mmol L1 arachidonic acid (AA, Bio/Data Corporation, Horsham, PA). The platelet

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aggregation response was recorded for 6 min and maximum aggregation values were used for analysis. Evaluation of the baseline anti-P2Y12 effect on each patient was also assessed using the quantification of the phosphorylation status of the vasodilator phosphoprotein (VASP) on whole blood using a standardized assay (platelet VASP/P2Y12, Biocytex, Marseille, France) on a FACSCalibur (BD Biosciences, San Jose, CA). An ADP-induced maximal platelet aggregation (ADP-Aggmax) of 40% was used as the cut-off above which the anti-P2Y12 effect could be sufficiently overcome by the addition of non-inhibited platelets [11]. An AA-induced maximal platelet aggregation (AA-Aggmax) of 20% was used as the cut-off above which the aspirin effect was sufficiently overcome by the addition of noninhibited platelets [12]. Statistics The main objective of this study was to determine the amount of potent platelets that would allow an ADP-Aggmax of at least 40% in 90% of prasugrel-treated patients (primary endpoint). The sample size required to estimate a proportion of 90% with a level of confidence of 95% and a precision of 10% (half width of the 95% CI) was 35 patients. Secondary endpoints included the determination of the amount of non-inhibited platelets that would allow an ADP-Aggmax of at least 40% in 90% of patients treated with ticagrelor and that would allow an AA-Aggmax of at least 20% in 90% in each patient group. The effect of the addition of non-inhibited platelets on maximal platelet aggregation (Aggmax), induced by two strong platelet agonists such as the thrombin mimetic TRAP-6 and collagen, was also evaluated. Categorical data are presented as frequencies and percentages. Continuous normally distributed variables are expressed as mean ± standard deviation (SD) whereas the time between last dose and samples collection is reported as median and interquartile range (IQR). Differences between the two groups of treatment were assessed with the chi square test or the Fisher’s exact test, as appropriate, for categorical variables, with the t-test for continuous normally distributed data and the Mann–Whitney test for time between last dose and samples collection. Aggmax per non-inhibited platelet dose are presented as mean ± SD. The proportion of patients reaching the primary or secondary endpoints (ADP-Aggmax  40% or AA-Aggmax  20%) was calculated for each non-inhibited platelet dose. Confidence intervals were estimated using the Clopper Pearson method. The non-inhibited platelet supplementation effects on ADP-Aggmax, TRAP-Aggmax, AA-Aggmax, and COL-Aggmax were assessed using linear mixed effects models with a random effect of patients on intercept. When there was no linear relationship between noninhibited platelet mixture and Aggmax (as was the case for AAAggmax and COL-Aggmax), statistical analyses were performed on two sets of conditions so that a linear relation could be assumed on each partition. Statistical analysis was performed using R software (Vienna, Austria, URL http://www.R-project.org/). A two-tailed P-value of 0.05 was considered significant for all analyses.

Results Patient characteristics From October 2011 to December 2013, 32 consecutive ACS patients treated with prasugrel and 15 consecutive ACS patients treated with ticagrelor were enrolled and included in this study. At inclusion, all patients were on aspirin, with 28 (87.5%) of the prasugrel group and 14 (93.3%) of the ticagrelor group having

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received a LD (250 mg aspirin) in the previous 24 h; at the discretion of the physician, a few patients who were on a chronic aspirin treatment (100 mg/d), did not received an aspirin LD. Patients’ characteristics are shown in Table I. The majority (53.1%) of patients treated with prasugrel were admitted for a STEMI, while patients treated with ticagrelor were admitted for an unstable angina or, a NSTEMI. More patients were smokers in the prasugrel group (53%) compared with the ticagrelor group (13%, P ¼ 0.023), probably due to the fact that STEMI were in the prasugrel group [13]. Within 24 h after the LD, 81% of patients in the prasugrel group received a 10-mg maintenance dose (MD), while 93% of patients in the ticagrelor group received a 90-mg MD, so that Table I. Participants’ baseline characteristics. Prasugrel group (n ¼ 32)

Ticagrelor group (n ¼ 15)

p Value

58 ± 9 26 (81%) 82.3 ± 16.3 19 (59%) 17 (53%) 5 (16%) 17 (53%) 9 (28%)

64 ± 11 11 (73%) 86.4 ± 13.5 9 (60%) 10 (67%) 4 (27%) 2 (13%) 2 (13%)

0.084 0.704 0.366 1 0.576 0.438 0.023 0.461

Demographics Age (years) Male Body weight (kg) Hyperlipidemia Hypertension Diabetes mellitus Smoking FHCAD Medication Statin b blockers ACE inhibitors PPIs Aspirin before ACS ACS NSTEMI STEMI Unstable angina

23 22 13 4 10

(72%) (69%) (41%) (13%) (31%)

13 (40.6%) 17 (53.1%) 2 (6.3%)

10 9 7 2 6

(67%) (60%) (47%) (13%) (40%)

0.742 0.795 0.941 1 0.795

13 (86.7%) 0 (0%) 2 (13.3%)

0.008 0.001 0.583

Data are mean ± SD, or n (%). ACE, Angiotensin-converting enzyme; ACS, acute coronary syndrome; FHCAD, family history of coronary artery disease; NSTEMI, non-STsegment elevation myocardial infarction; PPIs, proton pump inhibitors; STEMI, ST-segment elevation myocardial infarction.

samples were collected at a median time of 4.3 h (IQR: 1.9–6.0) after the last dose (LD or MD) of prasugrel and 5.5 h (IQR: 4.3– 5.9) after the last dose of ticagrelor, P ¼ 0.25. All but two patients had a platelet reactivity index (PRI) according to the VASP assay below 50% (one patient in the prasugrel group (PRI ¼ 74.6%) and one in the ticagrelor group (PRI ¼ 62.9%)), confirming proper intake and absorption of the drugs in the large majority of patients. Mean platelet counts of P-PRP and C-PRP were 372 ± 89 G L1 and 435 ± 59 G L1, respectively. The PRP-20% mixture contained 63 ± 13 G L1 of non-inhibited platelets, PRP40% contained 109 ± 21 G L1 of non-inhibited platelets; PRP60% contained 145 ± 26 G L1 of non-inhibited platelets; and PRP-80% contained 174 ± 29 G L1 of non-inhibited platelets. Mean ADP-Aggmax, AA-Aggmax, TRAP-Aggmax, and COLAggmax were of 79.1 ± 6.9%, 81.6 ± 9.7%, 79.0 ± 7.3%, and 81.4 ± 7.3%, respectively, for the healthy subjects throughout the study. Impact of addition of non-inhibited platelets The impact on platelet aggregation of the progressive addition of non-inhibited platelets into P-PRP is described in Table II. ADP-Aggmax Targeting the pharmacodynamic effect of P2Y12 inhibitors on platelet reactivity, ADP-Aggmax was successfully analyzed for 30 patients in the prasugrel group and for all patients in the ticagrelor group. In the prasugrel group, ADP-Aggmax increased linearly from 32.1 ± 8.4% to 63.9 ± 11.5% in association with the incremental addition of non-inhibited platelets, corresponding to an average ADP-Aggmax increase of 7.9% (95% CI [7.1; 8.8], p50.001) per each 20% increase in the ratio (Figure 1A). This dose-response effect was not observed in the ticagrelor group, with an average change of ADP-Aggmax of 0.7% (95% CI [0.5; 1.9], p ¼ 0.3) per each 20% increase in the ratio (Figure 1B). The difference in the impact of non-inhibited platelet supplementation on ADP-Aggmax between the two groups of patients was significant (p50.001). The main objective of the study (ADPAggmax  40% in 90% of patients) was reached with a ratio of

Table II. Impact of non-inhibited platelet supplementation on platelet aggregation.

P-PRP ADP-Aggmax (%) Prasugrel 32.1 ± 8.4 Ticagrelor 41.8 ± 9.1 AA-Aggmax (%) Prasugrel 9.8 ± 8.9 Ticagrelor

13.3 ± 6.9

TRAP-Aggmax (%) Prasugrel 32.8 ± 12.7 Ticagrelor 39.8 ± 10.2 COL-Aggmax (%) Prasugrel 39.1 ± 12.6 Ticagrelor

40.3 ± 10.7

Average increase per 20% of non-inhibited platelets*

p Value*

PRP-20%

PRP-40%

PRP-60%

PRP-80%

46.6 ± 9.6 47.7 ± 9.6

47.4 ± 12 41.9 ± 8.9

62.2 ± 9.5 49.2 ± 9.9

63.9 ± 11.5 44.6 ± 10.4

7.9 ± 0.44 0.7 ± 0.6

50.001 0.3

51.3 ± 19.5

57.9 ± 19.1

66.2 ± 19.4

67.5 ± 17.2

58.5 ± 9.4

64.1 ± 11

69.7 ± 6.9

68.9 ± 13.3

41.5 ± 3.1y 4.0 ± 1.2z 45.2 ± 4.6y 2.4 ± 1.8z

50.001y 50.001z 50.001y 0.2z

44.7 ± 15.1 42.6 ± 10.3

43.6 ± 21.9 37.7 ± 12.2

56.5 ± 21 41 ± 13.7

56.8 ± 20.1 38.6 ± 15.9

5.8 ± 0.6 0.4 ± 0.8

50.001 0.6

71 ± 11.7

70 ± 11.1

76.2 ± 7.1

76.7 ± 12.3

71.9 ± 6.7

70.3 ± 13.7

31. 9 ± 2.4y 3.4 ± 1.2z 27.7 ± 3.3y 1.2 ± 1.6z

50.001y 0.005z 50.001y 0.5z

68 ± 11.8

67.9 ± 7.9

Prasugrel vs. ticagrelor groups p Value* 50.001

0.5y 0.4z

50.001 0.3y 0.3z

Data are mean ± SD. *Results assessed using a linear mixed effects model (with random effects on patients; fixed effects are the treatment and non-inhibited platelet dose factors with an interaction term between the latter). yResults from P-PRP to PRP-20%. zResults for PRP from 20% to 80%

DOI: 10.3109/09537104.2015.1035247

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Figure 1. Impact of non-inhibited platelet supplementation on ADP-Aggmax. The? graph represents the evolution of ADPinduced maximal aggregation according to the ratio of non-inhibited platelet to inhibited platelet in patients treated with prasugrel (A) and ticagrelor (B).

60%, corresponding to 145 ± 26 G L1 of non-inhibited platelets: 100% of patients (95% CI [88.4; 100]) of prasugrel-treated P-PRP had an ADP-Aggmax  40%. In the ticagrelor group, none of the platelet supplementation was associated with at least 90% of patients reaching ADP-Aggmax  40%. AA-Aggmax Targeting the pharmacodynamic effect of aspirin on platelet reactivity, AA-Aggmax increased progressively in both prasugrel and ticagrelor groups, in association with the incremental addition of non-inhibited platelets. As the trend here was clearly not linear, we dichotomized the non-inhibited platelet effect from 0% to 20% platelet addition and from 20% to 80%, as described in the Methods section. The effect of the addition of non-inhibited platelets was strong, with a ratio of 20% (corresponding to 63 ± 13 G L1 of non-inhibited platelets) associated with an AAAggmax increase of 41.5% (95% CI [35.2; 47.8], p50.001) and 45.2% (95% CI [36.0; 54.4], p50.001) in the prasugrel and ticagrelor groups, respectively. This effect was not significantly different between the two groups (p ¼ 0.5). The effects of further non-inhibited platelets were much smaller — from PRP-40% to PRP-80% — with an average AA-Aggmax increase of 4.0% (95% CI [1.6; 6.5], p ¼ 0.001) and 2.4% (95% CI [1.1; 5.8], p ¼ 0.2) per each 20% increase in the ratio in prasugrel and ticagrelor groups, respectively. This effect was not statistically different between the two groups (p ¼ 0.4). A ratio of 40% (corresponding to 109 ± 21 G L1 of non-inhibited platelets) was sufficient to obtain an AA-Aggmax  20% in 93.8% (95% CI [79.2; 99.2]) of patients treated with prasugrel. A ratio of 20% (corresponding to 63 ± 13 G L1 of non-inhibited platelets) was sufficient to obtain an AA-Aggmax  20% in 100% (95% CI [78.2; 100.0]) of patients treated with ticagrelor. TRAP-Aggmax TRAP-Aggmax is a powerful platelet agonist. Its results mirrored those of ADP-Aggmax in both groups. In the prasugrel group, there was a significant average increase in TRAP-Aggmax of 5.8% (95% CI [4.7; 6.9], p50.001) per each 20% increase in the ratio.

However, this dose-response effect was not significant in the ticagrelor group (0.4% (95% CI [1.9; 1.1], p ¼ 0.6). COL-Aggmax A ratio of 20% (corresponding to 63 ± 13 G L1 of non-inhibited platelets) led to an important increase in platelet aggregation in both groups: 31.9% (95% CI [27.0; 36.8], p50.001) patients treated with prasugrel and 27.7% (95% CI [21.0; 34.5], p50.001) in those treated with ticagrelor. This mirrored the results of AAAgmax. The addition of larger proportions of platelets had only a minor incremental pharmacodynamic impact. The effects of the first 20% of non-inhibited platelets and of additional proportions of platelets were not different between the two groups (p ¼ 0.3).

Discussion This study’s main findings are: (1) platelet reactivity in patients treated with prasugrel can be significantly improved in at least 90% of patients by reaching a ratio of non-inhibited platelets to inhibited platelets equal to 60% (corresponding to 145 ± 26 G L1 of non-inhibited platelets) or more, owing to a dose-response effect; and (2) the impact of non-inhibited platelet supplementation is significantly different in patients treated with ticagrelor, with no evidence of a dose-response effect. As expected [14, 15], the effects of ASA are easily cancelled out by a ratio of 20–40%. These observations provide an insight on the minimal platelet transfusion requirement for the practical management of major bleeding in cardiovascular patients treated with next generation P2Y12 inhibitors. Excessive bleeding is a life-threatening complication associated with antiplatelet drugs. Because prasugrel binds to the P2Y12 receptor irreversibly, recovery of platelet function after drug discontinuation depends on platelet turnover. Due to the more intense antiplatelet effect of prasugrel, recovery of platelet reactivity after discontinuation is slower than with clopidogrel, and it is recommended that prasugrel be withdrawn seven days before any elective invasive surgical procedure, compared with five days before for clopidogrel [16, 17]. Conversely, the recovery of platelet function is faster after ticagrelor discontinuation than after clopidogrel [6], balancing its more intense

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pharmacodynamic effect. A five-day discontinuation policy before an invasive surgical procedure is recommended for ticagrelor [18]. In cases of life-threatening spontaneous bleeding or emergency surgical procedures, the immediate reversal of antiplatelet effects is of utmost importance. Although platelet transfusions are widely used in these clinical settings, their impact on platelet function recovery has scarcely been studied. This ex vivo study was thus performed to assess the recovery of platelet function in PRP samples from patients with an ACS, who had received a LD of prasugrel or ticagrelor in association with ASA. We added increasing amounts of non-inhibited platelets to patients’ PRP taken from 6 to 24 h after a LD. Assuming that the transfusion of one platelet concentrate should increase the platelet count by 30–40 G L1, the different mixtures used in this study (20%, 40%, 60%, and 80%) would correspond to the transfusion of 1, 2, 3, or 4 platelet concentrates, respectively, in a patient with an hematocrit around 40% (1 platelet concentrate ¼ 5 platelet units). Although ADP and AA are specific agonists for testing platelet inhibition induced by anti-P2Y12 inhibitors (such as prasugrel and ticagrelor) and ASA, respectively, others agonists were also tested as it is known that cross-interactions and co-stimuli occurring in vivo are important for platelet activation and thrombus stability [19]. Platelet aggregation induced by collagen is especially dependent on secondary mediators released from activated platelets, including thromboxane A2 [20]. This may explain why this study’s results regarding COL-Aggmax mirrored those obtained with AA. There is a correlation between the degree of P2Y12 inhibition and low dose TRAP-induced platelet aggregation [21] that may thus explain the results regarding TRAPAggmax mirroring those obtained with ADP-Aggmax. The primary endpoint of 40% of ADP-Aggmax was chosen since values below this threshold have been associated with an increased risk of bleeding, with severe coagulopathy requiring multiple platelet and red blood cell transfusions [22]. The same cut-off was also identified in patients treated with ASA and clopidogrel and was associated with an increased risk of major and minor non-CABG-related bleeding [11]. It must be acknowledged that there is no definitive consensus on a threshold of platelet reactivity to ADP associated with bleeding risk, as recent studies have used different tests of platelet function with different methods [23, 24]. Our study indicated that a ratio of non-inhibited platelets to inhibited platelets equal to 60% was necessary in the prasugrel group to reach the primary endpoint of at least 90% of patients with ADP-Aggmax  40%. The difference of recovery induced by fresh platelets supplementation was more obvious between 40% and 60% rather than 20% and 40%. This may be related to a threshold effect where a minimal amount of functional platelets is required to obtain an irreversible and full aggregation profile. These data suggest that three platelet concentrates might be necessary in clinical practice to overcome the antiplatelet effect of prasugrel. The dose of non-inhibited platelets required to overcome ADPinduced aggregation in patients treated with prasugrel appears to be greater than the dose required for those treated with clopidogrel. Indeed, in a similar study, Vilahur et al. found that doses of 40–50% of additional platelets fully reversed platelet inhibition in healthy volunteers treated with clopidogrel [25]. It is of note that the amount of non-inhibited platelets which needed to be added to overcome prasugrel’s effects in this study implied that there were no active metabolite left. Indeed, prasugrel’s active metabolite is not stable in vitro and has a very short half-life [26]; even if some blood samples were collected shortly after the first MD, prasugrel’s active metabolite was no longer effective in P-PRP when we prepared the mixture with C-PRP. Thus, the results obtained in the prasugrel group

Platelets, 2015; 26(4): 324–330

reflect the effect of platelet supplementation given at least 6 h after the last drug intake. In an ex vivo study in healthy subjects receiving an oral prasugrel LD, the earliest that freshly added platelets were not inhibited by prasugrel’s active metabolite was 6 h, while the amount of circulating prasugrel’s active metabolite at 2 h was sufficient to inhibit the added functional platelets [27]. Hence, the proportion of non-inhibited platelet to overcome the prasugrel inhibitory effect changes over the time, and depends from the interval time between the last prasugrel dose and platelet transfusion. Platelet transfusions would be the most effective at least 6 h after prasugrel intake and the amount of platelet transfusion necessary to overcome the effect of prasugrel might have to be greater when given earlier than 6 h after the last prasugrel dosing. Conversely to what was evidenced in prasugrel-treated patients, there was no change in ADP-induced aggregation in patients treated with ticagrelor when non-inhibited platelets were added. This finding could be explained by the specific pharmacological properties of ticagrelor. Although we did not measure the active metabolite of ticagrelor (AR-C124910XX), it is known that its plasma concentration remains above 100 ng/ml between each dosing [4], allowing platelet inhibition [28]. As ARC124910XX binds to platelet P2Y12 receptors in a reversible manner, an equilibrium between the amount of AR-C124910XX on platelets and in plasma occurs; the AR-C124910XX left in the plasma is thus available to inhibit any platelets added, thus reducing the benefits of a platelet transfusion. Moreover, the plasma concentrations of both ticagrelor and its active metabolite remain much higher (5.2-fold and 7.7-fold, respectively) than the concentration required to induce half the maximum platelet effect (EC50) for at least 24 h [4]. This observation is consistent with recent clinical observations that we and others described recently reporting biological and clinical inefficacy of platelet transfusions in ticagrelor-treated patients with life-threating bleeding [29, 30]. Studies based on multiple electrode aggregometry testing on whole blood showed that even a high dose of non-inhibited platelets had a limited effect in restoring platelet reactivity to ADP in patients treated with ticagrelor compared with clopidogrel [31, 32]. In the study by Hobl et al. [32], adding PRP at a ratio of 1:3 restored basal platelet aggregation to ADP in less than half of the patients, indicating that the majority of patients would need more than two platelet concentrates, but no higher ratios were tested in this study. Contrary to our results, platelet supplementation enhanced TRAP-induced aggregation in ticagrelor-treated patients based on the multiple electrode aggregometry assay [31]. This difference may be due to the different concentrations of TRAP-6 used to induce aggregation: in our study, platelet reactivity was challenged with low dose of TRAP-6 compared with higher concentrations in the multiple electrode aggregometry assay. The present study also highlighted a significant variability in terms of platelet inhibition at baseline and during recovery after non-inhibited platelet supplementation for patients treated with prasugrel or ticagrelor. This variability is in line with previous reports [33, 34]. We did not found any correlation between ADPAggmax and factors such as body weight, age, sex, concomitant medication, and delay between last dose intake and blood sampling (data not shown). However, this study was not designed to specifically address the issue of the variability of nextgeneration P2Y12 inhibitors. Of note, prasugrel achieved a more profound inhibition of platelet function than ticagrelor. This is in line with a recent study [35], whereas another one found no significant difference between the potency of the two drugs during the first 24 h [36]. Easy monitoring of platelet function may facilitate the clinical management of patients with an underlying cardiovascular

Prasugrel/ticagrelor and platelet supplementation

DOI: 10.3109/09537104.2015.1035247

thrombotic risk who are then exposed to a hemorrhagic risk when undergoing an invasive surgical procedure. In the near future, platelet transfusions may well be individualized and guided by the results of platelet function assays [37]. Several platelet function assays are available, but none can currently be recommended because of their weak prognostic value for bleeding and the lack of well-conducted studies [38, 39]. Thus, in the present study, we used light transmittance aggregometry that is still the goldstandard for platelet testing against which all other assays are compared. This study has noteworthy limitations. Due to the slow recruitment of participants, the study stopped before the inclusion of 35 patients required to reach a precision of 10% in estimating the proportion of patients reaching the primary endpoint. Nevertheless, with 30 patients in the prasugrel group, this precision was 11%, quite close to the planned precision. Second, we used freshly prepared PRP and the quality of platelets may differ from that of allogenic platelet concentrates [40]. Finally, the ability of non-inhibited platelet supplementation to overcome ADP-Aggmax in patients treated with ticagrelor was one of this study’s secondary objectives and it cannot be ruled out that the absence of a significant effect from platelet supplementation in this group might be due to a lack of power. However, considering the upper bound of the 95% CI (1.9%) for the ADPAggmax increase, it is unlikely that a larger number of patients in the ticagrelor group would have changed the overall conclusion to a relevant extent. Moreover, the impact of non-inhibited platelet supplementation in patients treated with prasugrel clearly and significantly differs from that of patients treated with ticagrelor (p50.001). In conclusion, in patients admitted for an ACS with recent prasugrel LD, a dose-dependent ex vivo non-inhibited platelet supplementation overcame the inhibitory effect of prasugrel; a ratio of non-inhibited platelets to inhibited platelets equal to 60% (corresponding to the transfusion of three platelet concentrates) was necessary to reach an ADP-Aggmax  40% in more than 90% of patients. There was no evidence of such a reversal effect with non-inhibited platelet supplementation in ACS patients treated with ticagrelor. These findings provide important clinical insights for interventional cardiologists’ daily dealings for the selection of the appropriate anti-P2Y12 inhibitor that may differ according to patient’s bleeding risk, as it suggests that platelet transfusion may be more effective in blunting bleeding in patients treated with prasugrel, than those treated with ticagrelor. Studies are now required for rapid clinical testing of this laboratory observation.

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Acknowledgements We thank Severine Nolli and Nicolas Lorenzon for their excellent technical assistance and Youssef Daali for helpful discussions.

Declaration of interest This study was supported by the department of Anesthesiology, Pharmacology and Intensive Care, Geneva University Hospitals, Geneva, Switzerland. Pierre Fontana is supported by the Swiss National Science Foundation (grant No 320030_144150). Pierre Fontana and Fanny Bonhomme received an unrestricted grant from Astra Zeneca.

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Impact of non-inhibited platelet supplementation on platelet reactivity in patients treated with prasugrel or ticagrelor for an acute coronary syndrome: An ex vivo study.

Managing bleeding in patients receiving P2Y12 inhibitors is challenging. Few data are available regarding the efficacy of platelet transfusion in pati...
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