Circulating reticulated platelets over time in patients with myocardial infarction treated with prasugrel or ticagrelor

J Thromb Thrombolysis DOI 10.1007/s19-014-1156-4 Circulating reticulated platelets over time in patients with myocardial infarction treated with prasugrel or ticagrelor Alon Eisen Hila Lerman-Shivek Leor Perl Eldad Rechavia Dorit Leshem-Lev Noa Zemer-Wassercug Oshrat Dadush Shirit Kazum Pablo Codner Ran Kornowski Eli I. Lev Ó Springer Science+Business Media New York 2014 Abstract Reticulated platelets (RP) are young, hyperactive platelets that are increased during situations of enhanced platelet turnover such as acute myocardial infarction (AMI). The dynamics of RP levels after AMI is not established. We aimed to characterize the levels of circulating RP over time in patients with AMI. Patients with AMI treated with ticagrelor or prasugrel who underwent percutaneous coronary intervention (PCI) were tested for circulating RP using flow cytometry with Thiazole orange staining at 3 time points at 2 4 days, 30 60 days and 1 year post PCI. Platelet reactivity was assessed using the VerifyNow P 2 Y 12 assay at these time points (results in platelet reactivity units PRU). Thirty-five patients were included in the study (mean age 62.6 ± 9.1 years, 82.9 % Alon Eisen and Hila Lerman-Shivek contributed equally to the study and are co-first authors of this paper. A. Eisen L. Perl E. Rechavia N. Zemer-Wassercug S. Kazum P. Codner R. Kornowski E. I. Lev (&) Cardiology Department, Rabin Medical Center, Petah Tikva, Israel e-mail: elil@clalit.org.il; elev@tmhs.org A. Eisen L. Perl E. Rechavia N. Zemer-Wassercug S. Kazum P. Codner R. Kornowski E. I. Lev The Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel H. Lerman-Shivek Hasharon Hospital, Rabin Medical Center, Petah Tikva, Israel H. Lerman-Shivek Clinical Pharmacy Department, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel D. Leshem-Lev O. Dadush The Felsenstein Medical Research Institute, Rabin Medical Center, Petah Tikva, Israel males). Median RP levels were similar at the first and second time points (17.5 %, IQR 25 75: 10.8 22.4 % and 14.9 %, IQR 25 75: 9.7 26.8 %, respectively; p = 0.75). However, RP levels after 1 year were significantly lower as compared to the first and second time points (10.5 % (IQR 25 75: 5.3 18.1 %), p = 0.005 and p = 0.01, respectively). Residual platelet reactivity was very low at all 3 time points (median PRU 25, IQR 25 75: 7 53) and did not change significantly between them (p = 0.66). No significant correlation was found between levels of RP and PRU at any given time point. RP levels of patients with AMI treated with prasugrel or ticagrelor decrease over time after the acute event. However, RP levels over time do not correlate well with residual platelet reactivity. Keywords Acute coronary syndrome Antiplatelet therapy Reticulated platelets Abbreviations AMI Acute myocardial infarction PCI Percutaneous coronary intervention RP Reticulated platelets PRU P 2 Y 12 reaction units ACS Acute coronary syndrome Introduction Acute coronary syndrome (ACS) is associated with increased platelet reactivity and platelet turnover during the acute event. Data on platelet reactivity as time elapses after the acute episode are based mainly on clopidogrel treated patients, and demonstrate that platelet reactivity tends to

A. Eisen et al. either decrease or remain relatively stable over time, depending on the time course and assessment methods [1 3]. Furthermore, high on-treatment platelet reactivity, as assessed mainly during the acute episode, is associated with increased cardiovascular adverse events [4 8]. The new P 2 Y 12 inhibitors, prasugrel and ticagrelor, provide more potent and persistent platelet inhibition, as compared to clopidogrel [9, 10]. Several studies investigating platelet reactivity under prasugrel or ticagrelor treatment in patients with ST elevation myocardial infarction (STEMI) revealed strong and consistent inhibition over time, with very low variability in platelet inhibition [11 13]. In this context, perhaps other, more sensitive, markers of platelet reactivity may demonstrate more variability in residual reactivity despite the marked platelet inhibition. Reticulated platelets (RP) represent young, hyperactive platelets, with increased mean volume and a greater number of dense granules, as compared to older platelets [14]. RP have been shown to increase in situations of increased platelet turnover, such as ACS [15] and stroke [16]. We have previously shown that the proportion of circulating RP inversely correlates with clopidogrel responsiveness in patients with stable coronary artery disease [17] and more recently, that RP levels strongly correlates with higher platelet reactivity in prasugrel treated patients with STEMI [11]. However, there are no data regarding the dynamics of RP levels over time in patients after AMI. This study aimed to characterize the levels of circulating RP over time in patients with AMI treated with prasugrel or ticagrelor. cases, ticagrelor was the drug of choice. Contraindication to prasugrel and ticagrelor included a history of intracranial hemorrhage, active pathological bleeding, co-administration with strong CYP3A4 inhibitors, need for chronic anticoagulation and moderate to severe hepatic impairment. In these cases clopidogrel was the anti-platelet drug used and these patients were excluded from the present study. Other exclusion criteria included hemodynamic instability, thrombocytopenia (\100,000 cells/mm 3 ), anemia (hemoglobin \10 g/dl) and renal failure (creatinine [2.5 mg/dl). Study protocol All patients were treated with prasugrel 60 mg or ticagrelor 180 mg and aspirin 325 mg before the PCI, and received daily prasugrel 10 mg or ticagrelor 90 mg twice daily with aspirin 100 mg thereafter through all study period. Adjunctive treatment during PCI (heparin, bivalirudin and/ or glycoprotein IIB/IIIA inhibitor- eptifibatide) was given according to physician discretion. Blood samples were collected from each patient at three time points (1) 2 4 days post-pci just prior to hospital discharge; (2) 30 60 days post-pci; (3) 1-year after the acute event. At each time-point, RP levels were determined by using a flow cytometry assay with Thiazole orange staining and platelet reactivity was determined using the VerifyNow P 2 Y 12 assay. Platelets count, mean platelet volume and hemoglobin level were also documented at baseline using routine complete blood count test taken after PCI. The overall study design is summarized in Fig. 1. Methods A prospective cohort study, conducted from July 2012 through April 2014, at the Rabin Medical Center, Petah Tikva, Israel (Beilinson and Hasharon hospitals). The study was approved by the Ethics Committee of the Rabin Medical Center in accordance with the Declaration of Helsinki and all patients provided written informed consent. Patients All patients aged 18 75, admitted to the hospital with AMI, either ST- or non ST- segment elevation. All patients who were treated with either prasugrel or ticagrelor and underwent percutaneous coronary intervention (PCI) during index hospitalization were considered eligible for study participation. According to institute protocol, all patients with AMI are treated with either prasugrel or ticagrelor unless contraindicated. Contraindication to prasugrel treatment included patients aged[75 years, weight\60 kg or a history of stroke or transient ischemic attack. In these Fig. 1 Study design

Reticulated platelets after myocardial infarction Reticulated platelets RPs were measured with a previously described flow cytometry assay using FACScan flow cytometer (Becton Dickinson, San Jose, CA) and the fluorescent RNA dye, Thiazole orange (TO; ReticCount, Becton Dickinson, Franklin Lakes, NJ) [16]. Five ll of whole blood were added to a tube containing 1 ml of 0.1 % Thiazole orange and a second tube containing 1 ml of Isoflo phosphate buffer saline (Beckman-Coulter) as a control. After incubation for 30 min in the dark, the tubes were spun at 1,200 g for 2.5 min to form a cellular pellet. The supernatant was discarded, and the pellet was resuspended in 1 ml of Isoflo phosphate buffer saline. Within 1 h, flow cytometry using FACScan flow cytometer (Becton Dickinson, San Jose, CA) was performed counting 10,000 stained platelets in the platelet gate. The platelets demonstrating an increase in the mean fluorescence intensity beyond a threshold margin set at \1 % of all platelets at baseline were counted as RP and expressed as a percentage of total platelets counted. Platelet reactivity Platelet reactivity was evaluated by previously validated assay. VerifyNow P 2 Y 12 (Accumetrics, San Diego, CA) is a rapid platelet-function cartridge-based assay designed to directly measure drug effect on the P 2 Y 12 receptor. The assay contains 20 lmol ADP and 22 nmol prostaglandin E1 to reduce the activation contribution from ADP binding to P 2 Y 1 receptors. Fibrinogen-coated microparticles are used in the VerifyNow P 2 Y 12 cartridge to bind to available platelet receptors. The VerifyNow instrument measures platelet-induced aggregation as an increase in light transmittance and is expressed in P 2 Y 12 reaction units (PRU). With this assay, a higher PRU reflects greater ADP-mediated platelet reactivity. Statistical analysis Continous variables were evaluated for normal distribution using Kolmogorov Smirnov test and histogram. Non-normally distributed variables were described using median and interquartile range (IQR). Categorical variables were described using frequency and percentage. Correlations between continuous variables were evaluated using Spearman s correlation coefficient. Friedman test was used to evaluate difference between PRU and RP between the 3 time points. Wilcoxon signed ranks test was used for post hoc tests. All statistical tests were 2-tailed. Analyses were performed using SPSS version 21 (SPSS, Inc. Chicago, Illinois). Statistical significance was set at p \ 0.05. Results Baseline characteristics Forty-five patients with AMI (ST and non ST segment elevation) were included in the initial baseline analysis. Of these, 10 patients had missing RP data at one of the time points and thus 35 patients with complete data were included in the present analysis. Table 1 describes the Table 1 Baseline characteristics Variable (n = 35) Demographic characteristics Age (years) 62.3 ± 9.1 Female (%) 17.1 Body mass index (kg/m 2 ) 27.8 ± 4.7 Clinical characteristics Type 2 diabetes mellitus (%) 22.9 Hypertension (%) 57.1 Smoking (%) a 28.6 Family History of CAD (%) 28.6 Hyperlipidemia (%) 60.0 Prior myocardial infarction (%) 20.0 Prior PCI 20.0 Prior CABG (%) 5.7 Prior P 2 Y 12 inhibitors upon admission 0 Current myocardial infarction NSTEMI 60.0 Ticagrelor 60.0 Prasugrel 40.0 PCI to LAD 40.0 PCI to RCA 42.9 DES 71.4 Peri-procedural GPIIB-IIIA inhibitors (%) 14.3 Laboratory findings Hemoglobin (g/dl) 13.7 ± 1.1 Platelets (910 3 /mm 3 ) 206.9 ± 54.4 Mean platelet volume (fl) 8.0 ± 0.9 Creatinine (mg/dl) 0.9 ± 0.2 Medications at discharge Statins (%) 100.0 b-blockers (%) 83.0 ACE-inhibitors (%) 94.3 Proton pump inhibitors (%) 40.0 a Includes current and former smokers. Categorical variables are expressed as n (%), while continuous variables are expressed as mean ± standard deviation ACE angiotensin-converting enzyme, CAD coronary artery disease, CABG coronary artery bypass graft surgery, DES drug eluting stent, GP glycoprotein, NSTEMI non ST segment elevation myocardial infarction, PCI percutaneous coronary intervention, LAD left anterior descending, RCA right coronary artery

A. Eisen et al. Fig. 2 Reticulated platelets levels (expressed as a percentage of total platelets count) in 3 time points baseline characteristics of the cohort. The mean age was 62.6 ± 9.1 years with a male predominance (82.9 %). Twenty-one patients (60.0 %) were admitted due to non STEMI and the rest with STEMI. A minority of patients (14.3 %) received peri-procedural glycoprotein IIB IIIA blockade. All patients had undergone PCI during hospitalization, mainly to the left anterior descending artery (40.0 %) and right coronary artery (42.9 %). Drug eluting stent was implanted in the majority of patients (71.4 %). The majority of patients were discharged with an aspirin, angiotensin converting enzyme inhibitor, beta blocker and a statin. RP levels Median RP levels were similar in the first and second time points (17.5 %, IQR 25 75: 10.8 22.4 % and 14.9 %, IQR 25 75: 9.7 26.8 %, respectively; p = 0.75). Median RP levels at the third time point were 10.5 % (IQR 25 75: 5.3 18.1 %). RP levels after 1 year were significantly lower as compared to the first and second time points (p = 0.005 and p = 0.01, respectively). Dynamics of RP levels over time are depicted in Fig. 2. Platelet reactivity Most patients in our study exhibited very low platelet reactivity already at the first time point with a median PRU of 25.0 (IQR 25 75: 7.0 43.0). PRU levels were similar at all 3 time points as depicted in Fig. 3 (p = 0.66) with a median PRU of 25.0 (IQR 25 75: 7.0 53.0) at all 3 time points taken together. Only 2 patients had a PRU level [100 at the first time point, whereas, none of the patients had a PRU level above 100 at the third time point. None of Fig. 3 Platelet reactivity (expressed by P 2 Y 12 reactivity units) in 3 time points the patients had high on-treatment platelet reactivity (previously defined by C208 PRU) at any of the time points. Correlation between RP levels and PRU No significant correlation was demonstrated between levels of RP and PRU at any given time point (Spearman s r = 0.21, -0.02 and 0.37 for the first, second and third time points, respectively). Discussion To our knowledge, this is the first study that examined the dynamics of RP levels over time in patients with AMI. The main finding of the study is that during AMI, RP levels are elevated and remain relatively stable during the first few months after the acute event. However, following this early period RP levels decrease significantly over time. RPs are young, relatively large platelets which are more physiologically active and contain mrna [18 20], which enables the expression of increased amounts of ADP receptors and intra-granular proteins. These intrinsic platelet products may explain the observed increased reactivity and reduced responsiveness to antiplatelet therapy [16, 21]. Recently we have demonstrated that levels of RP (2 4 and 30 days post AMI) are correlated with higher platelet reactivity, as assessed by VerifyNow P 2 Y 12 assay and by multiple electrode aggregometry, in STEMI Patients [11]. However, in the current study we did not observe a correlation between RP levels and platelet reactivity as assessed by the VerifyNow P 2 Y 12 assay. This observation may have several possible explanations. First,

Reticulated platelets after myocardial infarction all study patients had very low PRU levels under treatment with prasugrel or ticagrelor, and there was limited variability in PRU levels during the different time points. We believe the low PRU levels with limited variability under treatment with potent anti-platelet drugs, may have limited the potential association between residual platelet reactivity and the proportion of RPs. Second, platelet reactivity was assessed solely by the verifynow P 2 Y 12 assay which may not be sensitive enough to observe relatively small differences in platelet reactivity under treatment with potent platelet inhibitors. Studies that have associated high on-treatment platelet reactivity with adverse cardiovascular outcomes have been mostly performed in the era of clopidogrel treatment for ACS and were based mainly on platelet function as measured once during the acute episode. Only few studies investigated the dynamics in platelet function over time after the acute episode and their clinical implications [1 3]. Several recent studies have investigated platelet reactivity over time in patients treated with prasugrel and ticagrelor and the results mainly demonstrate strong and consistent platelet inhibition during time with very low variability in platelet inhibition, in particular in patients treated with ticagrelor [12]. We, therefore, sought to examine other aspects of platelet biology, focusing on the dynamics of RP after AMI. Our results suggest that relying solely on PRU as a measure of platelet reactivity in patients treated with either prasugrel or ticagrelor may not fully reflect the dynamics and profile of platelet biology over time. In this context, RP levels may perhaps be used as an adjunct marker for assessing platelet function and biology. This study may have important clinical implications that deserve further investigation. It was recently demonstrated that higher RP levels are associated with cardiovascular mortality in ACS patients treated with clopidogrel [22]. Whether this holds true for ACS patients treated with prasugrel and ticagrelor is beyond the scope of this manuscript. However, If indeed levels of RPs are elevated in the early period after an AMI and then gradually decrease, it may be possible to treat patients with ACS with potent platelet inhibitors (prasugrel or ticagrelor) in this early period, and then consider switching to clopidogrel in the latter period, in order to reduce the risk of bleeding, and possibly also cost. Another potential implication is that RP levels may be used as an adjunct thrombotic risk marker. It remains to be established whether patients who display a significant reduction in RP levels over time are at reduced risk for cardiovascular events compared with patients in whom RP levels remain relatively stable, and also whether the association between RP levels and cardiovascular risk in patients with ACS is also significant for patients treated with prasugrel or ticagrelor. Study limitations The study findings should be considered in the context of several limitations. First, the number of patients enrolled is relatively small and the population is heterogeneous, including ST and non ST segment elevation AMI patients. The study sample is too small to assess variables and subgroups of interest for example the association between Glycoprotein IIb/IIIa inhibitor treatment and RP levels. Furthermore, the rate of patients lost to follow-up (*23 %) at 1-year post AMI may introduce additional bias. RP testing lacks protocol standardization and TO labeling is not entirely mrna specific. Furthermore, platelet function was assessed solely by the Verify-Now P 2 Y 12 assay and not using other methods such as the gold standard lighttransmission aggregometry. Finally, the study was not powered to assess clinical endpoints and the clinical implications of our finding remains to be investigated in future studies. Conclusions In patients with AMI treated with prasugrel or ticagrelor RP levels decrease over time after the acute event, but do not correlate well with platelet reactivity. Conflict of interest of interest. References The authors declare that they have no conflict 1. Campo G, Parrinello G, Ferraresi P et al (2011) Prospective evaluation of on-clopidogrel platelet reactivity over time in patients treated with percutaneous coronary intervention relationship with gene polymorphisms and clinical outcome. J Am Coll Cardiol 57(25):2474 2483 2. 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