604 Original article
High on-treatment platelet reactivity in patients with ischemic cerebrovascular disease: assessment of prevalence and stability over time using four platelet function tests Eva Jovera, Jose´ M. Rodrı´guezb, Agustina Bernalc, Ana B. Arroyoc, Juan A. Iniestab, Isabel Sa´nchez Guiu´c, Constantino Martı´nezc, Vicente Vicentec, Marı´a L. Lozanoc,M and Jose´ Riverac,M High on-treatment platelet reactivity (HTPR), referred to as a higher than expected platelet reactivity in patients under antiplatelet therapy, could influence outcome in cerebrovascular disease (CVD), but its prevalence and its stability over time is uncertain. Platelet reactivity was assessed in 18 patients with ischemic stroke/transient ischemic attack (TIA) 7 days (D7) and 90 days (D90) after prescription of clopidogrel, using four methods: light transmission aggregometry with 5 mmol/l ADP (LTA-ADP), vasodilator-stimulated phosphoprotein (VASP), Verify Now P2Y12 and platelet function analyzer (PFA) P2Y. HTPR was defined as LTA-ADP more than 46%; PFA-100-P2Y closure time less than 106 s; VerifyNow P2Y12, PRU greater than 235, VASP, PRI greater than 50%. Patients displayed, both at D7 and D90, a marked inhibition of platelet reactivity towards ADP in all tests as compared with reference levels. Correlations between the results obtained with all the tests at D7 and D90 and between measurements on each day in each test were low-to-moderate. The prevalence of HTPR for all the tests was 40% at D7 and 42% at D90. There was a moderate degree of agreement (k statistic < 0.5) between tests with regard to categorizing patients as HTPR/NoHTPR (D7 and D90). The on-clopidogrel platelet reactivity phenotype, HTPR/No-HTPR, remained stable in 55–72% of
Introduction Ischemic cerebrovascular disease (CVD), including ischemic stroke or transient ischemic attack (TIA), is a highly recurrent disease and one of the main causes of morbidity and mortality worldwide [1]. Antiplatelet therapy is a first-line option for the secondary prevention of acute ischemic stroke/TIA and its long-term treatment [2–4]. Aspirin is the most commonly prescribed treatment in CVD, but the use of clopidogrel monotherapy, aspirin and dipyridamole, and aspirin and clopidogrel has also been reported in several studies [5–9]. Recently, the new concept of high on-treatment platelet reactivity (HTPR) has emerged in the cardiovascular field. This refers to the limited degree of inhibition of platelet function, as assessed by various assays, compared with the inhibition expected using antiplatelet therapy [10,11]. Indeed, HTPR has been associated with poor cardiovascular outcomes and might be of clinical value for identifying patients with a high risk of recurrent vascular events who may benefit from intensified antiplatelet 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
patients, depending on the test. A high prevalence of HTPR is found among CVD patients treated with clopidogrel and this platelet reactivity phenotype remains over time. There is poor agreement between the different platelet function tests for categorizing the platelet reactivity phenotype in these patients. The new PFA-100 P2Y equals other platelet function assays for evaluating HTPR in CVD. Blood Coagul Fibrinolysis 25:604–611 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2014, 25:604–611 Keywords: clopidogrel, ischemic stroke, on-treatment platelet reactivity, platelet function testing, transient ischemic attack a Hospital Universitario Virgen de la Arrixaca, bHospital Universitario Reina Sofı´a and cCentro Regional de Hemodonacio´n, University of Murcia, IMIB, Murcia, Spain
Correspondence to Jose´ Rivera, PhD, Centro Regional de Hemodonacio´n, C/ Ronda de Garay S/N. 30003, Murcia, Spain Tel: +34968341990; fax: +34968261914; e-mail: [email protected] Both Marı´a L. Lozano and Jose´ Rivera contributed equally to the writing of this
article. Received 2 September 2013 Revised 22 February 2014 Accepted 28 February 2014
treatment [12]. However, in the setting of CVD, there is much less information available on the prevalence of exvivo nonappropriate responsiveness to antiplatelet therapies or HTPR [13,14]. The heterogeneous cause of stroke/ TIA [15] compared with ischemic heart disease makes necessary to identify HTPR in those particular scenarios. In this study, we have used different platelet function tests to investigate the HTPR phenotype in unselected patients with ischemic stroke/TIA, being treated with clopidogrel monotherapy or with a combination of clopidogrel and aspirin. A double evaluation of platelet function, 1 week and 3 months after the start of clopidogrel treatment, was performed in all patients in order to evaluate the stability over time of the HTPR phenotype.
Materials and methods Patients
This study involved 18 consecutive white patients diagnosed with ischemic stroke or TIA according to the DOI:10.1097/MBC.0000000000000118
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High on-treatment platelet reactivity in stroke/TIA Jover et al. 605
TOAST criteria [15]. Upon hospital admission, patients were prescribed clopidogrel (75 mg/day), either as a monotherapy or as a complement to previously prescribed aspirin (100–300 mg/day) at the discretion of the attending neurologist. Exclusion criteria to enter the study were contraindication for clopidogrel therapy, active infection, inflammatory disease, thrombocytopenia (A; rs4244285) and CYP2C1917 (806C>T; rs12248560) [16], as additional demographical-clinical data. The study complies with the Helsinki Declaration and was approved by the ethics committee of Hospital Universitario Reina Sofia (28 April 2010). All patients gave written informed consent to participate. Blood sampling and platelet function assays
Platelet function testing was performed at two different time points after the initiation of clopidogrel therapy: Day 7 (D7) while the patient was still in hospital and day 90 (D90) after the patient had been discharged. On both days, venous blood samples were collected after overnight fasting and before taking the daily dose of clopidogrel in order to detect the on-treatment platelet reactivity at the theoretical trough level of the clopidogrel active metabolite. Light transmittance aggregometry (LTA) was performed in platelet-rich plasma (PRP) (D7: 295.2 82.1 109 platelets/l; range 150–438 109/l; D90: 293.8 77.9 109platelets/l; range 176–428 109/l) (Aggrecorder II; Menarini Diagnostics, Florence, Italy) essentially as previously described [16,17], using the following agonists: 5 mmol/l ADP (LTA-ADP) (DiaMed, Cressier, Switzerland), 1.6 mmol/l arachidonic acid (LTA-AA) (DiaMed) and 25 mmol/l thrombin receptor activating peptide (TRAP) (LTA-TRAP) (Sigma-Aldrich Quı´mica SL, Madrid, Spain). LTA-TRAP was used as an intraassay positive control of each patient’s platelet aggregation status, as this response is mediated by the thrombin receptor protease-activating receptor-1 (PAR-1) and is not blocked by aspirin or clopidogrel. An LTA-ADP of at least 46% defined on-clopidogrel HTPR, as proposed in cardiovascular patients [11], and LTA-AA at least 20%, was considered to reflect biochemical aspirin resistance. Verify Now (Accumetrics, San Diego, California, USA), a point-of-care assay evaluating platelet aggregation in whole blood [18], with P2Y12 cartridges was also used according to the manufacturer’s instructions. On-clopidogrel HTPR in this test was defined as PRU of at least 235 [11]. The novel INNOVANCE Platelet function Analyzer 100 P2Y cartridge (PFA P2Y) (Siemens Healthcare
Diagnostics Products GmbH, Marburg, Germany) was employed to assess specific ADP P2Y12-blockage on in-vitro high-shear stress-dependent platelet function [19], following the manufacturer’s test procedure (B4170G22C0502–1199-SD/ST; Siemens). Essentially, in this assay, citrated blood is automatically aspired at vacuum-induced high shear rates (5000–6000 s1) through a microscopic aperture (150 mm diameter) in a membrane coated with 20 mg ADP, 5 ng PGE1 and 431 mg calcium dichloride dehydrate. This agonist mix induces P2Y12-mediated platelet activation and aggregation, leading to the formation of a platelet plug that closes the aperture, thus stopping the blood flow. The results are given as the time taken to occlude the aperture or closure time in seconds. Following the manufacturer proposal, HTPR on this system was defined as closure time less than 106 s. Finally, we use a commercial vasodilator-stimulated phosphoprotein (VASP) kit (Biocytex Inc, Marseille, France) to assess ex-vivo P2Y12 blockage [20] by flow cytometry (FacsCalibur, Becton Dickinson, Franklin Lakes, New Jersey, USA). The extent of VASP phosphorylation (VASP-ADP) was assessed by difference in the mean fluorescence intensity (MFI) values between samples incubated with PGE1 in the absence or presence of ADP and expressed as a platelet reactivity index (PRI) [PRI(%) ¼ {(MFIPGE1) – (MFIPGE1þADP)/(MFIPGE1)} 100], which inversely correlated with the clopidogrelmediated P2Y12inhibition. PRI at least 50% was the cutoff used to define HTPR [11]. As a control of the functionality of the VASP pathway, in all patients, we also assessed the clopidogrel-independent VASP phosphorylation upon stimulation of blood with PGE1 in the absence or presence of 5 mmol/l epinephrine (VASP-EPI). Statistical analysis
Variables are presented as mean standard deviation or counts (percentages). Statistical analysis was performed using SPSS 15.0 for Windows (SPSS, Inc., Chicago, Illinois, USA) and P values of less than 0.05 were accepted as statistically significant. Normal distribution of continuous data was assessed by Kolmogorov– Smirnov test. For comparison of variables, between days or assays, x2, paired t-student or Wilcoxon tests were used as appropriate. Correlations between quantitative results obtained with the various platelet function assays at D7 or D90, and between the results obtained at D7 and D90 with the same test, were performed using Pearson’s or Spearman’s correlation, as indicated. To compare patient groups with or without HTPR within a continuous variable, we used an unpaired Student’s t-test or the Mann–Whitney U test, as appropriate. Cohen’s kappa statistic (k) [21] was used to evaluate the agreement between the different platelet assays for categorizing patients with and without HTPR.
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606 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 6
Demographic and clinical characteristics of the eighteen patients with cerebrovascular disease included in the study
Table 1
Parameter
after clopidogrel introduction (75 mg/day), at the discretion of their neurologist. All patients displayed normal blood cell counts and haematocrit at D7 and D90 (Table 1). Genotyping revealed three (16.7%) and two (11.1%) polymorphic allele carriers for CYP2C192/A and CYP2C1917/T, respectively (Table 1). Both genotype distributions fulfilled the Hardy–Weinberg equilibrium (P ¼ 0.362 and P ¼ 0.557, respectively).
Mean SD or n (%) 72.4 9.9 9 (50.0) 14 (77.8) 12 (66.7) 9 (50.0) 4 (22.2) 2 (11.1) 48.9 14.0 106.3 36.1 152.4 119.7
Age Male sex Hypertension Diabetes mellitus Hypercholesterolemia Smoking habit Alcohol consumption HDL (mg/dl) LDL (mg/dl) Triglycerides (mg/dl) Event type (TOAST criteria) TIA Large-artery atherosclerosis Small-vessel occlusion (lacunar) Cardioembolic stroke Stroke of undetermined cause Concomitant diseases Prior stroke/TIA Atrial fibrillation Carotid stenosis PVD Drug regimen Statin Pump proton inhibitor Nonsteroid anti-inflammatory Oral anticoagulant Blood cell measurements Platelet count (x109/l) MPV (fl) Hematocrit (%) Antiplatelet therapy Aspirin and clopidogrel Clopidogrel CYP2C19 genotype CYP2C19M2 G/G CYP2C19M2 M/A CYP2C19M17 C/C CYP2C19M17 M/T
8 4 4 1 1
(44.4) (22.2) (22.2) (5.6) (5.6)
6 4 10 7
(33.3) (22.2) (55.5) (38.9)
(50) (11.1) (11.1) (5.6) D7 198.1 39.3 8.25 0.94 41.9 4.3 D7 11 (61.1) 7 (38.9)
Early patient’s classification for on-clopidogrel platelet reactivity at day 7 is maintained after 3 months under clopidogrel therapy
All patients also displayed normal platelet reactivity to TRAP (LTA-TRAP) and epinephrine (VASP-EPI) (not shown), which are agonists activating signalling pathways nonspecifically targeted by clopidogrel or aspirin. This indicated no abnormalities in basal platelet reactivity nor in the cyclic AMP pathway, which may have influenced the acquisition of a HTRP or No-HTPR phenotype in the patients.
9 2 2 1
15 3 16 2
There was high interindividual variability in the platelet response to ADP with all the assays, both at D7 and D90 (Table 2). We found no significant differences between platelet reactivity measured at D7 and D90, with the exception of LTA-AA probably due to the suspension of concomitant aspirin in half of the patients. However, the correlation between measurements for on-clopidogrel platelet reactivity at D7 and D90 was moderate but significant only for Verify Now P2Y12 and PFA P2Y (Table 2).
D90 200.8 48.3 8.09 1.13 41.1 4.9 D90 6 (33.3) 12 (66.7)
(83.3) (16.7) (88.9) (11.1)
Dual antiplatelet therapy leads to lower platelet reactivity
Stroke classification for stroke at entry was performed according to TOAST [15]. MPV, mean platelet volume; PVD, peripheral vascular disease; TIA, transient ischemic attack.
When patients were divided according to their aspirin status, those patients being treated with concomitant aspirin and clopidogrel displayed a trend towards lower platelet reactivity in most tests, which reached statistical significance for LTA-AA at D7 (16.8 8.2 vs. 86.1 10.1, P < 0.0001) and at D90 (18.6 10.8 vs. 56.3 37.7, P ¼ 0.006).
Results Baseline demographic and clinical characteristics of patients are detailed in Table 1. The qualifying clinical event was TIA in eight (44.4%) patients and stroke of distinct cause in the remaining patients. There was a high prevalence of vascular risk factors. The previously prescribed concomitant drugs, such as statins, were not changed during the study (Table 1). Moreover, aspirin treatment was continued in six (33.3%) of the patients
High prevalence of HTPR among stroke/TIA patients
We have found a high prevalence of HTPR in our cohort. On the basis of LTA-AA, 36.3% patients (four out of 11)
Table 2 Platelet responsiveness to arachidonic acid and ADP as measured by means of different platelet function assays in the patients with cerebrovascular disease being treated either with clopidogrel monotherapy or with clopidogrel and concomitant aspirin
LTA-AA, 1.6 mmol/l (%) LTA-ADP, 5 mmol/l (%) Verify Now P2Y12 (PRU) PFA P2YM (s) VASP-ADP, PRI (%)
D7
D90
P
Correlation coefficient
P
Healthy untreated (n)
43.8 35.9 41.8 13.2 232.6 94.2 171.7 108.5 42.2 25.6
66.7 34.4 49.2 23.2 251.0 82.7 154.1 99.9 34.9 27.0
0.010 0.203 0.297 0.474 0.347
0.513 0.239 0.672 0.522 0.216
0.029 0.339 0.002 0.026 0.389
92.8 5.4 (62) 87.9 7.6 (73) 332.4 9.7 (5) 61,2 14,5 (10) 83.5 5.5 (46)
Values shown are mean SD. Normal distribution of all these continuous variables was confirmed by Kolmogorov–Smirnov statistic. Measurements were performed at days 7 (D7) and 90 (D90) after prescription of clopidogrel (75 mg/day). A paired Student’s t-test was used to assess significant differences between values at days 7 and 90. Pearson´s correlation between results on both days is also shown. Correlation coefficients were interpreted as 0–0.2 no correlation; 0.2–0.4 low correlation; 0.4–0.8 moderate correlation; >0.8 good correlation. AA, arachidonic acid; LTA, light transmission aggregometry; PRI, platelet reactivity index; PRU, P2Y12 reaction units; VASP, vasodilator-stimulated phosphoprotein phosphorylation assay.
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at D7 (LTA-AA ¼ 25.6 5.6%) and 33.3% (two out of six) at D90 (LTA-AA ¼ 56.5 15.5%) showed an inadequate response to aspirin.
Fig. 2
On-clopidogrel HTPR was found in nine (50%) patients according to LTA-ADP more than 46% at D7. This HTPR phenotype remained at D90 in only five of them (55.5%), whereas four non-HTRP patients at D7 became HTRP at D90 (Fig. 1). There was poor agreement between the platelet reactivity phenotype (HTPR/NoHTPR) found at D7 and that obtained at D90 (k statistic ¼ 0.111, P ¼ 0.637).
PFA P2Y, closure time (s)
High on-treatment platelet reactivity in stroke/TIA Jover et al. 607
By means of PFA P2Y, we found that seven out of 18 (38.9%) patients displayed on-clopidogrel HTPR at D7 (closure time 66.8 10.9 s), and 86% of them maintained HTPR at D90 (Fig. 2). In the group of 11 patients with No-HTPR at D7 (closure time 238.5 85.6 s), four (36%) became HTPR at D90 (Fig. 2). For this test, there was moderate but significant agreement between the platelet reactivity phenotype (HTPR/No-HTPR) found at D7 and D90 (k statistic ¼ 0.458, P ¼ 0.040). Using the Verify Now P2Y12 assay, there were 10 (55.5%) patients displaying on-clopidogrel HTPR phenotype (304.3 44.7 PRU, n ¼ 10) at D7, and seven (70%) still showed HTPR at D90 (Fig. 3). Of the eight (44.4%) No-HTPR at D7 (143.0 49.6 PRU, n ¼ 8), half of them became HTPR at D90. Thus, 61.1% patients kept their on-clopidogrel platelet reactivity phenotype at D7 and D90, with a fair but not significant agreement between D7 and D90 (k statistic ¼ 0.203, P ¼ 0.387).
ADP [5 µM]-induced platelet aggregation (%)
Fig. 1
100
300
200
100
0
D7
D90
No-HTPR, (n = 11)
D7
D90
HTPR, (n = 7)
Platelet reactivity measured with the novel INNOVANCE PFA P2Y test in patients with cerebrovascular disease treated with clopidogrel (75 mg/day). The plot shows the evolution of the closure time in the PFA P2Y system, from D7 to D90 after starting treatment with clopidogrel (75 mg/day). Results from patients identified at D7 as HTPR or No-HTPR are shown separately. Dotted line shows the closure time (106 s) below which patients were considered to have HTPR (high on treatment platelet reactivity).
In the VASP-ADP assay, 10 out of 18 (55.6%) patients displayed No-HTPR at D7 (PRI 24.7 13.7), based on PRI greater than 50, and this phenotype remained stable at D90 in 90% of them (Fig. 4). The remaining eight (44.4%) patients displayed HTPR at D7 (PRI 64.2 10.6) and only half of them maintained this HTPR at D90 (Fig. 4). We found a trend for moderate agreement between the platelet reactivity phenotype found at D7 and D90 (k statistic ¼ 0.416, P ¼ 0.060). Noteworthy, HTPR categorization of patients at D7, by LTA-ADP or Verify Now, was consistently associated with higher platelet counts [(platelets 109 /l); HTPR vs. No-HTPR; LTA-ADP: 218.0 37.1 n ¼ 9 vs. 178.1 31.7, n ¼ 9; P ¼ 0.026; Verify Now: 215.4 40.1 n ¼ 10 vs. 176.4 26.6, n ¼ 8; P ¼ 0.031]. Interestingly, HTPR categorization at D7 by PFA P2Y and VASP assays was associated with higher mean platelet volume (MPV) [(fL); HTPR vs. No-HTPR: PFA P2Y 8.78 0.31 n ¼ 7 vs. 7.90 1.05 n ¼ 11, P ¼ 0.023; VASP: 8.95 0.54 n ¼ 8 vs. 7.69 0.80 n ¼ 10, P ¼ 0.002].
75
50
25
0 D7
D90
No-HTPR, (n = 9)
D7
D90
HTPR, (n = 9)
The plots show, for each patient, the change in the aggregation response induced with ADP at 5 mmol/l (a) or 10 mmol/l (B), from D7 to D90 after starting treatment with clopidogrel (75 mg/day). The results from patients identified at D7 as HTPR or No-HTPR are plotted separately. Dotted line corresponds to the cut-off value defining HTPR (high on treatment platelet reactivity).
Correlation and agreement among platelet function assays
Moderate correlation was consistently observed between measurements with these four assays evaluating onclopidogrel platelet reactivity towards ADP. As shown in Table 3, PFA P2Y and VASP-ADP were correlated significantly both at D7 (r ¼ 0.565, P ¼ 0.01) and D90 (r ¼ 0.546, P ¼ 0.05). In contrast, PFA P2Y and Verify Now were significantly correlated only at D7 (r ¼ 0.605,
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608 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 6
Fig. 3
No-HTPR according to their preestablished cut-off values. Thus, LTA-ADP significantly agreed with Verify Now at D7 (k ¼ 0.444, P < 0.05) and with PFA P2Y at D90 (k ¼ 0.444, P < 0.05). PFA P2Y also agreed moderately and significantly with Verify Now (D7 k ¼ 0.458; D90 k ¼ 0.430; P < 0.05) and with VASP (D7 k ¼ 0.430; D90 k ¼ 0.471; P < 0.05). Lastly, the VASP test agreed significantly with the Verify Now only at D90 (k ¼ 0.393; P < 0.05).
Verify now P2Y12, PRU
400
300
200
Discussion
100
0 D7
D90
No-HTPR, (n = 8)
D7
D90
HTPR, (n = 10)
Platelet reactivity measured with the point of care Verify Now P2Y12 in patients in patients with cerebrovascular disease treated with clopidogrel (75 mg/day). The plot shows the change in platelet reactivity measured in the patients by means of the Verify Now P2Y12 test at D7 and D90 after starting clopidogrel therapy. Results from patients identified at D7 as HTPR or No-HTPR are shown separately. The dotted line is the level of platelet reactivity in this assay (235 PRU) that was used to discriminate patients with HTPR (high on treatment platelet reactivity).
P ¼ 0.01). Noteworthy, correlation of LTA-ADP with any of the other assays was weak and did not reach statistical significance. A moderate concordance was found between the assays with regard to the categorization of patients as HTPR or
Fig. 4
VASP-ADP, PRI (%)
100
75
The major findings of this study are a high prevalence of HTPR in ischemic CVD being treated with clopidogrel, around 40% overall, and that the on-clopidogrel platelet reactivity phenotype, HTPR or No-HTPR, is stable over 3 months of treatment in 55–70% of CVD patients. These findings agree with few previous studies evaluating inadequate responsiveness to clopidogrel therapy in the CVD setting [22–26] and with the stability in platelet reactivity that has recently been reported in cardiological patients under chronic clopidogrel treatment [27]. Moreover, our study further confirms that outcome of on-treatment platelet reactivity and subsequent categorization of CVD patients as HTPR or No-HTPR would be largely influenced by many factors including the test of platelet function used and the cutoff value defining the HTPR phenotype. This is first indicated by the fact that correlations between platelet reactivity measurements at D7 and D90 were moderate and only significant for Verify Now and PFA assays but not for LTA-ADP and VASP tests (Table 2), which may reflect higher interassay variability in the latest tests than in semiautomatic points-of-care. Second, the inter-test correlation for platelet reactivity was consistently significant at D7 and D90 only between the PFA P2Y and VASP-ADP tests, although weak-to-moderate significant correlations between the results of other assays were seen solely at D7 or at D90 (Table 3). As stated above, all four assays used here demonstrated consistently a high prevalence of HTPR in ischemic CVD after a week and 3 months of treatment with a standard dose of clopidogrel.
50
25
0 D7
D90
No-HTPR, (n = 10)
D7
D90
HTPR, (n = 8)
Platelet reactivity to ADP measured with the VASP assay in patients with cerebrovascular disease treated with clopidogrel (75 mg/day). Plot shows the change in the platelet reactivity index (PRI) in VASP test from D7 to D90 after starting treatment with clopidogrel. Results from patients classified at D7 as HTPR (high on treatment platelet reactivity) and No-HTPR according to a cut-off values of PRI > 50% (dotted line) are shown separately.
Thus, LTA with ADP 5 mmol/l, cut-off of LTA-ADP greater than 46% [11], led to a 50% prevalence of HTPR in our cohort. This is close to the 44% of clopidogrel nonresponders found in a retrospective analysis of 142 neurological patients taking clopidogrel for the secondary prophylaxis of cerebrovascular events [22]. A similarly high HTPR prevalence, based on LTA-ADP, was recently reported in a clopidogrel tailoring study in ischaemic stroke/TIA patients [24]. In contrast, other recent studies using platelet aggregometry have reported prevalence of just around 20% of on-clopidogrel HTPR in patients with ischemic stroke [14,23]. Likely, the different clinical background of the patients and interlaboratory variations in sample handling and assay
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High on-treatment platelet reactivity in stroke/TIA Jover et al. 609
Correlation between platelet reactivity to ADP measured with different platelet function tests in patients with cerebrovascular disease at days 7 (D7) and 90 (D90) of clopidogrel (75 mg/day) treatment
Table 3
D7 LTA–ADP
PFA P2Y –0.307
LTA–ADP PFA P2Y
–0.432
Verify Now P2Y12
0.397
–0.267
VASP–ADP
0.125
–0.546*
Verify Now P2Y12 VASP–ADP 0.389
0.289
–0.605**
–0.565* 0.294
0.092
D90 Values are Spearman´s correlation coefficients between values of platelet reactivity obtained with the different assays at day 7 (D7, above the dotted arrow) and at day 90 (D90, below the dotted arrow). Correlation coefficients were interpreted as 0–0.2 no correlation; 0.2–0.4 low correlation; 0.4–0.8 moderate correlation; >0.8 good correlation. LTA-ADP, light transmission aggregometry induced with 5 mmol/l ADP; Verify Now P2Y12, Verify Now cartridge stimulated with ADP; VASP, vasodilatorstimulated phosphoprotein (VASP) phosphorylation assay; MCorrelation is significant at 0.05 level. MMCorrelation is significant at 0.01 level.
conditions account for much of the differences among studies assessing on-clopidogrel HTPR in CVD patients by platelet aggregation assays. Using the Verify Now P2Y12 assay and the cut-off of PRU greater than 235, we found a 60% prevalence of HTPR in our cohort, whereas a recent study in ischemic CVD patients under clopidogrel monotherapy reported a 44% prevalence of HTPR defined as PRU greater than 194 [25]. Using the last criterion, our prevalence of HTPR rose to 80%. Interestingly, 61% of the patients maintained the HTPR phenotype over time. Furthermore, in two recent studies that defined HTPR as less than 20% inhibition of platelet aggregation induced by ADP, calculated automatically by the Verify Now device from comparison with iso-TRAP induced aggregation, the prevalence of on-clopidogrel HTPR were 26 and 54% [28,29]. If we use this definition, the prevalence of HTPR in our cohort of ischemic CVD patients rises to 55%. To the best of our knowledge, this is the first study using the new INNOVANCE PFA P2Y cartridge to monitor clopidogrel inhibition of platelet function in CVD patients. With this novel test, the prevalence of HTPR, defined as closure time less than 106 s, was around 40% in our patients, which is much lower than that previously reported using PFA-100 with COL-EPI and COL-ADP cartridges [25,30–32]. There is also scarce information regarding monitorization of on-treatment platelet function in CVD patients, by means of the VASP assay that specifically assesses the effect of P2Y12 blockage by clopidogrel at the intraplatelet signalling cascade [20]. Here, the VASP provided a 30–45% prevalence of HTPR, comparable to that observed with the other assays. Importantly, we observed that if distribution of the platelet reactivity phenotype, that is HTPR or NoHTPR, is categorized according to the intensity of the
antiplatelet therapy given to patients, that is clopidogrel vs. concomitant aspirin and clopidogrel, all assays, except Verify Now P2Y12, showed a trend at D90 towards more HTPR in patients following clopidogrel monotherapy (data not shown). In concordance, more potent inhibition of platelet activity following dual antiplatelet therapy after a recent stroke was also reported in the PLUTOStroke Trial [33] and other recent studies in CVD patients [14,22]. Dual antiplatelet therapy lowers platelet reactivity by compensating the ADP-induced TXA2 generation in human platelets [34], but, in contrast to the cardiology scenario, available guidelines do not recommend combined therapy with clopidogrel and aspirin in CVD patients [2–4]. Regarding the categorization of patient’s platelet phenotype as HTPR or No-HTPR, it is remarkable that we found, at best, a moderate degree of agreement (k statistic) between D7 and D90 for each assay, and between the different methods both in D7 and D90, and these agreements were highly dependent on the definition of HTPR in each test. For instance, for the Verify Now P2Y12, there was weak and nonsignificant agreement between D7 and D90 based on a cut-off of PRU of more than 235 (k ¼ 0.203, P ¼ 0.387), but moderate and significant (k ¼ 0.491, P ¼ 0.016) using a cut-off of PRU of more than 167. Other authors have previously shown lack of reproducibility over time and discordance between LTA, Verify Now and PFA-100, in identifying HTPR among CVD patients being treated with aspirin or clopidogrel [13,22,23,25,28]. Noteworthy, of the assays used in this study, the novel PFA P2Y test provides the best agreement in the categorization of patients as HTPR or No-HTPR between D7 and D90, and with the result of others tests, thus supporting that INNOVANCE PFA-100 P2Y is at least comparable to other platelet function assays for evaluating on-clopidogrel platelet reactivity in CVD patients.
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610 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 6
The mechanisms associated with the phenomenon of HTPR in CVD and other clinical settings are complex and multifactorial, including noncompliance, concomitant diseases, interference with other drugs or genetic differences in enzymes mediating drug absorption and metabolism and in platelet receptors [35,36]. Indeed, HTPR could result either from insufficient inhibition of normal baseline platelet reactivity or from adequate inhibition of hyper-reactive platelets [10]. Moreover, factors not commonly related with platelet´s reactivity to agonist may play a role in HTPR. Thus, we observed that CVD patients with HTPR, based on different tests, had significantly higher platelet counts and/or MPV than those without HTPR. This finding agrees with a recent study in the same type of patients [25] and supports the idea that larger and increased numbers of platelets genetically determined could influence platelet reactivity and the clinical outcome in cardiovascular and CVD patients [37]. Furthermore, up to 67% of our patients have diabetes mellitus, which has been recently identified as a major risk factor for on-clopidogrel HTPR in acute ischemic stroke patients [26] and had been previously associated with higher MPV. As no overrepresentation of the variant alleles CYP2C192/A or CYP2C1917/T was observed in this cohort as compared with our general population [16], it is unlikely that our findings have been influenced by the CYP2C19 genotype. Compliance is a major issue for the response to antiplatelet therapy. In our study, medication compliance during clinical admittance was assured (D7), but it could not be guaranteed at D90, despite the phone confirmation to ensure medication compliance during, at least, the previous 10 days. Finally, the clinical impact of HTPR observed over time with distinct tests in this small cohort of ischemic CVD patients is uncertain. Obviously, the small sample size is a major limitation that precludes identifying which test and cut-off, if any, is best for predicting ischemic or bleeding complications in a CVD setting. Anecdotally, we reviewed clinical records after 1 year of the patient inclusion and found that three (16.7%) patients suffered adverse outcomes (one TIA, one non-ST elevation acute coronary syndrome and one large artery stroke). At D90, these patients displayed HTPR by Verify Now and PFA P2Y, which may, with caution, suggest that CVD patients with HTPR are at an increased risk of recurrent ischemia. Indeed, recent studies in CVD or cardiovascular settings tailoring antiplatelet therapy according to platelet function tests [12,24,38] have provided controversial results.
weak-to-moderate agreement between assays. For the first time, we show that INNOVANCE PFA-100 P2Y and VASP assays are not any worse than LTA or Verify Now P2Y12 for the purpose of platelet function monitoring in CVD patients treated with clopidogrel. Although there is much evidence of worse outcome in patients with coronary syndrome and HTPR, prospective studies are required to elucidate the clinical relevance of HTPR in ischemic stroke/TIA patients.
Acknowledgements This work was funded by the Instituto de Salud Carlos III (ISCIII, PI10/02594, PI11/00566), RIC RD12/0042/0050 (ISCIII and FEDER) and Fundacio´n Se´neca (07703/ GERM/07). I.S.G. is a fellowship from ISCIII (FI10/ 00535). C.M. is an investigator from Fundacio´n para la Formacio´n e Investigacio´n Sanitarias de la Regio´n de Murcia (FFIS) Conflicts of interest
The INNOVANCE PFA P2Y cartridges used in the study were generously provided free of charge by (Siemens Healthcare Diagnostics SL, Barcelona, Spain). The authors declare no financial or other conflict of interest.
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This study demonstrates that a significant proportion of ischemic CVD patients who start therapy with clopidogrel have an HTPR phenotype that seems to be stable over time. The prevalence of HTPR in these CVD patients is, however, highly test-dependent, with only
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High on-treatment platelet reactivity in patients with ischemic cerebrovascular disease: assessment of prevalence and stability over time using four platelet function tests Eva Jovera, Jose´ M. Rodrı´guezb, Agustina Bernalc, Ana B. Arroyoc, Juan A. Iniestab, Isabel Sa´nchez Guiu´c, Constantino Martı´nezc, Vicente Vicentec, Marı´a L. Lozanoc,M and Jose´ Riverac,M High on-treatment platelet reactivity (HTPR), referred to as a higher than expected platelet reactivity in patients under antiplatelet therapy, could influence outcome in cerebrovascular disease (CVD), but its prevalence and its stability over time is uncertain. Platelet reactivity was assessed in 18 patients with ischemic stroke/transient ischemic attack (TIA) 7 days (D7) and 90 days (D90) after prescription of clopidogrel, using four methods: light transmission aggregometry with 5 mmol/l ADP (LTA-ADP), vasodilator-stimulated phosphoprotein (VASP), Verify Now P2Y12 and platelet function analyzer (PFA) P2Y. HTPR was defined as LTA-ADP more than 46%; PFA-100-P2Y closure time less than 106 s; VerifyNow P2Y12, PRU greater than 235, VASP, PRI greater than 50%. Patients displayed, both at D7 and D90, a marked inhibition of platelet reactivity towards ADP in all tests as compared with reference levels. Correlations between the results obtained with all the tests at D7 and D90 and between measurements on each day in each test were low-to-moderate. The prevalence of HTPR for all the tests was 40% at D7 and 42% at D90. There was a moderate degree of agreement (k statistic < 0.5) between tests with regard to categorizing patients as HTPR/NoHTPR (D7 and D90). The on-clopidogrel platelet reactivity phenotype, HTPR/No-HTPR, remained stable in 55–72% of
Introduction Ischemic cerebrovascular disease (CVD), including ischemic stroke or transient ischemic attack (TIA), is a highly recurrent disease and one of the main causes of morbidity and mortality worldwide [1]. Antiplatelet therapy is a first-line option for the secondary prevention of acute ischemic stroke/TIA and its long-term treatment [2–4]. Aspirin is the most commonly prescribed treatment in CVD, but the use of clopidogrel monotherapy, aspirin and dipyridamole, and aspirin and clopidogrel has also been reported in several studies [5–9]. Recently, the new concept of high on-treatment platelet reactivity (HTPR) has emerged in the cardiovascular field. This refers to the limited degree of inhibition of platelet function, as assessed by various assays, compared with the inhibition expected using antiplatelet therapy [10,11]. Indeed, HTPR has been associated with poor cardiovascular outcomes and might be of clinical value for identifying patients with a high risk of recurrent vascular events who may benefit from intensified antiplatelet 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
patients, depending on the test. A high prevalence of HTPR is found among CVD patients treated with clopidogrel and this platelet reactivity phenotype remains over time. There is poor agreement between the different platelet function tests for categorizing the platelet reactivity phenotype in these patients. The new PFA-100 P2Y equals other platelet function assays for evaluating HTPR in CVD. Blood Coagul Fibrinolysis 25:604–611 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2014, 25:604–611 Keywords: clopidogrel, ischemic stroke, on-treatment platelet reactivity, platelet function testing, transient ischemic attack a Hospital Universitario Virgen de la Arrixaca, bHospital Universitario Reina Sofı´a and cCentro Regional de Hemodonacio´n, University of Murcia, IMIB, Murcia, Spain
Correspondence to Jose´ Rivera, PhD, Centro Regional de Hemodonacio´n, C/ Ronda de Garay S/N. 30003, Murcia, Spain Tel: +34968341990; fax: +34968261914; e-mail: [email protected] Both Marı´a L. Lozano and Jose´ Rivera contributed equally to the writing of this
article. Received 2 September 2013 Revised 22 February 2014 Accepted 28 February 2014
treatment [12]. However, in the setting of CVD, there is much less information available on the prevalence of exvivo nonappropriate responsiveness to antiplatelet therapies or HTPR [13,14]. The heterogeneous cause of stroke/ TIA [15] compared with ischemic heart disease makes necessary to identify HTPR in those particular scenarios. In this study, we have used different platelet function tests to investigate the HTPR phenotype in unselected patients with ischemic stroke/TIA, being treated with clopidogrel monotherapy or with a combination of clopidogrel and aspirin. A double evaluation of platelet function, 1 week and 3 months after the start of clopidogrel treatment, was performed in all patients in order to evaluate the stability over time of the HTPR phenotype.
Materials and methods Patients
This study involved 18 consecutive white patients diagnosed with ischemic stroke or TIA according to the DOI:10.1097/MBC.0000000000000118
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High on-treatment platelet reactivity in stroke/TIA Jover et al. 605
TOAST criteria [15]. Upon hospital admission, patients were prescribed clopidogrel (75 mg/day), either as a monotherapy or as a complement to previously prescribed aspirin (100–300 mg/day) at the discretion of the attending neurologist. Exclusion criteria to enter the study were contraindication for clopidogrel therapy, active infection, inflammatory disease, thrombocytopenia (A; rs4244285) and CYP2C1917 (806C>T; rs12248560) [16], as additional demographical-clinical data. The study complies with the Helsinki Declaration and was approved by the ethics committee of Hospital Universitario Reina Sofia (28 April 2010). All patients gave written informed consent to participate. Blood sampling and platelet function assays
Platelet function testing was performed at two different time points after the initiation of clopidogrel therapy: Day 7 (D7) while the patient was still in hospital and day 90 (D90) after the patient had been discharged. On both days, venous blood samples were collected after overnight fasting and before taking the daily dose of clopidogrel in order to detect the on-treatment platelet reactivity at the theoretical trough level of the clopidogrel active metabolite. Light transmittance aggregometry (LTA) was performed in platelet-rich plasma (PRP) (D7: 295.2 82.1 109 platelets/l; range 150–438 109/l; D90: 293.8 77.9 109platelets/l; range 176–428 109/l) (Aggrecorder II; Menarini Diagnostics, Florence, Italy) essentially as previously described [16,17], using the following agonists: 5 mmol/l ADP (LTA-ADP) (DiaMed, Cressier, Switzerland), 1.6 mmol/l arachidonic acid (LTA-AA) (DiaMed) and 25 mmol/l thrombin receptor activating peptide (TRAP) (LTA-TRAP) (Sigma-Aldrich Quı´mica SL, Madrid, Spain). LTA-TRAP was used as an intraassay positive control of each patient’s platelet aggregation status, as this response is mediated by the thrombin receptor protease-activating receptor-1 (PAR-1) and is not blocked by aspirin or clopidogrel. An LTA-ADP of at least 46% defined on-clopidogrel HTPR, as proposed in cardiovascular patients [11], and LTA-AA at least 20%, was considered to reflect biochemical aspirin resistance. Verify Now (Accumetrics, San Diego, California, USA), a point-of-care assay evaluating platelet aggregation in whole blood [18], with P2Y12 cartridges was also used according to the manufacturer’s instructions. On-clopidogrel HTPR in this test was defined as PRU of at least 235 [11]. The novel INNOVANCE Platelet function Analyzer 100 P2Y cartridge (PFA P2Y) (Siemens Healthcare
Diagnostics Products GmbH, Marburg, Germany) was employed to assess specific ADP P2Y12-blockage on in-vitro high-shear stress-dependent platelet function [19], following the manufacturer’s test procedure (B4170G22C0502–1199-SD/ST; Siemens). Essentially, in this assay, citrated blood is automatically aspired at vacuum-induced high shear rates (5000–6000 s1) through a microscopic aperture (150 mm diameter) in a membrane coated with 20 mg ADP, 5 ng PGE1 and 431 mg calcium dichloride dehydrate. This agonist mix induces P2Y12-mediated platelet activation and aggregation, leading to the formation of a platelet plug that closes the aperture, thus stopping the blood flow. The results are given as the time taken to occlude the aperture or closure time in seconds. Following the manufacturer proposal, HTPR on this system was defined as closure time less than 106 s. Finally, we use a commercial vasodilator-stimulated phosphoprotein (VASP) kit (Biocytex Inc, Marseille, France) to assess ex-vivo P2Y12 blockage [20] by flow cytometry (FacsCalibur, Becton Dickinson, Franklin Lakes, New Jersey, USA). The extent of VASP phosphorylation (VASP-ADP) was assessed by difference in the mean fluorescence intensity (MFI) values between samples incubated with PGE1 in the absence or presence of ADP and expressed as a platelet reactivity index (PRI) [PRI(%) ¼ {(MFIPGE1) – (MFIPGE1þADP)/(MFIPGE1)} 100], which inversely correlated with the clopidogrelmediated P2Y12inhibition. PRI at least 50% was the cutoff used to define HTPR [11]. As a control of the functionality of the VASP pathway, in all patients, we also assessed the clopidogrel-independent VASP phosphorylation upon stimulation of blood with PGE1 in the absence or presence of 5 mmol/l epinephrine (VASP-EPI). Statistical analysis
Variables are presented as mean standard deviation or counts (percentages). Statistical analysis was performed using SPSS 15.0 for Windows (SPSS, Inc., Chicago, Illinois, USA) and P values of less than 0.05 were accepted as statistically significant. Normal distribution of continuous data was assessed by Kolmogorov– Smirnov test. For comparison of variables, between days or assays, x2, paired t-student or Wilcoxon tests were used as appropriate. Correlations between quantitative results obtained with the various platelet function assays at D7 or D90, and between the results obtained at D7 and D90 with the same test, were performed using Pearson’s or Spearman’s correlation, as indicated. To compare patient groups with or without HTPR within a continuous variable, we used an unpaired Student’s t-test or the Mann–Whitney U test, as appropriate. Cohen’s kappa statistic (k) [21] was used to evaluate the agreement between the different platelet assays for categorizing patients with and without HTPR.
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606 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 6
Demographic and clinical characteristics of the eighteen patients with cerebrovascular disease included in the study
Table 1
Parameter
after clopidogrel introduction (75 mg/day), at the discretion of their neurologist. All patients displayed normal blood cell counts and haematocrit at D7 and D90 (Table 1). Genotyping revealed three (16.7%) and two (11.1%) polymorphic allele carriers for CYP2C192/A and CYP2C1917/T, respectively (Table 1). Both genotype distributions fulfilled the Hardy–Weinberg equilibrium (P ¼ 0.362 and P ¼ 0.557, respectively).
Mean SD or n (%) 72.4 9.9 9 (50.0) 14 (77.8) 12 (66.7) 9 (50.0) 4 (22.2) 2 (11.1) 48.9 14.0 106.3 36.1 152.4 119.7
Age Male sex Hypertension Diabetes mellitus Hypercholesterolemia Smoking habit Alcohol consumption HDL (mg/dl) LDL (mg/dl) Triglycerides (mg/dl) Event type (TOAST criteria) TIA Large-artery atherosclerosis Small-vessel occlusion (lacunar) Cardioembolic stroke Stroke of undetermined cause Concomitant diseases Prior stroke/TIA Atrial fibrillation Carotid stenosis PVD Drug regimen Statin Pump proton inhibitor Nonsteroid anti-inflammatory Oral anticoagulant Blood cell measurements Platelet count (x109/l) MPV (fl) Hematocrit (%) Antiplatelet therapy Aspirin and clopidogrel Clopidogrel CYP2C19 genotype CYP2C19M2 G/G CYP2C19M2 M/A CYP2C19M17 C/C CYP2C19M17 M/T
8 4 4 1 1
(44.4) (22.2) (22.2) (5.6) (5.6)
6 4 10 7
(33.3) (22.2) (55.5) (38.9)
(50) (11.1) (11.1) (5.6) D7 198.1 39.3 8.25 0.94 41.9 4.3 D7 11 (61.1) 7 (38.9)
Early patient’s classification for on-clopidogrel platelet reactivity at day 7 is maintained after 3 months under clopidogrel therapy
All patients also displayed normal platelet reactivity to TRAP (LTA-TRAP) and epinephrine (VASP-EPI) (not shown), which are agonists activating signalling pathways nonspecifically targeted by clopidogrel or aspirin. This indicated no abnormalities in basal platelet reactivity nor in the cyclic AMP pathway, which may have influenced the acquisition of a HTRP or No-HTPR phenotype in the patients.
9 2 2 1
15 3 16 2
There was high interindividual variability in the platelet response to ADP with all the assays, both at D7 and D90 (Table 2). We found no significant differences between platelet reactivity measured at D7 and D90, with the exception of LTA-AA probably due to the suspension of concomitant aspirin in half of the patients. However, the correlation between measurements for on-clopidogrel platelet reactivity at D7 and D90 was moderate but significant only for Verify Now P2Y12 and PFA P2Y (Table 2).
D90 200.8 48.3 8.09 1.13 41.1 4.9 D90 6 (33.3) 12 (66.7)
(83.3) (16.7) (88.9) (11.1)
Dual antiplatelet therapy leads to lower platelet reactivity
Stroke classification for stroke at entry was performed according to TOAST [15]. MPV, mean platelet volume; PVD, peripheral vascular disease; TIA, transient ischemic attack.
When patients were divided according to their aspirin status, those patients being treated with concomitant aspirin and clopidogrel displayed a trend towards lower platelet reactivity in most tests, which reached statistical significance for LTA-AA at D7 (16.8 8.2 vs. 86.1 10.1, P < 0.0001) and at D90 (18.6 10.8 vs. 56.3 37.7, P ¼ 0.006).
Results Baseline demographic and clinical characteristics of patients are detailed in Table 1. The qualifying clinical event was TIA in eight (44.4%) patients and stroke of distinct cause in the remaining patients. There was a high prevalence of vascular risk factors. The previously prescribed concomitant drugs, such as statins, were not changed during the study (Table 1). Moreover, aspirin treatment was continued in six (33.3%) of the patients
High prevalence of HTPR among stroke/TIA patients
We have found a high prevalence of HTPR in our cohort. On the basis of LTA-AA, 36.3% patients (four out of 11)
Table 2 Platelet responsiveness to arachidonic acid and ADP as measured by means of different platelet function assays in the patients with cerebrovascular disease being treated either with clopidogrel monotherapy or with clopidogrel and concomitant aspirin
LTA-AA, 1.6 mmol/l (%) LTA-ADP, 5 mmol/l (%) Verify Now P2Y12 (PRU) PFA P2YM (s) VASP-ADP, PRI (%)
D7
D90
P
Correlation coefficient
P
Healthy untreated (n)
43.8 35.9 41.8 13.2 232.6 94.2 171.7 108.5 42.2 25.6
66.7 34.4 49.2 23.2 251.0 82.7 154.1 99.9 34.9 27.0
0.010 0.203 0.297 0.474 0.347
0.513 0.239 0.672 0.522 0.216
0.029 0.339 0.002 0.026 0.389
92.8 5.4 (62) 87.9 7.6 (73) 332.4 9.7 (5) 61,2 14,5 (10) 83.5 5.5 (46)
Values shown are mean SD. Normal distribution of all these continuous variables was confirmed by Kolmogorov–Smirnov statistic. Measurements were performed at days 7 (D7) and 90 (D90) after prescription of clopidogrel (75 mg/day). A paired Student’s t-test was used to assess significant differences between values at days 7 and 90. Pearson´s correlation between results on both days is also shown. Correlation coefficients were interpreted as 0–0.2 no correlation; 0.2–0.4 low correlation; 0.4–0.8 moderate correlation; >0.8 good correlation. AA, arachidonic acid; LTA, light transmission aggregometry; PRI, platelet reactivity index; PRU, P2Y12 reaction units; VASP, vasodilator-stimulated phosphoprotein phosphorylation assay.
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at D7 (LTA-AA ¼ 25.6 5.6%) and 33.3% (two out of six) at D90 (LTA-AA ¼ 56.5 15.5%) showed an inadequate response to aspirin.
Fig. 2
On-clopidogrel HTPR was found in nine (50%) patients according to LTA-ADP more than 46% at D7. This HTPR phenotype remained at D90 in only five of them (55.5%), whereas four non-HTRP patients at D7 became HTRP at D90 (Fig. 1). There was poor agreement between the platelet reactivity phenotype (HTPR/NoHTPR) found at D7 and that obtained at D90 (k statistic ¼ 0.111, P ¼ 0.637).
PFA P2Y, closure time (s)
High on-treatment platelet reactivity in stroke/TIA Jover et al. 607
By means of PFA P2Y, we found that seven out of 18 (38.9%) patients displayed on-clopidogrel HTPR at D7 (closure time 66.8 10.9 s), and 86% of them maintained HTPR at D90 (Fig. 2). In the group of 11 patients with No-HTPR at D7 (closure time 238.5 85.6 s), four (36%) became HTPR at D90 (Fig. 2). For this test, there was moderate but significant agreement between the platelet reactivity phenotype (HTPR/No-HTPR) found at D7 and D90 (k statistic ¼ 0.458, P ¼ 0.040). Using the Verify Now P2Y12 assay, there were 10 (55.5%) patients displaying on-clopidogrel HTPR phenotype (304.3 44.7 PRU, n ¼ 10) at D7, and seven (70%) still showed HTPR at D90 (Fig. 3). Of the eight (44.4%) No-HTPR at D7 (143.0 49.6 PRU, n ¼ 8), half of them became HTPR at D90. Thus, 61.1% patients kept their on-clopidogrel platelet reactivity phenotype at D7 and D90, with a fair but not significant agreement between D7 and D90 (k statistic ¼ 0.203, P ¼ 0.387).
ADP [5 µM]-induced platelet aggregation (%)
Fig. 1
100
300
200
100
0
D7
D90
No-HTPR, (n = 11)
D7
D90
HTPR, (n = 7)
Platelet reactivity measured with the novel INNOVANCE PFA P2Y test in patients with cerebrovascular disease treated with clopidogrel (75 mg/day). The plot shows the evolution of the closure time in the PFA P2Y system, from D7 to D90 after starting treatment with clopidogrel (75 mg/day). Results from patients identified at D7 as HTPR or No-HTPR are shown separately. Dotted line shows the closure time (106 s) below which patients were considered to have HTPR (high on treatment platelet reactivity).
In the VASP-ADP assay, 10 out of 18 (55.6%) patients displayed No-HTPR at D7 (PRI 24.7 13.7), based on PRI greater than 50, and this phenotype remained stable at D90 in 90% of them (Fig. 4). The remaining eight (44.4%) patients displayed HTPR at D7 (PRI 64.2 10.6) and only half of them maintained this HTPR at D90 (Fig. 4). We found a trend for moderate agreement between the platelet reactivity phenotype found at D7 and D90 (k statistic ¼ 0.416, P ¼ 0.060). Noteworthy, HTPR categorization of patients at D7, by LTA-ADP or Verify Now, was consistently associated with higher platelet counts [(platelets 109 /l); HTPR vs. No-HTPR; LTA-ADP: 218.0 37.1 n ¼ 9 vs. 178.1 31.7, n ¼ 9; P ¼ 0.026; Verify Now: 215.4 40.1 n ¼ 10 vs. 176.4 26.6, n ¼ 8; P ¼ 0.031]. Interestingly, HTPR categorization at D7 by PFA P2Y and VASP assays was associated with higher mean platelet volume (MPV) [(fL); HTPR vs. No-HTPR: PFA P2Y 8.78 0.31 n ¼ 7 vs. 7.90 1.05 n ¼ 11, P ¼ 0.023; VASP: 8.95 0.54 n ¼ 8 vs. 7.69 0.80 n ¼ 10, P ¼ 0.002].
75
50
25
0 D7
D90
No-HTPR, (n = 9)
D7
D90
HTPR, (n = 9)
The plots show, for each patient, the change in the aggregation response induced with ADP at 5 mmol/l (a) or 10 mmol/l (B), from D7 to D90 after starting treatment with clopidogrel (75 mg/day). The results from patients identified at D7 as HTPR or No-HTPR are plotted separately. Dotted line corresponds to the cut-off value defining HTPR (high on treatment platelet reactivity).
Correlation and agreement among platelet function assays
Moderate correlation was consistently observed between measurements with these four assays evaluating onclopidogrel platelet reactivity towards ADP. As shown in Table 3, PFA P2Y and VASP-ADP were correlated significantly both at D7 (r ¼ 0.565, P ¼ 0.01) and D90 (r ¼ 0.546, P ¼ 0.05). In contrast, PFA P2Y and Verify Now were significantly correlated only at D7 (r ¼ 0.605,
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608 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 6
Fig. 3
No-HTPR according to their preestablished cut-off values. Thus, LTA-ADP significantly agreed with Verify Now at D7 (k ¼ 0.444, P < 0.05) and with PFA P2Y at D90 (k ¼ 0.444, P < 0.05). PFA P2Y also agreed moderately and significantly with Verify Now (D7 k ¼ 0.458; D90 k ¼ 0.430; P < 0.05) and with VASP (D7 k ¼ 0.430; D90 k ¼ 0.471; P < 0.05). Lastly, the VASP test agreed significantly with the Verify Now only at D90 (k ¼ 0.393; P < 0.05).
Verify now P2Y12, PRU
400
300
200
Discussion
100
0 D7
D90
No-HTPR, (n = 8)
D7
D90
HTPR, (n = 10)
Platelet reactivity measured with the point of care Verify Now P2Y12 in patients in patients with cerebrovascular disease treated with clopidogrel (75 mg/day). The plot shows the change in platelet reactivity measured in the patients by means of the Verify Now P2Y12 test at D7 and D90 after starting clopidogrel therapy. Results from patients identified at D7 as HTPR or No-HTPR are shown separately. The dotted line is the level of platelet reactivity in this assay (235 PRU) that was used to discriminate patients with HTPR (high on treatment platelet reactivity).
P ¼ 0.01). Noteworthy, correlation of LTA-ADP with any of the other assays was weak and did not reach statistical significance. A moderate concordance was found between the assays with regard to the categorization of patients as HTPR or
Fig. 4
VASP-ADP, PRI (%)
100
75
The major findings of this study are a high prevalence of HTPR in ischemic CVD being treated with clopidogrel, around 40% overall, and that the on-clopidogrel platelet reactivity phenotype, HTPR or No-HTPR, is stable over 3 months of treatment in 55–70% of CVD patients. These findings agree with few previous studies evaluating inadequate responsiveness to clopidogrel therapy in the CVD setting [22–26] and with the stability in platelet reactivity that has recently been reported in cardiological patients under chronic clopidogrel treatment [27]. Moreover, our study further confirms that outcome of on-treatment platelet reactivity and subsequent categorization of CVD patients as HTPR or No-HTPR would be largely influenced by many factors including the test of platelet function used and the cutoff value defining the HTPR phenotype. This is first indicated by the fact that correlations between platelet reactivity measurements at D7 and D90 were moderate and only significant for Verify Now and PFA assays but not for LTA-ADP and VASP tests (Table 2), which may reflect higher interassay variability in the latest tests than in semiautomatic points-of-care. Second, the inter-test correlation for platelet reactivity was consistently significant at D7 and D90 only between the PFA P2Y and VASP-ADP tests, although weak-to-moderate significant correlations between the results of other assays were seen solely at D7 or at D90 (Table 3). As stated above, all four assays used here demonstrated consistently a high prevalence of HTPR in ischemic CVD after a week and 3 months of treatment with a standard dose of clopidogrel.
50
25
0 D7
D90
No-HTPR, (n = 10)
D7
D90
HTPR, (n = 8)
Platelet reactivity to ADP measured with the VASP assay in patients with cerebrovascular disease treated with clopidogrel (75 mg/day). Plot shows the change in the platelet reactivity index (PRI) in VASP test from D7 to D90 after starting treatment with clopidogrel. Results from patients classified at D7 as HTPR (high on treatment platelet reactivity) and No-HTPR according to a cut-off values of PRI > 50% (dotted line) are shown separately.
Thus, LTA with ADP 5 mmol/l, cut-off of LTA-ADP greater than 46% [11], led to a 50% prevalence of HTPR in our cohort. This is close to the 44% of clopidogrel nonresponders found in a retrospective analysis of 142 neurological patients taking clopidogrel for the secondary prophylaxis of cerebrovascular events [22]. A similarly high HTPR prevalence, based on LTA-ADP, was recently reported in a clopidogrel tailoring study in ischaemic stroke/TIA patients [24]. In contrast, other recent studies using platelet aggregometry have reported prevalence of just around 20% of on-clopidogrel HTPR in patients with ischemic stroke [14,23]. Likely, the different clinical background of the patients and interlaboratory variations in sample handling and assay
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High on-treatment platelet reactivity in stroke/TIA Jover et al. 609
Correlation between platelet reactivity to ADP measured with different platelet function tests in patients with cerebrovascular disease at days 7 (D7) and 90 (D90) of clopidogrel (75 mg/day) treatment
Table 3
D7 LTA–ADP
PFA P2Y –0.307
LTA–ADP PFA P2Y
–0.432
Verify Now P2Y12
0.397
–0.267
VASP–ADP
0.125
–0.546*
Verify Now P2Y12 VASP–ADP 0.389
0.289
–0.605**
–0.565* 0.294
0.092
D90 Values are Spearman´s correlation coefficients between values of platelet reactivity obtained with the different assays at day 7 (D7, above the dotted arrow) and at day 90 (D90, below the dotted arrow). Correlation coefficients were interpreted as 0–0.2 no correlation; 0.2–0.4 low correlation; 0.4–0.8 moderate correlation; >0.8 good correlation. LTA-ADP, light transmission aggregometry induced with 5 mmol/l ADP; Verify Now P2Y12, Verify Now cartridge stimulated with ADP; VASP, vasodilatorstimulated phosphoprotein (VASP) phosphorylation assay; MCorrelation is significant at 0.05 level. MMCorrelation is significant at 0.01 level.
conditions account for much of the differences among studies assessing on-clopidogrel HTPR in CVD patients by platelet aggregation assays. Using the Verify Now P2Y12 assay and the cut-off of PRU greater than 235, we found a 60% prevalence of HTPR in our cohort, whereas a recent study in ischemic CVD patients under clopidogrel monotherapy reported a 44% prevalence of HTPR defined as PRU greater than 194 [25]. Using the last criterion, our prevalence of HTPR rose to 80%. Interestingly, 61% of the patients maintained the HTPR phenotype over time. Furthermore, in two recent studies that defined HTPR as less than 20% inhibition of platelet aggregation induced by ADP, calculated automatically by the Verify Now device from comparison with iso-TRAP induced aggregation, the prevalence of on-clopidogrel HTPR were 26 and 54% [28,29]. If we use this definition, the prevalence of HTPR in our cohort of ischemic CVD patients rises to 55%. To the best of our knowledge, this is the first study using the new INNOVANCE PFA P2Y cartridge to monitor clopidogrel inhibition of platelet function in CVD patients. With this novel test, the prevalence of HTPR, defined as closure time less than 106 s, was around 40% in our patients, which is much lower than that previously reported using PFA-100 with COL-EPI and COL-ADP cartridges [25,30–32]. There is also scarce information regarding monitorization of on-treatment platelet function in CVD patients, by means of the VASP assay that specifically assesses the effect of P2Y12 blockage by clopidogrel at the intraplatelet signalling cascade [20]. Here, the VASP provided a 30–45% prevalence of HTPR, comparable to that observed with the other assays. Importantly, we observed that if distribution of the platelet reactivity phenotype, that is HTPR or NoHTPR, is categorized according to the intensity of the
antiplatelet therapy given to patients, that is clopidogrel vs. concomitant aspirin and clopidogrel, all assays, except Verify Now P2Y12, showed a trend at D90 towards more HTPR in patients following clopidogrel monotherapy (data not shown). In concordance, more potent inhibition of platelet activity following dual antiplatelet therapy after a recent stroke was also reported in the PLUTOStroke Trial [33] and other recent studies in CVD patients [14,22]. Dual antiplatelet therapy lowers platelet reactivity by compensating the ADP-induced TXA2 generation in human platelets [34], but, in contrast to the cardiology scenario, available guidelines do not recommend combined therapy with clopidogrel and aspirin in CVD patients [2–4]. Regarding the categorization of patient’s platelet phenotype as HTPR or No-HTPR, it is remarkable that we found, at best, a moderate degree of agreement (k statistic) between D7 and D90 for each assay, and between the different methods both in D7 and D90, and these agreements were highly dependent on the definition of HTPR in each test. For instance, for the Verify Now P2Y12, there was weak and nonsignificant agreement between D7 and D90 based on a cut-off of PRU of more than 235 (k ¼ 0.203, P ¼ 0.387), but moderate and significant (k ¼ 0.491, P ¼ 0.016) using a cut-off of PRU of more than 167. Other authors have previously shown lack of reproducibility over time and discordance between LTA, Verify Now and PFA-100, in identifying HTPR among CVD patients being treated with aspirin or clopidogrel [13,22,23,25,28]. Noteworthy, of the assays used in this study, the novel PFA P2Y test provides the best agreement in the categorization of patients as HTPR or No-HTPR between D7 and D90, and with the result of others tests, thus supporting that INNOVANCE PFA-100 P2Y is at least comparable to other platelet function assays for evaluating on-clopidogrel platelet reactivity in CVD patients.
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610 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 6
The mechanisms associated with the phenomenon of HTPR in CVD and other clinical settings are complex and multifactorial, including noncompliance, concomitant diseases, interference with other drugs or genetic differences in enzymes mediating drug absorption and metabolism and in platelet receptors [35,36]. Indeed, HTPR could result either from insufficient inhibition of normal baseline platelet reactivity or from adequate inhibition of hyper-reactive platelets [10]. Moreover, factors not commonly related with platelet´s reactivity to agonist may play a role in HTPR. Thus, we observed that CVD patients with HTPR, based on different tests, had significantly higher platelet counts and/or MPV than those without HTPR. This finding agrees with a recent study in the same type of patients [25] and supports the idea that larger and increased numbers of platelets genetically determined could influence platelet reactivity and the clinical outcome in cardiovascular and CVD patients [37]. Furthermore, up to 67% of our patients have diabetes mellitus, which has been recently identified as a major risk factor for on-clopidogrel HTPR in acute ischemic stroke patients [26] and had been previously associated with higher MPV. As no overrepresentation of the variant alleles CYP2C192/A or CYP2C1917/T was observed in this cohort as compared with our general population [16], it is unlikely that our findings have been influenced by the CYP2C19 genotype. Compliance is a major issue for the response to antiplatelet therapy. In our study, medication compliance during clinical admittance was assured (D7), but it could not be guaranteed at D90, despite the phone confirmation to ensure medication compliance during, at least, the previous 10 days. Finally, the clinical impact of HTPR observed over time with distinct tests in this small cohort of ischemic CVD patients is uncertain. Obviously, the small sample size is a major limitation that precludes identifying which test and cut-off, if any, is best for predicting ischemic or bleeding complications in a CVD setting. Anecdotally, we reviewed clinical records after 1 year of the patient inclusion and found that three (16.7%) patients suffered adverse outcomes (one TIA, one non-ST elevation acute coronary syndrome and one large artery stroke). At D90, these patients displayed HTPR by Verify Now and PFA P2Y, which may, with caution, suggest that CVD patients with HTPR are at an increased risk of recurrent ischemia. Indeed, recent studies in CVD or cardiovascular settings tailoring antiplatelet therapy according to platelet function tests [12,24,38] have provided controversial results.
weak-to-moderate agreement between assays. For the first time, we show that INNOVANCE PFA-100 P2Y and VASP assays are not any worse than LTA or Verify Now P2Y12 for the purpose of platelet function monitoring in CVD patients treated with clopidogrel. Although there is much evidence of worse outcome in patients with coronary syndrome and HTPR, prospective studies are required to elucidate the clinical relevance of HTPR in ischemic stroke/TIA patients.
Acknowledgements This work was funded by the Instituto de Salud Carlos III (ISCIII, PI10/02594, PI11/00566), RIC RD12/0042/0050 (ISCIII and FEDER) and Fundacio´n Se´neca (07703/ GERM/07). I.S.G. is a fellowship from ISCIII (FI10/ 00535). C.M. is an investigator from Fundacio´n para la Formacio´n e Investigacio´n Sanitarias de la Regio´n de Murcia (FFIS) Conflicts of interest
The INNOVANCE PFA P2Y cartridges used in the study were generously provided free of charge by (Siemens Healthcare Diagnostics SL, Barcelona, Spain). The authors declare no financial or other conflict of interest.
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This study demonstrates that a significant proportion of ischemic CVD patients who start therapy with clopidogrel have an HTPR phenotype that seems to be stable over time. The prevalence of HTPR in these CVD patients is, however, highly test-dependent, with only
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