http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, 2015; 26(1): 17–24 ! 2015 Informa UK Ltd. DOI: 10.3109/09537104.2013.870334
ORIGINAL ARTICLE
Variation in thromboxane B2 concentrations in serum and plasma in patients taking regular aspirin before and after clopidogrel therapy Richard I. S. Good1, Anne McGarrity1, Rory Sheehan2, Tina E. James2, Helen Miller1, Jonathan Stephens3, Stuart Watkins4, Alex McConnachie5, Alison H. Goodall2, & Keith G. Oldroyd4
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
1
BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK, 2Department of Cardiovascular Sciences, NIHR Cardiovascular Biomedical Research Unit, Glenfield Hospital, University of Leicester, Leicester, UK, 3Department of Haematology, National Health Service Blood and Transplant Centre, University of Cambridge, Cambridge, UK, 4Department of Cardiology, West of Scotland Regional Heart & Lung Centre, Golden Jubilee National Hospital, Glasgow, UK, and 5Robertson Centre for Biostatistics, University of Glasgow, Glasgow, UK
Abstract
Keywords
Dual antiplatelet therapy with aspirin and a P2Y12 antagonist is widely prescribed for the prevention of thrombotic events in patients with an acute coronary syndrome or undergoing percutaneous coronary intervention (PCI). It is recognised that there is inter-individual variation in the antiplatelet effects of both drugs. Recent data also suggest that P2Y12 antagonists can affect the response to aspirin. A direct indicator of the effect of aspirin on platelets is their ability to generate thromboxane, which if measured as the difference between the level of thromboxane B2 in serum and plasma ([TxB2]S-P) avoids the confounding effect of endogenous TxB2 production from other cells. We therefore analysed [TxB2]S-P as a measure of aspirin response in a group of 123 patients undergoing elective PCI before and after the introduction of clopidogrel. In a subgroup of 40 patients taking aspirin alone, we compared [TxB2]S-P and VerifyNow Aspirin for the assessment of aspirin response. There was a wide variation in plasma and serum TxB2 concentrations both before and after clopidogrel therapy but only 3.5% of patients had residual serum concentration of TxB2 4 10 ng/ml. There was a strong correlation between the pre and post clopidogrel levels of TxB2 (r 0.78; p ¼ 0.001) and no significant difference in [TxB2]S-P. There was no correlation between the magnitude of response to clopidogrel response and the generation of thromboxane B2. Correlation between [TxB2]S-P and VerifyNow Aspirin was poor. We conclude that the use of a P2Y12 antagonist does not influence the effect of aspirin on the ability of platelets to generate thromboxane. Therefore, measurement of TxB2 levels in serum, after subtracting the contribution from plasma, provides a measure of the response to aspirin in patients taking dual antiplatelet therapy.
Aspirin, coronary artery disease, P2Y12 antagonists, platelet function assays, thromboxane
Introduction Dual antiplatelet therapy (DAPT), with aspirin and a P2Y12 antagonist, is recommended for the majority of patients suffering an acute coronary syndrome (ACS) or undergoing percutaneous coronary intervention (PCI) [1, 2]. There has been much recent focus on the inter-individual variation in the response to P2Y12 antagonists [3–5], but there is also evidence to suggest variation in the individual antiplatelet effects of aspirin is associated with increased cardiovascular risk [6–9]. The optimal method of measuring response to aspirin remains elusive, with poor correlations between the more commonly reported methods [10, 11]. Thus, incorporating measurement of aspirin response into clinical practice is not recommended at this time [1].
Correspondence: Professor Keith G Oldroyd, Consultant Interventional Cardiologist, Director of Research and Development, Department of Cardiology, West of Scotland Regional Heart & Lung Centre, Golden Jubilee National Hospital, Agamemnon Street, Glasgow G81 4DY, UK. Tel: +44 141 951 5180. E-mail: [email protected]
History Received 19 August 2013 Revised 22 November 2013 Accepted 25 November 2013 Published online 16 January 2014
It has been suggested that there is an association between poor response to aspirin and clopidogrel [12–15]. However, Fontana et al. [16] showed no association between response to aspirin monotherapy and clopidogrel monotherapy in healthy volunteers, and Lordkipandze et al. [17] have shown only weak correlation between assays measuring aspirin and clopidogrel response in patients with coronary disease taking both therapies. In addition, data from the CHARISMA study have suggested that levels of urinary 11-dehydro-TxB2 are not influenced by the addition of clopidogrel to regular low dose aspirin [18]. However, there are data to suggest that ADP contributes to the response of platelets to thromboxane [19], P2Y12 inhibition influences thromboxane A2 (TxA2) production and the addition of aspirin augments the antiplatelet effects of partial P2Y12 inhibition [20, 21]. In the majority of compliant patients taking aspirin therapy, serum concentrations of thromboxane are low compared to healthy, non-aspirinated subjects [13, 22, 23]. Despite this, these low levels of serum thromboxane may be clinically important as it has been shown that 499% reduction in serum TxB2 is required to fully block its effect on platelet aggregation [24, 25]. In addition, these small residual concentrations are
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
18
R. I. S. Good et al.
greater in patients with the metabolic syndrome, coronary artery disease and in patients taking lower doses of aspirin [22, 26]. This may reflect TxA2 production from non-platelet sources, including white blood cells and endothelial cells which have been shown to be elevated in patients with vascular disease, particularly at the time of an acute coronary event [27]. In light of this, and to better assess the residual ability of platelets to generate thromboxane in patients with cardiovascular disease taking low dose aspirin therapy, we measured concentrations of TxB2 in samples of plasma ([TxB2]P) and serum ([TxB2]S) taken in parallel, and calculated their difference ([TxB2]S-P). This provides a measure of the capacity of platelets to generate TxB2 and removes the potential confounding effects of plasma TxB2 from non-platelet sources. To ensure maximal activation of the platelets, and to minimise inter-individual variation, serum was prepared from blood collected into tubes containing exogenous thrombin. Breet et al. [15] found VerifyNow Aspirin was more predictive of future cardiovascular events following PCI than light transmission aggregometry in patients taking both aspirin and clopidogrel. We therefore sought to compare [TxB2]S-P with VerifyNow Aspirin for the assessment of aspirin response. In a subgroup of patients, we reassessed [TxB2]S-P and VerifyNow Aspirin following the introduction of clopidogrel therapy, to investigate whether these assays may allow measurement of the antiplatelet effects of aspirin in patients taking additional P2Y12 receptor inhibition. In addition, we investigated whether response to clopidogrel, measured using VerifyNow P2Y12 or VASP-PRI, influenced any change in [TxB2]S-P seen following the introduction of this additional antiplatelet therapy.
Methods Patient selection We sampled a cross-section of patients with known coronary artery disease taking regular low dose aspirin (75 mg/day) for at least 5 days and no other antithrombotic or anticoagulant agent (pre-clopidogrel). Patients were recruited in our centre from outpatient clinics, cardiology in-patient wards and the cardiac catheterisation day care unit. All patients were between 18 and 85 years and were able to provide informed consent. Patients were excluded if they were receiving dipyridamole or NSAID therapy, were pregnant, anaemic (Hb 510 g/dl), had a platelet count 5100 or 4500 109/l or a personal or family history of a bleeding disorder. Patients were excluded if they were non-compliant with aspirin therapy, as assessed by direct questioning at the time of recruitment. Ethical approval was granted by the West Research Ethics Committee, Glasgow. A subgroup of patients scheduled for percutaneous coronary intervention (PCI) subsequently received clopidogrel in preparation for the procedure. In this group, further samples were collected following the introduction of clopidogrel (postclopidogrel), prior to commencing PCI. Patients were deemed to be fully loaded on clopidogrel therapy if they had received either; (i) a 600 mg loading dose 42 hours previously, (ii) a 300 mg loading dose 424 hours previously or (iii) 75 mg/day of clopidogrel for at least 5 days. For comparison, [TxB2]S-P was assessed in healthy, nonaspirinated subjects recruited through the National Blood Service clinic, Cambridge, UK as part of the Bloodomics programme [28]. Sample collection For all control and pre-clopidogrel samples, blood was drawn from a large calibre vein in the antecubital fossa using a 21G
Platelets, 2015; 26(1): 17–24
butterfly needle and vacutainer system. Samples were collected between the hours of 08:00 and 17:00 and patients were asked to sit for 10 minutes prior to sampling. Use of a tourniquet was kept to a minimum, and care was taken to avoid unnecessary trauma to the vein or agitation of the sample. Blood was collected in strict sequential order; an initial sample of 3.5 ml was collected into a 3.2% sodium citrate tube and discarded a further 3.5 ml was collected into 3.2% sodium citrate to prepare plasma, and 3 ml was collected in a glass vacutainer tube containing thrombin to prepare serum. For patients tested using the VerifyNow Aspirin assay an additional 2 ml 3.2% sodium citrate tube (Accumetrics, USA) was collected prior to the thrombin-containing sample. All samples were immediately mixed by gently inverting five times. Following the introduction of clopidogrel, the second series of samples (post-clopidogrel) was collected either by venepuncture, or following the insertion of an arterial sheath at the time of coronary angiography with additional 3.2% sodium citrate tubes for the assessment of response to clopidogrel using VASP-PRI and VerifyNow P2Y12. Thromboxane B2 assay Patient samples: within 30 minutes of collection the 3.2% sodium citrate blood tube was centrifuged for 30 minutes at 1500 g (3000 rpm) and the plasma supernatant stored in 0.5 ml aliquots at 80 C until use. The thrombin containing serum tube was left at room temperature for 1 hour after which it was centrifuged at 1500 g (3000 rpm) for 10 minutes and the serum supernatant collected in 0.5 ml aliquots and immediately stored at 80 C. Control samples: within 30 minutes of collection the citrated blood tube was centrifuged at 1550 g (3100 rpm) for 15 minutes. Plasma was removed and re-centrifuged at 9300 g (10 000 rpm) for 10 minutes after which it was stored in aliquots at 80 C until use. Serum thrombin tubes were left at room temperature for 1 hour, after which they were centrifuged at 1550 g (3100 rpm) for 15 minutes. Serum was removed and re-centrifuged at 9300 g (10 000 rpm) for 10 minutes after which it was stored in aliquots at 80 C until use. The level of TxB2 in the plasma and serum samples was analysed by ELISA (R&D Systems Europe; cat. no. KGE011) following the manufacturer’s instructions. Platelet thromboxane generation ([TxB2]S-P) was calculated by subtracting the levels of TxB2 in the paired plasma sample ([TxB2]P) from the level in the thrombin-serum sample ([TxB2]S). VerifyNow Aspirin and P2Y12 assay The Ultegra rapid platelet function analyser (RPFA) and VerifyNow Aspirin assay cartridges were purchased from Accumetrics Inc. (San Diego, CA) and used according to manufacturer’s instructions [29]. VerifyNow Aspirin results are expressed as aspirin reaction units (ARUs). At the time of the study, the manufacturer recommended a qualitative measure of aspirin ‘‘resistance’’ with a cut off of 4550 ARU. As recommended by the manufacturer’s guidelines, the 2 ml 3.2% sodium citrate samples were stored at room temperature for at least 30 minutes before assaying and all samples were analysed between 30 minutes and 2 hours of acquisition. VASP-PRI assay Measurement of phosphorylated VASP in platelets was performed by flow cytometry using a commercially available assay (VASP/ P2Y12, Biocytex, France) [30]. Reagents were prepared as instructed by the manufacturer and each box was used within 1 month of opening. The samples were stored at room temperature until processing and all assays were carried out within 24 hours of
Thromboxane generation on dual anti-platelet therapy
DOI: 10.3109/09537104.2013.870334
sampling. Samples were run on a four channel BD FACSCalibur flow cytometer.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
Statistical methods This was an exploratory study to assess the variation in thromboxane concentrations in this group of patients. In particular, we were interested in the effects of clopidogrel on the measured concentration of TxB2. We sought a sample size of 80 patients with samples before and after clopidogrel treatment. Although no formal power calculation was undertaken prior to this study, using the standard deviation of log10[TxB2]S-P for our pre-clopidogrel samples (0.34), in our cohort of 69 patients had an 80% power to detect a difference on a log scale of 0.116, or a relative difference of 12% between samples before and after clopidogrel. The distribution of results are summarised as the median, interquartile range and range of values for each assay. Samples with negative values of [TxB2]S-P were excluded on the basis of biological implausibility. TxB2 concentrations showed a strongly positively skewed distribution. Logarithmic transformations produced a distribution of concentrations approaching normality. This transformed data was used in simple linear regression analysis to investigate whether any baseline characteristic was associated with [TxB2]S-P. Patients characteristics with a significant correlation were subsequently included in a stepwise, multiple regression model. Correlations between log transformed [TxB2]P, [TxB2]S and [TxB2]S-P before and after clopidogrel,
19
and between log10[TxB2]S-P and VerifyNow Aspirin data were calculated using Pearson’s correlation coefficient. Differences between log transformed [TxB2]P, [TxB2]S and [TxB2]S-P before and after clopidogrel were investigated using paired sample t-tests. Significance was taken at the p50.05 level for all statistical tests.
Results In total, 123 patients were recruited for the assessment of [TxB2]S-P on aspirin monotherapy. Study recruitment and sampling are shown in Figure 1 and the demographics of the patients are shown in Table I, with a particular focus on those parameters most likely to influence the response to aspirin therapy. Four thromboxane samples taken prior to the introduction of clopidogrel were erroneously processed. An additional four samples produced plasma levels of TxB2 that were higher than those of serum. This was deemed to be biologically implausible and these samples were excluded from further analysis. This left 115 paired serum and plasma samples from patients taking aspirin alone available for analysis. A summary of thromboxane concentrations before the introduction of clopidogrel is given in Table II (Panel a). One hundred and four healthy controls [median age 46, range 20–63; male 80/104 (77%)] taking no antiplatelet or anticoagulant treatment had [TxB2] measured in both serum and plasma. These results are summarised in Table II (Panel a) and shown graphically in Figure 2.
Figure 1. Flow diagram of patient recruitment and sampling.
20
R. I. S. Good et al.
Platelets, 2015; 26(1): 17–24
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
In the 115 patients undergoing PCI, levels of TxB2 were 510 ng/ml in both plasma (n ¼ 114; 99%) and serum (n ¼ 110; 96%) samples (Figure 2). Residual platelet thromboxane generation, as shown by [TxB2]S-P, was 510 ng/ml in 110 (96%) of the patients. These levels were significantly lower than in healthy subjects not on aspirin where only one subject had [TxB]S-P 51 ng/ml and 71/104 (68%) had [TxB2]S-P 410 ng/ml (Figure 2). Simple linear regression of the patients’ demographic data showed significant associations between log10[TxB2]S-P prior to the introduction of clopidogrel and age, statin therapy, current smoking and creatinine (Table I). When these variables were included in a stepwise multiple regression analysis, age alone was the only independent negative predictor of pre-clopidogrel log10[TxB2]S-P (regression coefficient 0.015, 95% CI 0.027 to 0.003, p ¼ 0.013; n ¼ 115). No such association was found between age and [TxB2]S-P in control subjects.
Table I. Patient demographics and blood parameters of 123 patients sampled prior to the introduction of clopidogrel. Linear regression (N ¼ 115)* N ¼ 123 Patient characteristic Age (years) Mean SD Male (%) BMI (kg/m2) Mean SD Medical history Hypertension (%) Diabetes (%) Dyslipidaemia (%) Previous MI (%) Smoking status Current Medication Statin Beta blocker ACE inhibitor Blood parameters Haemoglobin (g/dl) (Mean SD) Platelet count (109/l) (Mean SD) Creatinine (mmol/l) (Mean SD)
Coefficient (95% CI)
p
A subgroup of 40 patients was tested for response to aspirin using VerifyNow Aspirin prior to the introduction of clopidogrel (Table II, Panel b). Of these 40 patients, 36 had [TxB2]S-P assay results available for comparison. Two patients met the definition of aspirin resistance (ARU 4 550) according to the VerifyNow assay. There was a poor correlation between VerifyNow Aspirin ARU and [TxB2]S-P (r ¼ 0.40; p50.001, Figure 3), but the patient with the highest residual generation of TxB2 (36 ng/ml) also had the highest activity in the VerifyNow Aspirin assay (653 ARU). The remaining patient deemed aspirin resistant by VerifyNow had a modest residual [TxB2]S-P of 0.98 ng/ml. These two samples are shown in Figure 3 by the solid and dotted arrows respectively. Thromboxane B2 was measured in a second sample obtained from 79/115 patients following the introduction of clopidogrel. Of these, four samples were erroneously processed and were not able to be analysed and a further six samples produced plasma concentrations of TxB2 greater than that in the serum sample and were again excluded on the basis of biological implausibility (Figure 1). This left a total of 69 patients with paired plasma and serum TxB2 concentrations before and after clopidogrel therapy. Of these, 42/69 (61%) had received clopidogrel 75 mg/day for45 days, 18/69 (26%) 300 mg424 hours previously and 9/69 (13%) 600 mg42 hours but524 hours prior to sampling. The majority 54/69 (78%) were venous samples and the remaining
64.4 10.2 0.015 (0.025 to 0.006) 0.002 81 (66) 0.003 (0.211 to 0.216) 0.980 27.7 4.4 69 17 81 33
(56.1) (13.8) (65.9) (26.8)
0.020 (0.003 to 0.043) 0.161 0.084 0.051 0.098
(0.362 (0.377 (0.265 (0.130
to to to to
0.084
0.041) 0.209) 0.162) 0.326)
0.117 0.570 0.635 0.397
25 (20.3)
0.281 (0.035 to 0.527)
0.026
107 (87) 89 (72.4) 47 (38.2)
0.300 (0.005 to 0.596) 0.003 (0.224 to 0.229) 0.063 (0.271 to 0.144)
0.046 0.982 0.545
13.8 1.4
0.011 (0.122 to 0.100)
0.838
0.002 (50.001 to 0.004)
0.188
0.007 (0.013 to 0.001)
0.030
253 69 92 20
*Simple linear regression using each patients characteristic as a predictor and log10[TxB2]S-P as the dependent variable (n ¼ 115).
Figure 2. Concentration of thromboxane B2 [TxB2] in plasma (p), serum (s) and serum–plasma (s–p) in patients taking aspirin (n ¼ 115) and healthy controls (n ¼ 104). Upper dashed line represents a cut off of [TxB2]S-P 410 ng/ml. The lower dashed line represents a residual level of thromboxane generation 41 ng/ml.
Table II. Summary statistics of (a) [TxB2] in plasma and serum in controls and patients before and after clopidogrel therapy and (b) VerifyNow P2Y12, VASP-PRI and VerifyNow Aspirin results in patients before and after clopidogrel therapy. Controls (n ¼ 104)
Panel a [TxB2]P (pg/ml) [TxB2]S (pg/ml) [TxB2]S-P (pg/ml) Panel b VerifyNow P2Y12 (%inhibition) VASP-PRI (%activation) VerifyNow Aspirin (ARU)
Pre-clopidogrel (n ¼ 115)
Post-clopidogrel (n ¼ 69)
Median
IQR
Range
Median
IQR
Range
Median
IQR
Range
229 18 667 18 299
236 21 639 21 793
43–5946 481–354 708 380–354 531
110 773 646
148 1018 842
9–16 620 85–36 344 19–30 511
136 673 491
138 701 612
16–5301 200–36 344 119–31 043
11 82 428
11 1–40 8 29–100 (n ¼ 40) 94 366–653
45 58 382
41 6–97 31 1–88 (n ¼ 9) 28 350–424
DOI: 10.3109/09537104.2013.870334
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
15 samples (22%) were collected from the arterial sheath at the time of angiography. There was a wide range of response to clopidogrel measured by either VerifyNow P2Y12 (%inhibition) or VASP-PRI (%activation; Table II, Panel b) with a moderate correlation between the two assays (Figure 4; r ¼ 0.76; p50.001). Neither the clopidogrel loading regimen, nor method of sample collection influenced [TxB2]S-P, VerifyNow P2Y12 or VASP-PRI results (p40.05 for all; data not shown). Concentrations of [TxB2]P, [TxB2]S and [TxB2]S-P before and after clopidogrel therapy showed very little change (Table II, Panel a and Figure 5) and were highly correlated (r 0.78; p50.001 for all). Comparing the log transformed concentrations of thromboxane before and after clopidogrel using paired sample t-tests, there was a small but significant reduction in log10[TxB2]P (mean change 0.062, 95% CI 0.005–0.120, p ¼ 0.035), a trend towards a lower log10[TxB2]S (mean change 0.062, 95% CI
Figure 3. Correlation of [TxB2]S-P and VerifyNow Aspirin assays. The highest [TxB2]S-P (arrowhead) was seen in one of two patients deemed aspirin resistant by the VerifyNow Aspirin assay.
Figure 5. Thromboxane B2 concentrations [TxB2] in plasma (p), serum(s) and serum– plasma (s–p) before (Pre) and after (Post) the introduction of clopidogrel. *Paired sample t-tests of the difference in log10[TxB2] before and after clopidogrel.
Thromboxane generation on dual anti-platelet therapy
21
0.005 to 0.129, p ¼ 0.069) but no significant difference in log10[TxB2]S-P, (mean change 0.041, 95% CI 0.040 to 0.123, p ¼ 0.311). As shown in Figure 6, the magnitude of response to clopidogrel measured using VASP-PRI did not correlate with the change in [TxB2]S-P following the introduction of clopidogrel (r ¼ 0.02, p ¼ 0.99), and the findings were similar when measured by the VerifyNow P2Y12 % inhibition (r ¼ 0.02, p ¼ 0.97). In 9/40 patients with repeat testing using VerifyNow Aspirin before and after the introduction of clopidogrel, all patients showed a reduction in ARU and the difference was statistically significant despite the small sample size (p ¼ 0.004; Figure 7).
Discussion Recent focus on platelet testing in CAD patients has been on near patient testing, and largely on assessing the efficacy of P2Y12 antagonists [3–5]. However, there remains a need to monitor the
Figure 4. Correlation between clopidogrel response assessed by VerifyNow P2Y12 (% inhibition) and VASP-PRI.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
22
R. I. S. Good et al.
Figure 6. Clopidogrel response measured by VASP-PRI does not influence D[TxB2]S-P observed following the introduction of this additional antiplatelet agent.
Figure 7. VerifyNow Aspirin before and after the introduction of clopidogrel (n ¼ 9). p Value is for a paired sample t-test.
efficacy of aspirin, especially in patients receiving DAPT. This observational study, in a routine clinical setting, used [TxB2]S-P as a marker of the residual ability of platelets to generate thromboxane to assess the efficacy of aspirin in patients receiving single and dual antiplatelet therapy. We have shown that the efficacy of aspirin measured by [TxB2]S-P is unaffected by additional P2Y12 antagonism. The demographics of the patients recruited to this study are comparable with those seen in contemporary studies of coronary artery disease [31]. There was a measurable variation in the concentration of TxB2 in both plasma and serum with the majority of patients having [TxB2]S-P levels below 10 ng/ml. There was a highly positive skew of thromboxane B2 concentrations and the normalisation of [TxB2]S-P concentrations following
Platelets, 2015; 26(1): 17–24
logarithmic transformation. This exploratory study does not attempt to define aspirin ‘‘resistance’’; however, these data do demonstrate a significant overlap of [TxB2]S-P levels between aspirinated patients and normal controls taking no antiplatelet therapy. This may help guide future studies to investigate whether there is a threshold of residual thromboxane generation that is predictive of adverse clinical events. Age alone was found to be an independent negative predictor of [TxB2]S-P This is in contrast to previously published data, using urinary 11-dehydro TxB2, suggesting an increased ability to generate thromboxane, with increasing age [32]. Our study demonstrates strong and significant correlations between [TxB2] concentrations before and after clopidogrel. There was no significant difference in [TxB2]S-P following the introduction of this additional P2Y12 receptor inhibitor, thus supporting the use of [TxB2]S-P as a measure of aspirin response in patient taking DAPT. As further support for this, we confirmed that the magnitude of response to clopidogrel assessed using either the bedside (VerifyNow P2Y12) or laboratory based (VASP-PRI) assay does not have any influence on the small change in [TxB2]S-P after the introduction of clopidogrel. As newer, more potent and uniform ADP receptor antagonists have entered clinical practice, it is encouraging that these assay results may remain relevant for patients taking aspirin with either prasugrel or ticagrelor. Although measurement of [TxB2] in platelet rich plasma (PRP) following stimulation with arachidonic acid has been used for the measurement of aspirin response [33], the generation of thromboxane in these circumstances may be affected by sample manipulation and selective loss of larger platelets during the preparation of PRP. In addition, this method does not account for background TxB2 production from non-platelet pathways [27, 34]. It has also been shown that P2Y12 inhibitors alone may reduce the production of thromboxane in PRP by comparable amounts to aspirin [20]. In contrast, our method of measuring TxB2 generation avoids these potential confounders through maximally stimulating platelets in the whole blood milieu prior to serum sample preparation and measuring the background level of TxB2 in plasma generated from unstimulated samples. An additional finding of importance was a poor correlation between [TxB2]S-P and VerifyNow Aspirin. This highlights the difficulties encountered in the field of measuring response to aspirin [35]. We remain uncertain of the best test, and metaanalyses suggesting association with increased cardiovascular risk are hampered by the myriad of assays being used, and the poor correlation between these assays [9–11, 36, 37]. The additional challenge of measuring response to aspirin in patients taking a P2Y12 inhibitor is highlighted by the data obtained using VerifyNow Aspirin. In nine of our patients sampled before and after the addition of clopidogrel to aspirin we found significantly reduced ARU values suggesting the manufacturer’s recommended cut-off for aspirin ‘‘resistance’’ of 550ARU cannot be applied in the setting of DAPT. In keeping with our findings, Lordkipandze et al. [17] showed only 5/85 (6%) of patients with stable CAD had an impaired response to aspirin assessed by VerifyNow Aspirin on DAPT. In the recent ARCTIC study only 46/1213 patients (3.9%) were aspirin ‘‘resistant’’ according to VerifyNow Aspirin when taking DAPT [5]. This compares with previously published data suggesting an incidence of aspirin ‘resistance’ of 17–19% [8, 10] on aspirin monotherapy using VerifyNow Aspirin. Breet et al. [15] found the cut-off for VerifyNow Aspirin high on-aspirin platelet reactivity on DAPT that best predicted clinical outcome was 454ARU, a much lower value than recommended by the manufacturer. In conclusion, the aggregation based platelet function assay VerifyNow Aspirin appears to be significantly influenced by
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
DOI: 10.3109/09537104.2013.870334
additional P2Y12 inhibition. As such we believe that [TxB2]S-P may offer the most appropriate method of assessing aspirin response in patients taking an additional P2Y12 inhibitor and warrants further investigation as a predictor of clinical outcome and in studies of response to antiplatelet therapy. For example, a large population with previous DES implantation may provide an opportunity to assess whether residual thromboxane generation is clinically relevant as we know that a number of these patients have delayed neointimal coverage and persistent malapposition which may cause very late stent thrombosis [38]. However, we acknowledge that measurement of [TxB2]S-P is time consuming and requires expertise so is not suited to studies requiring rapid results. The current study has several limitations. We studied a relatively small number of patients, particularly post clopidogrel therapy, and further studies would be warranted to confirm our findings. Although patients were questioned at the time of recruitment, no formal testing was performed to confirm compliance [39]. In addition, the accuracy of measurement of thromboxane generation may have been improved by collecting plasma samples into citrated tubes containing indomethacin to prevent additional activation of platelets during sample processing.
Declaration of interest The authors have no conflicts of interest related to this research. This research was supported by a British Heart Foundation Junior Research Fellowship (FS/06/031).
References 1. Hamm CW, Bassand JP, Agewall S, Bax J, Boersma E, Bueno H, Caso P, Dudek D, Gielen S, Huber K, et al. ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: The Task Force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J 2011;32: 2999–3054. 2. Wijns W, Kolh P, Danchin N, Di Mario C, Falk V, Folliguet T, Garg S, Huber K, James S, et al. Guidelines on myocardial revascularization. Eur Heart J 2010;31:2501–2555. 3. Price MJ, Angiolillo DJ, Teirstein PS, Lillie E, Manoukian SV, Berger PB, Tanguay JF, Cannon CP, Topol EJ. Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: A time-dependent analysis of the Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety (GRAVITAS) trial. Circulation 2011;124:1132–1137. 4. Trenk D, Stone GW, Gawaz M, Kastrati A, Angiolillo DJ, Muller U, Richardt G, Jakubowski JA, Neumann FJ. A randomized trial of prasugrel versus clopidogrel in patients with high platelet reactivity on clopidogrel after elective percutaneous coronary intervention with implantation of drug-eluting stents: Results of the TRIGGERPCI (Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy With Prasugrel) study. J Am Coll Cardiol 2012;59:2159–2164. 5. Collet JP, Cuisset T, Range G, Cayla G, Elhadad S, Pouillot C, Henry P, Motreff P, Carrie´ D, Boueri Z, et al. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. N Engl J Med 2012; 367:2100–2109. 6. Eikelboom JW, Hirsh J, Weitz JI, Johnston M, Yi Q, Yusuf S. Aspirin-resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke, or cardiovascular death in patients at high risk for cardiovascular events. Circulation 2002;105:1650–1655. 7. Gum PA, Kottke-Marchant K, Welsh PA, White J, Topol EJ. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease.[see comment]. J Am Coll Cardiol 2003;41:961–965. 8. Chen WH, Lee PY, Ng W, Tse HF, Lau CP. Aspirin resistance is associated with a high incidence of myonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment. J Am Coll Cardiol 2004;43:1122–1126.
Thromboxane generation on dual anti-platelet therapy
23
9. Snoep JD, Hovens MM, Eikenboom JC, van der Bom JG, Huisman MV. Association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events: A systematic review and meta-analysis. Arch Intern Med 2007;167:1593–1599. 10. Harrison P, Segal H, Blasbery K, Furtado C, Silver L, Rothwell PM. Screening for aspirin responsiveness after transient ischemic attack and stroke: Comparison of 2 point-of-care platelet function tests with optical aggregometry. Stroke 2005;36:1001–1005. 11. Lordkipanidze M, Pharand C, Schampaert E, Turgeon J, Palisaitis DA, Diodati JG. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur Heart J 2007;28:1702–1708. 12. Lev EI, Patel RT, Maresh KJ, Guthikonda S, Granada J, DeLao T, Bray PF, Kleiman NS. Aspirin and clopidogrel drug response in patients undergoing percutaneous coronary intervention: The role of dual drug resistance. J Am Coll Cardiol 2006;47:27–33. 13. Ohmori T, Yatomi Y, Nonaka T, Kobayashi Y, Madoiwa S, Mimuro J, Ozaki Y, Sakata Y. Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity: Involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients. J Thromb Haemost 2006;4:1271–1278. 14. Cuisset T, Frere C, Quilici J, Morange PE, Camoin L, Bali L, Lambert M, Juhan-Vague I, Alessi MC, Bonnet JL. Relationship between aspirin and clopidogrel responses in acute coronary syndrome and clinical predictors of non response. Thromb Res 2009;123:597–603. 15. Breet NJ, van Werkum JW, Bouman HJ, Kelder JC, Harmsze AM, Hackeng CM, ten Berg JM. High on-treatment platelet reactivity to both aspirin and clopidogrel is associated with the highest risk of adverse events following percutaneous coronary intervention. Heart 2011;97:983–990. 16. Fontana P, Nolli S, Reber G, de Moerloose P. Biological effects of aspirin and clopidogrel in a randomized cross-over study in 96 healthy volunteers. J Thromb Haemost 2006;4:813–819. 17. Lordkipanidze M, Diodati JG, Schampaert E, Palisaitis DA, Pharand C. Prevalence of unresponsiveness to aspirin and/or clopidogrel in patients with stable coronary heart disease. Am J Cardiol 2009;104: 1189–1193. 18. Eikelboom JW, Hankey GJ, Thom J, Bhatt DL, Steg PG, Montalescot G, Johnston SC, Steinhubl SR, Mak KH, Easton JD, et al. Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA) Investigators. Incomplete inhibition of thromboxane biosynthesis by acetylsalicylic acid: Determinants and effect on cardiovascular risk. Circulation 2008;118:1705–1712. 19. Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Biol Chem 1999;274:29108–29114. 20. Armstrong PC, Dhanji AR, Tucker AT, Mitchell JA, Warner TD. Reduction of platelet thromboxane A2 production ex vivo and in vivo by clopidogrel therapy. J Thromb Haemost 2010;8:613–615. 21. Kirkby NS, Leadbeater PD, Chan MV, Nylander S, Mitchell JA, Warner TD. Antiplatelet effects of aspirin vary with level of P2Y12 receptor blockade supplied by either ticagrelor or prasugrel. J Thromb Haemost 2011;9:2103–2105. 22. Frelinger AL III, Furman MI, Linden MD, Li Y, Fox ML, Barnard MR, Michelson AD. Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independent pathway: A 700patient study of aspirin resistance. Circulation 2006;113:2888–2896. 23. Santilli F, Rocca B, De Cristofaro R, Lattanzio S, Pietrangelo L, Habib A, Pettinella C, Recchiuti A, Ferrante E, Ciabattoni G, et al. Platelet cyclooxygenase inhibition by low-dose aspirin is not reflected consistently by platelet function assays: Implications for aspirin ‘‘resistance". J Am Coll Cardiol 2009;53:667–677. 24. Reilly IA, FitzGerald GA. Inhibition of thromboxane formation in vivo and ex vivo: Implications for therapy with platelet inhibitory drugs. Blood 1987;69:180–186. 25. Maree AO, Curtin RJ, Dooley M, Conroy RM, Crean P, Cox D, Fitzgerald DJ. Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease. J Am Coll Cardiol 2005; 46:1258–1263. 26. Smith JP, Haddad EV, Taylor MB, Oram D, Blakemore D, Chen Q, Boutaud O, Oates JA. Suboptimal inhibition of platelet cyclooxygenase-1 by aspirin in metabolic syndrome. Hypertension 2012;59: 719–725.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
24
R. I. S. Good et al.
27. Karim S, Habib A, Levy-Toledano S, Maclouf J. Cyclooxygenase-1 and -2 of endothelial cells utilize exogenous or endogenous arachidonic acid for transcellular production of thromboxane. J Biol Chem 1996;271:12042–12048. 28. Jones CI, Garner SF, Angenent W, Bernard A, Berzuini C, Burns P, Farndale RW, Hogwood J, Rankin A, Stephens JC, et al. Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus. J Thromb Haemost 2007;5: 1756–1765. 29. Michelson AD. Methods for the measurement of platelet function. Am J Cardiol 2009;103:20A–26A. 30. Aleil B, Ravanat C, Cazenave JP, Rochoux G, Heitz A, Gachet C. Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases. J Thromb Haemost 2005;3:85–92. 31. Mahaffey KW, Wojdyla DM, Carroll K, Becker RC, Storey RF, Angiolillo DJ, Held C, Cannon CP, James S, Pieper KS, et al. Ticagrelor compared with clopidogrel by geographic region in the platelet inhibition and patient outcomes (PLATO) trial. Circulation 2011;124:544–554. 32. Faraday N, Becker DM, Yanek LR, Herrera-Galeano JE, Segal JB, Moy TF, Bray PF, Becker LC. Relation between atherosclerosis risk factors and aspirin resistance in a primary prevention population. Am J Cardiol 2006;98:774–779.
Platelets, 2015; 26(1): 17–24
33. Chan MV, Armstrong PC, Papalia F, Kirkby NS, Warner TD. Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing. Platelets 2011;22:485–494. 34. Faraday N, Scharpf RB, Dodd-o JM, Martinez EA, Rosenfeld BA, Dorman T. Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin. Anesthesiology 2001;94:145–151. 35. Patrono C, Rocca B. Drug insight: Aspirin resistance–fact or fashion? Nat Clin Pract Cardiovasc Med 2007;4:42–50. 36. Krasopoulos G, Brister SJ, Beattie WS, Buchanan MR. Aspirin ‘‘resistance’’ and risk of cardiovascular morbidity: systematic review and meta-analysis. BMJ 2008;336:195–198. 37. Sofi F, Marcucci R, Gori AM, Abbate R, Gensini GF. Residual platelet reactivity on aspirin therapy and recurrent cardiovascular events – a meta-analysis. Int J Cardiol 2008;128:166–171. 38. Kang S, Lee CW, Song H, Ahn J, Kim W, Lee J, Park DW, Lee SW, Kim YH, Mintz GS, et al. OCT analysis in patients with very late stent thrombosis. JACC Cardiovasc Imaging 2013;6: 695–703. 39. Schwartz KA, Schwartz DE, Ghosheh K, Reeves MJ, Barber K, DeFranco A. Compliance as a critical consideration in patients who appear to be resistant to aspirin after healing of myocardial infarction. Am J Cardiol 2005;95:973–975.
ORIGINAL ARTICLE
Variation in thromboxane B2 concentrations in serum and plasma in patients taking regular aspirin before and after clopidogrel therapy Richard I. S. Good1, Anne McGarrity1, Rory Sheehan2, Tina E. James2, Helen Miller1, Jonathan Stephens3, Stuart Watkins4, Alex McConnachie5, Alison H. Goodall2, & Keith G. Oldroyd4
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
1
BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK, 2Department of Cardiovascular Sciences, NIHR Cardiovascular Biomedical Research Unit, Glenfield Hospital, University of Leicester, Leicester, UK, 3Department of Haematology, National Health Service Blood and Transplant Centre, University of Cambridge, Cambridge, UK, 4Department of Cardiology, West of Scotland Regional Heart & Lung Centre, Golden Jubilee National Hospital, Glasgow, UK, and 5Robertson Centre for Biostatistics, University of Glasgow, Glasgow, UK
Abstract
Keywords
Dual antiplatelet therapy with aspirin and a P2Y12 antagonist is widely prescribed for the prevention of thrombotic events in patients with an acute coronary syndrome or undergoing percutaneous coronary intervention (PCI). It is recognised that there is inter-individual variation in the antiplatelet effects of both drugs. Recent data also suggest that P2Y12 antagonists can affect the response to aspirin. A direct indicator of the effect of aspirin on platelets is their ability to generate thromboxane, which if measured as the difference between the level of thromboxane B2 in serum and plasma ([TxB2]S-P) avoids the confounding effect of endogenous TxB2 production from other cells. We therefore analysed [TxB2]S-P as a measure of aspirin response in a group of 123 patients undergoing elective PCI before and after the introduction of clopidogrel. In a subgroup of 40 patients taking aspirin alone, we compared [TxB2]S-P and VerifyNow Aspirin for the assessment of aspirin response. There was a wide variation in plasma and serum TxB2 concentrations both before and after clopidogrel therapy but only 3.5% of patients had residual serum concentration of TxB2 4 10 ng/ml. There was a strong correlation between the pre and post clopidogrel levels of TxB2 (r 0.78; p ¼ 0.001) and no significant difference in [TxB2]S-P. There was no correlation between the magnitude of response to clopidogrel response and the generation of thromboxane B2. Correlation between [TxB2]S-P and VerifyNow Aspirin was poor. We conclude that the use of a P2Y12 antagonist does not influence the effect of aspirin on the ability of platelets to generate thromboxane. Therefore, measurement of TxB2 levels in serum, after subtracting the contribution from plasma, provides a measure of the response to aspirin in patients taking dual antiplatelet therapy.
Aspirin, coronary artery disease, P2Y12 antagonists, platelet function assays, thromboxane
Introduction Dual antiplatelet therapy (DAPT), with aspirin and a P2Y12 antagonist, is recommended for the majority of patients suffering an acute coronary syndrome (ACS) or undergoing percutaneous coronary intervention (PCI) [1, 2]. There has been much recent focus on the inter-individual variation in the response to P2Y12 antagonists [3–5], but there is also evidence to suggest variation in the individual antiplatelet effects of aspirin is associated with increased cardiovascular risk [6–9]. The optimal method of measuring response to aspirin remains elusive, with poor correlations between the more commonly reported methods [10, 11]. Thus, incorporating measurement of aspirin response into clinical practice is not recommended at this time [1].
Correspondence: Professor Keith G Oldroyd, Consultant Interventional Cardiologist, Director of Research and Development, Department of Cardiology, West of Scotland Regional Heart & Lung Centre, Golden Jubilee National Hospital, Agamemnon Street, Glasgow G81 4DY, UK. Tel: +44 141 951 5180. E-mail: [email protected]
History Received 19 August 2013 Revised 22 November 2013 Accepted 25 November 2013 Published online 16 January 2014
It has been suggested that there is an association between poor response to aspirin and clopidogrel [12–15]. However, Fontana et al. [16] showed no association between response to aspirin monotherapy and clopidogrel monotherapy in healthy volunteers, and Lordkipandze et al. [17] have shown only weak correlation between assays measuring aspirin and clopidogrel response in patients with coronary disease taking both therapies. In addition, data from the CHARISMA study have suggested that levels of urinary 11-dehydro-TxB2 are not influenced by the addition of clopidogrel to regular low dose aspirin [18]. However, there are data to suggest that ADP contributes to the response of platelets to thromboxane [19], P2Y12 inhibition influences thromboxane A2 (TxA2) production and the addition of aspirin augments the antiplatelet effects of partial P2Y12 inhibition [20, 21]. In the majority of compliant patients taking aspirin therapy, serum concentrations of thromboxane are low compared to healthy, non-aspirinated subjects [13, 22, 23]. Despite this, these low levels of serum thromboxane may be clinically important as it has been shown that 499% reduction in serum TxB2 is required to fully block its effect on platelet aggregation [24, 25]. In addition, these small residual concentrations are
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
18
R. I. S. Good et al.
greater in patients with the metabolic syndrome, coronary artery disease and in patients taking lower doses of aspirin [22, 26]. This may reflect TxA2 production from non-platelet sources, including white blood cells and endothelial cells which have been shown to be elevated in patients with vascular disease, particularly at the time of an acute coronary event [27]. In light of this, and to better assess the residual ability of platelets to generate thromboxane in patients with cardiovascular disease taking low dose aspirin therapy, we measured concentrations of TxB2 in samples of plasma ([TxB2]P) and serum ([TxB2]S) taken in parallel, and calculated their difference ([TxB2]S-P). This provides a measure of the capacity of platelets to generate TxB2 and removes the potential confounding effects of plasma TxB2 from non-platelet sources. To ensure maximal activation of the platelets, and to minimise inter-individual variation, serum was prepared from blood collected into tubes containing exogenous thrombin. Breet et al. [15] found VerifyNow Aspirin was more predictive of future cardiovascular events following PCI than light transmission aggregometry in patients taking both aspirin and clopidogrel. We therefore sought to compare [TxB2]S-P with VerifyNow Aspirin for the assessment of aspirin response. In a subgroup of patients, we reassessed [TxB2]S-P and VerifyNow Aspirin following the introduction of clopidogrel therapy, to investigate whether these assays may allow measurement of the antiplatelet effects of aspirin in patients taking additional P2Y12 receptor inhibition. In addition, we investigated whether response to clopidogrel, measured using VerifyNow P2Y12 or VASP-PRI, influenced any change in [TxB2]S-P seen following the introduction of this additional antiplatelet therapy.
Methods Patient selection We sampled a cross-section of patients with known coronary artery disease taking regular low dose aspirin (75 mg/day) for at least 5 days and no other antithrombotic or anticoagulant agent (pre-clopidogrel). Patients were recruited in our centre from outpatient clinics, cardiology in-patient wards and the cardiac catheterisation day care unit. All patients were between 18 and 85 years and were able to provide informed consent. Patients were excluded if they were receiving dipyridamole or NSAID therapy, were pregnant, anaemic (Hb 510 g/dl), had a platelet count 5100 or 4500 109/l or a personal or family history of a bleeding disorder. Patients were excluded if they were non-compliant with aspirin therapy, as assessed by direct questioning at the time of recruitment. Ethical approval was granted by the West Research Ethics Committee, Glasgow. A subgroup of patients scheduled for percutaneous coronary intervention (PCI) subsequently received clopidogrel in preparation for the procedure. In this group, further samples were collected following the introduction of clopidogrel (postclopidogrel), prior to commencing PCI. Patients were deemed to be fully loaded on clopidogrel therapy if they had received either; (i) a 600 mg loading dose 42 hours previously, (ii) a 300 mg loading dose 424 hours previously or (iii) 75 mg/day of clopidogrel for at least 5 days. For comparison, [TxB2]S-P was assessed in healthy, nonaspirinated subjects recruited through the National Blood Service clinic, Cambridge, UK as part of the Bloodomics programme [28]. Sample collection For all control and pre-clopidogrel samples, blood was drawn from a large calibre vein in the antecubital fossa using a 21G
Platelets, 2015; 26(1): 17–24
butterfly needle and vacutainer system. Samples were collected between the hours of 08:00 and 17:00 and patients were asked to sit for 10 minutes prior to sampling. Use of a tourniquet was kept to a minimum, and care was taken to avoid unnecessary trauma to the vein or agitation of the sample. Blood was collected in strict sequential order; an initial sample of 3.5 ml was collected into a 3.2% sodium citrate tube and discarded a further 3.5 ml was collected into 3.2% sodium citrate to prepare plasma, and 3 ml was collected in a glass vacutainer tube containing thrombin to prepare serum. For patients tested using the VerifyNow Aspirin assay an additional 2 ml 3.2% sodium citrate tube (Accumetrics, USA) was collected prior to the thrombin-containing sample. All samples were immediately mixed by gently inverting five times. Following the introduction of clopidogrel, the second series of samples (post-clopidogrel) was collected either by venepuncture, or following the insertion of an arterial sheath at the time of coronary angiography with additional 3.2% sodium citrate tubes for the assessment of response to clopidogrel using VASP-PRI and VerifyNow P2Y12. Thromboxane B2 assay Patient samples: within 30 minutes of collection the 3.2% sodium citrate blood tube was centrifuged for 30 minutes at 1500 g (3000 rpm) and the plasma supernatant stored in 0.5 ml aliquots at 80 C until use. The thrombin containing serum tube was left at room temperature for 1 hour after which it was centrifuged at 1500 g (3000 rpm) for 10 minutes and the serum supernatant collected in 0.5 ml aliquots and immediately stored at 80 C. Control samples: within 30 minutes of collection the citrated blood tube was centrifuged at 1550 g (3100 rpm) for 15 minutes. Plasma was removed and re-centrifuged at 9300 g (10 000 rpm) for 10 minutes after which it was stored in aliquots at 80 C until use. Serum thrombin tubes were left at room temperature for 1 hour, after which they were centrifuged at 1550 g (3100 rpm) for 15 minutes. Serum was removed and re-centrifuged at 9300 g (10 000 rpm) for 10 minutes after which it was stored in aliquots at 80 C until use. The level of TxB2 in the plasma and serum samples was analysed by ELISA (R&D Systems Europe; cat. no. KGE011) following the manufacturer’s instructions. Platelet thromboxane generation ([TxB2]S-P) was calculated by subtracting the levels of TxB2 in the paired plasma sample ([TxB2]P) from the level in the thrombin-serum sample ([TxB2]S). VerifyNow Aspirin and P2Y12 assay The Ultegra rapid platelet function analyser (RPFA) and VerifyNow Aspirin assay cartridges were purchased from Accumetrics Inc. (San Diego, CA) and used according to manufacturer’s instructions [29]. VerifyNow Aspirin results are expressed as aspirin reaction units (ARUs). At the time of the study, the manufacturer recommended a qualitative measure of aspirin ‘‘resistance’’ with a cut off of 4550 ARU. As recommended by the manufacturer’s guidelines, the 2 ml 3.2% sodium citrate samples were stored at room temperature for at least 30 minutes before assaying and all samples were analysed between 30 minutes and 2 hours of acquisition. VASP-PRI assay Measurement of phosphorylated VASP in platelets was performed by flow cytometry using a commercially available assay (VASP/ P2Y12, Biocytex, France) [30]. Reagents were prepared as instructed by the manufacturer and each box was used within 1 month of opening. The samples were stored at room temperature until processing and all assays were carried out within 24 hours of
Thromboxane generation on dual anti-platelet therapy
DOI: 10.3109/09537104.2013.870334
sampling. Samples were run on a four channel BD FACSCalibur flow cytometer.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
Statistical methods This was an exploratory study to assess the variation in thromboxane concentrations in this group of patients. In particular, we were interested in the effects of clopidogrel on the measured concentration of TxB2. We sought a sample size of 80 patients with samples before and after clopidogrel treatment. Although no formal power calculation was undertaken prior to this study, using the standard deviation of log10[TxB2]S-P for our pre-clopidogrel samples (0.34), in our cohort of 69 patients had an 80% power to detect a difference on a log scale of 0.116, or a relative difference of 12% between samples before and after clopidogrel. The distribution of results are summarised as the median, interquartile range and range of values for each assay. Samples with negative values of [TxB2]S-P were excluded on the basis of biological implausibility. TxB2 concentrations showed a strongly positively skewed distribution. Logarithmic transformations produced a distribution of concentrations approaching normality. This transformed data was used in simple linear regression analysis to investigate whether any baseline characteristic was associated with [TxB2]S-P. Patients characteristics with a significant correlation were subsequently included in a stepwise, multiple regression model. Correlations between log transformed [TxB2]P, [TxB2]S and [TxB2]S-P before and after clopidogrel,
19
and between log10[TxB2]S-P and VerifyNow Aspirin data were calculated using Pearson’s correlation coefficient. Differences between log transformed [TxB2]P, [TxB2]S and [TxB2]S-P before and after clopidogrel were investigated using paired sample t-tests. Significance was taken at the p50.05 level for all statistical tests.
Results In total, 123 patients were recruited for the assessment of [TxB2]S-P on aspirin monotherapy. Study recruitment and sampling are shown in Figure 1 and the demographics of the patients are shown in Table I, with a particular focus on those parameters most likely to influence the response to aspirin therapy. Four thromboxane samples taken prior to the introduction of clopidogrel were erroneously processed. An additional four samples produced plasma levels of TxB2 that were higher than those of serum. This was deemed to be biologically implausible and these samples were excluded from further analysis. This left 115 paired serum and plasma samples from patients taking aspirin alone available for analysis. A summary of thromboxane concentrations before the introduction of clopidogrel is given in Table II (Panel a). One hundred and four healthy controls [median age 46, range 20–63; male 80/104 (77%)] taking no antiplatelet or anticoagulant treatment had [TxB2] measured in both serum and plasma. These results are summarised in Table II (Panel a) and shown graphically in Figure 2.
Figure 1. Flow diagram of patient recruitment and sampling.
20
R. I. S. Good et al.
Platelets, 2015; 26(1): 17–24
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
In the 115 patients undergoing PCI, levels of TxB2 were 510 ng/ml in both plasma (n ¼ 114; 99%) and serum (n ¼ 110; 96%) samples (Figure 2). Residual platelet thromboxane generation, as shown by [TxB2]S-P, was 510 ng/ml in 110 (96%) of the patients. These levels were significantly lower than in healthy subjects not on aspirin where only one subject had [TxB]S-P 51 ng/ml and 71/104 (68%) had [TxB2]S-P 410 ng/ml (Figure 2). Simple linear regression of the patients’ demographic data showed significant associations between log10[TxB2]S-P prior to the introduction of clopidogrel and age, statin therapy, current smoking and creatinine (Table I). When these variables were included in a stepwise multiple regression analysis, age alone was the only independent negative predictor of pre-clopidogrel log10[TxB2]S-P (regression coefficient 0.015, 95% CI 0.027 to 0.003, p ¼ 0.013; n ¼ 115). No such association was found between age and [TxB2]S-P in control subjects.
Table I. Patient demographics and blood parameters of 123 patients sampled prior to the introduction of clopidogrel. Linear regression (N ¼ 115)* N ¼ 123 Patient characteristic Age (years) Mean SD Male (%) BMI (kg/m2) Mean SD Medical history Hypertension (%) Diabetes (%) Dyslipidaemia (%) Previous MI (%) Smoking status Current Medication Statin Beta blocker ACE inhibitor Blood parameters Haemoglobin (g/dl) (Mean SD) Platelet count (109/l) (Mean SD) Creatinine (mmol/l) (Mean SD)
Coefficient (95% CI)
p
A subgroup of 40 patients was tested for response to aspirin using VerifyNow Aspirin prior to the introduction of clopidogrel (Table II, Panel b). Of these 40 patients, 36 had [TxB2]S-P assay results available for comparison. Two patients met the definition of aspirin resistance (ARU 4 550) according to the VerifyNow assay. There was a poor correlation between VerifyNow Aspirin ARU and [TxB2]S-P (r ¼ 0.40; p50.001, Figure 3), but the patient with the highest residual generation of TxB2 (36 ng/ml) also had the highest activity in the VerifyNow Aspirin assay (653 ARU). The remaining patient deemed aspirin resistant by VerifyNow had a modest residual [TxB2]S-P of 0.98 ng/ml. These two samples are shown in Figure 3 by the solid and dotted arrows respectively. Thromboxane B2 was measured in a second sample obtained from 79/115 patients following the introduction of clopidogrel. Of these, four samples were erroneously processed and were not able to be analysed and a further six samples produced plasma concentrations of TxB2 greater than that in the serum sample and were again excluded on the basis of biological implausibility (Figure 1). This left a total of 69 patients with paired plasma and serum TxB2 concentrations before and after clopidogrel therapy. Of these, 42/69 (61%) had received clopidogrel 75 mg/day for45 days, 18/69 (26%) 300 mg424 hours previously and 9/69 (13%) 600 mg42 hours but524 hours prior to sampling. The majority 54/69 (78%) were venous samples and the remaining
64.4 10.2 0.015 (0.025 to 0.006) 0.002 81 (66) 0.003 (0.211 to 0.216) 0.980 27.7 4.4 69 17 81 33
(56.1) (13.8) (65.9) (26.8)
0.020 (0.003 to 0.043) 0.161 0.084 0.051 0.098
(0.362 (0.377 (0.265 (0.130
to to to to
0.084
0.041) 0.209) 0.162) 0.326)
0.117 0.570 0.635 0.397
25 (20.3)
0.281 (0.035 to 0.527)
0.026
107 (87) 89 (72.4) 47 (38.2)
0.300 (0.005 to 0.596) 0.003 (0.224 to 0.229) 0.063 (0.271 to 0.144)
0.046 0.982 0.545
13.8 1.4
0.011 (0.122 to 0.100)
0.838
0.002 (50.001 to 0.004)
0.188
0.007 (0.013 to 0.001)
0.030
253 69 92 20
*Simple linear regression using each patients characteristic as a predictor and log10[TxB2]S-P as the dependent variable (n ¼ 115).
Figure 2. Concentration of thromboxane B2 [TxB2] in plasma (p), serum (s) and serum–plasma (s–p) in patients taking aspirin (n ¼ 115) and healthy controls (n ¼ 104). Upper dashed line represents a cut off of [TxB2]S-P 410 ng/ml. The lower dashed line represents a residual level of thromboxane generation 41 ng/ml.
Table II. Summary statistics of (a) [TxB2] in plasma and serum in controls and patients before and after clopidogrel therapy and (b) VerifyNow P2Y12, VASP-PRI and VerifyNow Aspirin results in patients before and after clopidogrel therapy. Controls (n ¼ 104)
Panel a [TxB2]P (pg/ml) [TxB2]S (pg/ml) [TxB2]S-P (pg/ml) Panel b VerifyNow P2Y12 (%inhibition) VASP-PRI (%activation) VerifyNow Aspirin (ARU)
Pre-clopidogrel (n ¼ 115)
Post-clopidogrel (n ¼ 69)
Median
IQR
Range
Median
IQR
Range
Median
IQR
Range
229 18 667 18 299
236 21 639 21 793
43–5946 481–354 708 380–354 531
110 773 646
148 1018 842
9–16 620 85–36 344 19–30 511
136 673 491
138 701 612
16–5301 200–36 344 119–31 043
11 82 428
11 1–40 8 29–100 (n ¼ 40) 94 366–653
45 58 382
41 6–97 31 1–88 (n ¼ 9) 28 350–424
DOI: 10.3109/09537104.2013.870334
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
15 samples (22%) were collected from the arterial sheath at the time of angiography. There was a wide range of response to clopidogrel measured by either VerifyNow P2Y12 (%inhibition) or VASP-PRI (%activation; Table II, Panel b) with a moderate correlation between the two assays (Figure 4; r ¼ 0.76; p50.001). Neither the clopidogrel loading regimen, nor method of sample collection influenced [TxB2]S-P, VerifyNow P2Y12 or VASP-PRI results (p40.05 for all; data not shown). Concentrations of [TxB2]P, [TxB2]S and [TxB2]S-P before and after clopidogrel therapy showed very little change (Table II, Panel a and Figure 5) and were highly correlated (r 0.78; p50.001 for all). Comparing the log transformed concentrations of thromboxane before and after clopidogrel using paired sample t-tests, there was a small but significant reduction in log10[TxB2]P (mean change 0.062, 95% CI 0.005–0.120, p ¼ 0.035), a trend towards a lower log10[TxB2]S (mean change 0.062, 95% CI
Figure 3. Correlation of [TxB2]S-P and VerifyNow Aspirin assays. The highest [TxB2]S-P (arrowhead) was seen in one of two patients deemed aspirin resistant by the VerifyNow Aspirin assay.
Figure 5. Thromboxane B2 concentrations [TxB2] in plasma (p), serum(s) and serum– plasma (s–p) before (Pre) and after (Post) the introduction of clopidogrel. *Paired sample t-tests of the difference in log10[TxB2] before and after clopidogrel.
Thromboxane generation on dual anti-platelet therapy
21
0.005 to 0.129, p ¼ 0.069) but no significant difference in log10[TxB2]S-P, (mean change 0.041, 95% CI 0.040 to 0.123, p ¼ 0.311). As shown in Figure 6, the magnitude of response to clopidogrel measured using VASP-PRI did not correlate with the change in [TxB2]S-P following the introduction of clopidogrel (r ¼ 0.02, p ¼ 0.99), and the findings were similar when measured by the VerifyNow P2Y12 % inhibition (r ¼ 0.02, p ¼ 0.97). In 9/40 patients with repeat testing using VerifyNow Aspirin before and after the introduction of clopidogrel, all patients showed a reduction in ARU and the difference was statistically significant despite the small sample size (p ¼ 0.004; Figure 7).
Discussion Recent focus on platelet testing in CAD patients has been on near patient testing, and largely on assessing the efficacy of P2Y12 antagonists [3–5]. However, there remains a need to monitor the
Figure 4. Correlation between clopidogrel response assessed by VerifyNow P2Y12 (% inhibition) and VASP-PRI.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
22
R. I. S. Good et al.
Figure 6. Clopidogrel response measured by VASP-PRI does not influence D[TxB2]S-P observed following the introduction of this additional antiplatelet agent.
Figure 7. VerifyNow Aspirin before and after the introduction of clopidogrel (n ¼ 9). p Value is for a paired sample t-test.
efficacy of aspirin, especially in patients receiving DAPT. This observational study, in a routine clinical setting, used [TxB2]S-P as a marker of the residual ability of platelets to generate thromboxane to assess the efficacy of aspirin in patients receiving single and dual antiplatelet therapy. We have shown that the efficacy of aspirin measured by [TxB2]S-P is unaffected by additional P2Y12 antagonism. The demographics of the patients recruited to this study are comparable with those seen in contemporary studies of coronary artery disease [31]. There was a measurable variation in the concentration of TxB2 in both plasma and serum with the majority of patients having [TxB2]S-P levels below 10 ng/ml. There was a highly positive skew of thromboxane B2 concentrations and the normalisation of [TxB2]S-P concentrations following
Platelets, 2015; 26(1): 17–24
logarithmic transformation. This exploratory study does not attempt to define aspirin ‘‘resistance’’; however, these data do demonstrate a significant overlap of [TxB2]S-P levels between aspirinated patients and normal controls taking no antiplatelet therapy. This may help guide future studies to investigate whether there is a threshold of residual thromboxane generation that is predictive of adverse clinical events. Age alone was found to be an independent negative predictor of [TxB2]S-P This is in contrast to previously published data, using urinary 11-dehydro TxB2, suggesting an increased ability to generate thromboxane, with increasing age [32]. Our study demonstrates strong and significant correlations between [TxB2] concentrations before and after clopidogrel. There was no significant difference in [TxB2]S-P following the introduction of this additional P2Y12 receptor inhibitor, thus supporting the use of [TxB2]S-P as a measure of aspirin response in patient taking DAPT. As further support for this, we confirmed that the magnitude of response to clopidogrel assessed using either the bedside (VerifyNow P2Y12) or laboratory based (VASP-PRI) assay does not have any influence on the small change in [TxB2]S-P after the introduction of clopidogrel. As newer, more potent and uniform ADP receptor antagonists have entered clinical practice, it is encouraging that these assay results may remain relevant for patients taking aspirin with either prasugrel or ticagrelor. Although measurement of [TxB2] in platelet rich plasma (PRP) following stimulation with arachidonic acid has been used for the measurement of aspirin response [33], the generation of thromboxane in these circumstances may be affected by sample manipulation and selective loss of larger platelets during the preparation of PRP. In addition, this method does not account for background TxB2 production from non-platelet pathways [27, 34]. It has also been shown that P2Y12 inhibitors alone may reduce the production of thromboxane in PRP by comparable amounts to aspirin [20]. In contrast, our method of measuring TxB2 generation avoids these potential confounders through maximally stimulating platelets in the whole blood milieu prior to serum sample preparation and measuring the background level of TxB2 in plasma generated from unstimulated samples. An additional finding of importance was a poor correlation between [TxB2]S-P and VerifyNow Aspirin. This highlights the difficulties encountered in the field of measuring response to aspirin [35]. We remain uncertain of the best test, and metaanalyses suggesting association with increased cardiovascular risk are hampered by the myriad of assays being used, and the poor correlation between these assays [9–11, 36, 37]. The additional challenge of measuring response to aspirin in patients taking a P2Y12 inhibitor is highlighted by the data obtained using VerifyNow Aspirin. In nine of our patients sampled before and after the addition of clopidogrel to aspirin we found significantly reduced ARU values suggesting the manufacturer’s recommended cut-off for aspirin ‘‘resistance’’ of 550ARU cannot be applied in the setting of DAPT. In keeping with our findings, Lordkipandze et al. [17] showed only 5/85 (6%) of patients with stable CAD had an impaired response to aspirin assessed by VerifyNow Aspirin on DAPT. In the recent ARCTIC study only 46/1213 patients (3.9%) were aspirin ‘‘resistant’’ according to VerifyNow Aspirin when taking DAPT [5]. This compares with previously published data suggesting an incidence of aspirin ‘resistance’ of 17–19% [8, 10] on aspirin monotherapy using VerifyNow Aspirin. Breet et al. [15] found the cut-off for VerifyNow Aspirin high on-aspirin platelet reactivity on DAPT that best predicted clinical outcome was 454ARU, a much lower value than recommended by the manufacturer. In conclusion, the aggregation based platelet function assay VerifyNow Aspirin appears to be significantly influenced by
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
DOI: 10.3109/09537104.2013.870334
additional P2Y12 inhibition. As such we believe that [TxB2]S-P may offer the most appropriate method of assessing aspirin response in patients taking an additional P2Y12 inhibitor and warrants further investigation as a predictor of clinical outcome and in studies of response to antiplatelet therapy. For example, a large population with previous DES implantation may provide an opportunity to assess whether residual thromboxane generation is clinically relevant as we know that a number of these patients have delayed neointimal coverage and persistent malapposition which may cause very late stent thrombosis [38]. However, we acknowledge that measurement of [TxB2]S-P is time consuming and requires expertise so is not suited to studies requiring rapid results. The current study has several limitations. We studied a relatively small number of patients, particularly post clopidogrel therapy, and further studies would be warranted to confirm our findings. Although patients were questioned at the time of recruitment, no formal testing was performed to confirm compliance [39]. In addition, the accuracy of measurement of thromboxane generation may have been improved by collecting plasma samples into citrated tubes containing indomethacin to prevent additional activation of platelets during sample processing.
Declaration of interest The authors have no conflicts of interest related to this research. This research was supported by a British Heart Foundation Junior Research Fellowship (FS/06/031).
References 1. Hamm CW, Bassand JP, Agewall S, Bax J, Boersma E, Bueno H, Caso P, Dudek D, Gielen S, Huber K, et al. ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: The Task Force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J 2011;32: 2999–3054. 2. Wijns W, Kolh P, Danchin N, Di Mario C, Falk V, Folliguet T, Garg S, Huber K, James S, et al. Guidelines on myocardial revascularization. Eur Heart J 2010;31:2501–2555. 3. Price MJ, Angiolillo DJ, Teirstein PS, Lillie E, Manoukian SV, Berger PB, Tanguay JF, Cannon CP, Topol EJ. Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: A time-dependent analysis of the Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety (GRAVITAS) trial. Circulation 2011;124:1132–1137. 4. Trenk D, Stone GW, Gawaz M, Kastrati A, Angiolillo DJ, Muller U, Richardt G, Jakubowski JA, Neumann FJ. A randomized trial of prasugrel versus clopidogrel in patients with high platelet reactivity on clopidogrel after elective percutaneous coronary intervention with implantation of drug-eluting stents: Results of the TRIGGERPCI (Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy With Prasugrel) study. J Am Coll Cardiol 2012;59:2159–2164. 5. Collet JP, Cuisset T, Range G, Cayla G, Elhadad S, Pouillot C, Henry P, Motreff P, Carrie´ D, Boueri Z, et al. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. N Engl J Med 2012; 367:2100–2109. 6. Eikelboom JW, Hirsh J, Weitz JI, Johnston M, Yi Q, Yusuf S. Aspirin-resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke, or cardiovascular death in patients at high risk for cardiovascular events. Circulation 2002;105:1650–1655. 7. Gum PA, Kottke-Marchant K, Welsh PA, White J, Topol EJ. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease.[see comment]. J Am Coll Cardiol 2003;41:961–965. 8. Chen WH, Lee PY, Ng W, Tse HF, Lau CP. Aspirin resistance is associated with a high incidence of myonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment. J Am Coll Cardiol 2004;43:1122–1126.
Thromboxane generation on dual anti-platelet therapy
23
9. Snoep JD, Hovens MM, Eikenboom JC, van der Bom JG, Huisman MV. Association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events: A systematic review and meta-analysis. Arch Intern Med 2007;167:1593–1599. 10. Harrison P, Segal H, Blasbery K, Furtado C, Silver L, Rothwell PM. Screening for aspirin responsiveness after transient ischemic attack and stroke: Comparison of 2 point-of-care platelet function tests with optical aggregometry. Stroke 2005;36:1001–1005. 11. Lordkipanidze M, Pharand C, Schampaert E, Turgeon J, Palisaitis DA, Diodati JG. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur Heart J 2007;28:1702–1708. 12. Lev EI, Patel RT, Maresh KJ, Guthikonda S, Granada J, DeLao T, Bray PF, Kleiman NS. Aspirin and clopidogrel drug response in patients undergoing percutaneous coronary intervention: The role of dual drug resistance. J Am Coll Cardiol 2006;47:27–33. 13. Ohmori T, Yatomi Y, Nonaka T, Kobayashi Y, Madoiwa S, Mimuro J, Ozaki Y, Sakata Y. Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity: Involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients. J Thromb Haemost 2006;4:1271–1278. 14. Cuisset T, Frere C, Quilici J, Morange PE, Camoin L, Bali L, Lambert M, Juhan-Vague I, Alessi MC, Bonnet JL. Relationship between aspirin and clopidogrel responses in acute coronary syndrome and clinical predictors of non response. Thromb Res 2009;123:597–603. 15. Breet NJ, van Werkum JW, Bouman HJ, Kelder JC, Harmsze AM, Hackeng CM, ten Berg JM. High on-treatment platelet reactivity to both aspirin and clopidogrel is associated with the highest risk of adverse events following percutaneous coronary intervention. Heart 2011;97:983–990. 16. Fontana P, Nolli S, Reber G, de Moerloose P. Biological effects of aspirin and clopidogrel in a randomized cross-over study in 96 healthy volunteers. J Thromb Haemost 2006;4:813–819. 17. Lordkipanidze M, Diodati JG, Schampaert E, Palisaitis DA, Pharand C. Prevalence of unresponsiveness to aspirin and/or clopidogrel in patients with stable coronary heart disease. Am J Cardiol 2009;104: 1189–1193. 18. Eikelboom JW, Hankey GJ, Thom J, Bhatt DL, Steg PG, Montalescot G, Johnston SC, Steinhubl SR, Mak KH, Easton JD, et al. Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA) Investigators. Incomplete inhibition of thromboxane biosynthesis by acetylsalicylic acid: Determinants and effect on cardiovascular risk. Circulation 2008;118:1705–1712. 19. Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Biol Chem 1999;274:29108–29114. 20. Armstrong PC, Dhanji AR, Tucker AT, Mitchell JA, Warner TD. Reduction of platelet thromboxane A2 production ex vivo and in vivo by clopidogrel therapy. J Thromb Haemost 2010;8:613–615. 21. Kirkby NS, Leadbeater PD, Chan MV, Nylander S, Mitchell JA, Warner TD. Antiplatelet effects of aspirin vary with level of P2Y12 receptor blockade supplied by either ticagrelor or prasugrel. J Thromb Haemost 2011;9:2103–2105. 22. Frelinger AL III, Furman MI, Linden MD, Li Y, Fox ML, Barnard MR, Michelson AD. Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independent pathway: A 700patient study of aspirin resistance. Circulation 2006;113:2888–2896. 23. Santilli F, Rocca B, De Cristofaro R, Lattanzio S, Pietrangelo L, Habib A, Pettinella C, Recchiuti A, Ferrante E, Ciabattoni G, et al. Platelet cyclooxygenase inhibition by low-dose aspirin is not reflected consistently by platelet function assays: Implications for aspirin ‘‘resistance". J Am Coll Cardiol 2009;53:667–677. 24. Reilly IA, FitzGerald GA. Inhibition of thromboxane formation in vivo and ex vivo: Implications for therapy with platelet inhibitory drugs. Blood 1987;69:180–186. 25. Maree AO, Curtin RJ, Dooley M, Conroy RM, Crean P, Cox D, Fitzgerald DJ. Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease. J Am Coll Cardiol 2005; 46:1258–1263. 26. Smith JP, Haddad EV, Taylor MB, Oram D, Blakemore D, Chen Q, Boutaud O, Oates JA. Suboptimal inhibition of platelet cyclooxygenase-1 by aspirin in metabolic syndrome. Hypertension 2012;59: 719–725.
Platelets Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
24
R. I. S. Good et al.
27. Karim S, Habib A, Levy-Toledano S, Maclouf J. Cyclooxygenase-1 and -2 of endothelial cells utilize exogenous or endogenous arachidonic acid for transcellular production of thromboxane. J Biol Chem 1996;271:12042–12048. 28. Jones CI, Garner SF, Angenent W, Bernard A, Berzuini C, Burns P, Farndale RW, Hogwood J, Rankin A, Stephens JC, et al. Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus. J Thromb Haemost 2007;5: 1756–1765. 29. Michelson AD. Methods for the measurement of platelet function. Am J Cardiol 2009;103:20A–26A. 30. Aleil B, Ravanat C, Cazenave JP, Rochoux G, Heitz A, Gachet C. Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases. J Thromb Haemost 2005;3:85–92. 31. Mahaffey KW, Wojdyla DM, Carroll K, Becker RC, Storey RF, Angiolillo DJ, Held C, Cannon CP, James S, Pieper KS, et al. Ticagrelor compared with clopidogrel by geographic region in the platelet inhibition and patient outcomes (PLATO) trial. Circulation 2011;124:544–554. 32. Faraday N, Becker DM, Yanek LR, Herrera-Galeano JE, Segal JB, Moy TF, Bray PF, Becker LC. Relation between atherosclerosis risk factors and aspirin resistance in a primary prevention population. Am J Cardiol 2006;98:774–779.
Platelets, 2015; 26(1): 17–24
33. Chan MV, Armstrong PC, Papalia F, Kirkby NS, Warner TD. Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing. Platelets 2011;22:485–494. 34. Faraday N, Scharpf RB, Dodd-o JM, Martinez EA, Rosenfeld BA, Dorman T. Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin. Anesthesiology 2001;94:145–151. 35. Patrono C, Rocca B. Drug insight: Aspirin resistance–fact or fashion? Nat Clin Pract Cardiovasc Med 2007;4:42–50. 36. Krasopoulos G, Brister SJ, Beattie WS, Buchanan MR. Aspirin ‘‘resistance’’ and risk of cardiovascular morbidity: systematic review and meta-analysis. BMJ 2008;336:195–198. 37. Sofi F, Marcucci R, Gori AM, Abbate R, Gensini GF. Residual platelet reactivity on aspirin therapy and recurrent cardiovascular events – a meta-analysis. Int J Cardiol 2008;128:166–171. 38. Kang S, Lee CW, Song H, Ahn J, Kim W, Lee J, Park DW, Lee SW, Kim YH, Mintz GS, et al. OCT analysis in patients with very late stent thrombosis. JACC Cardiovasc Imaging 2013;6: 695–703. 39. Schwartz KA, Schwartz DE, Ghosheh K, Reeves MJ, Barber K, DeFranco A. Compliance as a critical consideration in patients who appear to be resistant to aspirin after healing of myocardial infarction. Am J Cardiol 2005;95:973–975.
