Author + information
- Received July 25, 2008
- Revision received October 8, 2008
- Accepted October 14, 2008
- Published online February 24, 2009.
- Francesca Santilli, MD⁎,
- Bianca Rocca, MD, PhD†,
- Raimondo De Cristofaro, MD‡,
- Stefano Lattanzio, BSc⁎,
- Laura Pietrangelo, BSc⁎,
- Aida Habib, PhD§,
- Caterina Pettinella, PhD⁎,
- Antonio Recchiuti, PhD⁎,
- Elisabetta Ferrante, BSc⁎,
- Giovanni Ciabattoni, MD⁎,
- Giovanni Davì, MD⁎ and
- Carlo Patrono, MD†,⁎ ()
- ↵⁎Reprint requests and correspondence:
Prof. Carlo Patrono, Department of Pharmacology, Catholic University School of Medicine, Largo F. Vito 1 00168 Rome, Italy
Objective This study was conducted to assess the thromboxane (TX) dependence of biochemical and functional indexes used to monitor the effect of low-dose aspirin.
Background Functional assays of the antiplatelet effects of low-dose aspirin variably reflect the TX-dependent component of platelet aggregation. Previous studies of aspirin resistance were typically based on a single determination of platelet aggregation.
Methods We assessed the TXB2dependence of biochemical and functional indexes, as well as their intersubject and intrasubject variability during administration of the drug and after its withdrawal in 48 healthy volunteers randomized to receive aspirin 100 mg daily for 1 to 8 weeks.
Results Serum TXB2was uniformly suppressed by 99% of baseline. Urinary 11-dehydro-TXB2, arachidonic acid-induced aggregation, and VerifyNow Aspirin (Accumetrics Inc., San Diego, California) showed stable, incomplete inhibition (65%, 80%, and 35%, respectively). Adenosine diphosphate- and collagen-induced aggregation was highly variable and poorly affected by aspirin, with an apparent time-dependent reversal. Inhibition of platelet cyclooxygenase activity was nonlinearly related to inhibition of platelet aggregation. Platelet function largely recovered by day 3 post-aspirin, independently of treatment duration. With any functional assay, occasionally “resistant” subjects were found to be “responders” on previous or subsequent determinations.
Conclusions Platelet cyclooxygenase activity, as reflected by serum TXB2levels, is uniformly and persistently suppressed by low-dose aspirin in healthy subjects. However, the effect of aspirin is variably detected by functional assays, potentially leading to misclassification of “responder” as “resistant” phenotypes owing to poor reproducibility of functional measurements. The nonlinearity of the relationship between inhibition of TX production and inhibition of platelet function has important clinical implications.
Low-dose aspirin can prevent up to 25% of fatal and nonfatal vascular events in high-risk patients (1,2). Patients may experience recurrent events while on aspirin because of the multifactorial nature of atherothrombosis (3). Such a treatment failure is often inappropriately referred to as aspirin “resistance” (4). This term has been used also to indicate incomplete inhibition of platelet function by low-dose aspirin, with widely variable estimates of its incidence and inconclusive data on its clinical significance (4).
Low-dose aspirin inhibits thromboxane (TX)-dependent platelet function through permanent inactivation of the cyclooxygenase (COX) activity of prostaglandin H synthase 1 (also referred to as COX-1) (2). This represents the best-characterized mechanism of action, which fully accounts for the unique pharmacodynamics of aspirin as an antiplatelet agent and adequately explains the saturability of its cardioprotective effects at low doses (1,2). It is already well known that the various methods used to quantitate the antiplatelet effect of aspirin variably reflect the aspirin-sensitive TX-dependent component of platelet aggregation (3). Moreover, different platelet function tests correlate poorly among themselves (5). However, whether the relative contributions of TX-dependent and -independent pathways are constant for any given assay is largely unknown. This is particularly relevant to the current debate on aspirin “resistance” because characterization of “resistant” versus “responder” status is typically based on a single determination of platelet function, with the underlying assumption that this determination represents a stable phenotype (6,7).
The present study assessed the TX dependence of biochemical and functional indexes commonly used to monitor the antiplatelet effect of low-dose aspirin, as well as their intersubject and intrasubject variability during administration of the drug and after its withdrawal, in healthy volunteers. Moreover, we tested whether the antiplatelet response to low-dose aspirin may vary as a function of time because of accelerated thrombocytopoiesis (8), expression of COX-2 in newly formed platelets (9), or partial recovery of COX-1 activity (10).
Design of the study
We enrolled 48 healthy Caucasian subjects (25 women and 23 men; age range 19 to 38 years, mean 26.2 ± 4.5 years). Exclusion criteria were atherothrombotic disease, bleeding history, gastroduodenal ulcer, aspirin intolerance, obesity, diabetes, cigarette smoking, dyslipidemia, hypertension, and pregnancy. Subjects were randomized to 1 of 8 groups, according to treatment duration, ranging from 1 to 8 weeks. Each participant received enteric-coated aspirin (Cardioaspirina, Bayer, Milan, Italy) 100 mg once daily and was instructed to take tablets at the same time of the day. None of the participants had taken vitamin supplements, aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), or other antiplatelet drugs in the preceding 10 days. NSAIDs were not allowed during the study.
Blood and urine samples were collected after an overnight fast before starting aspirin (baseline), at the end of each week on aspirin, and at days 1, 2, 3, and 7 after withdrawal. Aspirin was provided at each visit for the subsequent week. Compliance was measured by pill count. The protocol was approved by the Ethics Committee, and written informed consent was obtained from each participant.
One milliliter of blood without anticoagulant was transferred into a glass tube, incubated for 1 hour at 37°C, and centrifuged at 1,200 gfor 10 min. The supernatant serum was stored at −20°C until assayed for TXB2(11). Urinary 11-dehydro-TXB2was measured by a previously validated radioimmunoassay (12).
Platelet function assays
Optical aggregation was measured in platelet-rich plasma obtained from citrated blood (0.105 M) using a dual-channel aggregometer (Chrono-Log 490, Chrono-Log, Havertown, Pennsylvania) and stimulated by 1 mM arachidonic acid (AA) (Cayman Chemical Company, Ann Arbor, Michigan), 10 μg/ml collagen (Mascia Brunelli, Milan, Italy), or 10 μM adenosine diphosphate (ADP) (Mascia Brunelli). These agonists and their concentrations were chosen based on previous aspirin “resistance” studies (4,13). Maximal aggregation (Tmax) was expressed as % of maximal light transmittance.
VerifyNow Aspirin, kindly provided by Dr. Robert Hillman (Accumetrics Inc., San Diego, California), was used on citrated whole blood, and aggregation was expressed as aspirin response units, as specified by the manufacturer.
Platelet COX-2, reticulated platelets, and thrombopoietin measurements
COX-2 expression was assessed by immunocytochemistry (9) using anti–COX-2 polyclonal antibodies (9) and by flow cytometry on washed and permeabilized platelets (9,14) using anti-CD61 and anti–COX-2 fluorescein-conjugated monoclonal antibodies (Cayman Chemical Company). Reticulated platelets were measured by the thiazole-orange technique (9). Plasma thrombopoietin was measured using a commercial kit (R&D Systems, Minneapolis, Minnesota).
Data were analyzed by parametric or nonparametric methods according to their distribution. Comparisons were made using analysis of variance or the Kruskal-Wallis test. Correlations were assessed by the Pearson or Spearman rank test. Differences between baseline and on-treatment values were analyzed with the ttest or Wilcoxon signed-rank test. Comparisons of time-course curves after aspirin withdrawal were analyzed by 2-factor repeated measurements analysis of variance with the post hoc Holm-Sidak test for pairwise comparisons (p < 0.05). An unpaired ttest was performed to compare measurements of serum TXB2or urinary 11-dehydro-TXB2, below or above response thresholds defined for platelet functional assays. Data are reported as mean ± SD. Significance was defined as p < 0.05. All tests were 2-tailed; analyses were performed using SPSS (version 13.0, SSPS Inc., Chicago, Illinois) and SigmaStat 3.1 (Systat Software Inc., Hounslow, United Kingdom). The intrasubject coefficient of variation (CV) was the ratio of the SD to the mean value of each variable in 1 subject at all time points during treatment. Corrections for correlated observations within subjects were not made.
Experimental data were fitted using Grafit software (Erithacus Software, Staines, United Kingdom). Different equations were used, and the Ftest indicated the best fitting equation. Data of post-aspirin recoveries were analyzed by a first-order rate equation:(1)in which R(t) is the percent recovery at time t, Rmaxis the maximum recovery (percentage), k is the first-order rate constant, and Off is the percentage value of the considered parameter at day = 0.
However, Equation 1could not properly describe serum TXB2recovery, and the following equation was used:(2)in which R(t) is the recovery at time t, Rmaxis the maximum recovery, k is the rate constant, and Ts is the time corresponding to the maximum rate of recovery.
The percent inhibition of serum TXB2was analyzed as a function of the corresponding inhibition of other assays. The shape of the plot showed a complex exponential, rather than linear, relationship, with an initial linear and subsequent steep exponential dependence. Thus, data were analyzed using the following equation.(3)in which a is the slope of the linear part of the curve and b is the base of the exponential part of the fit.
The 8 groups were comparable for all baseline measurements (data not shown). The mean interval between aspirin intake and blood sampling was 14 ± 4 h (n = 47), without differences among groups. One subject dropped out on day 4 for an unrelated cause, and 1 subject could not be evaluated at weeks 1, 2, and 6, 1 at weeks 4 and 5, and 1 at week 7 during the study. Full compliance was recorded by pill counting.
ADP-induced aggregation was significantly reduced by aspirin (40% maximal inhibition by week 3) but displayed substantial interindividual variability (Fig. 1A).It also showed time-dependent recovery, with Tmaxvalues at weeks 7 and 8 comparable to those at baseline (Fig. 1A). However, whereas baseline platelet aggregation was irreversible, aggregation at weeks 7 and 8 was consistently reversible (Figs. 1 and 2).⇓Furthermore, light transmittance values of the aggregometric tracings recorded at 6 min showed similar results and trends compared with Tmaxvalues (data not shown). Tmaxand 6-min light transmittance values were positively correlated (r = 0.72; p < 0.00001). The intrasubject CV of percent inhibition of Tmaxon aspirin was 70 ± 71% (n = 47; range 7% to 320%).
Tmaxvalues of collagen-induced aggregation showed approximately 50% inhibition by week 3 of treatment (Fig. 1B), with wide interindividual variability. The intrasubject CV of percent inhibition was 47 ± 44% (n = 47; range 25% to 249%).
AA-induced aggregation showed stable inhibition over the 8-week treatment, averaging 80% (range 50% to 97%) (Fig. 1C). The intrasubject CV of percent of inhibition was 18 ± 9% (n = 47; range 1% to 41%).
The VerifyNow Aspirin assay showed a steady 30% to 35% inhibition over the 8-week treatment (Fig. 1D). The intrasubject CV of percent inhibition was 19 ± 18% (n = 47; range 2% to 55%).
Serum TXB2was steadily suppressed over 8 weeks (Fig. 1E), absolute values being consistently <10 ng/ml (maximum 8.7 ng/ml). The average percent inhibition was constantly above 99%, without significant intergroup differences: 1-week treatment caused 99.3 ± 0.7% (range 96.3% to 99.9%) inhibition, and 8-week treatment produced 99.6 ± 0.3% (range 99% to 99.9%) inhibition. The intrasubject CV of percent inhibition was 0.3 ± 0.1% (n = 47; range 0.02% to 2%).
Levels of urinary 11-dehydro-TXB2were similarly reduced in the 8 groups (Fig. 1F): 1-week aspirin caused 66 ± 18% (range 46% to 93%) reduction, and 8-week treatment resulted in 61 ± 14% (range 42% to 74%) reduction. The intrasubject CV of percent inhibition was 21 ± 11% (n = 47; range 10% to 54%). No statistically significant sex-related differences were observed during aspirin intake for any of the assays (data not shown).
Recovery of platelet function by optical aggregation and VerifyNow Aspirin followed first-order kinetics and reached approximately 70% of the relative function at day 3 post-aspirin, without significant differences between 1 week and 8 weeks of treatment (Figs 3Aand 3B, and data not shown).
In contrast, initial recovery of serum TXB2levels differed among groups. At days 1 and 2 following aspirin withdrawal, TXB2values were similar in the subjects treated for 1 and 2 weeks and significantly higher than the corresponding values of longer treatment groups (3 to 8 weeks). Exposure to aspirin for at least 3 weeks showed a 2-day delay before detectable recovery (Fig. 3C). The overall kinetics of TXB2recovery showed a complex sigmoidal pattern, not appropriately described by the first-order kinetics of the other assays (Fig. 3), confirmed by Ftesting (p < 0.001 vs. first-order fitting). Whereas the values obtained with platelet functional assays had largely recovered by day 3 post-aspirin (Figs. 3A and 3B), day 3 TXB2values still averaged 45% of baseline (Figs. 3C and 3D); full recovery occurred by day 7 post-aspirin (Fig. 3C).
Correlations between percent inhibition of the different assays included measurements during and post-aspirin, which allowed the investigation of these relationships over a broader range of values. Platelet aggregation, VerifyNow Aspirin, and urinary 11-dehydro-TXB2were all linearly correlated, the highest coefficients being observed among AA-induced aggregation, VerifyNow Aspirin, and 11-dehydro-TXB2(Figs. 4Aand 4B, Table 1).However, a linear model could not properly describe the correlation between percent inhibition of serum TXB2and other assays, which displayed a complex relationship. This relationship showed an initial linear and subsequent steep exponential dependence (Ftesting: p < 0.001 vs. linear fitting) (Figs. 4C to 4F). The nonlinear relationship between percent inhibition of serum TXB2and urinary 11-dehydro-TXB2showed that for 0 to 97% of COX inhibition, TX biosynthesis in vivo was linearly inhibited by <40% and that >97% suppression of serum TXB2was necessary to maximally reduce TX metabolite excretion (Figs. 4E and 4F).
We next investigated whether serum TXB2and urinary 11-dehydro-TXB2levels differed in association with functional measurements defined as “responder” or “resistant,” using arbitrary thresholds, as described in previous studies (4,13). No statistically significant differences in TX-related indexes were observed between measurements below or above these thresholds (Fig. 5).Furthermore, with any assay, occasionally “resistant” subjects were found to be “responders” on previous or subsequent determinations (Fig. 6).
Aspirin did not modify plasma thrombopoietin levels, peripheral platelet counts, mean platelet volume, or platelet distribution width (Fig. 7Aand data not shown). During 8 weeks of aspirin exposure, the percentage of reticulated platelets, the youngest and mRNA-richest platelets (15), was not modified (Fig. 7B). Similarly, the fraction of platelets expressing COX-2 was not altered by aspirin (Figs. 7C to 7E).
Although initial evaluation of aspirin as an antithrombotic agent was based on functional assays predicting the requirement of relatively high doses and 3 or 4 times daily dosing regimens (16), further development of low-dose aspirin was largely based on biochemical assays reflecting its mechanism of action (17–19). The latter successfully predicted 2 novel features of the drug, that is, an optimal once-daily regimen and saturability of the antithrombotic effect at low doses. Interest in optical platelet aggregation was resurrected by the first description of so-called aspirin “resistance” in 1994 (20). Based on more than 500 publications on the topic (4), it has been proposed that aspirin “resistance” represents a true clinical diagnosis, requiring a change in antiplatelet therapy (21). Intrinsic to this suggestion is the underlying assumption that a single determination of agonist-induced platelet aggregation can determine whether aspirin has fully inhibited platelet COX activity and can define a stable “resistant” or “nonresponder” phenotype based on arbitrary thresholds of functional response (4,6,13).
We compared different functional and biochemical assays for their capacities to detect the antiplatelet effect of aspirin, 100 mg given orally for 1 to 8 weeks to 48 healthy subjects. Novel aspects of the study design included up to 8 repeated measurements in the same subjects to assess reproducibility of the various assays, as well as analysis of the recovery following withdrawal. The latter allowed characterization of aspirin's kinetics and also provided data on a wide range of inhibitory values of platelet COX activity to fully describe the relationship between inhibition of enzyme activity and inhibition of TX-dependent platelet function.
We found that platelet COX activity, as reflected by serum TXB2values (11,22), and TX-dependent platelet function, as reflected by AA-induced platelet aggregation and VerifyNow Aspirin values, were uniformly suppressed during prolonged exposure to aspirin, encompassing up to 5 to 6 platelet regeneration cycles and well beyond the steady-state of its pharmacodynamic effect at 100 mg daily (22). We found no evidence of time-dependent changes in several indexes of thrombocytopoiesis, including the percentage of reticulated or COX-2–expressing platelets, a phenomenon that transiently characterizes newly formed platelets (9,15).
Among the biochemical and functional assays, serum TXB2had the highest signal-to-noise ratio and the lowest interindividual and intraindividual variabilities. Aspirin, although an effective inhibitor of platelet TX production (11,18,22), is often considered a weak platelet inhibitor because of its limited effects on aggregation by high concentrations of ADP or collagen. This may account for some of the variability of the response to these agonists, which, in contrast to AA, activate platelets through both TX-dependent and -independent pathways (3). The paradoxical increase in light transmittance with persistence of reversible aggregation, recorded at 7 and 8 weeks on aspirin, might reflect complex physical properties of the ADP-induced turbidimetric signal (23,24), rather than a real platelet recovery, as indicated by the stable inhibition of TX-dependent aggregation. However, whether the paradoxical increase in Tmaxreflects a change in the ratio of small to large platelet aggregates or a change in the signaling pathway for the first phase of ADP-induced aggregation remains to be established.
In contrast to measurements of serum TXB2, for which every measurement of more than 200 values during aspirin intake was suppressed by at least 97% versus baseline, measurements of various functional indexes categorized according to previously described thresholds (21,25), identified 1.4% to 30% of “nonresponder” samples. However, inspection of prior or subsequent determinations performed in the same subjects clearly identified the fluctuating nature of this apparent “nonresponder” phenotype, most likely reflecting the relatively poor intrasubject reproducibility of functional measurements (4,26). These findings clearly indicate that functional assays cannot predict which individuals have effective inhibition of platelet TX production in response to aspirin.
One major limitation of the present study is that it was carried out in healthy volunteers. It might be argued that platelet aggregation patterns may be different in patients with vascular disease. However, the practical implications of the 2 major findings of the present study would still apply to both populations. In fact, the variability of the TX-independent component of the different aggregation signals and the instability of the “resistant” phenotype are likely to be amplified, rather than diminished, by cardiovascular diseases. Therefore, establishing the pattern in a normal population constitutes a sound and necessary basis to interpret observations in patients.
Given the requirement for >97% suppression of platelet COX activity to maximally inhibit TX-dependent platelet function, less than adequate suppression caused by noncompliance or pharmacodynamic interactions with some traditional NSAIDs (27,28) is best reflected by serum TXB2.
Based on the average effects of different doses of aspirin, up to 2,600 mg daily, and a selective TX synthase inhibitor, Reilly and FitzGerald (29) previously described the strikingly nonlinear relationship between inhibition of serum TXB2ex vivo and inhibition of TX biosynthesis in vivo and predicted that virtually complete suppression of the biosynthetic capacity of platelets is required to have a measurable impact on TX-dependent platelet function. Our detailed analysis of such a relationship provides direct evidence and kinetic interpretation for this hypothesis.
Inhibition of platelet COX activity, explored both on and off treatment, was nonlinearly related to inhibition of TX-dependent platelet function, leading to faster functional recovery following aspirin withdrawal than predicted by the rate of platelet turnover. Thus, 3 days after stopping aspirin, AA-induced platelet aggregation and VerifyNow Aspirin had recovered approximately 60% and 80% of baseline values, respectively. This finding may have clinical implications for the adequacy of recommended timing of drug withdrawal before surgical/invasive procedures in aspirin-treated patients (30,31). The nonlinearity of the relationship between inhibition of the TX biosynthetic capacity and inhibition of TX-dependent platelet function enables some recovery of platelet function at 48 hours after drug withdrawal, a phenomenon that may be substantial in some subjects because of the interindividual variability in platelet turnover. This may have contributed to the less than expected reduction in vascular complications associated with the alternate-day regimen of aspirin 100 mg used in the Women's Health Study (32). Additional studies in clinical settings characterized by enhanced platelet turnover are clearly warranted.
We conclude that in healthy subjects: 1) platelet COX-1 activity is uniformly and persistently suppressed by low-dose aspirin; 2) this effect is variably detected by platelet function assays, potentially leading to the erroneous diagnosis of aspirin “resistance,” particularly when based on a single determination; and 3) the nonlinearity of the relationship between inhibition of TXA2production and inhibition of platelet function allows rapid recovery of TXA2-dependent activation upon aspirin withdrawal.
The authors thank Daniela Basilico for her expert editorial assistance, Professor Franco Ranelletti for platelet imaging and immunohistochemical support, and Giovanna Petrucci for technical assistance.
Supported by the European Commission FP6 funding (LSHM-CT-2004-005033). Dr. Davì has received research grant support from Bayer, Sanofi-Aventis, and Servier. Dr. Patrono has received research grants from Bayer and Servier and lecture and consulting fees from AstraZeneca, Bayer, Eli-Lilly, Schering-Plough, Sanofi-Aventis, and Servier. Dr. Rocca has received honoraria from Nycomed and Bristol-Myers Squibb. Drs. Santilli and Rocca contributed equally to this work.
- Abbreviations and Acronyms
- arachidonic acid
- adenosine diphosphate
- coefficient of variation
- nonsteroidal anti-inflammatory drug
- maximal aggregation
- Received July 25, 2008.
- Revision received October 8, 2008.
- Accepted October 14, 2008.
- American College of Cardiology Foundation
- Antithrombotic Trialists' Collaboration
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