Author + information
- Received June 12, 2009
- Accepted August 10, 2009
- Published online January 12, 2010.
- Eli I. Lev, MD*,†,* ( )(, )
- Alejandro Solodky, MD*,†,
- Naama Harel*,
- Aviv Mager, MD*,†,
- David Brosh, MD*,†,
- Abid Assali, MD*,†,
- Milton Roller, MD*,†,
- Alexander Battler, MD*,†,
- Neal S. Kleiman, MD‡ and
- Ran Kornowski, MD*,†
- ↵*Reprint requests and correspondence:
Dr. Eli I. Lev, Rabin Medical Center, The Sackler Faculty of Medicine, 39 Jabotinsky Street, 49100 Petah Tikva, Israel
Objectives The aim of this study was to evaluate whether addition of omega-3 fatty acids or increase in aspirin dose improves response to low-dose aspirin among patients who are aspirin resistant.
Background Low response to aspirin has been associated with adverse cardiovascular events. However, there is no established therapeutic approach to overcome aspirin resistance. Omega-3 fatty acids decrease the availability of platelet arachidonic acid (AA) and indirectly thromboxane A2 formation.
Methods Patients (n = 485) with stable coronary artery disease taking low-dose aspirin (75 to 162 mg) for at least 1 week were screened for aspirin response with the VerifyNow Aspirin assay (Accumetrics, San Diego, California). Further testing was performed by platelet aggregation. Aspirin resistance was defined by ≥2 of 3 criteria: VerifyNow score ≥550, 0.5-mg/ml AA-induced aggregation ≥20%, and 10-μmol/l adenosine diphosphate (ADP)-induced aggregation ≥70%. Thirty patients (6.2%) were found to be aspirin resistant and randomized to receive either low-dose aspirin + omega-3 fatty acids (4 capsules daily) or aspirin 325 mg daily. After 30 days of treatment patients were re-tested.
Results Both groups (n = 15 each) had similar clinical characteristics. After treatment significant reductions in AA- and ADP-induced aggregation and the VerifyNow score were observed in both groups. Plasma levels of thromboxane B2 were also reduced in both groups (56.8% reduction in the omega-3 fatty acids group, and 39.6% decrease in the aspirin group). Twelve patients (80%) who received omega-3 fatty acids and 11 patients (73%) who received aspirin 325 mg were no longer aspirin resistant after treatment.
Conclusions Treatment of aspirin-resistant patients by adding omega-3 fatty acids or increasing the aspirin dose seems to improve response to aspirin and effectively reduces platelet reactivity.
In recent years it has become apparent that the biologic response to aspirin therapy is not uniform; rather, there is wide interindividual variability in the response to aspirin (1,2). The prevalence of low response or “resistance” to the antiplatelet effects of aspirin is estimated to be <1% to 45%, depending on the assay used, cutoff value chosen, and population tested (3–8). Low response to aspirin has been associated with adverse clinical outcomes in patients with cardiovascular disease or cardiovascular risk factors (2,4,7,9) and with an increase in markers of myonecrosis after percutaneous coronary intervention (PCI) (5,6). Furthermore, 2 recent meta-analyses encompassing 15 to 20 studies and totaling almost 3,000 patients showed that aspirin resistance was associated with an odds ratio of almost 4 for development of cardiovascular events (10,11). Despite the emerging clinical evidence, there is no established treatment paradigm for the management of patients who are resistant to the effects of aspirin.
One potential approach to manage aspirin resistance is to increase the drug dose. Most patients receiving long-term aspirin therapy after an acute myocardial infarction (MI) or PCI are treated with low-dose aspirin (75 to 162 mg/day). Therefore, resistant patients could potentially be treated by increasing the dose to 325 mg/day. However, the Antithrombotic Trialists' Collaboration meta-analysis has shown that there is no difference in clinical efficacy for prevention of vascular events when comparing aspirin doses of 75 to 325 mg and even higher doses (12). Conversely, Lee et al. (13) have shown that, among patients with stable coronary artery disease (CAD), low aspirin dose (80 to 100 mg) was associated with the highest prevalence of resistance compared with higher doses. Furthermore, Gurbel et al. (14) reported dose-dependent effects of aspirin on platelet function when using assays not based on arachidonic acid (AA). It is not known, however, whether increasing the dose in resistant patients taking low-dose aspirin would improve the response to the drug.
Another possible approach to the management of aspirin resistance is to add an additional substance that would inhibit the cyclooxygenase (COX)-1 pathway. Omega-3 fatty acids, which are components of dietary fish oil, might fulfill this role. Ingestion of fish oils leads to replacement of n-6 fatty acids (such as AA) by n-3 fatty acids in the cell membranes of various cells (15,16). In platelets this process leads to a reduction in the availability of platelet membrane AA, along with an increase in eicosapentaenoic acid (EPA) (15). The latter substance can compete with AA as a substrate of the COX-1 pathway. The net effect is an increase in the ratio of EPA to AA and indirectly a reduction in thromboxane A2 formation from AA (15,16). Studies of antiplatelet effects of fish oil, however, have been inconsistent (16,17). In patients naïve to aspirin, a daily dose of 3 to 4 g omega-3 fatty acids given over several weeks seems to have mild antiplatelet effects, such as weak inhibition of collagen-induced platelet aggregation and mild prolongation of bleeding time (16–18).
Recently Larson et al. (19) reported that omega-3 fatty acids alone had no significant effect on platelet aggregation but when combined with aspirin had a synergistic inhibitory effect. It should be noted that addition of omega-3 fatty acids (1 to 4 g/day) to aspirin in patients with cardiovascular disease has not been associated with an increased risk of bleeding complications (20,21). Given the mechanism of action of omega-3 fatty acids and the report by Larson et al. (19), we hypothesized that omega-3 fatty acids might act in synergy with aspirin among patients with low response to aspirin. Therefore, our aim was to evaluate whether adding omega-3 fatty acids or increasing the aspirin dose would improve the response to low-dose aspirin among patients who are aspirin resistant.
Stable patients with CAD, ages 18 to 80 years, who have been treated with aspirin at a dose of 75 to 162 mg for at least 1 week were included. Exclusion criteria were PCI or an acute coronary syndrome within 1 month of enrollment; treatment with glycoprotein IIb/IIIa inhibitors or thrombolytic agents within 1 month of enrollment; concomitant treatment with omega-3 fatty acid-containing products; concomitant treatment with ibuprofen, naproxene, or warfarin; thrombocytopenia (<100 × 103 cells/mm3); anemia (hemoglobin <10 g/dl); or renal insufficiency (creatinine >2.5 mg/dl). The study was approved by the Investigational Review Board (Helsinki committee) of the Rabin Medical Center, Israel, and all subjects provided written informed consent.
Screening for aspirin resistance
The VerifyNow Aspirin assay (Accumetrics, San Diego, California) was used for the purpose of screening for aspirin resistance. This assay is a point-of care, turbidometric-based system that uses citrate-anticoagulated blood and cartridges containing fibrinogen-coated beads and AA as the platelet agonist. Results are expressed as aspirin reaction units (ARU). According to the manufacturer's recommendation, a cutoff of ≥550 ARU indicates resistance to the antiplatelet effects of aspirin. However, from our previous cohort of 150 patients (5), a cutoff level of 550 ARU had a sensitivity of 70% and specificity of 90% for identification of aspirin resistance when compared with AA-induced platelet aggregation. Therefore we used a cutoff level of 500 ARU for screening, which was associated with sensitivity of 90% and specificity of 75% in our previous cohort. Patients with an ARU >500 underwent further testing by turbidimetric platelet aggregation in response to AA and adenosine diphosphate (ADP) (Fig. 1).Our aim was to identify 30 patients with aspirin resistance (Fig. 1).
Definition of aspirin resistance
Aspirin resistance was defined by the presence of at least 2 of the following 3 criteria: 0.5-mg/ml AA-induced platelet aggregation ≥20%, 10-μmol/l ADP-induced aggregation ≥70%, and VerifyNow ARU ≥550 (in contrast to the screening process in which an ARU >500 was used as the cutoff) (5). In case of concomitant treatment with clopidogrel (approximately 5% of the patients screened) aspirin resistance was defined on the basis of 2 of 2 criteria (AA-induced aggregation and VerifyNow Aspirin).
Randomization of aspirin-resistant patients
Patients found to be aspirin resistant were randomized 1:1 into 2 groups (n = 15 each): 1) treatment with the original dose of aspirin and, in addition, omega-3 fatty acids—4 capsules/day taken 2 in the morning and 2 in the evening, each capsule containing 360 mg EPA and 240 mg DHA (Omega Max 3, SupHerb, Israel), for a period of 30 days; and 2) treatment with aspirin 325 mg daily for a period of 30 days.
In addition, a control group of 15 patients was randomly selected from the patients with an ARU >500 who did not fulfill the criteria for aspirin resistance (Fig. 1). These patients continued to receive the same treatment (including the original aspirin dose), without change. The 2 study groups and control group underwent testing for response to aspirin after 30 days of treatment with the corresponding regimen.
All 30 patients included in the 2 treatment groups were invited to participate in a follow-up phase performed approximately 1 year (13 ± 4 months) after the 30-day treatment period. Patients were tested at conditions identical to the first baseline sample, after discontinuing the corresponding treatment (omega-3 fatty acids supplementation or high-dose aspirin) and reverting back to the original low-dose aspirin. The purpose of this phase was to examine the stability of aspirin response at the baseline conditions. Some of the patients were treated with low-dose aspirin when contacted; others were asked to take low-dose aspirin (without omega-3 fatty acids) for a period of at least 1 week before testing. A total of 24 patients (12 in each group) participated in the follow-up phase. The other 6 patients were not available or interested in participating or had significant changes in their medical management.
All blood samples were obtained from an antecubital vein, with a 21-gauge needle, and collected in tubes containing 3.2% citrate. The tubes were filled to capacity and gently mixed. The blood samples were processed within 2 h of blood collection.
Evaluation of response to aspirin
In addition to the VerifyNow Aspirin assay, the following tests were performed at all time points.
Light Transmittance (Turbidimetric) Platelet Aggregation
Light transmittance (turbidimetric) platelet aggregation was performed in platelet-rich plasma with a platelet count adjusted to approximately 250 × 103/mm3. Platelets were stimulated with 0.5 mg/ml (1.6 mmol/l) AA and 5- and 10-μmol/l ADP. Aggregation was performed with a BioData PAP-4 platelet aggregometer (BioData, Horsham, Pennsylvania). The extent of aggregation was defined as the maximal light transmission ≤6 min after addition of the agonist, with platelet-poor plasma used as reference.
Plasma Levels of Thromboxane B2 (TXB2)
Plasma levels of TXB2 were determined from frozen plasma samples (kept at −70°C until testing), with an enzyme immunoassay, according to the manufacturer's instructions (Cayman Chemical Co., Ann Arbor, Michigan).
Compliance at the initial interview
Compliance to aspirin (taken ≥1 week) was verified separately by the patient's attending physician, study nurse, and study physician. In cases of uncertainty, patients were not enrolled. Two patients with an ARU >500 had an exceptionally high level of AA-induced aggregation (>40%) and were asked to be re-tested after 1 week of aspirin treatment (1 patient became responsive and was withdrawn from the study; the other remained resistant but with lower levels of AA-induced aggregation than originally measured). In the 2 study groups, compliance to the specific regimen was verified by phone call after 2 weeks of treatment.
Continuous variables are presented as mean ± SD. Comparisons between aspirin-resistant and responsive patients (Table 1)were performed by unpaired Student ttests and chi-square tests for continuous and categorical variables, respectively. Intragroup comparisons (between the 2 time points) were performed by paired Student ttests. Intergroup comparisons (between the 2 study groups) of the magnitude of change induced by the specific treatment were performed by 2-factor analysis of variance with repeated measures (for group- and time-dependence). Analyses were performed with SPSS version 11.0 statistical software (SPSS, Inc., Chicago, Illinois), and statistical significance was set at p < 0.05.
To achieve the pre-specified goal of 30 patients with aspirin resistance (as defined by ≥2 of 3 criteria), screening of 485 patients was required (aspirin resistance rate of 6.2%) (Fig. 1). Patients with aspirin resistance had similar clinical characteristics to those of aspirin-responsive patients, except for a higher proportion of women among resistant patients (33.3% vs. 16%, p = 0.02) (Table 1). Patients with aspirin resistance were also treated with similar medications to aspirin-responsive patients (Table 1). It should be noted that concomitant clopidogrel treatment was not contraindicated. Among the 22 patients treated with clopidogrel, 2 had an ARU >500, but neither satisfied any criteria for aspirin resistance and therefore did not enter the study groups (they were also not included in the control group).
Platelet reactivity and response to aspirin in the study groups
The 30 patients with aspirin resistance were randomized to receive either omega-3 fatty acids (omega group, n = 15) or aspirin 325 mg (aspirin group, n = 15) for a period of 30 days. Clinical characteristics and concomitant medical treatment were similar among the 2 groups and did not significantly differ from the characteristics of the total aspirin-resistant cohort (presented in Table 1). In both the omega and aspirin groups, significant reductions in platelet aggregation and the VerifyNow Aspirin score were observed after 30 days of treatment (Table 2).Plasma levels of TXB2 were also reduced in both groups after 30 days of treatment (57% relative reduction in TXB2 levels in the omega group, and a 40% decrease in the aspirin group) (Fig. 2).Comparison of the magnitude of reductions in platelet reactivity and TXB2 levels between the 2 groups revealed that there were no significant intergroup differences, except for a trend for difference in 10-μmol/l ADP-induced platelet aggregation, with greater reduction in the omega group (relative reduction of 14.9% in the omega group vs. 6.9% in the aspirin group, p = 0.06) (Fig. 3).
Rates of aspirin resistance after treatment
After 30 days of treatment with omega-3 fatty acids, 3 patients (20%) remained resistant to aspirin (≥2 of 3 criteria), and 4 other patients fulfilled only 1 criterion for aspirin resistance (3 of them had ADP-induced aggregation ≥70%, and 1 had AA-induced platelet aggregation ≥20%). In the aspirin group after 30 days of treatment with aspirin 325 mg, 4 patients (26.7%) were still resistant to the effects of aspirin, and 5 other patients fulfilled 1 criterion for aspirin resistance (all had ADP-induced aggregation ≥70%). Thus, 80% of the patients in the omega group and 73% of the patients in the aspirin group were no longer resistant to aspirin, according to the definition employed in our study.
During the 30-day treatment period no bleeding episodes were reported in either group. One patient treated with omega-3 fatty acids reported belching for a few days that subsided gradually and did not require discontinuation of the drug.
Twenty-four patients (12 in each group) participated in this phase—performed approximately 1 year after the treatment period. As shown in Figure 4,in both groups platelet aggregation in response to AA and ADP increased significantly after discontinuation of the corresponding treatment regimen and returned to levels similar to the baseline levels (in both groups there were no significant differences between the baseline and follow-up levels in any of the aggregation assays). Similar findings were noted with the VerifyNow Aspirin assay (data not shown).
The control group consisted of 15 patients who had a VerifyNow ARU >500 but did not satisfy criteria for aspirin resistance. The purpose of the control group was to examine whether there is significant variability in response to aspirin over a 30-day period, without any change in medical treatment. In contrast to the study groups, there were no significant differences in platelet aggregation or VerifyNow score between the baseline and 30-day measurements in the control group (Table 3).
To our knowledge, this is the first study that examined potential therapeutic approaches to manage resistance to the antiplatelet effects of low-dose aspirin. Two approaches were evaluated: addition of omega-3 fatty acids or aspirin dose escalation (to 325 mg daily). We identified 30 patients with stable CAD who were aspirin resistant, on the basis of AA and ADP-induced platelet aggregation and the VerifyNow Aspirin assay criteria. These patients were randomized to receive 1 of the 2 treatment protocols for 30 days. Both therapeutic approaches were associated with a reduction in platelet aggregation, VerifyNow Aspirin score, and plasma TXB2 levels and therefore with improved response to aspirin. In addition, the majority of patients treated by both treatment protocols were no longer resistant to aspirin, according to the criteria employed in our study.
The prevalence of aspirin resistance found in our study (6.2%) is similar to that reported by Gum et al. (4) in patients with stable cardiovascular disease (5.2%). Both studies employed similar criteria for definition of aspirin resistance, although we also incorporated the VerifyNow Aspirin assay. Previous studies that reported higher rates of aspirin resistance (12% to 30%) studied somewhat different patient populations, such as patients undergoing PCI (5,6), and/or used different criteria for aspirin resistance (e.g., only the VerifyNow Aspirin assay) (6,7). Lower rates of aspirin resistance (<1% to 6%) were reported when using only AA-induced platelet aggregation as the criteria for resistance (14) and/or testing the effects of 325 mg aspirin (8). Our finding of a higher proportion of women among aspirin-resistant patients, compared with aspirin-responsive patients, is consistent with previous studies (4,6,13). Higher rates of aspirin resistance among women might contribute to the recently reported failure of aspirin to reduce the risk of a first MI in women, in contrast to its beneficial primary prevention effects in men (22).
Aspirin dose effect
Considerable research has been dedicated to study the dose-effect relationship of aspirin. In the Antithrombotic Trialists' Collaboration, a meta-analysis that included more than 200,000 patients, low-dose aspirin was associated with proportional risk reduction of vascular events similar to that of high-dose aspirin but with fewer gastrointestinal side effects and less bleeding (12). Furthermore, previous studies have shown that aspirin at low daily doses of 50 to 100 mg is an effective antithrombotic agent that generally causes efficient inhibition of COX-1 as demonstrated by ≥95% inhibition of serum TXB2 (23,24). However, Hart et al. (25) reported an incremental reduction in serum TXB2 levels with increasing doses of aspirin (81, 325, or 1,300 mg). In the ASPECT (Aspirin Induced Platelet Effect) study, Gurbel et al. (14) reported that dose-dependent effects of aspirin (81, 162, and 325 mg) were assay-dependent and were observed when using assays such as collagen- and ADP-induced platelet aggregation, PFA-100, and urinary 11-dehydroTXB2 but not AA-based assays. Therefore, although these studies indicate aspirin dose-response with respect to TXB2 synthesis (14,25), there does not seem to be a clear and consistent dose-effect relationship for aspirin, as assessed by platelet inhibition or clinical outcomes. This is in apparent disagreement with our study, which showed improved response to aspirin when increasing the dose from a low dose (75 to 162 mg) to 325 mg. However, in contrast to previous studies, we examined the effect of dose escalation only in patients with aspirin resistance, which might be a unique state. Lee et al. (13) have shown that low aspirin doses (≤100 mg) were associated with an increased prevalence of aspirin resistance compared with higher doses. Although the mechanisms for aspirin resistance are probably multiple and have not been well-established (24,26), in some cases there might be a contribution of reduced bioavailability of aspirin (e.g., reduced absorption) (26) or of increased platelet turnover and platelet hyper-reactivity (24,26,27). In both circumstances—reduced drug bioavailability, and increased platelet turnover—an increase in the dose of aspirin might be associated with better inhibition of its target—platelet COX-1.
Effect of omega-3 fatty acids on response to aspirin
Treatment of aspirin-resistant patients with omega-3 fatty acid supplementation was associated in our study with improved response to aspirin and reduced platelet reactivity, at least as effective as that achieved by increasing the aspirin dose. The mechanism of action probably involves a reduction in the availability of platelet membrane AA, along with an increase in EPA, which can compete with AA as a substrate of the COX-1 pathway (15,16). The increased ratio of EPA to AA likely leads to a reduction in thromboxane A2 formation from AA (15,16). Indeed, we have observed a 56.8% relative reduction in plasma TXB2 levels when omega-3 fatty acids were added to low-dose aspirin for 30 days. These results are supported by the study of Engström et al. (28), who showed that in 4 healthy volunteers addition of fish oil to low-dose aspirin augmented the inhibition of serum TXB2 by aspirin. Larson et al. (19) have also reported that omega-3 fatty acids enhance the antiplatelet effects of aspirin (measured by platelet aggregation) in healthy volunteers. We extended these findings to patients with aspirin resistance. It should be emphasized that the addition of omega-3 fatty acids (1 to 4 g/day) to aspirin in cardiovascular patients has not been associated with an increased risk of bleeding (20,21). Furthermore, omega-3 fatty-acid supplementation might have favorable effects in the secondary prevention of CAD and its complications (21,29,30). Taken together, the clinical and platelet function studies suggest that omega-3 fatty-acid supplementation might be a promising approach to optimize response to aspirin.
First, although we attempted to verify compliance to aspirin and the treatment regimens before and during the study, by several measures, we cannot absolutely rule out inadequate compliance. Second, unlike the 2 study groups, the control group did not consist of patients with aspirin resistance but rather patients with a VerifyNow Aspirin score >500 ARU who did not satisfy aspirin resistance criteria. Therefore, it is not a “classic” matched control group. However, it does indicate that in patients with stable CAD treated with low-dose aspirin there were no significant changes in the measured platelet indexes over a 30-day period. Therefore, the reductions in platelet reactivity and TXB2 levels we observed in the study groups are likely not the result of “spontaneous” fluctuations over time but are related to the effects of the therapeutic regimens given. Finally, the study groups are of relatively limited size for inter-group comparisons.
Conclusions and Clinical Implications
There is accruing evidence that resistance to the antiplatelet effects of aspirin is associated with an increased risk of adverse cardiovascular events (2,4–7,9–11). However, currently there is no established therapeutic approach to manage and overcome aspirin resistance in patients treated with low dose of the drug. We have demonstrated by using various platelet assays that either adding omega-3 fatty acids or increasing the aspirin dose can improve response to aspirin and reduce residual platelet reactivity in stable patients with CAD. Treatment with both regimens was associated with a reduction in aspirin-resistance rates. We did not examine the clinical impact of such therapeutic interventions or the impact of routine monitoring of aspirin response. Further larger studies are required to assess whether our findings can be translated to a clinical benefit in patients with cardiovascular disease found to be resistant to aspirin and treated with the therapeutic regimens tested in our study.
- Abbreviations and Acronyms
- arachidonic acid
- adenosine diphosphate
- aspirin reaction units
- coronary artery disease
- eicosapentaenoic acid
- myocardial infarction
- percutaneous coronary intervention
- thromboxane B2
- Received June 12, 2009.
- Accepted August 10, 2009.
- American College of Cardiology Foundation
- Eikelboom J.W.,
- Hirsh J.,
- Weitz J.I.,
- Johnston M.,
- Yi Q.,
- Yusuf S.
- Gum P.A.,
- Kottke-Marchant K.,
- Welsh P.A.,
- White J.,
- Topol E.J.
- Chen W.H.,
- Lee P.Y.,
- Ng W.,
- Tse H.F.,
- Lau C.P.
- Tantry U.S.,
- Bliden K.P.,
- Gurbel P.A.
- Eikelboom J.W.,
- Hankey G.J.,
- Thom J.,
- et al.
- Krasopoulos G.,
- Brister S.J.,
- Beattie W.S.,
- Buchanan M.R.
- Antithrombotic Trialists' Collaboration
- Gurbel P.A.,
- Bliden K.P.,
- DiChiara J.,
- et al.
- Knapp H.R.
- Mori T.A.,
- Beilin L.J.,
- Burke V.,
- Morris J.,
- Ritchie J.
- Maree A.O.,
- Fitzgerald D.J.
- Gasparyan A.Y.,
- Watson T.,
- Lip G.Y.