Treatment of Aspirin-Resistant Patients With Omega-3 Fatty Acids Versus Aspirin Dose Escalation

Patients

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).

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Figure 1

Algorithm of the Study

We aimed to identify 30 patients with aspirin resistance. Patients with stable coronary artery disease (CAD) (n = 485) were screened for resistance by the VerifyNow Aspirin assay (Accumetrics). Sixty-two patients (12.8%) had an aspirin reaction unit (ARU) score >500 and underwent further testing by platelet aggregation. Thirty patients (6.2%) were found to be resistant and were randomized to 1 of the 2 treatment groups. They were re-tested after 30 days of treatment. f/u = follow-up.

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.

Follow-up phase

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.

Blood sampling

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.

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.

Statistical analysis

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.

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).

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Figure 2

Plasma TXB2 Levels at Baseline and After 30 Days of Treatment

Plasma thromboxane B2 (TXB2) levels at baseline and after 30 days of treatment with omega-3 fatty acids (omega group) or 325 mg of aspirin (aspirin group). In both groups a significant reduction was noted from baseline after 30 days of treatment (57% relative reduction with p = 0.02 in the omega group, and 40% relative reduction with a p = 0.045 in the aspirin group). There were no significant differences between the groups at baseline or after treatment.

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Figure 3

Comparison of the Relative Reductions From Baseline to Post-Treatment in the 2 Groups

Comparison of the magnitude of relative reductions from baseline to post-treatment in platelet aggregation (Aggr) and the VerifyNow Aspirin assay (Accumetrics) score between the omega group and the aspirin group. There were no significant differences between the 2 groups, except for a tendency for greater reduction in 10-μmol/l adenosine diphosphate (ADP)-induced platelet aggregation in the omega group (p = 0.06). AA = arachidonic acid.

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.

Safety

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.

Follow-up phase

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).

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Figure 4

Platelet Aggregation at Baseline, After Treatment, and During Follow-Up

Platelet aggregation in response to arachidonic acid (AA) and adenosine diphosphate (ADP) in the omega and aspirin groups (12 patients in each group) at 3 time points: (A)baseline—low-dose aspirin (solid bars); (B)after 30 days of treatment with the corresponding regimen—omega-3 fatty acids or high-dose aspirin (gray bars); and (C)low-dose aspirin—after discontinuing the corresponding treatment regimens for at least 1 week (open bars—follow-up phase). In both groups, aggregation decreased after treatment and increased back to levels similar to the baseline levels after discontinuing the treatment regimens (p values for Bvs. Ctime points).

Control group

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).

Aspirin-resistance rate

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.

Study limitations

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.