American Heart Journal
Volume 151, Issue 5 , Pages 976.e7-976.e11, May 2006

Lack of effect of enteric coating on aspirin-induced inhibition of platelet aggregation in healthy volunteers☆

Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, OH

Received 18 August 2005; accepted 5 February 2006.

Article Outline

Background

Aspirin inhibits platelet aggregation and is widely used in the treatment of cardiovascular disease. Some individuals are less responsive to aspirin's antiplatelet effect, a phenomenon termed aspirin resistance. It is not known whether the antiplatelet effect is fully preserved with the enteric-coated (EC) formulation.

Methods

We performed a prospective randomized trial of 50 healthy volunteers using a crossover design to compare the EC with the standard aspirin formulations. The subjects received a 7-day course of each aspirin formulation (81-mg) (Bayer Corporation, Morristown, NJ) separated by a 3-week washout period. Platelet function was measured before and after each course using optical aggregometry (with arachidonic acid and adenosine diphosphate as agonists) and a point-of-care platelet assay.

Results

The assays were reproducible, and the variation in baseline platelet function was small to moderate between the subjects. There was no difference in the extent of platelet inhibition between the EC and standard formulations with any of the 3 assays. With the point-of-care platelet assay, the mean aspirin effect favoring the standard formulation (more aggregation inhibition) compared with the EC formulation was 1.6% ± 15.8% (P = .60 for difference between the formulations). The corresponding optical aggregometry values were −3.4% ± 39.5% (P = .97) and −1.4% ± 16.6% (P = .75) for arachidonic acid and adenosine diphosphate, respectively.

Conclusions

Compared with standard aspirin, EC aspirin appears to exhibit similar inhibition of platelet aggregation in healthy volunteers. Furthermore, point-of-care platelet assessment correlated well with the gold standard of laboratory-based optical platelet aggregometry.

 

Aspirin irreversibly acetylates serine 530 of the platelet cyclooxygenase-1 enzyme and ultimately inhibits the synthesis of thromboxane.1 This results in the inhibition of platelet activation and aggregation, essential steps in the pathophysiology of thrombosis and myocardial infarction.2 Aspirin has been shown in large randomized clinical trials to lower mortality and the risk of myocardial infarction both in primary and secondary prevention of coronary artery disease.3 Individual variation exists in the amount of aspirin's antiplatelet effect, and aspirin resistance, defined as failure of aspirin to inhibit platelet aggregation beyond a selected cutoff as detected by optical aggregometry, has been associated with increased incidence of adverse clinical outcomes.4, 5, 6 One side effect of aspirin therapy is an increased risk of gastrointestinal bleeding, not only mediated in part by aspirin's antiplatelet effect, but also by local effects on the gastric mucosa.7 The enteric-coated (EC) formulation may offset some of aspirin's increased gastrointestinal bleeding risk,8, 9 but it is not known whether the systemic antiplatelet effect is fully preserved with the EC formulation. We sought to determine in the BARE study whether the EC formulation would exhibit impaired platelet inhibition compared with the standard non-EC formulation.

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Methods 

A total of 50 healthy volunteers (25 men) were studied using a prospective, randomized, crossover design to compare the EC with the standard aspirin formulations. The recruitment was accomplished using poster advertising at the Cleveland Clinic Foundation. The inclusion criterion was age >18 years. The exclusion criteria included (1) aspirin, clopidogrel, cilostazol, vitamin E, cyclooxygenase-2 inhibitor, or the use of any nonsteroidal anti-inflammatory agent in the 21 days preceding the study period or during the study period (except for the study drugs); (2) history of hyperlipidemia, hypertension, cardiovascular disease, renal insufficiency, bleeding diatheses, peptic ulcer disease, or recent gastrointestinal bleeding (within 12 months); (3) platelet count <100000/μL or >500000/μL; (4) anemia (hematocrit <36.0 or 33.0 for menstruating women); and (5) positive urinary β-human chorionic gonadotropin screen for pregnancy (performed in female subjects).

The subjects filled out a questionnaire about demographic information, medical history, allergies, and medications (including any over-the-counter medications). Female subjects provided a urine sample for pregnancy testing, and all subjects underwent a measurement of complete blood count. A total of 25 subjects were randomized to start with EC aspirin, and the remaining 25 started with standard aspirin. The randomization of the order of aspirin formulations was carried out using a table of random numbers.

The protocol specified for the following intervention and data collection. First, the subjects underwent a determination of the baseline platelet function (with blood specimen obtained by venipuncture). Subjects randomized to start with EC formulation then received EC aspirin 81 mg (Bayer Corporation, Morristown, NJ) daily for 7 days, followed by a determination of post–aspirin-therapy platelet function. This was followed by a 21-day washout phase during which the subjects were instructed not to take any aspirin products or nonsteroidal anti-inflammatory agents (see the complete list in the previously mentioned exclusion criteria). They then underwent a repeat determination of baseline platelet function. After this, they received standard non-EC aspirin 81 mg (chewable formulation, Bayer Corporation) with instructions to swallow the tablet without chewing it, daily for 7 days, and then had post–aspirin-therapy platelet function determined. The same protocol, with the exception of the reversed order of the 2 formulations, was carried out for the 25 subjects who were randomized to start with the standard formulation. Neither the subjects nor the study clinical coordinator was blinded regarding the aspirin formulation (EC vs standard). However, all of the laboratory personnel who performed the assays were blinded.

The platelet function testing was performed using 3 different assays. The optical aggregometry assays used 10 μmol/L adenosine diphosphate (ADP) (aspirin effect with 10 μmol/L ADP has been observed in the past6) and 0.5 mg/mL arachidonic acid (AA) as agonists and used previously described methods.6 Aggregation was measured at 10 minutes and at peak aggregation and denoted as % aggregation. Also used was the commercially available point-of-care VerifyNow Aspirin Assay (Accumetrics, Inc, San Diego, CA), which has been used to test platelet function10, 11 and has been approved by the Food and Drug Administration. It is a turbidimetric-based optical detection system, which uses AA as the agonist and measures platelet aggregation as an increase in light transmittance. The platelet aggregation is reported in aspirin resistance units (ARUs) with values ≥550 ARUs corresponding to aspirin resistance. Response to aspirin in this study was defined as >60 ARUs decrease after the aspirin course (an arbitrarily selected value approximating 10% of the baseline ARU value). To assess intraindividual variation, a repeat measurement (with a separate blood sample) was performed for each subject and for each aspirin formulation using all 3 assays (for the VerifyNow assay, the repeat sample was performed only with the 2 baseline measurements).

The subjects were encouraged to report any side effects (including bleeding complications) or protocol violations during the 2 study phases or during the washout period. All subjects participated after providing informed consent. The study protocol and the consent form were approved by the Cleveland Clinic Foundation Institutional Review Board.

The study design allowed each subject to serve as his or her own control. The magnitude of the differential platelet aggregation inhibition using each assay was assessed for each subject: aspirin platelet effect = 100% × (aggregation before aspirin − aggregation after aspirin)/aggregation before aspirin. The aspirin platelet effect was compared with the standard and the EC groups using the Wilcoxon rank sum test. A P of .05 value was used as the cutoff for statistical significance.

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Results 

A total of 52 volunteers were evaluated, and 2 were excluded because of histories of hypertension and hyperlipidemia, respectively. The mean age ± SD of the 50 study subjects (25 men) was 35.7 ± 7.0 years. There were 4 instances of protocol violations, each representing a missed dose of daily aspirin, and each resulting in the aspirin course being extended so as to end with 7 consecutive days of aspirin therapy.

A number of the following descriptive results are reported with outliers excluded, but similar results were obtained when the analysis included all data. The mean baseline value of the point-of-care platelet assay was 653.5 ± 11.4 ARUs (n = 93). Including the 7 outliers, the mean equaled 640.3 ± 52.6 ARUs (n = 100), thus setting the reference range lower limit (2 SDs below the mean) at 536 ARUs. The point-of-care platelet assay exhibited a high degree of reproducibility: 94 (95.9%) of the 98 baseline recording pairs were within 30 ARUs (4.6% of 653.5) of each other (2 subjects did not have a repeat measurement). The mean difference within the pairs was 7.8 ± 8.0 ARUs (n = 95). The mean baseline optical aggregometry (maximal) values were 80.9% ± 8.6% aggregation (n = 93) and 83.9% ± 8.9% aggregation (n = 97) for AA and ADP, respectively. The optical aggregometry assays using AA and ADP also exhibited a high degree of reproducibility with 330 (82.9%) of 398 and 309 (77.3%) of 400 of the pairs within 5% aggregation of each other, respectively. The mean difference among the pairs was 2.8% ± 3.2% (n = 398) and 3.8% ± 3.6% (n = 400) for AA and ADP, respectively. In comparing the point-of-care platelet assay across all data points (Figure 1) with the optical aggregometry (maximal inhibition using AA), the Spearman rank correlation ρ equaled 0.74 (P < .0001, n = 200).

  • View full-size image.
  • Figure 1. 

    Regression plot for all data points for the point-of-care platelet assay (ARU) on the y-axis and optical aggregometry with AA (maximal inhibition, % aggregation) on the x-axis. The two clusters correspond to values before and after aspirin treatment.

Using the point-of-care platelet assay, most subjects did respond to aspirin, as evidenced by 96% (n = 100) of the aspirin regimens having a >60 ARU reduction after the aspirin course (baseline mean − posttherapy value). The mean aspirin-induced reduction was 213.9 ± 78.1 ARUs (n = 100), and excluding the 4 nonresponders, it was 223.5 ± 62.6 ARUs (n = 96), representing large variation in the size of the effect. Using optical aggregometry with AA, the mean aspirin-induced percentage of inhibition was 66.0% ± 18.6% (n = 100). The mean of the mean postaspirin maximal aggregation was 10.4% ± 6.5% (n = 100), indicating small variation in the size of the effect. Aggregometry with ADP was less potent with the mean aspirin-induced percent inhibition of 18.8% ± 10.4% (n = 100). The mean of the mean postaspirin maximal aggregation was 63.9% ± 12.1% (n = 100), corresponding to moderate variation in the size of the effect.

There was no difference in the primary end point of the study, that is, the extent of platelet inhibition between the EC and standard formulations. The mean aspirin effect favoring the standard formulation (more aggregation inhibition) compared with the EC formulation using the point-of-care platelet assay was 1.6% ± 15.8% (n = 50, P = .60), using the Wilcoxon rank sum test. This corresponded to a mean absolute difference of 9.5 ± 94.9 ARUs (n = 50, P = .56). A total of 9 subjects had at least 1 outlying baseline point-of-care platelet assay value, and if these subjects were excluded from the analysis, the corresponding values were 0.55% ± 10.9% (n = 41, P = .60) and 4.6 ± 70.6 ARUs (n = 41, P = .56) (Figure 2). From the evaluation of the 41 subjects with no outlying baseline values individually, and using an arbitrary cutoff value of the relative difference of ≥5%, 12 subjects exhibited impaired platelet inhibition with the EC formulation. However, 11 subjects exhibited augmented platelet inhibition with the EC formulation.

  • View full-size image.
  • Figure 2. 

    Enteric-coated and standard aspirin effect on platelet inhibition as measured by the point-of-care platelet assay. Open circles indicate individual subjects. Adjacent to them are the mean and SD values. BL, Baseline; STD, standard.

A similar pattern was evident with the optical aggregometry data (Figure 3). Using AA as agonist, the mean aspirin effect favoring the standard formulation (more aggregation inhibition) compared with the EC formulation was −3.4% ± 39.5% (n = 50, P = .97). Using ADP as agonist, the mean aspirin effect favoring the standard formulation (more aggregation inhibition) compared with the EC formulation was −1.4% ± 16.6% (n = 50, P = .75).

  • View full-size image.
  • Figure 3. 

    Enteric-coated and standard aspirin effect on platelet inhibition as measured by optical aggregometry. Open circles indicate individual subjects. Adjacent to them are the mean and SD values.

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Discussion 

The major finding of this study is that EC aspirin, at a daily dose of 81 mg in healthy volunteers, appears to inhibit platelet aggregation equally compared with the standard non-EC aspirin. The mean effect of the enteric coating across all subjects was very close to zero with each of the assays, and the pattern of the effect in individual subjects was balanced in a manner, suggesting that any variation is due to the play of chance and not to a true biologic effect.

This study also revealed that the point-of-care bedside platelet assay and the optical aggregometry assays using AA and ADP exhibited a high degree of reproducibility. The variation of the baseline point-of-care platelet assay platelet-function measurements between the subjects was low, with optical aggregometry assays exhibiting moderate variation. Most subjects responded to aspirin, but the size of the effect varied between subjects. The variation in the size of this effect was different based on the assay (point-of-care platelet assay > ADP > AA). It is not clear what this difference reflects. This study establishes the point-of-care platelet assay reference range for healthy volunteers with the lower cutoff at 536 ARUs.

Because aspirin is one of the most commonly used medications in the world, it is interesting that the appropriate dose has been controversial, and the effect of enteric coating has been largely unclear.12 We selected the dose of 81 mg for this study because of a recent trend in clinical practice to use 81 mg in primary and secondary prevention of coronary artery disease.13, 14 Prior work has shown that a daily aspirin dose of 30 mg maximally inhibits thromboxane synthesis, and doses of 81 and 325 mg inhibit platelet aggregation equally.1, 2, 15 It has also been shown that individual variation exists in the amount of aspirin's antiplatelet properties, affecting clinical outcomes.6, 11, 16, 17 However, it had not been known whether the enteric coating of the aspirin tablet affects its antiplatelet activity. This is an important consideration in the selection of the aspirin formulation given that the EC formulation may offset some of aspirin's increased gastrointestinal bleeding risk.8, 9, 18, 19, 20, 21

This study guides the selection of the aspirin formulation by establishing that among healthy volunteers, the antiplatelet properties of EC aspirin are similar to those of standard aspirin. Given that the EC formulation may reduce the gastrointestinal bleeding risk and may improve gastric tolerability associated with aspirin use, consideration should be given to using EC aspirin preferentially in chronic therapy.

Limitations 

The compliance of the subjects regarding the study drugs and the prohibited medications was assessed by self-reporting only with no measurement of salicylate levels. We therefore cannot fully exclude the possibility that noncompliance accounts for some of the results, especially in regard to the outlying values. Furthermore, this sample consisted of healthy volunteers, and whether the results would apply to patients with cardiovascular disease or with impaired gastric absorption is not addressed by this investigation, although such studies are ongoing.22 This study does not examine aspirin doses higher than 81 mg, and the possibility that enteric coating may alter platelet inhibition at higher aspirin doses cannot be excluded based on this study. Given the small sample size and the resultant limited power, we cannot exclude the possibility of a type 2 error.

Conclusions 

Compared with standard non-EC aspirin, EC aspirin seems to exhibit similar inhibition of platelet aggregation in healthy volunteers. Use of point-of-care platelet assays instead of optical platelet aggregometry may allow easier testing of aspirin's antiplatelet effects in the future, both for research and potentially clinical indications.

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References 

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☆ This study was funded by the Cleveland Clinic Foundation Research Programs Council and the Department of Cardiovascular Medicine. The VerifyNow assays were donated without charge by Accumetrics, Inc, San Diego, CA.

PII: S0002-8703(06)00140-2

doi:10.1016/j.ahj.2006.02.017

American Heart Journal
Volume 151, Issue 5 , Pages 976.e7-976.e11, May 2006

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