Time-Dependent Effects of Aspirin on Blood Pressure and Morning Platelet Reactivity: A Randomized Cross-Over Trial

An overview of the study design is depicted in Figure 1. A prospective, randomized, open-label, blinded end point (PROBE), 2-period crossover study was conducted at a single center in the Netherlands and registered at www.clinicaltrials.gov/ct2/show/NCT01379079. Benefits of the PROBE design and its validity for studies measuring ambulatory blood pressure have been previously documented.²² The study was conducted in accordance with the Declaration of Helsinki, approved by the Leiden University Medical Center (LUMC) Ethics Committee, and all subjects gave written informed consent.

Patients between 18 and 75 years of age using low-dose (80–100 mg) aspirin for secondary prevention of CVD were recruited from general practitioner practices around Leiden, the Netherlands. Exclusion criteria were baseline blood pressure (BP) <120/70 or >160/100 mm Hg, use of other antiplatelet or anticoagulant drugs, change of antihypertensive medication in the 3 months before baseline, use of nonsteroidal anti-inflammatory drugs, employment as shift worker, evidence of secondary arterial hypertension (eg, pheochromocytoma), and pregnancy.

Randomization was performed with a computer-generated randomization code by an independent person at the Department of Clinical Epidemiology of the LUMC and was inaccessible to the investigators. Eligible subjects were randomized (1:1 ratio) to take aspirin on awakening followed by aspirin at bedtime or the opposite order during 2 intervention periods of 3 months (Figure 1). The 2 intervention periods were not separated by a wash-out period because withholding aspirin to the included patients was considered unethical. The duration of each intervention period was analogous to previous studies.^9–11 All subjects received 100 mg effervescent aspirin (Carbasalate Calcium, Vemeda Manufacturing, the Netherlands). At the end of each intervention period, subjects visited the research site for 2 consecutive days. At day 1, 24-hour ambulatory blood pressure measurement (ABPM) was started between 8 to 12 AM, and subjects took aspirin at the same time as in the preceding 3 months. At day 2, subjects refrained from taking aspirin in the morning, ABPM was ended, and blood was drawn. The time of ABPM start at day 1 and blood draw at day 2 was similar for each participant at each visit.

To detect an interindividual difference of 3 mm Hg in blood pressure with 80% power at a 5% significance level, we calculated a required sample size of 250 patients. We assumed an intraindividual standard deviation of 12.9 mm Hg, as derived from a previous study.¹² Estimating a drop-out of 10% and invalid ABPM of 5%, we randomized 290 subjects. As planned on beforehand, platelet reactivity was measured in the first consecutive 160 patients, yielding a power of 90% to detect a difference of 17 ARU at a 5% significance level. For this calculation, we used an intraindividual standard deviation of 46.85 ARU.²⁴ Continuous characteristics are described as mean±standard deviation (SD) if normally distributed or as median (interquartile range [IQR]) if not normally distributed. Categorical variables are expressed as numbers (percentages). ABPM values were edited according to conventional criteria to remove measurement errors and outliers. Because sampling frequency was denser during the day (3×/hour) than during the night (2×/hour), we calculated a weighted overall mean BP, as suggested previously²⁵:

Mean day and night BP was calculated as

The start of day- and nighttimes was obtained from diaries. The primary end point was assessed in a primary and secondary analysis population. The primary analysis population included all subjects who were randomized and completed measurements of end points. The secondary analysis population excluded subjects with ≥1 invalid ABPM, change of antihypertensive medication, or compliance <90%. Paired t-tests were performed to analyze day, night, and overall mean BP after intake of aspirin on awakening and at bedtime. Additionally, linear mixed models were used to assess treatment effects and period or carry-over effects. Subgroup analyses were prespecified for users of β-blockers, inhibitors of the renin–angiotensin system (users versus nonusers), users of no- versus ≥1 blood pressure lowering drugs, and subjects with baseline systolic BP of >140 versus ≤140 mm Hg.

The secondary end point platelet reactivity was analyzed with a paired t-test and linear mixed models. Subjects who forgot to take aspirin on the day before platelet reactivity measurements (n=3) were excluded from analysis. Subgroup analyses were prespecified for diabetic subjects, current smokers (yes versus no), and mean platelet volume values (divided into quartiles). Although not prespecified, an additional subgroup analysis for body mass index was performed because obesity, as a marker for metabolic syndrome, may be associated with platelet reactivity.²⁶ Side effects and patient preferences were analyzed descriptively and using McNemar’s test. All analyses were performed in SPSS 20.0 (IBM corp., USA) and were 2-sided, with a level of significance of 0.05.

Between June 2011 and March 2013, 3479 subjects were screened at 30 general practitioner practices, of whom 1704 did not meet inclusion criteria, primarily because of age >75 years (n=1080) and use of other platelet inhibiting drugs (n=386; Figure 2). A total of 290 subjects were randomized, and baseline characteristics were similar between groups (Table 1). Study follow-up was discontinued by 26 subjects, primarily because study participation was too aggravating (18/26; 70%). Primary and secondary analysis populations comprised 263 and 150 subjects, respectively, for assessment of the primary end point. Measurements for the secondary end point platelet reactivity were complete for 136 subjects. Compliance as measured by electronic pill boxes and pill count was high and similar with aspirin intake on awakening (99% [97%–100%] and 100% [100%–100%], respectively) and intake at bedtime (98% [94%–100%] and 100% [100%–100%]).

The circadian 24-hour ABPM profile after 3 months aspirin intake on awakening and 3 months intake at bedtime is depicted in Figure 3. The mean (SD) 24-hour systolic and diastolic blood pressures were 127 (12) and 79 (9) mm Hg with aspirin intake on awakening, whereas these were 127 (12) and 78 (8) with aspirin at bedtime. This resulted in differences of −0.1 mm Hg (95% confidence interval, −1.0 to 0.9) and −0.6 mm Hg (95% confidence interval, −1.2 to 0.0). Furthermore, systolic and diastolic blood pressures during day- and nighttime did not differ by the timing of aspirin intake (Table 2). Mixed model analysis showed the same results and no evidence for carry-over or period effects (data not shown). Additionally, findings among subgroups of subjects using or not using β-blockers, angiotensin inhibitors, blood pressure lowering drugs in general, or subjects with baseline office BP >140 or ≤140 mm Hg were similar to the overall results (Table S1 in the online-only Data Supplement). Finally, in the secondary analysis, comprising only patients with valid ABPM at both visits who did not change their antihypertensive medication between visit 2 and 3 and were ≥90% compliant as registered with electronic pill boxes, aspirin intake at bedtime was not associated with a reduction of mean 24-hour blood pressure or day- and nighttime blood pressure (Table S2).

Three subjects forgot to take aspirin on the day before platelet reactivity measurements and were excluded from this analysis. In the remaining 133 subjects, aspirin intake at bedtime reduced morning platelet reactivity (mean difference −22 ARU [95% confidence interval −35 to −9]; P=0.001; Figure 4). Subgroup analysis showed that, besides in subjects with diabetes mellitus, aspirin intake at bedtime reduced platelet reactivity in all subgroups (Table S3).

Three subjects did not complete the study because of side effects (Table S4). The frequency of well-known aspirin side effects (dyspepsia, nausea, heartburn) was similar between aspirin intake on awakening and at bedtime (Table S5).

After completion of the study, 53/264 (20%) preferred to switch to another time of aspirin intake than before study entry. A total of 32/264 (12%) switched from intake on awakening to intake at bedtime and 21/264 (8%) from at bedtime to on awakening. So, no clear patient preference was present for time of intake.

Multiple previous studies, mostly from a single source in this field, reported a blood pressure lowering effect of bedtime aspirin intake.^6–11,13,27 Subsequently, our group found a biological plausible mechanism underlying this phenomenon: compared with intake on awakening, bedtime aspirin intake reduced plasma renin activity and cortisol, dopamine, and norepinephrine excretions over 24 hours.¹² So, the finding that aspirin intake at bedtime compared with intake on awakening does not reduce blood pressure is in contrast with these previous studies. This may be explained by differences in study populations. First, previous studies included subjects who did not use blood pressure lowering drugs, such as β-blockers or inhibitors of the renin–angiotensin–aldosterone system. This is an important difference because the mechanism behind the time-dependent effect of aspirin on blood pressure was previously related to a reduction renin–angiotensin–aldosterone system and catecholamine activity over 24 hours.¹² However, we did not find an effect in both users and nonusers of β-blockers or renin–angiotensin–aldosterone system inhibitors. Even in the subgroup that did not use any blood pressure lowering drugs, there was no effect. Our findings corroborate those of an earlier study, which also did not find a blood pressure lowering effect of bedtime aspirin intake among treated hypertensive patients.²⁸ Second, patients in all previous studies did not use aspirin before study entry. In contrast, all patients in our study had a medical indication for aspirin use and had used aspirin for median 6 years. It is possible that the time-dependent effect of aspirin on blood pressure weakens over time because of increased arterial stiffening.²⁹ However, a potential blood pressure lowering effect of bedtime aspirin intake would only be clinically relevant in patients already using aspirin for CVD prevention, and we are the first in this field to include this clinically relevant patient group. Given the absence of a blood pressure lowering effect of bedtime aspirin in any subgroup of our study, in our opinion, no further studies are needed to assess the blood pressure lowering effect of bedtime aspirin in patients using aspirin for CVD prevention.

The circadian rhythm of platelet reactivity and its relation with the morning peak of cardiovascular events has been thoroughly studied.^15,30 Previous authors suggested that platelet inhibition during these high risk morning hours could be optimized by aspirin intake at bedtime.^20,21 Subsequent studies clearly showed that the antiplatelet effect of aspirin declines during the 24-hour dosing interval.^18,19,31 In our study, we compared platelet function 12 hours after aspirin intake (bedtime intake) with 24 hours after intake (morning intake). So, a decline in platelet activity during morning hours could have been expected. Nevertheless, to the best of our knowledge, this has never been evaluated in a clinical trial. Additionally, reducing platelet reactivity during the high risk morning hours could be clinically relevant for patients with CVD.

Previously, we studied the time-dependent effect of aspirin on morning platelet reactivity in healthy subjects.³² The results of this study confirm these findings for patients using aspirin on a daily basis. Our study suggests that morning platelet reactivity can be reduced by taking aspirin at bedtime instead of on awakening. This effect was homogeneously present in all subgroups, except in diabetic subjects. However, the size of this subgroup was too small (n=18) to rule out any effect in diabetic patients. Additionally, diabetic patients have higher platelet turnover, and twice daily dosing of aspirin yields more effective platelet inhibition over the whole day in diabetic patients.^33,34 The reduction of platelet reactivity during the vulnerable morning hours might be beneficial for patients with CVD, who have higher platelet turnover, and of which in 25%, platelet reactivity is inadequately inhibited 24 hours after aspirin intake.^31,35

It has been shown that the risk for recurrent cardiovascular events is increased in patients with higher VerifyNow-aspirin platelet reactivity values.^36,37 Stable CVD patients with platelet reactivity >550 ARU had an absolute risk of 15.6% for developing the composite cardiovascular end point, whereas this was only 5.3% in patients with ARU values <550.³⁷ In another study, the absolute risk for the primary end point (all-cause death and recurrent cardiovascular events) was 13.3% in patients >454 ARU and 5.9% in patients <454 ARU.³⁶ These observational studies suggest that a reduction in platelet reactivity could result in clinical benefit for patients with CVD. Because the CVD morning peak is a multifactorial process, we do not expect that bedtime aspirin would abolish the CVD morning peak completely.³⁸ Still, given the high prevalence of CVD, already a modest reduction of the morning peak would lead to a large absolute benefit. For example, 280.000 recurrent cardiovascular events occur in the United States (US) every year, with a known excess of 40% during the morning hours.³⁹ If aspirin intake at bedtime would reduce this morning peak by 20%, it would lead to an absolute reduction of 4853 recurrent events each year in the United States alone. So, switching to bedtime aspirin intake is a simple and possible effective intervention. Future studies should evaluate whether this indeed translates in a reduction of cardiovascular events.

The major strength of our study is its crossover design, which yields high statistical power and enables comparison of treatment effects within each patient. Furthermore, this is the first study in this field which registered the actual time of aspirin intake with electronic pill boxes, which is of major importance to study time-dependent effects.

The main limitation of our study is that only 150/263 (57%) patients complied perfectly with the study protocol. This was mainly because of invalid ABPM (n=57) or compliance of <90% within the prespecified time of intake (n=42). However, sensitivity analysis among patients with complete follow-up and compliance revealed the same results with narrow confidence intervals (Table S2). Regarding platelet reactivity measurements, it is a limitation that we measured platelet reactivity at only 1 time point during the morning, although comparability within subjects was optimized by drawing blood at the same time at each visit. A large proportion of the potentially eligible patients did not respond or did not want to participate in this study. However, the included patients resembled a general CVD population with regard to age, sex, medical history, and medication use.

In this study, bedtime aspirin did not reduce blood pressure in patients with stable CVD using low-dose aspirin on a daily basis. So, we would not recommend switching to bedtime intake of aspirin to reduce blood pressure in those patients. Yet, bedtime aspirin intake did reduce platelet reactivity during morning hours. Future studies are needed to assess the effect of this simple intervention on the excess of cardiovascular events during morning hours.