The human endogenous circadian system causes greatest platelet activation during the biological morning independent of behaviors

Abstract

Background: Platelets are involved in the thromboses that are central to myocardial infarctions and ischemic strokes. Such adverse cardiovascular events have day/night patterns with peaks in the morning (~9 AM), potentially related to endogenous circadian clock control of platelet activation. The objective was to test if the human endogenous circadian system influences (1) platelet function and (2) platelet response to standardized behavioral stressors. We also aimed to compare the magnitude of any effects on platelet function caused by the circadian system with that caused by varied standardized behavioral stressors, including mental arithmetic, passive postural tilt and mild cycling exercise.

Methodology/principal findings: We studied 12 healthy adults (6 female) who lived in individual laboratory suites in dim light for 240 h, with all behaviors scheduled on a 20-h recurring cycle to permit assessment of endogenous circadian function independent from environmental and behavioral effects including the sleep/wake cycle. Circadian phase was assessed from core body temperature. There were highly significant endogenous circadian rhythms in platelet surface activated glycoprotein (GP) IIb-IIIa, GPIb and P-selectin (6-17% peak-trough amplitudes; p ≤ 0.01). These circadian peaks occurred at a circadian phase corresponding to 8-9 AM. Platelet count, ATP release, aggregability, and plasma epinephrine also had significant circadian rhythms but with later peaks (corresponding to 3-8 PM). The circadian effects on the platelet activation markers were always larger than that of any of the three behavioral stressors.

Conclusions/significance: These data demonstrate robust effects of the endogenous circadian system on platelet activation in humans--independent of the sleep/wake cycle, other behavioral influences and the environment. The 9 AM timing of the circadian peaks of the three platelet surface markers, including platelet surface activated GPIIb-IIIa, the final common pathway of platelet aggregation, suggests that endogenous circadian influences on platelet function could contribute to the morning peak in adverse cardiovascular events as seen in many epidemiological studies.

Figure 1. Protocol.

Rasterplot, including two baseline days, twelve 20-h cycles, and discharge day of an example subject with a habitual bedtime of midnight. Blue bars, baseline and discharge wake episodes in normal room light (∼90 lux); solid back bars, scheduled sleep (0 lux); gray bars, wake episodes in dim light (∼1.8 lux); red bars, timing of the test batteries; dotted line illustrates the circadian core body temperature minimum throughout the protocol, with an average circadian period of 24.09h in these subjects. Each test battery consisted of a mental, tilt, and exercise stress (S) test, each preceded and followed by a baseline (B) and recovery (R) episode. The timing of the blood draws is indicated as red filled circles.

Figure 2. Independent influence of circadian cycle on platelet function.

Cosinor models of circadian rhythms in platelet function. Cosinor and other statistical analysis were performed on the 360° data sets, whereas data are double plotted (2 identical circadian cycles) to aid visualization of rhythmicity (the second circadian cycle data were not used for analyses). Each of the platelet surface markers of platelet activation (activated GPIIb-IIIa, P-selectin, and GPIb) and platelet size had significant endogenous circadian rhythms with circadian peaks corresponding to the vulnerable time of 6AM-noon (indicated by the orange bars). Platelet count, ATP release, WBA and plasma epinephrine also had significant circadian rhythms but with peaks later in the circadian cycle (3–8PM). The cosine models (black lines) and 95% confidence intervals (gray areas) are based on mixed model analyses and use precise circadian phase data. To show that these models adequately fit the actual data, we also plot average data grouped into 60 circadian degree windows with SEM error bars (averaged data are not double plotted). Bottom x-axes, circadian phase with 0° indicating the timing of the core body temperature minimum (average ∼4:30AM in these subjects); top x-axes, corresponding average clock time in these subjects; left y-axes, absolute values; right y-axes, percentage of each individual's mean across the protocol; P values, significance of circadian effect from cosinor analyses.

Figure 3. Independent influence of behavioral stressors on platelet function.

WBA, platelet count, and plasma epinephrine were increased by each of the three stressors, with a (partial) recovery during recovery. In contrast, while the platelet surface markers showed a gradual increase across the approximately 3-h test battery, there was no consistent increase and recovery caused by the three stressors. Mental, mental stress test; tilt, passive head up tilt table test; exercise, cycle exercise test; B, baseline; S, stress test; R, recovery; x-axes, minutes from first blood sample within test battery; left y-axes, absolute values; right y-axes, data expressed as a percentage of each individual’s mean values across the forced desynchrony protocol; error bars, SEM; P-values, significance for effect of time across full stress test battery (9 time points); *, significance for change between consecutive samples (from baseline to stress test and from stress test to recovery). Note platelet ATP release is not shown (see above text).