Effects of acute and chronic psychological stress on platelet aggregation in mice

Abstract

Although psychological stress has long been known to alter cardiovascular function, there have been few studies on the effect of psychological stress on platelets, which play a pivotal role in cardiovascular disease. In the present study, we investigated the effects of acute and chronic psychological stress on the aggregation of platelets and platelet cytosolic free calcium concentration ([Ca²⁺]_i). Mice were subjected to both transportation stress (exposure to novel environment, psychological stress) and restraint stress (psychological stress) for 2 h (acute stress) or 3 weeks (2 h/day) (chronic stress). In addition, adrenalectomized mice were subjected to similar chronic stress (both transportation and restraint stress for 3 weeks). The aggregation of platelets from mice and [Ca²⁺]_i was determined by light transmission assay and fura-2 fluorescence assay, respectively. Although acute stress had no effect on agonist-induced platelet aggregation, chronic stress enhanced the ability of the platelet agonists thrombin and ADP to stimulate platelet aggregation. However, chronic stress failed to enhance agonist-induced increase in [Ca²⁺]_i. Adrenalectomy blocked chronic stress-induced enhancement of platelet aggregation. These results suggest that chronic, but not acute, psychological stress enhances agonist-stimulated platelet aggregation independently of [Ca²⁺]_i increase, and the enhancement may be mediated by stress hormones secreted from the adrenal glands.

• Acute stress
• adrenalectomy
• chronic stress
• corticosterone
• cytosolic free calcium concentration
• platelet aggregation

Introduction

Psychological stress is known to alter the behavior and homeostatic state of animals by modulating nervous, endocrine and immunological function. Stress has also been reported to play a role in the development of cardiovascular disease, such as acute myocardial infarction (Rosengren et al., 2004; Willich et al., 1994) and coronary artery disease (Rozanski et al., 1999). The biological response to stress involves activation of both the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic-adrenomedullary system. Consequently, glucocorticoids and catecholamines are released from the cortex of the adrenal glands and sympathetic nerve terminals and adrenal medulla, respectively (Olfe et al., 2010). These stress hormones have both protective and damaging effects on the body. While they are essential for adaptation in the short term (allostasis), in the long term, stress hormones exact a cost (allostatic load) that can accelerate disease processes (McEwen, 1998; McEwen & Gianaros, 2011).

Platelets play an important role in hemostasis, and also participate in inflammation, immunoreactions and protection against infection (Smyth et al., 2009). However, platelets are involved in formation of thrombi, development of arteriosclerosis and cancer progression (McNicol & Israels, 2008). When released from the bone marrow, platelets circulate for an average lifetime of 7–10 days without significant interaction. Once platelets adhere to sites of vascular injury or are stimulated by agonists in plasma, such as thrombin and ADP, they are activated. Platelet activation results in rapid changes in platelet morphology, platelet aggregation and granule secretion. Ca²⁺ plays a critical role in platelet activation. Elevated Ca²⁺ activates multiple signaling events and molecules, including actin–myosin interaction, PKC, calmodulin, NO synthases and calcium-dependent proteases (Li et al., 2010). Enhanced platelet functions can predispose an individual to cardiovascular diseases (Harrison & Keeling, 2006) and platelets are essential to atherothrombosis, i.e. acute coronary syndrome and atherosclerotic stroke, which have been shown to be connected with psychological stress (McCabe et al., 2002; Watson et al., 1998).

Psychological stress has been reported to elevate circulating concentrations of IL-6, which has procoagulant effects on platelets (Yudkin et al., 2000). Grignani et al. (1992) reported that emotional stress (mental arithmetic for 10 min) produced a significant increase in platelet aggregation. On the other hand, Malyszko et al. (1994) and Takeda et al. (1992) showed that acute stress (water immersion restraint and cold restraint) caused a reduction in platelet aggregation. However, Knöfler et al. (1995) demonstrated that acute stress (electric footshock) had no effect on platelet aggregation. These discrepancies could be due to various differences between the studies, such as stress conditions and platelet aggregation measurement methods. To our knowledge, excepting the possible effects of plasma factors, the effects of acute and chronic stress on platelet aggregation have not yet been investigated. Therefore, in the present study, we investigated the effects of acute and chronic stress composed of transportation (novel environment) and restraint, which are regarded as models for psychological stress (Andersson et al., 2010; Grandin, 1997; Obernier & Baldwin, 2006), on agonist-induced platelet aggregation and cytosolic free calcium concentration ([Ca²⁺]_i) increases.

Methods

Chemicals

Thrombin and Fibrinogen were purchased from Sigma–Aldrich (St. Louis, MO). Fura-2-acetoxymethyl ester (fura-2-AM), HEPES and EGTA were supplied by Dojindo Laboratories (Kumamoto, Japan) and ADP by Wako Pure Chemicals (Osaka, Japan). Other chemicals were of reagent grade or the highest quality available.

Media

The standard incubation medium for the measurement of platelet aggregation and [Ca²⁺]_i contained the following: 125 mM NaCl, 5 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 5 mM NaHCO₃, 6 mM glucose, 1% Albumin and 25 mM HEPES, with the pH adjusted to 7.4.

Animals

The experiments were performed on male ddY strain mice (Japan SLC, Shizuoka, Japan). Mice (four to six per cage) were housed in polypropylene cages (265 mm in width × 425 mm in length × 155 mm in height) under standard light (lights on from 07:00 to 19:00 h) and temperature (22 ± 2 °C) conditions. Food and water were provided ad libitum, except during the period when restraint stress was applied. All experiments were approved by the Animal Research Committee of Obihiro University, and conducted in accordance with the Declaration of Helsinki and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health.

Stress protocol

Mice (9 weeks old) were randomly assigned to one of three experimental groups: a control group (Control), an acutely stressed group (Acute Stress) and a chronically stressed group (Chronic Stress) (Figure 1). Daily, mice in Chronic Stress were taken to a different room (room A), where each mouse was restrained by placing it in a well-ventilated polyethylene tube (40 mm in diameter × 80 mm in length) as described in our earlier study (Satoh et al., 2011). The restraint procedure was conducted for 2 h/day between 10:00 and 15:00 h for 3 weeks. The mice were kept in their home cages in the breeding room (room B) for 24 h following the final period of chronic stress and then taken to room A to be sacrificed. At 12 weeks old, mice in Acute Stress were restrained for 2 h from 08:00 h in a manner similar to that for Chronic Stress. The mice were kept in their home cages for 15 min following the restraint stress and then sacrificed (Satoh & Shimeki, 2010). During restraint stress, they were not physically compressed. The control animals were left undisturbed in their home cages until sacrifice. During the chronic stress experiment, the body weights and food and water intake of the mice were simultaneously recorded.

Figure 1. Schematic representation of the stress and adrenalectomy protocol. At 9 weeks old, mice in Chronic Stress were taken to a different room (room A) and restrained for 2 h/day between 10:00 and 15:00 h for 3 weeks. The mice were kept in their home cages in the breeding room (room B) for 24 h following the final period of chronic stress and then sacrificed in room A. At 12 weeks old, mice in Acute Stress were restrained in room A for 2 h from 08:00 h. The mice were kept in their home cages in room A for 15 min following the restraint stress and then sacrificed. The control animals were left undisturbed in their home cages until sacrifice. At 7 weeks old, mice in Sham and ADX were operated on. The mice were kept in their home cages for 2 weeks following the surgery and were used for the chronic stress protocol.

Adrenalectomy

Mice (7 weeks old) were randomly assigned to one of two experimental groups: a sham-operated group (Sham) and an adrenalectomized group (ADX) (Figure 1). Mice in ADX were anesthetized with pentobarbital. The skin on the back was shaved and disinfected and an incision was made above the spinal cord. Through incisions in the muscle layer at the left and right of the midline, the adrenals were removed from the surrounding fat tissues. The muscle layer and the skin were closed using simple sutures. Mice in Sham were treated similarly, excepting the actual removal of the adrenals. After surgery, the mice in ADX were given free access to 0.9% NaCl in addition to normal drinking water. At 9 weeks old, mice in Sham and ADX were used for the chronic stress experiment, whose protocol was identical to the initial stress protocol performed in non-operated mice.

Corticosterone determination

To avoid fluctuations in plasma corticosterone levels due to circadian rhythms, blood samples were taken between 08:00 and 10:00 h on the day of sacrifice. Collected blood was centrifuged and separated plasma frozen at −80 °C until analysis. Total plasma corticosterone concentrations were measured as described by Glick et al. (1964). Briefly, 900 µl of isooctane was added to 200 µl of each plasma sample with thorough mixing. After centrifugation at 300 × g for 5 min, the top isooctane layer was discarded. Following the addition of 900 µl of chloroform, each sample was mixed well and centrifuged at 300 × g for 5 min. The top aqueous layer was discarded, 800 µl of the chloroform was removed and the sample was added to 320 µl of an acid–alcohol solution (65% H₂SO₄ and 35% ethanol) with thorough mixing. After centrifugation at 300 × g for 5 min, the bottom acid layer from each sample was transferred to a cuvette. The intensity of fluorescence of the samples was measured using a model FP-770 spectrophotofluorometer (Jasco, Tokyo, Japan) with excitation at 350 nm and measurement of emission at 520 nm. Corticosterone concentrations were calculated from a standard curve and expressed in ng/ml. All samples were measured in a single assay and the intra-assay coefficient of variation was 5%.

Preparation of mouse platelets

Blood was collected from the vena cava of anesthetized mice into syringes containing acid citrate-glucose (9:1, v/v). After centrifugation at 380 × g for 5 min at room temperature, the supernatant was centrifuged at 1200 × g for 5 min. The platelet pellets were suspended in 1 ml of standard medium and washed three times by centrifugation at 1200 × g for 5 min. The washed platelets were then suspended in standard medium and adjusted to ∼1.0 × 10⁸ platelets/ml.

Platelet aggregation determination

The platelet aggregation assay was performed by a modified version of the method of Born (1962). Aggregation was monitored by light transmission using a model CAF-110 spectrofluorometer (Jasco) with high-speed stirring (1000 rpm) at 37 °C. The maximum 100% line was set with buffer, and the 0% line was set with platelet suspension. Aliquots (0.5 ml) of washed platelets (1.0 × 10⁸ platelets/ml) were stimulated with thrombin or ADP after incubation for 10 min with 1 mM CaCl₂ and monitored for 18 min thereafter.

[Ca²⁺]_i determination

Intracellular [Ca²⁺] levels were measured by monitoring the intensity of fura-2 fluorescence. Briefly, a platelet suspension (1.0 × 10⁸ platelets/ml) was incubated for 30 min at 37 °C with 5 µM fura-2-AM dissolved in dimethyl sulfoxide. The platelets were washed three times and then resuspended in 5 ml of standard medium. Aliquots (0.5 ml) of the platelet suspension were placed in cuvettes and incubated for 10 min with 1 mM CaCl₂ before addition of thrombin or ADP. Measurement of fluorescence was initiated 4 min before addition of thrombin and continued for 18 min thereafter, while it was initiated 4 min before addition of ADP and continued for 8 min thereafter. Samples in the cuvettes were maintained at 37 °C and mixed with a magnetic stirrer. Fura-2 fluorescence was measured with a model CAF-100 spectrofluorometer (Jasco) using the ratio mode. Excitation wavelengths were 340 and 380 nm, and the emission wavelength was 500 nm. According to previous research, the fluorescence of extracellular fura-2 is quenched by Mn²⁺ and then fura-2 fluorescence is stable (Komulainen & Bondy, 1987). Therefore, for each platelet suspension, fluorescence due to extracellular fura-2 was determined through the addition of 50 μM Mn²⁺ and this was subtracted from the maximal fluorescence in order to calculate the free Ca²⁺ concentration of subsequent samples of the same suspension. [Ca²⁺]_i was calculated according to the method of Grynkiewicz et al. (1985).

Statistical analysis

All data are expressed as the means ± SEM. Statistically significant differences were assessed using the Student’s t-test for unpaired samples or, when three or more groups were compared, two-way ANOVA for repeated measures as appropriate using the Tukey’s post hoc test or Dunnett’s test. Differences between means were considered significant at p < 0.05.

Results

Body weight, food and water intake and plasma corticosterone concentration

To confirm the physiological efficacy of the stress manipulation, we measured mice body weights, food and water intake and plasma corticosterone concentrations. The body weight gain of mice in Chronic Stress was significantly lower than that of mice in Control during the course of the chronic stress experiment (F_1,476 = 97.78, p < 0.001). One day after the final period of chronic stress, the body weight gains compared to the weight measured prior to the first period in Control and Chronic Stress were 106.4 ± 0.4% and 102.2 ± 0.5%, respectively (p < 0.001, Figure 2A). Food and water intake measured in Chronic Stress during the chronic stress experiment was not significantly different from those of Control (food: F_1,60 = 0.02, p > 0.05; water: F_1,60 = 0.19, p > 0.05; Figure 2B and C). We also measured plasma corticosterone concentrations in Control, Acute Stress and Chronic Stress. There was a significant increase in plasma corticosterone level 15 min after acute stress compared to the control level (t₂₀ = 6.39, p < 0.001), but the level 1 day after chronic stress was not increased (t₂₁ = 1.79, p > 0.05, Figure 2D). The corticosterone level in Control was consistent with values reported by Silberman et al. (2003).

Figure 2. (A) Mouse body weight gain under chronic stress. Body weight gain was calculated on the basis of the weight before the first period of chronic stress (baseline). Data are mean ± SEM (n = 57–64): ***p < 0.001 versus Control. (B) Food and (C) water intake in mice under chronic stress. Food and water intake was measured per cage and transformed into per mouse. Data are mean ± SEM (n = 9–13). (D) Effects of acute and chronic stress on mouse plasma corticosterone concentration. Plasma corticosterone levels were measured under basal conditions and after restraint stress. In the chronic stressed mice, blood was collected 1 day after the final period of stress, while in the acute stressed mice, blood was collected 15 min after the stress period. Data are mean ± SEM (n = 11–12): ***p < 0.001 versus Control.

Platelet aggregation

There was no difference in number of platelets among Control, Acute Stress and Chronic Stress (F_2,28 = 2.45, p > 0.05). To investigate the effects of acute and chronic stress on platelet activity, we examined platelet aggregation in Control, Acute Stress and Chronic Stress. Platelets were stimulated with 0.01–0.5 U/ml thrombin or 0.1–10 µM ADP. Thrombin is a potent agonist for platelets, while ADP is a weak agonist. Thrombin and ADP increased platelet aggregation in a concentration-dependent manner (thrombin: F_2,129 = 259.76, p < 0.001; ADP: F_2,117 = 13.16, p < 0.001; Figure 3A and B). Chronic stress enhanced platelet aggregation induced by 0.05 U/ml thrombin (p < 0.001) and 10 μM ADP (p < 0.05), while acute stress had no effect on platelet aggregation (Figure 3A and B).

Figure 3. (A) Effects of acute and chronic stress on thrombin-induced mouse platelet aggregation. Washed platelets were stimulated with increasing concentrations of thrombin (0.01–0.5 U/ml) following incubation for 10 min with Ca²⁺. (B) Effects of acute and chronic stress on ADP-induced mouse platelet aggregation. Washed platelets were stimulated with increasing concentrations of ADP (0.1–10 µM) following incubation for 10 min with Ca²⁺. Data are mean ± SEM (Control and Chronic Stress: n = 20–23; Acute Stress: n = 6): *p < 0.05, ***p < 0.001 versus Control.

Platelet [Ca²⁺]_i

As chronic stress had enhanced platelet aggregation, we studied its effect on platelet [Ca²⁺]_i. To investigate the effect on platelet Ca²⁺ homeostasis, we measured resting [Ca²⁺]_i in platelets under basal conditions and after chronic stress. Platelet functions, such as aggregation, are regulated by Ca²⁺. The resting [Ca²⁺]_i level in Chronic Stress was slightly below that in Control (t₂₅ = 2.78, p < 0.05, Table 1). We examined the changes in platelet [Ca²⁺]_i induced by the agonists thrombin and ADP in Control and Chronic Stress. Thrombin and ADP caused a rapid and transient increase in [Ca²⁺]_i, producing a peak (Peak I), followed by a decline to a sustained, still-elevated plateau level (Phase II). Thrombin (0.01–0.5 U/ml) and ADP (0.1–10 µM) increased both Peak I and Phase II [Ca²⁺]_i in a concentration-dependent manner (thrombin Peak I: F_1,50 = 328.11, p < 0.001; thrombin Phase II: F_2,73 = 89.78, p < 0.001; ADP Peak I: F_2,71 = 36.4, p < 0.001; ADP Phase II: F_2,71 = 29.33, p < 0.001; Figure 4A and B). In Chronic Stress, increases at Peak I and Phase II [Ca²⁺]_i were not enhanced, on the other hand, there was a slight decrease in Peak I induced by 10 µM ADP (p < 0.05, Figure 4A and B).

Figure 4. Peak I and Phase II indicate the rapid transient rise in [Ca²⁺]_i and sustained rise in [Ca²⁺]_i after addition of platelet agonists, respectively. The net Δ[Ca²⁺]_i for Peak I and Phase II are the differences between the increase in [Ca²⁺]_i 10 s after addition of platelet agonists and the end of incubation with agonists, and the vehicle, respectively. (A) Effect of chronic stress on thrombin-induced [Ca²⁺]_i increase in mouse platelets. Fura-2-loaded platelets were incubated for 18 min with thrombin (0.01–0.5 U/ml). (B) Effect of chronic stress on ADP-induced [Ca²⁺]_i increase in mouse platelets. Fura-2-loaded platelets were incubated for 18 min with ADP (0.1–10 µM). Data are mean ± SEM (n = 13–14): *p < 0.05 versus Control.

Table 1. Effect of chronic stress on resting [Ca²⁺]_i in mouse platelets.

Group	Resting [Ca^2+]i (nM)
Control	101.9 ± 3.5
Chronic Stress	89.8 ± 2.3^a

Fura-2-loaded platelets were incubated without addition of platelet agonists and the fluorescence of extracellular fura-2 was quenched by Mn²⁺. Data are mean ± SEM (n = 13–14): ^ap < 0.05 versus Control.

Effect of adrenalectomy

There was no difference in number of platelets between Sham and ADX (t₁₉ = 0.95, p > 0.05). To explore the role of stress hormones from the adrenal glands on chronic stress-induced enhancement of platelet aggregation, we examined plasma corticosterone concentrations and platelet aggregation in the mice that had undergone adrenalectomy. We measured corticosterone concentrations in the plasma sampled 15 min after the first period of chronic stress in Sham and ADX. Single stress significantly increased the corticosterone level in Sham (t₁₅ = 11.19, p < 0.001), while it had no effect on the level in ADX (t₁₆ = 0.87, p > 0.05). The plasma corticosterone concentrations in Sham and ADX were 296.9 ± 11.2 ng/ml and 185.0 ± 12.1 ng/ml, respectively. We also examined platelet aggregation in Sham and ADX using 0.05 U/ml thrombin, which had enhanced platelet aggregation in the Chronic Stress. In non-stressed mice, thrombin had no significant effect on platelet aggregation for either Sham or ADX (F_2,38 = 1.41, p > 0.05). Chronic stress significantly enhanced platelet aggregation in Sham (t₂₀ = 5.96, p < 0.001, Figure 5), while in ADX, there was no significant difference in platelet aggregation between non-stressed and chronic stressed mice (t₂₁ = 1.74, p > 0.05).

Figure 5. Effect of ADX on chronic stress-induced enhancement of mouse platelet aggregation. Washed platelets were stimulated with increasing concentrations of 0.05 U/ml thrombin following incubation for 10 min with Ca²⁺. Data are mean ± SEM (Control and Chronic Stress: n = 20–23; Sham, Sham + Chronic Stress, ADX and ADX + Chronic Stress: n = 10–12): ***p < 0.001 versus Control or Sham.

Discussion

In the present study, we observed that the plasma corticosterone concentration was significantly elevated in ddY strain mice 15 min after single restraint stress. Physiological responses induced by stress involve activation of both the HPA axis and the sympathetic-adrenomedullary system, with consequent increments in plasma glucocorticoids and catecholamines, respectively. Therefore, the acute stress conditions in the present study were sufficient to induce a stress response in the mice and may have also increased plasma adrenaline/noradrenaline concentrations. In addition, we observed that at 1 day after the final session of the 3-week restraint stress period, the plasma corticosterone concentration was similar to that in the control condition.

In an earlier study, we showed that plasma corticosterone levels were increased at 15 min after the final period of repeated restraint stress (21 days) (Satoh et al., 2011). In this regard, McQuade et al. (2006) found that after restraint stress, plasma corticosterone levels in C57BL/6J strain mice dropped within 1 h to near-baseline levels and in response to repeated restraint stress (21 days), corticosterone levels measured shortly after the final period of restraint stress were significantly increased. Therefore, we assume that the chronic stress paradigm used in the present study produced repeated activation of the HPA axis over the period of exposure.

As chronic stress also significantly reduced body weight gain during the course of our experiments, the chronic stress conditions in the present study were sufficient to induce a stress response in the mice. However, chronic stress had no effect on food and water intake. Therefore, we speculate that the decrease in weight gain resulted from a change in metabolism. Although some studies have shown that stress-induced weight reduction depends on intake loss (Chotiwat & Harris, 2006; Kim & Han, 2006), this disagreement with our finding could be due to various differences between the studies, such as in rodent species, stress conditions and housing conditions.

We demonstrated that chronic stress significantly enhances agonist-induced platelet aggregation but acute stress has no effect on such aggregation. Thrombin stimulates platelet aggregation by protease-activated receptors, whereas ADP induces aggregation via P2Y receptors. Consequently, we assume that chronic stress affects these receptors’ signaling pathways. Regarding overall effects of acute stress on platelet functions, prior studies have produced conflicting results. Knöfler et al. (1995) reported that acute electric footshock had no effect on collagen-induced platelet aggregation in whole blood, which is in keeping with our results. However, other studies have found that acute water immersion restraint stress (Malyszko et al., 1994) and acute cold-restraint stress (Takeda et al., 1992) reduced collagen-induced aggregation in whole blood and ADP-induced aggregation in platelet rich plasma, respectively. This disagreement with our finding could be due to various differences between the studies, such as in rodent species, stress conditions and stimulating agents. In addition, in these studies, platelet aggregation was measured in whole blood or plasma. As there are a wide variety of factors in plasma which influence aggregation, these studies did not examine the direct effect of stress on platelet aggregability. In the present study, we measured platelet aggregation in buffer to examine the direct consequences of stress on platelets and demonstrated that platelets do not immediately respond to psychological stress. The lifespan of a platelet in blood is ∼1 week and platelets are produced in bone marrow during the period when chronic stress is applied. Although the number of platelets was not affected by chronic stress, we speculate that repeated stress impacts platelets in blood and/or the process of platelet formation in bone marrow to cause changes in platelet functions involved in hyperaggregability.

Ca²⁺ within the cytosol plays the role of second messenger, and in platelets, an increase in [Ca²⁺]_i is followed by aggregation. In this regard, platelet agonists released Ca²⁺ from the dense tubular system and lysosomes (Haynes, 1993). In the present study, we found that chronic stress failed to enhance a thrombin- or ADP-induced increase in [Ca²⁺]_i in platelets, which indicates that chronic stress-induced hyperaggregability occurs without enhancement of an increase in [Ca²⁺]_i. Ca²⁺-independent signaling pathways are capable of inducing fibrinogen receptor activation (Quinton et al., 2002) and dense granule secretion (Murugappan et al., 2004) in human platelets. Chronic stress could affect these pathways. A slight decrease in resting [Ca²⁺]_i was observed in platelets from chronic stressed mice. Platelet Ca²⁺ homeostasis is controlled by Na⁺/Ca²⁺ exchangers (Bose et al., 2002) and a multi-Ca²⁺-ATPase system (Chaabane et al., 2007), and one or both of these systems could be affected by chronic stress.

Adrenal glands secrete hormones such as catecholamines and glucocorticoids. In the present study, we observed that single restraint stress increased the plasma corticosterone concentration in the sham-operated mice, but not in the adrenalectomized mice. We also demonstrated that adrenalectomy blocks chronic stress-induced enhancement of aggregation. These results indicate that chronic psychological stress-induced hyperaggregability is mediated by stress hormones secreted from the adrenal glands. We consider candidates for stress hormones mediating such hyperaggregability to be glucocorticoids, catecholamines or both glucocorticoids and catecholamines. Although catecholamines can directly induce platelet aggregation and potentiate the effects of other agonists inducing aggregation (Anfossi & Trovati, 1996), the effects of chronic increases in catecholamine and glucocorticoid levels on platelet functions have still to be revealed.

Conclusions

The present study showed that chronic psychological stress enhances agonist-induced aggregation of mouse platelets but acute psychological stress has no effect on aggregation, and that chronic psychological stress-induced enhancement is independent of an increase in [Ca²⁺]_i, and mediated by stress hormones secreted from the adrenal glands. These findings are important to the elucidation of a causal relationship between psychological stress and thrombosis. Additional studies are now needed to clarify which stress hormone(s) mediate(s) chronic psychological stress-induced hyperaggregability and which platelet signaling pathway(s) is/are involved in such hyperaggregability.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This study was supported by grants from the Research Foundation of Obihiro University of Agriculture and Veterinary Medicine.