One night of sleep deprivation induces release of small extracellular vesicles into circulation and promotes platelet activation by small EVs

Abstract Extracellular vesicles (EVs) are emerging as key players in intercellular communication. Few studies have focused on EV levels in subjects with sleep disorders. Here, we aimed to explore the role of acute sleep deprivation on the quantity and functionality of circulating EVs, and their tissue distribution. EVs were isolated by ultracentrifugation from the plasma of volunteers and animals undergoing one night of sleep deprivation. Arterio‐venous shunt, FeCl3 thrombus test and thrombin‐induced platelet aggregation assay were conducted to evaluate the in vivo and in vitro bioactivity of small EVs. Western blotting was performed to measure the expression of EV proteins. The fate and distribution of circulating small EVs were determined by intravital imaging. We found that one night of sleep deprivation triggers release of small EVs into the circulation in both healthy individuals and animals. Injection of sleep deprivation‐liberated small EVs into animals increased thrombus formation and weight in thrombosis models. Also, sleep deprivation‐liberated small EVs promoted platelet aggregation induced by thrombin. Mechanistically, sleep deprivation increased the levels of HMGB1 protein in small EVs, which play important roles in platelet activation. Furthermore, we found sleep deprivation‐liberated small EVs are more readily localize in the liver. These data suggested that one night of sleep deprivation is a stress for small EV release, and small EVs released here may increase the risk of thrombosis. Further, small EVs may be implicated in long distance signalling during sleep deprivation‐mediated adaptation processes.


| INTRODUC TI ON
Extracellular vesicles (EVs) are a heterogeneous group of cellderived membranous structures. They are present in biological fluids and are involved in multiple physiological and pathological processes. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. 1,2 Peripheral blood contains a substantial amount of circulating EVs and their amount, composition and molecular profile reflects the physiological and pathophysiological condition of the body. Thus, EVs received recognition as potential biomarkers in semi-invasive diagnostics. 3 It is well documented that the number of EVs in the blood varies between individuals, and that EVs sub-populations can originate from several tissues and are highly dynamic. 4 In fact, the majority (about 25%) of blood-derived EVs are thought to originate from the megakaryocytes, that is, either from circulating platelets or directly from platelet precursor cells, which reside in the bone marrow, [5][6][7][8] and, to a lesser extent, from erythrocytes, endothelial cells, lung epithelial cells and cardiomyocytes. 9 Currently, EVs are commonly classified based on their intracellular origin. Thus, three principal populations of EVs are considered: apoptotic bodies, microvesicles (MVs) and exosomes. 10 The apoptotic bodies (size between 50-5000 nm) are released by cells undergoing apoptosis and characterized by permeable membrane.
MVs are directly shed from the plasma membrane of cells with a particle size of approximately 100-1000 nm. 10 Exosomes (around 100 nm) are secreted from multivesicular endosomes. 10 EVs act in cell-to-cell communication, delivering cargo from donor to recipient cells and modulating their physiological condition. In addition to a set of common EV proteins utilized for their characterization, EVs carry a pattern of biomolecules related to their mother cell. Their content includes protein, lipids, as well as nucleic acids, largely in the form of small RNAs. MV and exosome secretion occurs in constitutive and regulated fashion, controlled by Ca 2+ signalling in response to extracellular signals such as ATP (monocytes), neurotransmitters (oligodendrocytes), depolarization (neurons), thrombin receptor activation (platelets), lipopolysaccharides (dendritic cells) or by cell stress. 6,[11][12][13][14][15] Poor or insufficient sleep is a major public health problem affecting millions of people of all ages. 16 Sleep deprivation and deficiency have a high prevalence in modern societies. The National Sleep Foundation reported that less than half (44%) of all Americans receive a good night's sleep almost every night. 17 org.cn). All participants recruited from the graduate school students aged 23-28 years old provided written informed consent to participate before enrolment in the study. Exclusion criteria were diagnosed chronic cardiovascular or metabolic disease, psychiatric condition, sleep disorder or any other condition known to affect sleep including stressful condition; current or recent use of sleep medication (past 2 months). Descriptive characteristics of the participants are presented in Table 1. The subjects were asked to have habitual sleep 2 weeks before the experiment. In the sleep deprivation night, subjects were asked not to go to bed before 3 a.m. and not to wake up after 6 a.m. They were not allowed to consume caffeinated beverages and to smoke cigarettes before and during the task. Blood samples were collected before (7:00 a.m.) and after the sleep deprivation (7:00 a.m.).

| Experimental animals
Wistar rats and BALB/c-nu nude mice were purchased from Beijing

| Isolation and identification of plasma extracellular vesicles
Plasma extracellular vesicles were obtained by differential centrifugation as previously described with slight modifications. [25][26][27] Briefly, cells and cell debris were gradually removed from plasma at the speed of 300 g 10 min and 2000 g 10 min. Then, platelets and apoptotic bodies were removed at 10,000 g 10 min. Based on others' experiences, the MVs can be clearly pelleted down at the minimum "g" forces of 20,000-30,000 g (2 times 30 min), 28,29 accordingly, the 10,000 g 10 min in our protocol can only settle down platelets and apoptotic bodies. Then, the supernatants were filtered through a 0.2μm membrane (Thermo Fisher Scientific) and transferred to the ultracentrifugation tube, centrifuged for 70 min at the speed of 100,000 g to retain the precipitate which contains small EVs and contaminating proteins.
Then, the pellet was washed in PBS and centrifuged for another 70 min at the speed of 100,000 g to get small EVs. Each resultant pellet was finally re-suspended in PBS buffer for use or stored at −80°C.
The size and number of small EVs were determined by nanoparticle tracking analysis (NTA) with NTA analyzer (Zetaview). The small EVs were identified by transmission electronic microscope analysis.
Briefly, small EVs were fixed with 2.5% glutaraldehyde at 4°C overnight. After washing, vesicles were loaded onto formvar-coated grids, then negatively stained with aqueous phosphotungstic acid for 60 s and imaged with a transmission electron microscope (Jeol).

| Western blot analysis
For EV marker protein analysis, the small EVs isolated from equal volumes of plasma of volunteers and rats were subjected to Western blot analysis which was performed according to the standard protocol as previously described. 30 The following antibodies were used: rab-

| Arterio-venous shunt thrombosis model
The small EVs, isolated from equal volumes (2 ml

| FeCl 3 thrombus test
The small EVs, isolated from equal volumes (1 ml) of plasma in individuals pre-or post-SD and re-suspended in 100 μl of PBS, were injected into WT C57BL/6 mice by tail vein injection. Two hours after the injection, a ferric chloride (FeCl 3 )-induced carotid artery thrombosis model was established as described previously. 33 Briefly, mice were anaesthetised with 1% Pentobarbital Sodium for 0.5-1 h to separate bilateral common carotid arteries. 5% FeCl 3 solution was dripped on the filter paper, which covered the separated common carotid artery without damaging the surrounding tissues. After 15 min, the filter paper was removed, and the proximal common carotid artery was ligated. The common carotid artery was carefully cut-off with scissors and immediately embedded with OCT (Japanese Cherry Blossom).
Sections were stained with haematoxylin eosin. Observe and take pictures with IX51 microscope (Olympus).

| Platelet aggregation test
To determine the effect of SD-induced small EVs on platelet activation, we first prepared platelet rich plasma (PRP) from rats. In brief, after general anaesthesia, blood was collected from abdominal vein of male Wistar rats and anticoagulated with EDTA. The blood was centrifuged at 25°C for 10 min at 100 g to obtain the supernatant of PRP. Then, the blood was centrifuged at 25°C for 10 min at 1000 g, and platelet poor plasma (PPP) was obtained at room temperature.
Then, the platelet count of each PRP sample was measured using an

| Intravital imaging of transplanted EVs
Donor Wistar male rats were randomly assigned to either SD or SC groups. One night of sleep deprivation rat models were established as described above. Both SC and SD rats were sedated by isoflurane inhalation and blood was collected from abdominal vein followed by euthanasia via cervical dislocation. Small EVs isolation from the plasma of SC or SD rats was carried out as described above and labelled in 1 mM lipophilic carbocyanine DiOC18 (7)

| Statistical analysis
The normality of all values was conducted by using the Shapiro-Wilk test. The values of normality distributions were expressed as mean ± SEM. Median and interquartile range (IQR) were used for non-normally distributed data. The differences between two groups for animal studies were tested by independent t-test for normally distributed data and Mann-Whitney U test for non-normally distributed. The differences between the pre-and post-SD experiments were tested by paired t-test for normally distributed data and Wilcoxon signed ranks test for non-normally distributed. All of the statistical tests were performed with the GraphPad Prism software version 8.0, and p < 0.05 was considered to be statistically significant.

| Acute SD triggers release of small EVs into the circulation
To investigate the influence of one night of SD on the levels of circulating small EVs in plasma, we recruited 20 healthy (10 females, age 25 ± 5 years old) individuals who undergo one night of sleep deprivation. We collected venous blood samples (EDTA-anticoagulated blood) before (pre) and after (post) SD, and prepared plasma. To analyse the levels of small EVs in the plasma samples, we performed differential centrifugation at 10,000 g removing platelet remnants and apoptotic bodies, followed by filtration of the supernatant and ultracentrifugation at 100,000 g to collect small vesicles of a size below 200 nm, which include exosomes (here defined as small EVs).
Vesicles ~30-150 nm in diameter were observed by Transmission electron microscopy (TEM), which was consistent with previously reported characteristics of small EVs ( Figure 1A). The collected small EVs were analysed by NTA revealing particles with a mean size of 120 (SEM ± 1.3) nm in diameter ( Figure 1B,D). The total amount of particles increased in average 3.0 times directly after one night of SD ( Figure 1C). Next, we investigated the small EVs of 6 subjects by Western blotting with antibodies against the EV marker proteins ( Figure 1E) and quantified signal intensities ( Figure 1F). The numbers of the 4 universal EV marker proteins increased on average 3.9 times after SD. It is worth mentioning that we detected integrin αIIb found on platelets indicating the presence and increment of platelet-derived small EVs, based on the fact that the majority (about 25%) of blood-derived EVs are thought to originate from the megakaryocytes. 5 Next, we performed controlled animal experiments in rats to further confirm the effect of SD on the amount of small EVs in the circulation. As shown in Figure 2A,B, the amounts of small EVs increased in average 2.7 times in SD group compared with SC control group, and particle size was not altered in 2 groups ( Figure 2C). The EV marker proteins increased on average 2.1 times in SD group compared with control ( Figure 2D,E). The data was in consistent with human study and indicates that acute SD leads to an increase in circulating small EVs.

| Injection of sleep deprivation-liberated small EVs increased thrombus formation and weight in thrombosis models
Sleep disruptions were associated with prothrombotic changes and might be an independent risk factor for arterial thrombosis, which is associated with high cardiovascular morbidity and mortality. 34 Here, we investigated the effects of SD-liberated small EVs on thrombosis.
We collected venous blood samples before (pre) and after (post) SD from 8 participants, and isolated small EVs. Pre-exos and post-exos isolated from equal volumes of plasma were then intravenously administered to recipient animals. First, an arteriovenous shunt thrombosis model was used. As shown in Figure 3A, tail vein injection of SD post-liberated small EVs resulted in an increased thrombus dry weight compared with the control group injected with pre-liberated small EVs. Next, in order to further examine in vivo prothrombotic activities of SD-liberated small EVs, the FeCl 3 -induced arterial thrombosis model was used, which is sensitive to antiplatelet drugs. 35 As shown in Figure 3B, the formation of thrombus is increased in SD-liberated small EV group. These data suggested that sleep deprivation-liberated small EVs increased thrombus formation and weight in thrombosis models.

| Sleep deprivation-liberated small EVs promoted platelet aggregation induced by thrombin
To determine whether the prothrombotic effect of sleep deprivationliberated small EVs was related to alterations in platelet aggregation, we observed platelet aggregation induced by thrombin in vitro. We isolated plate-rich plasma and incubated with small EV, followed by activating it by thrombin. As shown in Figure 4, sleep deprivationliberated small EVs clearly promoted thrombin-induced rat platelet aggregation.

| Sleep deprivation increased the levels of HMGB1 protein in small EVs
Proteins from EVs are known to play important roles in various cellular functions. In this study, we evaluated the protein level of HMGB1, which is critical for regulating platelet activation, granule secretion, adhesion and spreading. 36 As shown in Figure 5, the levels of HMGB1 protein increased on average 3 times in SD postliberated small EVs compared with pre-liberated small EVs. The data suggested that the platelet aggregation and thrombus formation induced by SD-liberated small EVs might be attributable to the elevated HMGB1 protein level in small EVs.

| Sleep deprivation-liberated small EVs demonstrate tropism to the liver
It is reported that small EVs communicate a wide range of information to target cells. 37 Here, we hypothesized that small EVs participate in tissue crosstalk during sleep deprivation is the uptake of small EVs from SC and SD groups, respectively ( Figure 6A), indicating that small EVs liberated into circulation by sleep deprivation more readily concentrate in tissues in the abdominal viscera, with a small amount in the brain. We then carried out repeat experiments in mice receiving labelled small EVs from a single donor at a oneto-one ratio and observed fluorescent signal in the liver and brain ( Figure 6B). Importantly, we observed a significant increase in fluorescence in the livers of mice receiving small EVs from SD group versus control donors 8 h after injection ( Figure 6B,C), indicating that small EVs liberated into circulation during sleep deprivation more readily localize in the liver.

| DISCUSS ION
Sleep is integral to life. 38  It has been reported that there is a trend for platelet-derived vesiculation and fractionation, which may contribute to the recovery of platelet EVs in MV and small EV fractions. 51 Further studies are needed to illustrate the components of small EVs. On the contrary, EV levels in the circulation depend on speed of the clearance, currently thought to involve direct receptor binding of liver or spleen phagocytes to phosphatidylserine or opsonization protein on the EVs. 58 Another report shows that macrophages in the liver play a crucial role in the clearance of the exosomes from the circulation. 59 In our study, we observed that small EVs liberated into circulation during sleep deprivation are more readily localize in the liver, suggesting the elevated small EV numbers induced by the sleep-disordered stress might have a trend to be cleared in the liver to maintain the homeostasis of the body.
The present study has two limitations. First, the cellular source, comprehensive cargo alterations, targets and signalling components of SD-induced small EVs were not validated to uncover the mechanism of their potential role as mediators of health-injurious effects associated with sleep deprivation. Second, although we determined the subsequent distribution of small EVs, based on the fact that the release of small EVs in this study is an acute event, the dynamic changes of circulating small EVs remain to be elucidated.

| CON CLUS IONS
In summary, we demonstrated one night of sleep deprivation triggers release of small EVs into the circulation, which promote thrombosis formation and platelet activation, and more readily localize in the liver. These data suggested that one night of sleep deprivation is a stress for small EV release, and EVs released here may increase the risk of thrombosis. Further, EVs may be implicated in long distance signalling during sleep deprivation-mediated adaptation processes.

CO N FLI C T O F I NTE R E S T
We declare that we have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.