Changes in mean platelet volume and hematologic indices in patients with panic disorder due to oxidative stress.

Abstract Objective Cardiovascular disorders are common in patients with panic disorder (PD), usually mediated by platelets. The present study was conducted to evaluate oxidative stress conditions and complete analysis of blood cells in patients with PD. Setting and Sample Population Sixty healthy individuals and 60 patients were included in the study. Whole blood and serum samples were obtained from patients and controls. Materials & Method Hematological studies, including blood cells count, hemoglobin, and hematocrit, were carried out on whole blood samples. In addition, oxidative stress indices including total antioxidant capacity, free oxygen species, and malondialdehyde concentration were measured in serum samples. Results Results showed that patients with PD had a significant increase in mean platelet volume index (MPV), platelet distribution width (PDW), red blood cell distribution width (RDW), and mean corpuscular hemoglobin concentration (MCHC) compared with healthy subjects (p < .05). Also, oxidative stress indices were significantly elevated in patients with PD compared with control group (p < .05). Conclusion Elevated MPV is a hematologic indicator for patients with PD. This disorder may be caused by impaired serotonin metabolism, resulting in increased oxidative stress, as well as in platelet serotonin transporters. Regarding elevated oxidative stress, the risk of cardiovascular complications is high in patients with PD.


| INTRODUC TI ON
Panic disorder (PD) is one of the most common serious disorders in the field of psychological illness characterized by sudden and unexpected attacks (Freire, Zugliani, Garcia, & Nardi, 2016). Symptoms of a panic attack include palpitations, sweating, shaking, shortness of breath, and fear of losing control (APA, 2000). Serotonergic system disorders and their receptors are thought to be effective in the neurobiology of the disease (Maron & Shlik, 2006). Although clinical and experimental researches, brain scans, and genetic and drug studies have confirmed this issue, but two hypotheses that justify it are contradictory. First hypothesis implies elevated serotonin level or activity whereas second hypothesis refers to decreased serotonin level or activity. The first hypothesis seems to be stronger because of numerous studies (Bell & Nutt, 1998;Iversen, 1984).
Serotonin is a derivative of tryptophan that is produced in the nervous system at the terminus of serotonergic neurons in the gastrointestinal tract. Also, serotonin is stored in platelet granules during their production. The role of serotonin in the platelet activity and aggregation has been established. Serotonin transporters in platelets and neurons are quite similar, and platelets are considered as a suitable storage source for serotonin. In addition, studies have shown that the levels of serotonin storage in cerebrospinal fluid and platelets are correlated (Audhya, Adams, & Johansen, 2012;Mercado & Kilic, 2010).
Platelets are taught to be a good tool to reflect metabolic disorders in the nervous system. In order to evaluate platelet functions, there are several laboratory parameters including platelet count (Plt), mean platelet volume index (MPV), and platelet distribution width (PDW), which latter is more suitable (Camacho & Dimsdale, 2000;Huczek et al., 2005). Studies have linked the increase in MPV index with cardiovascular disease such as myocardial infarction and ischemia. Researchers have suggested that elevated MPV is an indicator of 6-month mortality risk for heart failure patients (Canan et al., 2012;Stratz et al., 2008).
Studies have shown that platelets in peripheral blood reflect serotonin metabolism in the nervous system and hence the function of serotonergic neurons. Serotonin is an important factor in the pathophysiology of neurodegenerative diseases such as anxiety and plays an important role in platelet aggregation (Camacho & Dimsdale, 2000).
Platelets at their surface express a variety of serotonin receptors, including 5-HT2A and 5HT3. Serotonin transporter expression has also been reported in platelet membranes. Studies have shown that serotonin as a procoagulant is effective in thrombosis. These effects are usually mediated by serotonin reuptake inhibitor (SSRI) drugs used in patients with PD (Galan et al., 2009;Markovitz, Shuster, Chitwood, May, & Tolbert, 2000;Purselle & Nemeroff, 2003).
Various abnormalities of serotonin receptors have been identified in patients with PD, and it has been concluded that increased serotonin production occurs as a result of serotonin transporter dysfunction, leading to anxiety and fear in response to amygdala stimulation (Ataoglu & Canan, 2009;Gogcegoz Gul, Eryilmaz, Ozten, & Hizli Sayar, 2014). Conversely, serotonergic agents are used to relieve symptoms of PD. As serotonin levels increase, its catabo- As a result, the activity of oxidase enzymes induces the process of producing free radicals which can elevate oxidative stress production. Increased oxidative stress elevates the risk of cardiovascular disease directly and also indirectly through the activation of platelets to produce thrombosis (Freedman, 2008).
Imbalance between oxidant and antioxidant called as oxidative stress. We can assess the oxidative stress by assessment of some indexes such as malondialdehyde (MDA), reactive oxygen species (ROS), and total antioxidant capacity (TAC) in serum of patients.
Oxidative stress can cause many disorders such as damage to RBC, damage to DNA and proteins, alteration structure of LDL, and produced oxidized LDL. It elevates risk of cardiovascular disease (Amin et al., 2020;Flora, 2020).
The aim of the present study was to survey the changes in platelet and hematologic indices and their relation with oxidative stress in patients with PD in comparison with healthy subjects.

| ME THODS
The present case-control study was conducted for 9 months in a university-affiliated clinic under the code of ethics IR.SHMU.
REC.1397.90 at the Shahroud University of Medical Sciences. Sixty healthy subjects and sixty patients diagnosed by a neurologist were included in the study. The age range of the subjects was 18-45 years.
The study excluded patients with history of neurological disease, tobacco use, or any chronic illness.
All the participants signed the informed written consent and completed a demographic questionnaire. In addition, 5 cc venous blood samples were collected in an EDTA tube to get whole blood

Significant outcome
• These data in patients with PD highlight an altered oxidative stress due to serotonin metabolism.
• MPV, PDW, RDW, and MCHC promise as biomarkers for cardiovascular disorder in patients with PD.

Limitation
• The study may not have enough power to detect serotonin metabolites.
• The lack of validation experiments is a caveat. and a tube without anticoagulant to get serum. Hematologic tests were analyzed by Sysmex K21, and Plt, MPV, PDW, hemoglobin, and hematocrit were measured. In addition, oxidative stress indices including MDA, TAC, and ROS were measured using commercial kits (Razi Technologies) according to company guidelines.
Quantitative results were reported based on mean and standard deviation. Statistical analysis was performed by two-way independent t test and Fisher's exact test, and p ≤ .05 was considered significant.

| RE SULTS
The demographic survey of the present study showed that the mean age of the study population was 35.28 ± 10.23 which 37% of them were men (Table 1). Although more females participated in present study, there was no significant relationship between sex and disease. Table 2

| D ISCUSS I ON
The present study was designed to evaluate oxidative stress conditions and changes in platelet and hematologic indices in patients with PD. The current study found that oxidative stress is elevated in patients with PD, which was accompanied with changes in MPV, PDW, and RDW.
Since CSF collection is more invasive and difficult compared with plasma, evaluating peripheral blood platelets is used to investigate serotonin metabolism. Platelets are also used to monitor cellular signaling in neurodegenerative diseases as well as the serotonergic system (Camacho & Dimsdale, 2000). Although the platelet maturation process in bone marrow has been identified as an influencing factor on MPV, there is some evidence suggesting age and heterogeneity effect on MPV (May, Marques, Reddy, & Gangaraju, 2019

TA B L E 2 Hematologic indices in patients with panic disorder and healthy subjects
The results of this study showed that people with PD experience  (Martini et al., 2004).
In the present study, a significant decrease in MPV indicates impaired metabolism of serotonin and its transporters, which could also be a therapeutic guide in these patients, as lithium users are characterized by improvements in this parameter (Atagün et al., 2016).
On the other hand, PDW is strongly affected by platelet activity and MPV. Platelet volume increases with platelet activation and decreases with platelet discharge. Also, during peripheral platelet consumption and increase in bone marrow activity immature platelets appear in peripheral blood, which is a cause of increased RDW (Nishimura et al., 2018;Tang et al., 2017). TAC, so it can affect the RBC membrane and causes significant alterations of RDW and MCHC (Mehrpour et al., 2018).
In this regard, we found significant increase of MDA in patients with PD compared with healthy patients. MDA as a marker of lipid peroxidation due to oxidative stress can be helpful to determine the oxidative stress status in patients with PD (Amin et al., 2020).
Also, the number of platelets decreased significantly in the present study, indicating that activated platelets were removed from the peripheral circulation or replaced in the vascular lesions and therefore decreased in the collected specimens. This may be the reason why the bone marrow is activated and immature platelets are appeared in the blood and PDW are elevated (Asoglu et al., 2016;Kokacya et al., 2015).
The present study found a decreased trend of RBC levels in patients with PD, statistically not significant, which is probably due to increased oxidative stress and could be justified by increased RDW in these patients, as elevated RDW has been reported in depression patients (Asoglu et al., 2016;Bhad, 2017;Ransing, Patil, & Grigo, 2017

| CON CLUS ION
The present study showed that platelet activation in patients with PD is an inevitable complication. In addition, increased PDW and RDW indicate that the inflammatory system is active in these patients. Therefore, due to elevated oxidative stress and activated inflammatory system, these patients are at high risk of developing cardiovascular disease.

This study was supported by grant No 9751 from Shahroud
University of Medical Sciences.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R ' S CO NTR I B UTI O N
Maryam Naghipour Hamzekolaei contributed to conceptualization, data curation, and investigation; Mersedeh Tashakori contributed to investigation; Asghar Farajzadeh contributed to software facilitation; Seyed Shahrokh Aghayan contributed to methodology; Amir Atashi contributed to formal analysis; Maryam Yarmohammadi contributed to validation and visualization; Iman Sadeghi contributed to resource facilitation; Moslem Jafarisani contributed to project administration, writing-original draft, writing-review editing, and supervision.

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.