Major Depression is Associated with High Platelet Activity and Reactivity

Milovanovic M^1,2*

¹Department of Internal Medicine, Vrinnevi Hospital, Sweden

²Department of Social and Welfare, LinkÖping University, Sweden

*Corresponding author: Micha Milovanovic, Department of Internal Medicine, Vrinnevi Hospital, S-601 82 NorrkÖping, Sweden

Received: December 06, 2018; Accepted: February 01, 2019; Published: February 08, 2019

Abstract

Objective: Depression exists in mild, moderate and severe depressive attacks. Evidence show that the condition is a risk factor for the expansion of Cardiovascular Disease (CHD). Platelet reactivity is associated with CHD and may well be a thinkable connection between Major Depression (MD) and CHD. The current study examined platelet activity and reactivity in density separated platelets of patient with MD compared to a Control Group (CON) without MD.

Material and Methods: N=24 patients suffering from MD, all individuals were diagnosed with DSM-IV and ICD-10. N=52 healthy individuals without MD served as controls. A flow cytometer was used to measure platelet bound fibrinogen with and without platelet agonists (ADP and TRAP-6). Finally, platelet counts were carried out using a routine cell counter.

Results: When compared to CON, MD displayed a significant higher in vivo platelet bound fibrinogen i.e. no platelet agonists added (p<0.05). After adding of platelets agonists, MD showed significantly more reactive platelets (p<0.05).

Conclusion: Activated platelets are also a feature of CHD. It is therefore to presume that a common pathophysiology exists between these diseases. It is possible that both conditions activate same inflammatory reactions.

Introduction

Depression can be divided into mild, moderate and severe depressive episodes. The disease is frequent in a broad-spectrum of the population, and even more common in those who suffer from chronic disease [1]. Depression is considered as a risk factor for the development of Cardiovascular Disease (CHD) [2-4], although there are shared sentences if cardiovascular disease can cause depression [5]. However, there are evidence of clinical significance, after an acute myocardial infarction, almost 20% of patients obtain a Major Depressive (MD) disorder and additional 20% develop a minor depressive disorder [6]. Previous studies have shown that depression relates to more reactive platelets [7-11]. Platelet reactivity is also linked to CHD [12-17] and could therefore be a possible link between MD and CHD.

Platelets are essential in the hemostatic process by prevent bleeding which is the principal function of the cell. When activated, platelets change shape, redistribute Glycoprotein (GPIb) receptors and expose procoagulant phospholipids on their surface, and initiate the release reaction. The GPIIb/IIIa complex, which is also present on resting platelets, undergoes a conformational change leading to exposure of a binding site for fibrinogen. These activation-dependent platelet surface changes are detectable by whole blood flow cytometry using specific antibodies such as PAC-1 directed against the fibrinogen binding site of GPIIb/IIIa [18].

Circulating platelets devour a density span of 1.04-1.08 kg/l [19,20]. Platelet organelles are assumed to determine its density, high-density platelets are believed to have additional amount of a and dense granules [19]. There is no consensus regarding the density of platelets. Some studies show that the density of platelets increases with age [21,22], while other studies show the opposite [23,24]. Further studies show that density is constant during cell life [25-27]. The clinical effect of platelet heterogeneity has been researched for a lengthy period. Platelet density is boosted in combination with acute myocardial infarctions [28]. High-density platelets are connected with the activity of inflammatory bowel disease [29]. Low peak platelet density characterizes essential thrombocythemia [30]. Preeclampsia is related to large platelets having low peak density [31].

Platelet heterogeneity by investigating platelet density subpopulations in different clinical conditions has been our fundamental areas in our laboratory research. Surface bound fibrinogen in vivo reveal that high density platelets in contrast with the normal platelet population circulate “more activated”, these types of cells also have less P-selectin as an indication of platelet ex vivo a-granula liberation [32]. We have also confirmed that low density platelets circulate “more activated”, however, in difference to high density populations, light platelets contain additional P-selectin and a smaller amount of dense bodies [33]. Low-density platelet populations reveal low in vivo activity [34] and increased serotonin content in Alzheimer’s disease [35]. Normal platelet population of Fibromyalgia contain more serotonin [36], and is associated with elevated in vivo platelet activity [37].

The study aims to investigate if platelet activity and reactivity differ in MD compared to a control group (CON) without MD.

Material and Methods

Subjects

24 patients suffering from Major Depression (MD), aged 50 ± 14 years (mean ± SD) participated in the study. All patients were registered for the study when seeking care at the psychiatry clinic at the local hospital. All included individuals were diagnosed with DSMIV and ICD-10 i.e. the condition has lasted for at least two weeks. DSM-IV specifically emphasizes that the condition should have resulted in a change in the person’s condition. The patients who orally rejected were excluded from the study. 52 healthy individuals, aged 68 ± 4 years (mean + SD) were used as a Control-Group (CON). All of the CON-group were enrolled in the study after requesting medical care at a neighbouring health centre, everyone where free from any depression state. All of CON-group gave informed agreement. The study was sanctioned by the local ethics committee of LinkÖping University, Sweden (reg. number: 2012/269-32).

Laboratory investigations

Each individual donated two blood samples from the antecubital vein into Vacutainer™ tubes (Becton and Dickinson, New Jersey, U.S.A.). The blood of the first tube (7.5 ml) was anticoagulated by adding 2.5 ml platelet-inhibitory solution*, this test tube was used for analysis of platelet in vivo activity. Before measuring platelet in vivo activity, platelets had to be separated according to density, a linear Percoll™ (GE Healthcare Bio-Sciences AB, Sweden) gradient was used [32,33,38]. The subsequent ingredients were varied to prepare the two Percoll™ solutions (1.09 and 1.04 kg/L) for the gradient (Table 1).

Table 1: The subsequent ingredients were varied to prepare the two Percoll™ solutions (1.09 and 1.04 kg/L) for the gradient.




  
    Percoll^TM solutions
    1.09 kg/L
    1.04 kg/L
  
  
    Percoll^TM
    32.84 g
    8.88 g 
  
  
    H₂O
    11.42 g
    19.14 g
  
  
    Platelet-inhibitory solution* 
    4.50 g
    3.00 g



Table 1: The subsequent ingredients were varied to prepare the two Percoll™
solutions (1.09 and 1.04 kg/L) for the gradient.

The platelet inhibitory liquid* was made by a mixture of the same quantities of subsequent solutions:

1. 0.15 mol/L Na2 citrate# and 0.13 mmol/L Na3EDTA* (pH 7.4 at 25°C).

2. 0.001 g/L prostaglandin E₁ ^# and 1 mL 95% ethanol in H₂O. ^#Sigma-Aldrich, Missouri, U.S.A.

3. 2.7 mmol/L theophylline# dissolved in 0.15 mmol/L TRIS chloride buffer (pH 7.4 at 25°C). ^#Sigma-Aldrich, Missouri, U.S.A

A two-chamber gradient maker was put in use to manufacture linear gradients (Biorad, California, U.S.A.). The gradients were made in 50 mL test tubes guarding the density span of 1.09 kg/L to 1.04 kg/L. 7.63 g of the 1.09 kg/L Percoll™ mixture was covered in the lowest part of the test tube. Thereafter, 13.08 g of the 1.09 kg/L and 12.48 g of the 1.04 kg/L Percoll™ mixtures were engaged into two-chamber gradient maker to produce the gradient. Then, the 10 mL anticoagulated whole blood was thoughtfully covered on top of created 50 mL test tube including entirely produced gradient. The 50 mL test tube was then consequently centrifuged at 2767 g for 1½ hours. After centrifugation, the underside of the test tube was pierced and the content was segregated by gravity into 17 different density fractions [31]. All of these density fraction consists of about 2 mL of the test tube substance. In all fractions, platelet counts were analyzed with a CELL-DYN 4000 (Abbott Diagnostics, Illinois, U.S.A.). Platelet bound fibrinogen were measured with a Beckman Coulter EPICS XLMCL Flow Cytometer (Beckman Coulter, Inc., California, U.S.A.) [38]. Platelets were classified with a PE-conjugated antibody against GPIb (Dako AS, Denmark). Since no agonist was used to identify platelet-bound fibrinogen in this setting, it reflects the in vivo activity of this platelet.

The second tube, containing 3,5 ml venous blood anti-coagulated with 0.5 ml 0.129 M disodium citrate were used to measure platelet reactivity i.e. platelet bound fibrinogen with stimulation with platelet agonists. The agonists used were ADP (1.7 μmol/L and 8.5 μmol/L) (Sigma-Aldrich, Missouri, U.S.A.) and Thrombin Receptor Activating Peptide (TRAP-6) (57 μmol/L and 74 μmol/L) (Biotechnology Centre of Oslo, Norway). By this measurement it imitates in vitro platelet reactivity. The number of platelets (%) having more surface bound fibrinogen after stimulation than a negative control (containing 10 mmol/L EDTA and HEPES buffer) was applied as an investigational factor [39].

Statistics

Microsoft Excel^®, ver. 12.0.6514.5000 was employed for the statistical assessments. In text and tables are described mean values ± one Standard Deviation (SD). The unpaired Student’s t test was used for estimating quantitative data. p-values = 0.05 were utilized to specify significance.

Results

Platelet activity

The distribution of platelets in the 17 density fractions is shown in Figure 1. There was no demonstrated difference between the groups. Figure 2 shows in vivo platelet bound fibrinogen i.e. platelet activity in 17 different density fractions. MD had significantly more surface-bound fibrinogen among high and normal density platelets (fractions nos. 3-9) compared to CON, p<0.05. In vitro platelet bound fibrinogen, i.e. platelet reactivity is represented in Figure 3. After addition of platelets agonists, ADP (1.7 and 8.5 μmol/L) and TRAP- 6 (57 and 74 μmol/L) MD demonstrated significantly more reactive platelets when matched to CON, p<0.05. Finally, MD patients had larger platelets when evaluated against CON, i.e. Mean Platelet Volume (MPV), p<0.05 (data not shown).

Major Depression is Associated with High Platelet Activity and Reactivity

Abstract

Introduction

Material and Methods

Results

Discussion

Acknowledgement

References