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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institute of Medical Biochemistry, Vienna
Biocenter, University of Vienna; Institute for Histology and
Embryology, University of Vienna; and Institute of Cancer Research,
University of Vienna, Austria.
Lipid rafts are detergent-resistant, cholesterol- and
sphingolipid-rich membrane domains that are involved in important
cellular processes such as signal transduction and intracellular
trafficking. Stomatin, a major lipid-raft component of erythrocytes and
epithelial cells, is also an abundant platelet protein. Microscopical
methods and subcellular fractionation showed that stomatin is located mainly at the Stomatin (protein 7.2b, band 7.2), described as a
major protein component of the erythrocyte membrane,1-4
has been found to be absent from red cell membranes in patients with
overhydrated hereditary stomatocytosis.4,5 However,
because normal stomatin messenger RNA is present in the
reticulocytes6 of these patients and stomatocytosis does
not occur in stomatin knockout mice,7 the absence of
stomatin is an effect rather than the cause of the disease. Studies in
UAC epithelial cells revealed that stomatin forms high-order oligomers
and is associated with detergent-resistant membrane microdomains, which
are also termed lipid rafts.8,9 These characteristics of
stomatin and its unusual monotopic structure10 are
reminiscent of typical features of the caveolin proteins, which are
highly enriched at the cytoplasmic side of caveolae. In erythrocytes,
which do not express caveolins, stomatin and the distantly related
proteins flotillin-1 and flotillin-211 are the major
integral membrane proteins of lipid rafts, suggesting important, yet
distinct roles for these proteins at the interface between lipid rafts
and the cytoskeleton or signaling components.12
The concept of lipid rafts or membrane microdomains was originally
proposed to explain the vectorial transport of glycosyl phosphatidylinositol (GPI)-anchored proteins to the apical surface in
polarized cells.13,14 In the past decade, numerous studies have established the general characteristics of lipid
rafts.15-18 These microdomains contain mainly cholesterol
and sphingolipids as lipid constituents, which make them insoluble in
nonionic detergents, and are specifically enriched in different sets of
proteins, namely, GPI-anchored proteins such as Thy-1 and PrP;
palmitoylated proteins such as G proteins, Src family kinases, and
caveolins; tetraspanin proteolipids; and other signaling proteins.
However, lipid rafts are heterogeneous in their specific lipid and
protein content, and different types of rafts coexist at the plasma
membrane, even within close proximity. This was first shown by
Schnitzer et al,19 who separated caveolae, which are
morphologically and functionally distinct lipid microdomains of the
plasma membrane, from associated lipid rafts, which contain the bulk of
GPI-linked proteins.
Recently, confocal microscopy and biochemical analysis of the marker
proteins prominin and placental alkaline phosphatase revealed 2 distinct lipid microdomains in the microvilli of Madin-Darby canine
kidney cells.20 Moreover, a structural diversity of lipid rafts occupied by functionally different GPI-linked proteins has been
observed at the plasma membrane of neurons.21 However, lipid rafts are present not only at the plasma membrane but are also
found at internal membranes of the secretory14 or
endocytic22 pathways and at other intracellular
organelles, such as the Golgi complex23 and the
phagosomes.24 Hence, lipid rafts appear to be involved in
the complex network of intracellular membrane trafficking, an idea that
is supported by the various subcellular localizations of a specific
lipid-raft protein, depending on the cell type and state of
differentiation.23-26
Platelets are anuclear secretory blood cells that contain a complex
network of membrane structures, including the plasma membrane, the In this study, we showed that stomatin is an abundant platelet protein
located at the Chemicals
Antibodies
Identification of proteins Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining.33 Protein bands were excised, digested with trypsin, and identified by mass spectrometry (Bruker Reflex III matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF-MS]; Bruker Daltonics, Billerica, MA). The identity of the respective proteins was confirmed by Western blotting.Immunostaining for light microscopy For stomatin immunofluorescence staining on blood and bone marrow smears, cells were fixed in methanol at 4°C for 10 minutes, treated with 0.5% TX-100 in phosphate-buffered saline (PBS; pH 7.3) for 3 minutes at 4°C, and washed 3 times in PBS for 5 minutes each time. Blocking of unspecific binding sites and the staining protocol were carried out as described previously.34 The primary anti-stomatin antibody incubation was performed with the monoclonal antibody GARP-50 hybridoma supernatant, used either undiluted or diluted 1:3 with preincubation buffer, for 1 hour. Slides were examined and photographed with black and white film by using a Leitz fluorescence microscope.For immunocytochemistry, the cells were extracted with 0.5% TX-100 in PBS (pH 7.3) at 4°C for 3 minutes or 30 minutes and washed 3 times in PBS. The primary antibodies were detected by using the Chem-Mate horseradish peroxidase-diaminobenzidine system designed for automated immunostaining (DAKO, Glostrup, Denmark) with one modification. To improve the blocking, we used 3% hydrogen peroxide in 50% methanol for 3 minutes at room temperature to suppress endogenous peroxidase activity completely. After signal detection, the slides were dehydrated in ethanol and xylene and mounted with Eukitt (Merck, Darmstadt, Germany). Cells were photographed with a Nikon Mikrophot FX4 with black and white film. Immunoelectron microscopy Platelets were isolated from whole blood by density-gradient centrifugation through Lymphoprep (Nygaard, Denmark) and washed in PBS. For activation, platelets were incubated with 1 µM calcium ionophore A23187, as described previously.35 Resting or activated platelets were fixed for 30 minutes in 2% freshly depolymerized formaldehyde, washed, embedded, and sectioned as described previously.36 The sections were preincubated with PBS containing 5% bovine serum albumin and 0.5% Tween-20 (PBS-ST) for blocking of unspecific binding sites, and this was followed by incubation with monoclonal antibody GARP-50 at 22°C for 1 hour. The grids were rinsed 3 times, incubated with anti-mouse IgG linked to 10 nm colloidal gold (Biocell, Cardiff, United Kingdom) diluted 1:40 in PBS-ST (pH adjusted to 8.0), rinsed, counterstained, and examined as described previously.36For controls, we omitted the primary antibody or preabsorbed the specific antibody by incubation with a 100 to 1000 times molar excess of the N-terminal stomatin peptide for 20 minutes at 37°C. Both procedures abolished specific signal at the light and electron microscopical levels. While inspecting immunostained platelets, we recognized an increased cell-membrane labeling in activated platelets. To objectify this impression by morphometry on electron micrographs, we determined the grain density along the cell membrane (gold grains per membrane-length unit) in 10 resting and in 13 activated platelets and compared the 2 groups by using the Student t test. Isolation of platelets Whole blood (45 mL) was obtained from healthy donors by venipuncture and collected into heparin-treated tubes. Red cells and leukocytes were formed into pellets (by centrifugation at 200g for 15 minutes), the platelet-rich plasma was removed from the tubes, and 9 parts were mixed with 1 part acid-citrate-dextrose solution (25 g/L trisodium [Na3] citrate, 13.7 g/L citric acid, and 20 g/L glucose) as an anticoagulant. Platelets from this mixture were formed into pellets (2000g for 12 minutes) and resuspended in washing buffer (90 mM sodium chloride [NaCl], 5 mM potassium chloride [KCl], 36 mM Na3 citrate, and 10 mM EDTA). This washing step was repeated, remaining erythrocytes were removed by 1 or 2 quick spins, and the final platelet pellet was resuspended in the appropriate buffer for the various experiments. Buffer I (134 mM NaCl, 12 mM sodium bicarbonate, 2.9 mM KCl, 0.34 mM sodium phosphate dibasic, 1 mM magnesium chloride, 10 mM HEPES, and 5 mM glucose [pH 7.4]) was used for platelet homogenization. Buffer I containing 2 mM calcium chloride (CaCl2) was used for microvesicle isolation, calpain-inhibition experiments, flotation assays, and solubilization experiments. Typical platelet counts ranged from 0.5 to 2.0 × 109 cells/mL.Platelet homogenization and fractionation The platelets obtained from individual donors were resuspended in 1 mL buffer I (1-2 × 109 cells/mL) and mixed with 1 mL precooled 2 × Tris-citrate buffer (126 mM Tris-Cl, 190 mM NaCl, 10 mM KCl, and 24 mM citric acid [pH 6.5]) containing 2 mM PMSF, 2 µg/mL pepstatin A, 20 µg/mL leupeptin, and 20 µg/mL aprotinin. The platelet suspension was put on ice and homogenized in an Aminco French pressure cell (210 930 kg/m2). The platelet lysate was centrifuged (2000g for 10 minutes at 4°C) to obtain pellets of unhomogenized platelets. The supernatant was laid on top of a linear sucrose density gradient (30%-60% sucrose, 10 mM Tris-Cl, 150 mM NaCl, and 5 mM EDTA [pH 7.4]) and centrifuged (200 000g for 2 hours at 4°C) in an SW 40 rotor (Beckman Coulter, Fullerton, CA). Eighteen fractions (700 µL each) were collected from the top of the gradient, and the pellet at the bottom was resuspended in the 18th fraction. Aliquots from each fraction were mixed with reducing or nonreducing SDS-PAGE sample buffer, boiled for 3 minutes, and loaded on 11% polyacrylamide gels for analysis by Western blotting or silver staining.Preparation of lipid rafts Platelet pellets were resuspended in buffer I containing 2 mM CaCl2 (0.5-2 × 109 cells/mL) and lysed by addition of an equal volume of ice-cold TX-100 lysis buffer (2% TX-100, 100 mM Tris-Cl, 10 mM EGTA, 2 mM PMSF, 2 µg/mL pepstatin A, 20 µg/mL leupeptin, and 20 µg/mL aprotinin [pH 7.4]) or ice-cold CHAPS lysis buffer (TX-100 replaced by 2% CHAPS). The final detergent concentration was 1%, and lysates were mixed carefully and kept at 4°C for 20 minutes. The protein concentration ranged from 1.5 to 5.3 mg/mL. Subsequently, the lysates were mixed with 80% sucrose in Tris-buffered saline (TBS; 10 mM Tris-Cl and 150 mM NaCl [pH 7.4]) containing 1% TX-100 or 1% CHAPS to yield a final sucrose concentration of 50%. Then, 750 µL of the 50% sucrose layer was pipetted into SW 50 centrifuge tubes and overlaid with 750 µL each of 40%, 30%, 20%, and 10% sucrose in TBS [pH 7.4]. These sucrose step gradients were centrifuged in a precooled Beckman SW 50.1 rotor for 17 hours (230 000g at 4°C). Lipid rafts were visible as whitish bands at the 10% to 20% sucrose interface. Five fractions of 750 µL were collected from the top of the gradient, and the pellet at the bottom of the tube was resuspended in 750 µL TBS (pH 7.4). Aliquots of the fractions were mixed with SDS-PAGE sample buffer and boiled for 3 minutes. Equal volumes of the fractions were loaded on the gels and analyzed by Western blotting and silver staining as described previously.33 For silver staining, fraction 5, which contained by far the most protein, was diluted 1:10 with SDS-PAGE sample buffer.In one experiment, platelets resuspended in buffer I containing 2 mM
CaCl2 were treated with 0.5% m Solubilization experiments Platelets were resuspended in 0.5 mL buffer I containing 2 mM CaCl2 (2 × 109 cells/mL), and 100-µL aliquots were lysed by addition of 100 µL ice-cold lysis buffer containing the following detergents to yield the final concentrations indicated in parentheses: Lubrol (1%), CHAPS (1%), TX-100 (1%), OG (60 mM), and NP-40 (1%). The mixtures were incubated at 4°C for 20 minutes. Subsequently, insoluble material was formed into pellets by centrifugation (100 000g for 1 hour at 4°C). The pellet was resuspended in 200 µL of a mixture of equal volumes buffer I containing 2 mM CaCl2 and lysis buffer. The pellet and supernatant samples were mixed with SDS-PAGE sample buffer and boiled for 3 minutes. Equal volumes of these samples were analyzed by Western blotting.Isolation of microvesicles Platelets were resuspended in buffer I containing 2 mM CaCl2 (1-2 × 109 cells/mL) and activated by 4 µM calcium ionophore A23187 or 1 U/mL thrombin on a shaker for 30 minutes at 37°C. EDTA was added to produce a final concentration of 5 mM, and the platelet suspensions were centrifuged (2000g for 10 minutes) to form the platelets into pellets. The platelet pellets were resuspended in buffer I containing 5 mM EDTA. Microvesicles were formed into pellets from the supernatant (10 000g for 30 minutes at 4°C37), resuspended in buffer I containing 5 mM EDTA, centrifuged (2000g for 10 minutes) to remove contaminating platelets, formed into pellets again, and resuspended in buffer I containing 5 mM EDTA. The protein content of the corresponding platelet and microvesicle suspensions was determined with the DC protein assay, and equal protein amounts of platelets and microvesicles were analyzed by SDS-PAGE and Western blotting.Identification of calpain-cleavage products after platelet activation Platelets were resuspended in buffer I containing 2 mM CaCl2 (1 × 109 cells/mL). Aliquots (0.1 mL) of the platelet suspension were mixed with 1 µL of a 5 mg/mL dimethyl sulfoxide solution of the membrane-permeable calpain inhibitors E-64d38 and calpeptin,39 respectively, and incubated at 37°C for 30 minutes. Preincubated samples and respective controls were then activated by adding calcium ionophore A23187 (4 µM final concentration) at 37°C for 20 minutes. The samples were analyzed by SDS-PAGE and Western blotting.
To identify blood cells that express stomatin, we used the GARP-50
monoclonal antibody, which recognizes an N-terminal epitope of
stomatin, for immunocytochemical staining. In smear preparations, platelets revealed the highest signal density of all blood cells (Figure 1A). Typically, the label
distribution was heterogeneous, with most of the signal concentrated in
the center of the platelets. It consistently revealed a granular
pattern that was easier to recognize with immunofluorescence microscopy
(Figure 1B) than with immunocytochemistry (Figure 1A). Considerably
less staining was observed over the periphery of the platelets.
Megakaryocytes in bone marrow smears had intense and heterogeneous
labeling after GARP-50 immunofluorescence staining (Figure 1C).
Immunoelectron microscopy of resting platelets revealed that most of
the immunogold grains were located at the membrane of the
To examine the subcellular localization of stomatin more closely, we
analyzed platelet homogenates by using linear sucrose density-gradient
centrifugation to separate the different platelet organelles from the
bulk of cytosolic and plasma membrane proteins.40 Western
blot analysis of the gradient fractions revealed 2 distinct pools of
stomatin (Figure 2). Stomatin was partly
present in the low-density fractions, which contained most of the
cytoplasmic and plasma membrane proteins, and in fractions of higher
densities (~ 50% sucrose), in which intracellular granules are
expected. This distribution was paralleled by P-selectin and VWF, 2 marker proteins of
Because of the association of stomatin with lipid rafts in a variety of
cells, we wanted to know whether stomatin is also present in platelet
lipid rafts. To isolate lipid rafts, platelets were lysed in TX-100 on
ice and subjected to discontinuous density-gradient centrifugation. The
Western blot analysis shown in Figure 3A
demonstrates that stomatin and the flotillin proteins were partly
present in the low-density fractions, together with the Lyn protein.
The most abundant proteins of the lipid-raft fractions (fractions 1-3),
as shown on silver staining, were analyzed by MALDI-TOF-MS and
identified as stomatin, actin, and CD36 (Figure 3A). However, a large
amount of stomatin remained in the high-density fractions (fractions 4 and 5), indicating a high solubility in TX-100. Pretreatment of
platelets with the cholesterol-depleting agent m
The unusual high solubility of platelet stomatin in TX-100 at 4°C has
2 possible explanations: (1) a large part of stomatin is not present in
lipid rafts, (2) stomatin-specific lipid rafts are largely solubilized
by TX-100. To address this issue, we tested the solubility of platelet
stomatin in different detergents by using stringent centrifugation
conditions to recover the membrane skeleton and small lipid rafts in
the pellet fraction. Stomatin was predominantly solubilized by NP-40
and TX-100, poorly solubilized by OG, and virtually insoluble in Lubrol
and CHAPS (Figure 4). Flotillin-2 was
nearly insoluble in all tested detergents except OG. Notably, the mild
detergents Lubrol and CHAPS, though chemically quite different, have
both been used to isolate specific lipid rafts that would be dissolved
in TX-100.20,41 The insolubility of stomatin in Lubrol and
CHAPS therefore suggested a specific association of stomatin with lipid
rafts sensitive to TX-100.
We did observe a general TX-100 sensitivity of platelet lipid rafts at
low ratios of protein to detergent (< 2 mg/mL; data not shown);
however, at 5 mg protein/mL 1% TX-100, stomatin rafts appeared largely
soluble, whereas flotillin rafts were stable. Indeed, flotation
experiments with CHAPS lysates revealed that virtually all of the
stomatin was present in the low-density lipid-raft fraction (Figure
5B). This fraction also contained nearly
all of the proteins flotillin-1, flotillin-2, and CD36 and about 50% of the Lyn protein. In contrast, clathrin was detectable only in the
high-density soluble and pellet fractions.
The silver stain (Figure 5) showed that a distinct set of proteins was
present in the lipid-raft fraction. The strongest bands were analyzed
by MALDI-TOF-MS and identified as stomatin, flotillin-1, flotillin-2,
CD36, CD9,
Platelet activation results in fusion of the
Activation of platelets is known to result in the activation of
proteases and the degradation of several cytoskeletal and membrane
proteins.42-44 We found that degradation of stomatin and flotillin-2 depended on the presence of calcium and could be inhibited by leupeptin, an inhibitor of serine and cysteine proteases (data not
shown). More specifically, the membrane-permeable calpain inhibitors
E-64d and calpeptin almost completely inhibited stomatin cleavage and
significantly reduced the degradation of flotillin-2 and spectrin
(Figure 8). In contrast to these
proteins, flotillin-1 was not subject to activation-induced
degradation. Similar results (data not shown) were obtained when
platelets were permeabilized with saponin45 and incubated
with a calpain inhibitor peptide.46
In this study, we showed that stomatin is an abundant protein
constituent of platelets, which had the highest immunocytochemical signal density of all blood cells (Figure 1A). However, in contrast to
erythrocytes, in which stomatin is located exclusively at the (plasma)
membrane, stomatin in platelets was predominantly localized intracellularly rather than at the cell periphery.
Immunoelectron microscopy showed that most of the immunogold labeling
was in close proximity to Subcellular fractionation of platelet organelles revealed that
flotillin-1 and flotillin-2 were located predominantly at the platelet
plasma membrane or another light membrane, whereas stomatin was
associated mainly with Whereas stomatin was only partly present in lipid rafts, flotillin-2,
when isolated after TX-100 solubilization of platelets (Figure 3), was
present largely in the low-density fractions of the sucrose
gradient. As noted above, the stability of the platelet rafts
in TX-100 generally depended on a high ratio of protein to detergent
and decreased at concentrations below 2 mg protein/mL TX-100 solution.
However, the difference in solubility of stomatin and flotillin-2 at a
high protein concentration (5 mg/mL) suggested that these proteins are
associated with different types of lipid rafts varying in their degree
of TX-100 stability (Figures 3 and 4). About 50% to 70% of
flotillin-2 was associated with lipid rafts (Figure 3, lanes 2 and 3),
and 30% to 50% was found in the pellet and high-density fraction,
indicating a partial association with the cytoskeleton (Figure 3).
Pretreatment of platelets with 0.5% m In contrast to TX-100, the detergents CHAPS and Lubrol did not solubilize stomatin (Figure 4). We showed that the insolubility of stomatin in CHAPS was due to its association with lipid rafts, since virtually all of the stomatin was present in the low-density fractions (Figure 5, lanes 1 and 2) of the CHAPS flotation assay. Similarly, flotillin-1 and flotillin-2 were found exclusively in the low-density fractions. The altered flotation behavior of stomatin and the flotillins after CHAPS lysis indicates that lipid rafts are more stable in CHAPS than in TX-100 (Figures 3B and 5B). Remarkably, nearly 60% of the total cellular cholesterol was present in the lipid-raft fractions (Figure 5, lanes 1 and 2). Moreover, it is notable that platelet lipid rafts were generally stable in CHAPS, even at the very low ratio of protein to detergent of 0.6 mg protein/mL detergent solution (data not shown). Simons et al41 previously showed that neuronal lipid rafts are stable in CHAPS but dissolved in TX-100. The reduction in the amount of lipid-raft-associated actin after CHAPS lysis (compare silver stains shown in Figures 3A, 5A, and 6A and Western blots in 6B) indicates that this detergent disrupts the interaction between the actin cytoskeleton and the lipid rafts. Apart from stomatin and the flotillin proteins, the major protein
components of the CHAPS-insoluble membrane domains were identified as
One important conclusion that can be drawn from the flotation
experiments and the subcellular localization of stomatin is that the
We found that lipid rafts of different intracellular membrane origins
are recovered in the low-density fraction of the sucrose gradients,
thereby showing that careful biochemical and immunoelectron microscopical analyses are necessary to localize the various types of
lipid rafts within the complex membrane system of platelets. Different types of lipid rafts in platelets were also suggested by the
results of Waheed et al.48 Moreover, the fact that Lyn is
localized predominantly to coated vesicles33 indicates
that these clathrin-coated, endocytic vesicles might also contain lipid rafts. Thus, it remains to be determined whether the flotillin proteins
are located at lipid rafts of the plasma membrane, endocytic vesicles,
or another platelet organelle. Flotillin-1 was previously identified at
different subcellular locations depending on the cell type and state of
differentiation. It was originally described as a caveolar
protein25; however, in neurons26 and
erythrocytes,12 it is found in noncaveolar rafts at the
plasma membrane. In macrophages, flotillin-specific lipid rafts are
recruited to the phagosome,24 whereas in Chinese hamster
ovary, PC12, HeLa, and normal rat kidney cells, flotillin-1 is found
predominantly in the Golgi complex.23 Interestingly, on
differentiation of PC12 cells, flotillin-1 is relocated to the plasma
membrane.23 The exact subcellular location of the
flotillins in platelets remains to be determined; however, the results
of our subcellular-fractionation (Figure 2) and detergent-solubility experiments (Figure 4) exclude the possibility that they colocalize with stomatin to Platelets activated by calcium ionophore, thrombin, or collagen release microvesicles, which are thought to exert procoagulant activity at a distance from the site of platelet activation.37 Microvesicle shedding has been shown to depend on the activity of calpain by cleavage of key cytoskeleton and membrane protein components.42,54 In contrast to flotillin-1, stomatin and flotillin-2 were cleaved by calpain (Figure 8). However, whereas both flotillin proteins remained predominantly at the platelet plasma membrane after thrombin activation, stomatin became enriched in the released microvesicles (Figure 7). The specific behavior of stomatin and the flotillin proteins in the activation-induced microvesiculation process possibly reflects an underlying mechanism of lipid-raft sorting. Whereas flotillin-specific rafts are excluded from microvesicles, stomatin-specific rafts are sorted specifically into the budding microvesicle. The mechanism of this sorting process and its functional importance remain to be elucidated.
We thank Elisabeth Legenstein for performing the cholesterol assays, Edina Csaszar for mass spectrometric analyses, Dr Alexander Nader for bone marrow smears, and Dr Elisabeth Koller for helpful discussions.
Submitted July 19, 2001; accepted March 26, 2002.
Supported by grant P12907 from the Austrian Science Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Rainer Prohaska, Institute of Medical Biochemistry, University of Vienna, Vienna Biocenter, Dr Bohr-Gasse 9/3, A-1030 Vienna, Austria; e-mail: prohaska{at}bch.univie.ac.at.
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© 2002 by The American Society of Hematology.
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granules
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Abstract
Introduction
-granular membrane. The lipid-raft marker proteins flotillin-1 and flotillin-2 were also present in platelets but excluded
from 
3, and the glucose transporter GLUT-3.
Stomatin, the flotillins, and CD36 were exclusively present in this
lipid-raft fraction. Activation of platelets by calcium ionophore
A23187 or thrombin led to translocation of stomatin to the plasma
membrane, cleavage by calpain, and specific sorting into released
microvesicles. In conclusion, this study demonstrated the existence of
