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Investigation of the effects of 50 Hz magnetic fields
on platelet aggregation using a modified
aggregometer
Engin Sag
˘dilek
1,2
,Og
˘uz Sebik
1,3
&Gu
¨rbu
¨zC¸ elebi
1
1
Department of Biophysics, Faculty of Medicine, Ege University, Izmir, Turkey,
2
Department
of Biophysics, Faculty of Medicine, Uludag
˘University, Bursa, Turkey, and
3
Department of
Physiology, School of Medicine, Koc¸ University, Istanbul, Turkey
Purpose: Electromagnetic fields have various effects on intracellular calcium levels, free oxygen
radicals and various enzymes. The platelet activation pathway involves an increase in intracellular
calcium levels and protein kinase C activation; and free oxygen radicals play a mediating role in
this pathway. This study investigated whether 1 mT and 6 mT, 50 Hz magnetic fields had any
effects on platelet aggregation.
Materials and Methods: Blood from healthy volunteers was anticoagulated with either citrate or
heparin. Each sample was divided in half and assigned to exposure and control groups. Platelet
rich plasma samples in the exposure group were exposed to a 1 mT or a 6 mT, 50 Hz magnetic
field for 1.5 or 1 h, respectively. The samples from both exposure and control groups were
simultaneously evaluated using a modified optical aggregometer. Adenosine-diphosphate,
collagen, and epinephrine were used as inducing agents. The slopes of the aggregation curve,
the maximum values and the areas under the curves were recorded and compared.
Results: A significant effect was observed only in the 1 mT-citrate group. It was found that
magnetic field exposure significantly increased the maximum values and slopes of the collagen-
induced aggregations.
Conclusions: It was found that magnetic field exposure has an activating effect on platelet
aggregation.
Keywords Electromagnetic Fields, Platelet Aggregation, Optical Aggregometer
INTRODUCTION
Numerous studies have been conducted in recent years with the purpose of
elucidating the biological effects of electromagnetic fields. Various biological effects
were reported, including, but not limited to, effects on proliferation, differentiation,
apoptosis, deoxyribonucleic acid (DNA) damage/repair, and gene expression in both
normal and tumor cells (National Radiological Protection Board [NRPB], 2001;
Volpe, 2003; Vijayalaxmi and Obe, 2005; Blank, 2008; Blank and Goodman, 2008);
changes in the activity and concentration levels of reactive oxygen species in
leukocytes and other cell types (NRPB, 2001; Rosenspire et al., 2005; Seyhan and
Gu
¨ler, 2006); changes in the activity levels of some enzymes (Portaccio et al., 2003;
Address correspondence to Engin Sag
˘dilek, Uludag
˘U
¨niversitesi Tıp Faku
¨ltesi Biyofizik Anabilim
Dalı, 16059 Nilu
¨fer, Bursa, Turkey. E-mail: esagdilek@hotmail.com
Electromagnetic Biology and Medicine, 31(4): 382–393, 2012
Copyright QInforma Healthcare USA, Inc.
ISSN: 1536-8378 print / 1536-8386 online
DOI: 10.3109/15368378.2012.681822
382
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Simko and Mattsson, 2004; Morelli et al., 2005; Salamino et al., 2006); changes in the
stability and characteristics of biological membranes (Baureus et al., 2003; Volpe,
2003; Simko and Mattsson, 2004); and changes in intracellular calcium levels
(Dibirdik et al., 1998; Kristupaitis et al., 1998; Grassi et al., 2004; McCreary et al., 2006;
Piacentini et al., 2008). Despite the many studies conducted and many effects
reported, the exact mechanism of the interaction between electromagnetic fields
and biological systems still remains elusive.
Platelets are blood cells without nuclei that play a central role in hemostasis and
thrombosis having a lifespan of 8 –10 days. Sub-endothelial tissue that is exposed
due to endothelial damage and the von Willebrand Factor trigger the adhesion of the
platelets to the area of vascular damage. With the help of stimulating agents such as
collagen, ADP (adenosine diphosphate), epinephrine, thrombin, and serotonin, the
platelets in the area of injury change shape and begin secreting the contents of their
granules upon which thromboxane A
2
and platelet activating factor become
activated. The aggregating platelets get cross-linked with each other with fibrin
bridges and form the clot that serves as a mechanical plug (Hoffman et al., 2005).
Most of the receptors on platelets related to the aggregation inducing agents are
receptors bearing G proteins. Depending on the receptor, platelets are activated
though different pathways via different subtypes of G proteins. The main pathway for
platelet activation is the increase in intracellular calcium concentration and the
activation of protein kinase C (PKC) via the activation of phospholipase C (PLC)
(Hoffman et al., 2005). It is also known that radical oxygen species (ROS) play a role
in the regulation of platelet activation (Kro
¨tz et al., 2004; Olas and Wachowicz, 2007).
Previous studies on different cell types have reported that electromagnetic fields may
cause an increase in intracellular calcium concentrations, activate PKC, or increase
the concentration of ROS. Given the above facts, it is possible that extremely
low-frequency electromagnetic fields may have an activating effect on platelets.
This study investigated whether 1 mT and 6 mT, 50 Hz magnetic fields had any
effects on platelet aggregation. To this end, platelet-rich plasma samples were anti-
coagulated with either sodium citrate or heparin and exposed to 1 mT or 6 mT
magnetic fields at 50 Hz. The platelet aggregation profiles were then evaluated
with a modified optical aggregometer using ADP, collagen, and epinephrine as
inducing agents.
MATERIALS AND METHODS
The study was approved by Ege University Research Ethics Committee and was
conducted in compliance with its protocols. The study was conducted through the
period January–September 2009 in the laboratories of the Department of Biophysics
at Ege University Medical School (I
˙zmir, Turkey).
Subjects
The experiments were conducted on blood samples taken from 80 healthy volunteers
in the age range 19–51 years. The subjects and their families were screened for a
history of bleeding conditions or diseases, and it was made certain that they had not
smoked or taken any medication known to have an effect on platelets in the previous
ten days.
Collection of Blood Samples
Fasting blood samples were obtained from the subjects between 8.30 –10.00 AM.
Following the induction of a mild venous stasis in the upper arm, 20 ml of blood
(2 £10 ml) was drawn from the antecubital vein into test tubes containing the
Effects of 50 Hz MF on platelet aggregation 383
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anticoagulant using a 21 G needle. Heparinated tubes contained 5 U/ml heparin and
citrated tubes contained 0.0106 M sodium citrate (sodium citrate / blood ¼1/9).
Blood samples from only one subject were studied each day and only one type of
anticoagulant was used per day.
Preparation of Blood Samples
The 2 tubes of anticoagulated blood from the subject were poured into a 50 ml
tube and were mixed by turning the tube upside down three times. The blood
was then parted into 2 centrifuge tubes and centrifuged at 200g for 10 min. The
platelet rich plasma (PRP) from each tube was collected using a pipette and
poured in a tube. The PRP in this tube was mixed and separated into two equal
parts; one tube was marked as the exposure group (X) and the other was marked
as control (C). The tubes were sealed using parafilm. After the collection of the
PRP the remaining blood samples were centrifuged at 2000 g for 15 min and
the platelet poor plasma (PPP) samples were kept to be used for the calibration of
the optical aggregometer.
Magnetic Field Exposure
Magnetic field exposure was done using custom made Helmholtz coils (AYGEM
Plastik ve Elektrik San. ve Tic. Ltd. S¸ti. I
˙zmir, Turkey). The coils were built using a
2 mm copper wire wound on a circular frame of 50 cm diameter, with 350 turns in
each coil. The two coils were connected in series and the total resistance was
measured to be 7.2 ohms and inductance to be 274 mH. The city power was used to
supply electricity to the Helmholtz coils using an adjustable voltage transformer. A
current of 0.9 A (rms) was sufficient to create a magnetic field of 1 mT, and 10.4 A
(rms) for a magnetic field of 6 mT. The Helmoltz coils were powered 30 min before
the start of the experiments, in order for the wires to reach a stable temperature,
hence a stable resistance. The magnetic field intensity generated by the Helmholtz
coils was measured at the center of the coils once at the beginning and once at the
end of the exposure using a gaussmeter (F.W. BELL 5180, Sypris Test and
Measurement, Orlando, FL, USA). A maximum difference of ^0.05 mT was
measured between the two measurements.
The PRP samples in the exposure group were exposed to 1 mT-50Hz magnetic
field for 1.5 h or 6 mT-50 Hz magnetic field for 1 h. The PRP samples in the control
group were kept in the same ambient conditions for the duration of the exposure and
were not exposed to the magnetic field. The ambient magnetic field intensities were
measured to be in the range of 0.05– 0.15 mT direct current (DC) and 0.12 – 0.21 mT
alternative current (AC).
The ambient temperature was kept in the range of 20.9 ^2.6 C8. Temperature
measurements were made throughout the experiments around both exposure
and control groups (Digital Thermometer TPM-10, Ninghai BOTESHEN C, Ltd.,
Zhejiang, China). The average temperature changes between the beginning and
the end of the experiment were: 0.1 C8for the 1 mT exposure group, 0.2 C8for its
control group, 2.0 C8for the 6 mT exposure group, and 0.2 C8for its control
group. The 6 mT exposure experiment was limited to 1 hour, since longer exposure
durations resulted in higher temperature changes because of the heat dissipated
from the Helmholtz coils.
Experimental Groups
Using two different anti-coagulants and two different magnetic field intensities,
four experimental groups were formed. The names of the experimental groups,
number of subjects, the distribution of age and gender are presented in Table 1.
384 E. Sag
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Evaluation of Aggregation
Aggregation was measured using the Platelet Aggregation Profiler PAP-4CD
(Bio/Data, Horsham, PA, USA). In standard aggregometers, the plasma samples
are stirred using a magnetic stirrer which employs a magnetic stir bar inside the
tubes containing the samples. The stir bar is rotated by an external magnet attached
to a DC motor thus stirring the sample throughout the aggregation process (Fig. 1a).
For the aggregometer used in this study, the magnetic field intensities of the bar
magnets and the magnets attached to the DC motors were measured to be in the
range of 65 –75 mT around the poles, and 15 –25 mT along the sides. Since these
intensities were far above the exposure values to be used in the experiment
(1–6 mT), the aggregometer was modified so as to substitute its magnetic stirrer with
a custom made mechanical one. To this end, a platform that can move both
vertically and horizontally was mounted onto the aggregometer. Two pivot bearings
were fixed onto the platform and these bearings were connected to a DC motor with
adjustable speed. An optical reader was placed on one of the pivot bearings, and the
instantaneous speed was measured and displayed on a liquid crystal display (LCD)
screen in units of revolutions per minute (rpm). The mechanical and electronic
components of the mixer were manufactured in association with BAU Automation
Company, I
˙zmir, Turkey. The shafts and the stirring tips of the mixing unit were
manufactured at Ege University and were made of chrome. The stirring tips were of
the same shape and dimensions as the original magnetic stirring bars and were
welded to the chrome shafts. The shafts and the stirring tips were covered with a
transparent protective spray (1601, 3M Scotch, Austin, TX, USA). The magnets on the
DC motors of the aggregometer were removed. Plastic plugs were placed to the
bottom of the cuvettes in order to raise the sample tubes for 4 mm, since the samples
TABLE 1 Experimental groups.
1 mT-Heparin 1 mT-Citrate 6 mT-Heparin 6 mT-Citrate Total
Number of
subjects (n)
30 30 10 10 80
Female/Male 17/13 14/16 4/6 5/5 40/40
Age (mean ^sd) 34.9 ^7.9 33.1 ^6.7 32.3 ^6.2 35.2 ^6.5 34.0 ^7.1
(min-max) (20–48) (19 –51) (22 –43) (24– 49) (19–51)
mT: milliTesla
DC Motor
Optical
sensor
Stirring tip
Plastic
plug
DC Motor
Platelet rich
plasma
Stir bar
Light
source
Magnet
Coil
(a) (b)
FIGURE 1 The diagrams for a standard aggregometer (a) and the aggregometer modified for this
study (b).
Effects of 50 Hz MF on platelet aggregation 385
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were to be mixed from above mechanically rather than a stir bar from the bottom.
This step was necessary to create more room at the top of the sample tubes for
mixing to take place without interfering with the optical gadgetry of the instrument.
The unmodified and modified schematic diagrams of the aggregometer are shown
in Figs. 1a and b, respectively, and a photograph of the modified instrument is
shown in Fig. 2.
The aggregometer modified as such cannot start measuring the aggregation
process because it needs verification of the mixing and the rotation speed of the stir
bar in the sample to start. In the original design this verification is accomplished
with the help of a coil placed near the cuvette where a current is induced by the
spinning magnetic stir bar. In the modified aggregometer, this system was replaced
with a signal generator (PCGU 1000, 2 MHz USB PC Function Generator, Velleman
Instruments, Gavare, Belgium) which mimicked the current induced in the coil
in the original design and this current was fed to the relevant circuitry in the
aggregometer.
For the experiments the speed of the mixer was set at 900 rpm. The aggregometer
was calibrated to 100% light transmittance using the PPP sample. The exposure and
control PRP samples were randomly assigned to channel 1 and 2 of the aggregometer
and placed into the aggregometer cuvettes. Once the aggregometer was set to
measurement mode the inducing agent was added to the PRP samples. The stirring
tips were then lowered into the PRP samples, up to their determined positions.
The aggregation process was recorded by the aggregometer software.
ADP, collagen, and epinephrine were used as inducing agents (Bio/Data). The
final concentrations of the inducing agents were 4 mM for ADP, 20 mM for
epinephrine, 38 mg/ml for collagen when heparin was used as anticoagulant, and
95 mg/ml for collagen when sodium citrate was used. The experiments were run
FIGURE 2 The modified aggregometer with the external stirring system.
386 E. Sag
˘dilek et al.
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using two channels of the aggregometer, one randomly assigned to the control
group and the other to the exposure group. Experiments were conducted, first using
ADP, then collagen and last epinephrine as the inducing agent.
The parameters evaluated for the aggregation profiles are presented in Fig. 3.
The slope of the aggregation curve was calculated using the rising portion of the
0
20
10
50
40
80
70
30
90
100
60
10
986 7543
2
1
0
Primary
Aggregation (%)
Slope-1
Transition
time (s)
Slope-2
Maximum
Aggregation (%)
Epinephrine (heparin)
Area under
curve
Time (min)
(%) Aggregation
The point where
the inducing agent is
added
The point where the
stirring tips are lowered
into the PRP
The point where
the stirring tips
are removed
from the PRP
The aggregates
settling down
0
20
10
50
40
80
70
30
90
100
60
109867543210
Slope
ADP, Collagen, Epinephrine (Citrate)
Area under
curve
(%) Aggregation
Time (min)
The point where
the inducing agent
is added
The point where the
stirring tips are
lowered into the PRP
Maximum
aggregation (%)
The point where the
stirring tips are
removed from the PRP
The aggregates
settling down
(a)
(b)
FIGURE 3 The aggregation curves and the evaluation parameters acquired using ADP, collagen,
epinephrine (sodium citrate) (a) and epinephrine (heparin) (b).
Effects of 50 Hz MF on platelet aggregation 387
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aggregation curve as it rose from 10–20%. The maximum aggregation (%) was
determined from the point where the curve reached a peak and stayed there for
at least one minute. The area under the curve was determined as the integral from
the time the stirring tips enter the PRP to the time where the maximum aggregation
value is reached.
For aggregation curves having a biphasic nature, slope-1, primary aggregation
(%), transition time (s), and slope-2 (between 30 –40% aggregation) were also
measured (Fig. 3b).
The parameters evaluated were calculated using the computer software that
was bundled with the aggregometer.
All the procedures detailed above were completed within 2.5 h after the
blood samples were acquired (preparation of the PRP samples: 30 min, exposure:
60 –90 min, aggregation measurements: 30 min).
The workflow is presented as a flowchart in Fig. 4.
Statistical Analysis
The Paired Samples t-test was used to compare the control and exposure groups for
the 1 mT exposure experiments and the Wilcoxon signed-rank test was used for the
FIGURE 4 Flow diagram of experimental procedure.
388 E. Sag
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6 mT exposure experiments. The values of p lower than 0.05 were regarded as
statistically significant.
RESULTS
For PRP samples obtained from blood anti-coagulated with sodium citrate, exposure
to a 1 mT-50 Hz magnetic field for 1.5 h had a significant effect on the aggregation
parameters of collagen-induced aggregation. Exposure increased the slope of the
aggregation curve by 2.2 units on average ( p¼0.013) and increased the maximum
aggregation by 0.8% on average ( p¼0.033). No significant differences were observed
between the control and exposure groups for any other comparisons (Table 2).
Typical aggregation curves can be seen in Fig. 5.
In standard aggregometry, in measurements where aggregation is induced using
epinephrine, a biphasic aggregation curve, including primary and secondary
aggregation curves, is observed. In this study, where a modified aggregometer was
used, a biphasic curve was not observed for any of the PRP samples acquired from
blood samples anticoagulated with sodium citrate when epinephrine was used as the
inducing agent. For samples anticoagulated with heparin (n¼40), 26 of the
aggregation curves were of biphasic nature, whereas 14 were not. There was no
difference between the exposure and control groups for this parameter; either both
samples displayed a biphasic curve or neither did.
DISCUSSION
This study is the most comprehensive study on the effects of low-frequency
electromagnetic fields on platelet function in the literature up to date. It was found
that exposure to 1 mT-50 Hz magnetic field caused platelets to aggregate faster
and more when collagen was used as the inducing agent.
Two different anticoagulants were used in the study to be able to better differentiate
the possible effects of magnetic field exposure on platelet aggregation. Heparin is
a fast anti-coagulant that acts on antithrombin and inhibits mainly thrombin (F IIa)
and factors XIIa, XIa, Xa, and IXa. Sodium citrate, on the other hand, is a calcium
binding chelate; it prevents coagulation by binding to calcium, which plays an
active role in nearly all steps of the coagulation cascade (Hoffman et al., 2005).
It is remarkable that an effect was observed only for the 1 mT group when sodium
citrate was the anticoagulant and collagen was the inducing agent but not ADP or
epinephrine. When aggregation is induced by ADP or epinephrine, the pathway
leading to aggregation after the secondary messengers, is common to both inducers,
which includes the inhibition of adenylate cyclase and a decrease in cyclic adenosine
monophosphate (cAMP). Aggregation induced through the ADP P2Y
1
receptors
(ADP receptor subtype) starts with the activation of PLC
b
, which is followed by an
increase in intracellular calcium concentration and the activation of PKC.
Aggregation induced by collagen, on the other hand, starts with the activation of
PLC
g2
through tyrosine kinases, which again is followed by an increase in
intracellular calcium concentration and the activation of PKC (Hoffman et al., 2005).
The pathways activated by collagen and ADP, after activating different subtypes of
PLC, follow a similar track. In this sense, the activation of tyrosine kinases and PLC
g2
by magnetic exposure may explain our finding that the only effect we have found in
our experiments was for collagen-induced aggregations.
ROS also play a role in the regulation of platelet activation. It was reported that
exposure to collagen increased the superoxide anion (O
2
2
) and hydrogen peroxide
(H
2
O
2
) productionin platelets, and that ADP or thrombin had no sucheffect. ROS seems
Effects of 50 Hz MF on platelet aggregation 389
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to have a key function in the activation pathway of collagen-induced aggregation.
An increase in ROS concentrations leads to irreversible aggregation even when sub-
threshold concentrations of collagen are used, and a decrease in ROS concentrations
leads to an inhibition of aggregation (Pignatelli et al., 1998; Kro
¨tz et al., 2002).
Studies done on various cell types have reported that extremely low frequency
electromagnetic fields increase intracellular ROS concentrations (NRPB, 2001; Lupke
TABLE 2 The comparisons of the evaluated parameters for the exposure and control samples in all
experimental groups.
Exposure Control p
1 mT-Heparin (n ¼30)
ADP Slope 42.4 ^17.1 (16 –86) 42.3 ^15.9 (20–86) 0.854
Max Aggr (%) 44.7 ^14.8 (22.2 –74.6) 44.7 ^14.3 (19.2–76.2) 0.944
AuC 158.6 ^74.2 (57 –303) 157.0 ^75.4 (58–309) 0.419
Col Slope 31.2 ^11.0 (17 –57) 32.0 ^11.9 (16–64) 0.311
Max Agr (%) 47.2 ^13.5 (24.4 –70.4) 47.2 ^13.4 (24.6– 71.0) 0.949
AuC 182.6 ^60.5 (89 –343) 182.8 ^59.6 (87–322) 0.948
Epi Slope– 1 23.5 ^8.9 (7 –46) 23.1 ^9.0 (7 –45) 0.640
Pri Aggr (%) 17.8 ^15.9 (0 –47.2) 17.0 ^14.9 (0–45.4) 0.227
Transition time (s) 31.5 ^30.1 (0–91) 28.6 ^28.1 (0–99) 0.114
Slope–2 2.6 ^2.7 (0 –9) 3.0 ^2.9 (0–11) 0.118
Max Aggr (%) 42.9 ^14.3 (21.0 –73.4) 43.3 ^15.5 (15.2–71.6) 0.652
AuC 159.9 ^55.5 (51 –266) 162.9 ^60.1 (34–264) 0.413
1 mT-Citrate (n ¼30)
ADP Slope 36.0 ^10.0 (18 –54) 35.7 ^9.6 (16–51) 0.688
Max Aggr (%) 50.5 ^14.9 (17.6 –72.8) 50.9 ^14.6 (14.6–71.0) 0.604
AuC 189.0 ^71.7 (22 –298) 190.5 ^71.8 (20–293) 0.623
Col Slope 39.7 ^14.7 (16 –69) 37.5 ^14.2 (13–64) 0.013
Max Aggr (%) 54.9 ^14.1 (24.0 –80.4) 54.1 ^14.5 (21.6–77.4) 0.033
AuC 196.3 ^69.4 (53 –313) 189.8 ^70.6 (64–322) 0.175
Epi Slope 34.3 ^12.3 (6– 55) 33.6 ^12.6 (9 –53) 0.275
Max Aggr (%) 53.1 ^13.7 (21.0 –72.2) 52.7 ^14.2 (17.6–72.8) 0.387
AuC 201.3 ^63.6 (65 –297) 199.5 ^64.2 (57–288) 0.303
6 mT-Heparin (n ¼10)
ADP Slope 52.7 ^13.9 (30 –68) 52.0 ^14.6 (27–68) 0.355
Max Aggr (%) 53.0 ^8.7 (35.6 –63.6) 53.7 ^10.5 (32.6–71.0) 0.610
AuC 185.6 ^56.0 (88 –272) 188.3 ^60.3 (80–290) 0.341
Col Slope 36.5 ^16.5 (16 –61) 36.0 ^16.0 (14–63) 0.633
Max Aggr (%) 54.8 ^10.9 (38.4 –67.6) 55.6 ^11.5 (35.6–69.4) 0.326
AuC 204.5 ^63.8 (119 –290) 206.1 ^63.6 (115–287) 0.759
Epi Slope-1 23.4 ^7.2 (8 –36) 22.7 ^7.1 (8 –33) 0.590
Pri Aggr (%) 21.4 ^15.6 (0 –41.2) 21.3 ^15.7 (0–37.8) 0.833
Transition Time (s) 27.9 ^22.8 (0–62) 36.3 ^27.7 (0 –68) 0.075
Slope-2 1.9 ^1.5 (0 –4) 1.8 ^1.4 (0–3) 0.655
Max Aggr (%) 43.7 ^14.0 (15.8 –60.6) 43.9 ^13.2 (19.2–64.6) 0.683
AuC 155.5 ^67.0 (38 –274) 151.1 ^62.4 (47–236) 0.646
6 mT-Citrate (n ¼10)
ADP Slope 33.5 ^9.6 (19 –50) 33.5 ^12.6 (15–56) 0.944
Max Aggr (%) 45.6 ^11.2 (21.0 –60.0) 47.6 ^12.9 (22.2–70.0) 0.313
AuC 146.5 ^57.4 (52 –244) 156.0 ^71.9 (50–294) 0.357
Col Slope 35.2 ^8.4 (23 –48) 34.0 ^8.3 (21– 46) 0.356
Max Aggr (%) 50.7 ^11.4 (36.2 –70.4) 50.1 ^11.2 (33.2–67.6) 0.341
AuC 154.4 ^54.6 (85 –252) 149.2 ^54.6 (86–271) 0.507
Epi Slope 31.4 ^6.6 (22– 45) 30.8 ^7.6 (18 –44) 0.553
Max Aggr (%) 49.4 ^10.1 (36.2 –68.2) 49.4 ^11.1 (31.4–69.4) 0.859
AuC 150.5 ^50.4 (89 –244) 149.0 ^52.2 (88–240) 0.573
mT: milliTesla; ADP: Adenosine diphosphate; Col: Collagen; Epi: Epinephrine; Max Aggr: Maximum
aggregation; Pri Aggr: Primary aggregation; AuC: Area under curve.
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et al., 2004; Rosenspire et al., 2005; Seyhan and Gu
¨ler, 2006; Falone et al., 2007;
Morabito et al., 2010). It is probable that increased intracellular ROS concentrations
due to exposure are responsible for the effects reported in our study. This suggestion
is also plausible in the sense that the effects of exposure were seen only in collagen-
induced aggregations, where ROS are involved in the activation pathway.
In aggregometry, the inducing agents used to initiate aggregation make the
highest impact at the beginning of the aggregation process. In this sense, most of the
information regarding the inducing agent used and the pathway it activates is hidden
in the slope of the aggregation curve (Breddin, 2005). In our study, the most
FIGURE 5 Typical aggregation curves from 1 mT-Citrate group. ADP and collagen are the inducing
agents on top and bottom figures, respectively. Channel 1 (red) in both figures belongs to the
exposure sample and Channel 2 (green) belongs to the control sample.
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significant effect of magnetic field exposure was observed with respect to the slope of
the aggregation curve, when aggregation was induced using collagen. This may
indicate that the secondary messengers and the activation path related to collagen-
induced aggregation are possible places to investigate when searching for the
mechanisms of electromagnetic effects.
1 mT is an intensity that has been reported to have interactions of the cellular level
(Grassi et al., 2004; Wolf et al., 2005; Piacentini et al., 2008). Lower intensities were
also used in the literature, but ambient magnetic field intensities in the range of
0.05–0.15 mT DC and 0.12–0.21 mT AC prohibited us from using lower intensities in
our experimental setup.
An exposure intensity of 6 mT was used to control for the existence of an “effective
window” since it is previously established that magnetic fields effects may be limited
to a range of intensities or frequencies (Portaccio et al., 2005; Salamino et al., 2006).
The discrepancy between the results from 1 mT and 6 mT citrate groups may indicate
the existence of such an effect. On the other hand, the discrepancy may be
originating from the increase in temperature for the 6 mT exposure group (about
2
o
C), which may be significant enough to affect cell function. The heating of the
Helmholtz coils was also the initial factor that limited the 6 mT exposure time to one
hour instead of 1.5 h.
A novel stirring method was devised and used in this study; an interesting finding
was that for epinephrine-induced aggregations the familiar biphasic curve was not
observed for most of the aggregations. Further investigations are needed to elucidate
the cause and the mechanisms leading to this finding.
The elimination of the magnetic stirring system in an aggregometer to be replaced
by a non magnetic stirring system is a first in the literature and may act as a pioneer
to future platelet aggregation profiling studies.
CONCLUSIONS
The slopes and maximum values of the curves recorded from the collagen-induced
aggregations of PRP samples obtained from blood anti-coagulated with sodium
citrate were found to increase significantly upon exposure to a 1 mT-50 Hz magnetic
field for 1.5 h. The tyrosine kinases, PLC
g2
, and ROS which are involved in the platelet
activation pathway may be the probable targets of the magnetic fields.
Further studies are necessary using different methods of platelet function
assessment in order to elucidate the effects of extremely low-frequency electromagnetic
fields applied to platelets. Platelets provide a new vacant area of study for the
investigation of interaction of electromagnetic fields with biological systems.
ACKNOWLEDGEMENTS
This project was supported by Uludag
˘University Scientific Research Unit (Project
Number: T-2008/47) and Ege University Committee for Scientific Research Projects
(Project Number: 2008-TIP-013).
Declaration of Interest
The authors report no conflicts of interest. The authors alone are responsible for the
content and writing of this article.
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