Epigallocatechin-3-gallate has an anti-platelet effect in a cyclic AMP-dependent manner.

Abstract

In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation.
Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation.
EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation.
These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

Full text

Page 1

337
Journal of Atherosclerosis and Thrombosis　Vol.19, No.4
Original Article
Epigallocatechin-3-Gallate Has an Anti-Platelet Effect in a Cyclic
AMP-Dependent Manner
Woo-Jeong Ok1, Hyun-Jeong Cho2, Hyun-Hong Kim1, Dong-Ha Lee1, Hye-Yeon Kang2,
Hyuk-Woo Kwon1, Man Hee Rhee3, Mujo Kim4 and Hwa-Jin Park1
1Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering and Regional Research Center, Inje
University, Gyungnam, Korea
2Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon, Korea
3Laboratory of Veterinary Physiology & Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu, Korea
4Pharma Foods International Co. Ltd., Kyoto, Japan
Aim: In this study, we investigated the effect of (−)-epigallocatechin-3-gallate (EGCG) on cyclic
nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in colla-
gen (10 μg/mL)-stimulated platelet aggregation.
Methods: Washed platelets (108/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincu-
bated for 3 min at 37℃ in the presence of 2 mM exogenous CaCl2 with or without EGCG or other
materials, stimulated with collagen (10 μg/mL) for 5 min, and then used for the determination of
intracellular cytosolic Ca2＋ ([Ca2＋]i), thromboxane A2 (TXA2), adenosine 3’,5’-cyclic monophosphate
(cAMP), guanosine 3’,5’-cyclic monophosphate (cGMP), and VASP phosphorylation.
Results: EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both
[Ca2＋]i mobilization and TXA2 production. Of two aggregation-inhibiting molecules, cAMP and
cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated
cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMP-
specific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser157,
a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser239, a
cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-
Ser157 phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and
an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenyl-
ate cyclase activation and subsequently phosphorylates VASP-Ser157 through A-kinase activation to
inhibit [Ca2＋]i mobilization and TXA2 production on collagen-induced platelet aggregation.
Conclusions: These results strongly indicate that EGCG is a beneficial compound elevating cAMP
level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-
mediated thrombotic diseases.
J Atheroscler Thromb, 2012; 19:337-348.
Key words; (−)-Epigallocatechin-3-gallate, Adenosine 3’,5’-cyclic monophosphate, Intracellular cytosolic
Ca2＋, Thromboxane A2, Vasodilator-stimulated phosphoprotein-Ser157 phosphorylation
Introduction
Platelet aggregation is absolutely essential for the
formation of a haemostatic plug when normal blood
vessels are injured; however, the interactions between
platelets and collagen can also cause circulatory disor-
ders, such as thrombosis, atherosclerosis, and myocar-
dial infarction1). Accordingly, inhibition of the plate-
Address for correspondence: Hwa-Jin Park, Department of
Biomedical Laboratory Science, College of Biomedical Science
and Engineering and Regional Research Center, Inje University,
607, Obang-dong, Gimhae, Gyungnam 621-749, Republic of
Korea
E-mail: mlsjpark@inje.ac.kr
Received: June 1, 2011
Accepted for publication: October 18, 2011
Woo-Jeong Ok, Hyun-Jeong Cho and Hyun-Hong Kim contributed equally to this work.

Page 2

338
Ok et al.
loyl group at the 3’ position of catechin, which is
known to have anti-platelet13-16), anti-oxidative17),
anti-inflammatory18), anti-carcinogenic19), anti-hyper-
tensive20), and various biological effects21). In addition,
it is known that EGCG has anti-platelet activity by
inhibiting p38 mitogen-activated protein kinase and
extracellular signal-regulated kinase-1/213) and by
reducing thrombin-induced [Ca2＋]i increase via inhi-
bition of Syk and Lyn activities14). EGCG has also
been reported to inhibit collagen-induced PLC-r 2
activity and TXA2 production15); however, Ca2＋-antag-
onistic intracellular molecules such as cAMP and
cGMP were not determined in their previous
reports13-15). In particular, it is known that EGCG has
no effect on the elevation of cAMP in collagen-acti-
vated platelets16); however, in the present study, we
found that EGCG strongly stimulated the phosphory-
lation of VASP-Ser157 via the cAMP/A-kinase pathway
rather than the phosphorylation of VASP-Ser239 by the
cGMP/G-kinase pathway.
Materials and Methods
Materials
Collagen was obtained from the Chrono-Log
Corporation (Havertown, PA, USA). Fura 2-AM,
8-bromoadenosine-3’,5’-cyclic monophosphorothio-
ate, Rp-isomer (Rp-8-Br-cAMPS), 9-(tetrahydro-2´-
furyl)adenine (SQ22536), 1-ethyl-4-[(1-methylethyli-
dene)hydrazino] 1H-pyrazolo[3,4-b]pyridine-5-car-
boxylic acid (Etazolate), (−)-epigallocatechin-3-gallate
(EGCG), and other reagents were obtained from
Sigma Chemical Corporation (St. Louis, MO, USA).
cAMP, cGMP, and TXB2 enzyme immunoassay (EIA)
kits were purchased from GE Healthcare (Bucking-
hamshire, UK).
Preparation of Washed Rat Platelets
Blood was collected from Sprague-Dawley rats
(6-7 weeks old, male), and anti-coagulated with ACD
solution (0.8% citric acid, 2.2% sodium citrate,
2.45% glucose). Platelet-rich plasma was centrifuged
at 125×g for 10 min to remove red blood cells, and
the platelets were washed twice with washing buffer
(138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3,
0.36 mM NaH2PO4, 5.5 mM glucose, and 1 mM
EDTA, pH 7.4). The washed platelets were then
resuspended in suspension buffer (138 mM NaCl, 2.7
mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4,
0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH
7.4) to a final concentration of 5×108/mL. All of the
above procedures were carried out at 25℃ to avoid
platelet aggregation on cooling. The ethics committee
let-collagen interaction might be a promising
approach for the prevention of thrombosis. An impor-
tant role in the mechanism by which collagen induces
platelet aggregation is played by thromboxane A2
(TXA2) formation2), which also contributes to an
increase in cytosolic free Ca2＋ ([Ca2＋]i). An increase in
[Ca2＋]i activates both the Ca2＋/calmodulin-dependent
phosphorylation of myosin light chain and the diacyl-
glycerol (DG)-dependent phosphorylation of pleck-
strin to induce platelet aggregation3, 4). In addition,
DG can be hydrolyzed by DG lipase to produce ara-
chidonic acid (20:4), a precursor of TXA2, which is a
potent platelet aggregation agent generated from 20:4
liberated when phosphatidylinositol 4, 5-bisphosphate
(PIP2) is broken down by phospholipase C (PLC) in
collagen-, thrombin-, and ADP-activated platelets3-5).
Verapamil and theophylline have anti-platelet func-
tions that elevate the adenosine 3’,5’-cyclic mono-
phosphate (cAMP) level, and then decrease [Ca2＋]i, an
essential factor for platelet aggregation. Vasodilators
(such as molsidomine and nitroprusside) and guano-
sine 3’,5’-cyclic monophosphate (cGMP) phosphodi-
esterase (PDE) inhibitors (such as zaprinast and
erythro-9-[2-hydroxy-3-nonyl]adenine) elevate cGMP
levels in platelets5). It is believed that cGMP is pro-
duced via the activation of guanylate cyclase in the
presence or absence of nitric oxide (NO). NO, synthe-
sized in platelets, decreases agonist-elevated [Ca2＋]i6)
and has a role in inhibiting platelet activation7).
Accordingly, from the above-mentioned reports, it can
be seen that antiplatelet substances have activities to
inhibit [Ca2＋]i mobilization or TXA2 production,
aggregation-inducing molecules, and increase the pro-
duction of cAMP or cGMP, aggregation-inhibiting
molecules. It is known that the effects of cAMP and
cGMP on platelets are mediated via cAMP- and
cGMP-dependent protein kinases (A-kinase,
G-kinase), which phosphorylate substrate protein,
vasodilator-stimulated phosphoprotein (VASP),
involved in the platelet aggregation inhibitory path-
way8-10). VASP is composed of 46 kDa-dephosphopro-
tein and 50 kDa-phosphoprotein9). If VASP is phos-
phorylated by A-kinase or G-kinase, phosphorylation
of VASP (p-VASP) shifts from 46 kDa-dephosphopro-
tein to 50 kDa-phosphoprotein8, 9). Ser157 at 50 kDa
of VASP is phosphorylated by the cAMP/A-kinase
pathway, whereas Ser239 at 50 kDa of VASP is phos-
phorylated by the cGMP/G-kinase pathway11, 12);
therefore, phosphorylation of Ser157 or Ser239 at 50
kDa of VASP is a useful indicator for monitoring
cAMP/A-kinase and cGMP/G-kinase pathways.
A major catechin analogue, (−)-epigallocatechin-
3-gallate (EGCG, Fig.1A), from green tea has a gal-

Page 3

339
Anti-Platelet Effects of EGCG
sensitive, so the tube containing the platelet-rich
plasma was covered with aluminum foil during load-
ing. The fura 2-loaded washed platelets were prepared
using the procedure described above and platelets 108/
mL were preincubated for 3 min at 37℃ with various
concentrations of EGCG in the presence of 2 mM
CaCl2 and then stimulated with collagen (10 μg/mL)
for 5 min for evaluation of [Ca2＋]i. Fura 2 fluores-
cence was measured with a spectrofluorimeter (SFM
25; Bio-Teck Instrument, Italy) with an excitation
wavelength that was changed every 0.5 sec from 340
to 380 nm; the emission wavelength was set at 510
nm. The [Ca2＋]i values were calculated using the
method of Schaeffer22).
Measurement of TXB2
Washed platelets (108/mL) were preincubated
with or without EGCG for 3 min in the presence of 2
for animal experiments of Inje University (Gimhae,
Gyungnam, Korea) approved these animal experi-
ments.
Measurement of Platelet Aggregation
Washed platelets (108/mL) were preincubated for
3 min at 37℃ in the presence of 2 mM exogenous
CaCl2 with or without EGCG and then stimulated
with collagen (10 μg/mL) for 5 min. Aggregation was
monitored using an aggregometer (Chrono-Log,
Corp.) at a constant stirring speed of 1,000 rpm. Each
aggregation rate was evaluated as an increase in light
transmission. Suspension buffer was used as a refer-
ence. EGCG was dissolved in distilled water.
Determination of [Ca2＋]i
Platelet-rich plasma was incubated with 5 μM
fura 2-AM at 37℃ for 60 min. Fura 2-AM is light-
Fig.1. Effects of EGCG on collagen-stimulated platelet aggregation. (A) Chemical structure of EGCG from
green tea leaves. (B) The concentration threshold of collagen on platelet aggregation. Washed platelets
(108/mL) were stimulated with various doses of collagen for 5 min in the presence of 2 mM CaCl2 at
37℃. (C) Effects of EGCG pretreatment on collagen-stimulated platelet aggregation. Washed platelets
(108/mL) were preincubated with various concentrations of EGCG (1 to 50 μM) in the presence of 2
mM CaCl2 for 3 min at 37℃, and then stimulated with collagen (10 μg/mL) for 5 min. Platelet aggre-
gation (%) was recorded as an increase in light transmission. Inhibition by EGCG was recorded as a
percentage of the collagen-induced aggregation rate. Data are expressed as the means±S.E.M. (n=4).
＊p＜0.05. ＊ ＊p＜0.001.
90 90
90
n (%)
Inhibition
60
60
70
70
80
80
90
90
0
0
10
10
20
20
30
30
+
＋
1
1
Collagen
(10 μg/mL)
EGCG (μM)
EGCG (μM)
+
＋
-
+
＋
5
5
+
＋
10
10
+
＋
30
30
+
＋
50
50
Light tra
**
＊ ＊
**
＊ ＊
50
50
60
60
70
70
80
80
mission (%)
Light transm
1010
10
20
20
30
30
40
40
50
50
**
＊＊
**
＊ ＊
**
＊ ＊
00
0
10
10
20
20
30
30
40
40
EGCG (µM)
EGCG (μM)
0
0
0
0
10
10
30
30
50
50
*
＊
0
0
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
Collagen (μg)
Collagen (μg/mL)
AC
B
100
Light transmission (%)
Inhibition (%)
＊ ＊
＊ ＊
＊ ＊
＊
90
80
70
60
50
40
Light transmission (%)
Collagen
(10μg/mL)
－

Page 4

340
Ok et al.
was increased up to 81.5±2.4%; however, various
concentrations of EGCG (1 to 50 μM) significantly
reduced collagen-stimulated platelet aggregation in a
dose-dependent manner (Fig.1C).
Effects of EGCG on Regulation of Aggregation-
Inducing Molecules, [Ca2＋]i and TXA2
As shown in Fig.2A, when washed platelets (108/
mL) were stimulated by collagen (10 μg/mL), the
level of [Ca2＋]i increased from 48.6±9.2, the basal
level, to 90.2±8.3 nM; however, this was significantly
mM CaCl2, and activated for 5 min with collagen (10
μg/mL). The reactions were terminated by the addi-
tion of ice-cold EDTA (5 mM) and indomethacin (0.2
mM). The amount of TXB2, a stable metabolite of
TXA2, was determined with Synergy HT Multi-Model
Microplate Reader (BioTek Instruments, Winoosku,
VT, USA) using a TXB2 EIA kit.
Measurement of cAMP and cGMP
Washed platelets (108/mL) were preincubated for
3 min at 37℃ with various concentrations of EGCG
or other agonists in the presence of 2 mM CaCl2, and
then stimulated with collagen (10 μg/mL) for 5 min
for platelet aggregation. The aggregation was termi-
nated by the addition of 80% ice-cold ethanol. cAMP
and cGMP were measured Synergy HT Multi-Model
Microplate Reader (BioTek Instruments) using cAMP
and cGMP EIA kits.
Western Blot for Analysis of VASP Phosphorylation
Various volumes of platelet lysates containing the
same protein (15 μg) were used for analysis. Protein
concentrations were measured using a BCA protein
assay kit (Pierce Biotechnology, USA). An 8-10%
SDS-PAGE was used for electrophoresis and a PVDF
membrane was used for protein transfer from the gel.
The dilutions for anti-VASP, anti-phosphor-VASP
(Ser157), and anti-phosphor-VASP (Ser239), and anti-
rabbit IgG-HRP were 1:1000, 1:1000, 1:1000, and
1:10000, respectively. The membranes were visualized
using enhanced chemiluminescence (ECL). Blots were
analyzed using Quantity One, Ver. 4.5 (Bio-Rad, Her-
cules, CA, USA).
Statistical Analysis
The experimental results are expressed as the
means±S.E.M. and are accompanied by the number
of observations. Data were assessed by analysis of vari-
ance (ANOVA). If this analysis indicated significant
differences among the group means, then each group
was compared by the Newman-Keuls method. P ＜
0.05 was considered significant.
Results
Effects of EGCG on Collagen-Induced Platelet
Aggregation
The concentration of collagen-induced maximal
platelet aggregation was approximately 10 μg/mL
(Fig.1B)23); therefore, 10 μg collagen/mL was used as
the platelet agonist in this study. When washed plate-
lets (108/mL) were activated with 10 μg collagen/mL
in the presence of 2 mM CaCl2, the aggregation rate
Fig.2. Effects of EGCG on collagen-induced [Ca2＋]i mobili-
zation and TXA2 production. (A) Effects of EGCG on
collagen-induced [Ca2＋]i mobilization. Fura 2-loaded
platelets (108/mL) were preincubated with various
concentrations of EGCG in the presence of 2 mM
CaCl2 for 3 min at 37℃, and then collagen (10 μg/
mL) was added. [Ca2＋]i was determined as described
in “Materials and Methods”. (B) Effects of EGCG on
the production of TXB2 stimulated by collagen.
Washed platelets (108/mL) were preincubated with
EGCG for 3 min in the presence of 2 mM CaCl2, and
then stimulated with collagen (10 μg/mL). TXB2 was
determined as described in “Materials and Methods”.
The content of TXB2 was measured using a TXB2 EIA
kit. Data are expressed as the means±S.E.M. (n=4). ＊
＊p＜0.001.
100
90
80
70
60
50
40
30
20
10
0
＊ ＊
＊ ＊
＊ ＊
＊ ＊
＊ ＊
[Ca2＋]i (nM)
Collagen (10μg/mL)
EGCG (μM)
＋
－
＋
1
＋
5
＋
10
＋
30
＋
50
－
－
450
400
350
300
250
200
150
100
50
0
＊ ＊
TXB2 (ng/108 platelets)
Collagen (10μg/mL)
EGCG (50μM)
－
＋
＋
－
＋
＋
－
－
TXB2
(ng/108 platelets)

Inhibition (%)
Collagen (10μg/mL)
Collagen (10μg/mL)
＋EGCG (50μM)
356.1±46.9
37.3±0.4
0
89.5
A
B

Page 5

341
Anti-Platelet Effects of EGCG
reduced by various concentrations (1 to 50 μM) of
EGCG in a dose-dependent manner (83.5% inhibi-
tion at 50 μM). We next determined whether EGCG
blocks the production of TXA2 by collagen stimula-
tion. The TXA2 (determined as TXB2) level in intact
platelets was 4.0±0.1 ng/108 platelets, and this was
markedly increased to 356.1±46.9 ng/108 platelets in
collagen-stimulated platelets (Fig.2B); however,
EGCG potently reduced the production of TXA2 to
37.3±0.4 ng/108 platelets (89.5% inhibition at 50
μM). Because collagen-produced TXA2 resulted from
Ca2＋-dependent PLC activation, it is suggested that
the inhibition of TXA2 production (Fig.2B) by
EGCG might be associated with the inhibition of col-
lagen-elevated [Ca2＋]i (Fig.2A). Intracellular cAMP or
cGMP reduce platelet agonist-elevated [Ca2＋]i and
TXA2 to inhibit platelet aggregation24); therefore, we
next investigated whether EGCG up-regulates the cel-
lular level of cAMP or cGMP.
Effects of EGCG on Production of cGMP and cAMP,
Aggregation-Inhibiting Molecules
Collagen decreased intracellular cGMP from
1.40±0.15 pmoL/108 platelets (basal level) to 1.21±
0.15 pmoL/108 platelets in washed platelets (Table 1).
When the platelets were incubated in the presence of
both EGCG and collagen, cGMP by EGCG did not
increase in a dose-dependent manner (Fig.3A). The
cGMP level (1.43±0.04 pmoL/108 platelets) in the
presence of both EGCG (50 μM) and collagen (10
μg/mL) increased to 18.2 % as compared with that
(1.21±0.15 pmoL/108 platelets) in the presence of
collagen (10 μg/mL) alone (Table 1).
Collagen decreased intracellular cAMP from
3.89±0.18 pmoL/108 platelets (basal level) to 3.44±
0.43 pmoL/108 platelets in washed platelets (Table 1);
however, when the platelets were incubated in the
presence of both EGCG and collagen, cAMP by EGCG
increased in a dose-dependent manner (Fig.3B). The
cAMP level (6.60±0.04 pmoL/108 platelets) in the
Table 1. Changes of cAMP and cGMP
cAMP cGMPcAMP/cGMP
pmoL/108
platelets
Change
(%)
pmoL/108
platelets
Change
(%)
Ratio
Change
(%)
Base
EGCG (50 μM)
Collagen (10 μg/mL)
Collagen＋EGCG
3.89±0.18
5.31±0.80
3.44±0.43
6.60±0.04
0
＋36.51)
0
＋91.93)
1.40±0.15
1.30±0.10
1.21±0.15
1.43±0.04
0
−7.12)
0
＋18.24)
2.8
4.1
2.8
4.6
0
46.45)
0
64.36)
Data were from Fig.3. 1), 2), and 5) changes to base; 3), 4), and 6) changes to collagen.
Fig.3. Effects of EGCG on cGMP and cAMP production in
resting and collagen-stimulated platelets. (A) Effects of
EGCG on cGMP production in resting or collagen-
stimulated platelets. (B) Effects of EGCG on cAMP
production in resting or collagen-stimulated platelets.
Washed platelets (108/mL) were preincubated with or
without EGCG for 3 min in the presence of 2 mM
CaCl2 and then stimulated with collagen (10 μg/mL)
for 5 min at 37℃. The reactions were terminated by
adding 80% ice-cold ethanol. cGMP contents were
measured using EIA kits. Data are expressed as the
means±S.E.M. (n=4). ＊p＜0.05. ＊ ＊p＜0.001.
2.0
1.5
1.0
0.5
0.0
＊
＊
cGMP (pmoL/108 platelets)
Collagen (10μg/mL)
EGCG (μM)
＋
－
＋
1
＋
5
＋
10
＋
30
＋
50 50
－－
－
7
6
5
4
3
2
1
0
＊ ＊
＊
＊ ＊
＊ ＊
＊ ＊
＊ ＊
cAMP (pmoL/108 platelets)
Collagen (10μg/mL)
EGCG (μM)
＋
－
＋
1
＋
5
＋
10
＋
30
＋
5050
－－
－
A
B

Page 6

342
Ok et al.
platelet aggregation (Fig.4A lane 1, 2). Collagen (10
μg/mL) increased p-VASP (Ser157 and Ser239) at 50
kDa phosphoprotein of VASP (Fig.4A lane 2). It is
known that thrombin and collagen, agonists of plate-
lets, are involved in a feedback inhibition by elevating
p-VASP (Ser157 and Ser239)25). EGCG itself stimulated
the shift from 46 kDa dephosphoprotein of VASP to
50 kDa phosphoprotein of VASP, and markedly
increased the p-VASP (Ser157) only of VASP-50 kDa,
which is observed in collagen-induced platelet aggre-
presence of both EGCG (50 μM) and collagen (10
μg/mL) increased to 91.9% as compared with that
(3.44±0.43 pmoL/108 platelets) in the presence of
collagen (10 μg/mL) alone (Table 1).
Effects of EGCG on Phosphorylation at Ser157 or
Ser239 of VASP
In intact platelets, basal 46 kDa dephosphopro-
tein of VASP only was observed and was shifted to 50
kDa phosphoprotein of VASP in collagen-induced
Fig.4. Effects of EGCG on VASP phosphorylation of resting or collagen-stimulated platelets. (A) Effects of EGCG on
phosphorylation of VASP, VASP (Ser157), VASP (Ser239). Lane 1, intact platelets (base); lane 2, collagen (10 μg/
mL); lane 3, EGCG (50 μM); lane 4, collagen (10 μg/mL)＋EGCG (50 μM). (B) Changes of VASP (Ser157)
phosphorylation by EGCG. (C) Changes of VASP (Ser239) phosphorylation by EGCG. (D) Increase of p-VASP
(Ser157) or p-VASP (Ser239) by EGCG in collagen-stimulated platelets. Washed platelets (108/mL) were preincu-
bated with or without EGCG for 3 min in the presence of 2 mM CaCl2 and then stimulated with collagen (10
μg/mL) for 5 min at 37℃ in an aggregometer. The reactions were terminated by adding an equal volume of lysis
buffer. Proteins were extracted, separated by SDS-PAGE transferred to PVDF and immunoblotted with the indi-
cated corresponding antibodies, anti-phosphor-VASP Ser239 or Ser157. Blots were visualized by ECL plus kit
(Amersham, Buckinghamshire, UK) and the immunoblot panels are representative of 3-4 similar experiments.
Blots were analyzed using Quantity one, Ver, 4.5 program (Bio-rad, Hercules, CA, USA), and units are expressed
as density/mm2.
VASP
VASP
50 kDa
50 kDa
46 kDa
46 kDa
1250012500
12500
15000
15000
17500
17500
20000
20000
- 50kDa
p-VASP (Ser239) -
mm2)
(Blot intensity/
++
p-VASP (Ser157)
p-VASP (Ser157)
p-VASP (Ser239)
p-VASP (Ser239)
25002500
2500
5000
5000
7500
7500
10000
10000
Collagen (10 μg/mL)
Collagen (10μg/mL)
EGCG (50 μM)
EGCG (50μM)
-+
-
--
+
+
Collagen (10 μg/mL)
Collagen (10μg/mL)
EGCG (50 μM)
EGCG (50μM)
+
+
-
+
0
22500
20000
20000
17500
15000
12500
10000
7500
5000
2500
25000
25000
22500
14.0
14.0
16.0
16.0
18.0
18.0
ASP
ncrease % of p-VA
In
10000
7500
12500
15000
17500
SP (Ser157) - 50kDa
p-VAS
t intensity/mm2)
(Blot
6.0
6.0
8.0
8.0
10.0
10.0
12.0
12.0
Collagen (10 μg/mL)
Collagen (10μg/mL)
+
+
0
2500
0
5000
0.0
0.0
2.0
2.0
4.0
4.0
p-VASP (Ser157)
　P-VASP (Ser157)
p-VASP (Ser239)
　p-VASP (Ser239)
Collagen + EGCG
Collagen＋EGCG
P-VASP (Ser157) p-VASP (Ser239)
EGCG (50 μM)
EGCG (50μM)
-
+
AC
DB
p-VASP (Ser157)-50kDa
(Blot intensity/mm2)
＋
＋
＋
－
Increase % of p-VASP
0
p-VASP (Ser239)-50kDa
(Blot intensity/mm2)
＋
＋
＋
－
＋
－
－
＋
＋
50 kDa
50 kDa
＋
－
－
1234

Page 7

343
Anti-Platelet Effects of EGCG
Effects of EGCG on Platelet Aggregation and VASP
Phosphorylation in the Presence of cAMP-Depen-
dent Protein Kinase (A-kinase) Inhibitor Rp-8-Br-
cAMPS
The inhibitory action mode of platelet aggrega-
gation. The band density (21971 density/mm2) of
p-VASP (Ser157) by both EGCG and collagen was
increased to 16.6% as compared with that (18841
density/mm2) by collagen (Fig.4B, D). This suggests
that EGCG strongly phosphorylates VASP (Ser157),
indicating the relation to A-kinase activation by
EGCG-elevated cAMP. The band density (18050 den-
sity/mm2) of p-VASP (Ser239) by both EGCG and col-
lagen was increased to 5.5% as compared with that
(17188 density/mm2) by collagen (Fig.4C, D). The
phosphorylation of VASP (Ser239) by EGCG is much
slighter than p-VASP (Ser157) (Fig.4A lane 3, 4, C and
D). This suggests that EGCG slightly phosphorylates
VASP (Ser239), indicating a relation to G-kinase acti-
vation of cGMP elevated slightly by EGCG.
Effects of EGCG on cAMP Production and VASP
Phosphorylation in the Presence of Adenylate
Cyclase Inhibitor, SQ22536
The cAMP level is regulated by adenylate cyclase,
cAMP-specific phosphodiesterase (PDE4), cGMP-
stimulated phosphodiesterase (PDE2) and cGMP-
inhibited phosphodiesterase (PDE3) in platelets26).
Because EGCG hardly affected cGMP production in
both resting and collagen-stimulated platelets (Fig.3A),
it is inferred that EGCG-elevated cAMP was not regu-
lated by the inhibition of PDE2 hydrolyzing both
cAMP and cGMP, and PDE3 hydrolyzing cAMP
rather than cGMP. Accordingly, we set out to investi-
gate in this study whether the EGCG-mediated cAMP
level was regulated by enzymes of adenylate cyclase or
PDE4. For the first time, we used SQ22536, an ade-
nylate cyclase inhibitor. As a result, the EGCG-
increased cAMP level (6.6±0.1 pmoL/108 platelets)
was reduced in the presence of SQ22536 (10 μM) to
5.3±0.1 pmoL/108 platelets (Fig.5A). This suggests
that EGCG-elevated cAMP (Fig.3B) might be medi-
ated by the activity of adenylate cyclase, involved in
the phosphorylation of Ser157 at 50 kDa phosphopro-
tein of VASP via A-kinase. As shown in Fig.5B lane 5,
both collagen (10 μg/mL) and EGCG (50 μM)-phos-
phorylated VASP (Ser157) at 50 kDa [p-VASP (Ser157)]
were inhibited by SQ22536 (10 μM). The band den-
sity (25101±118 intensity/mm2) of p-VASP (Ser157)
phosphorylated by both collagen and EGCG was
decreased to 22.7% as compared with that (19396±
1471 intensity/mm2) in the presence of SQ22536
(Table 2, 3). The ratio (1.13) of p-VASP (Ser157)-50
kDa to VASP (50 kDa) phosphorylated by both colla-
gen and EGCG was decreased to about 30% as com-
pared with that (0.79) in the presence of SQ22536
(Table 2).
Fig.5. Effects of EGCG on cAMP production and VASP
phosphorylation in resting and collagen-stimulated
platelets. (A) Effects of EGCG with SQ22536, an ade-
nylate cylase inhibitor, on cAMP production in colla-
gen-stimulated platelets. Washed platelets (108/mL)
were preincubated with or without EGCG or SQ22536
for 3 min in the presence of 2 mM CaCl2 and then
stimulated with collagen (10 μg/mL) for 5 min at 37℃
in an aggregometer. The reactions were terminated by
adding 80% ice-cold ethanol. cAMP contents were
measured using EIA kits. Data are expressed as the
means±S.E.M. (n=4). ＊p＜0.05. ＊ ＊p＜0.001. (B)
Effects of EGCG with SQ22536, adenylate cyclase
inhibitor, on VASP (Ser157) phosphorylation [p-VASP
(Ser157)]. Lane 1, collagen (10 μg/mL); lane 2, EGCG
(50 μM); lane 3, collagen (10 μg/mL)＋EGCG (50
μM); lane 4, SQ22536 (10 μM); lane 5, collagen (10
μg/mL)＋EGCG (50 μM)＋SQ22536 (10 μM); lane
6, forskolin (10 μM). Proteins were extracted, separated
by SDS-PAGE transferred to PVDF and immunoblot-
ted with the indicated corresponding antibodies, anti-
phosphor VASP or Ser157. Blots were visualized by the
ECL plus kit (Amersham) and immunoblot panels are
representative of 3-4 similar experiments. Blots were
analyzed using Quantity One, Ver. 4.5 program (Bio-
rad), and units are expressed as intensity/mm2.
50kDa
46kDa
46 kDa
1
2
34
5
6
Collagen (10 μg/mL)
Collagen (10μg/mL)
EGCG (50 μM)
EGCG (50μM)
+-+
-
-++
-
p-VASP (Ser157)
p-VASP (Ser157)
+
+
-
-
50kDa
50 kDa
SQ22536 (10 μM)
SQ22536 (10μM)
---++-
Forskolin (10 μM)
Forskolin (10μM)
-----+
A
B
7
6
5
4
3
2
1
0
＊ ＊
＊ ＊
＊
＊
cAMP (pmoL/108 platelets)
Collagen (10μg/mL)
EGCG (50μM)
SQ22536 (10μM)
－
－
－
＋
－
＋
＋
＋
－
＋
＋
＋
＋
－
－
－
－
－
＋
－
－
＋
＋
－
－
－
50 kDa
＋
－
＋
－
－
＋
＋
＋
－
＋
－
－
－
VASP
123456

Page 8

344
Ok et al.
of both etazolate and EGCG, the cAMP level was
increased up to 6.2±0.6 pmoL/108 platelets (Fig.7).
This cAMP level (6.2±0.6 pmoL/108 platelets), how-
ever, was not synergistically increased as compared
with that (6.6±0.1 pmoL/108 platelets) increased by
EGCG in collagen-stimulated platelet aggregation.
These results suggest that EGCG-activated cAMP
enhancement (Fig.3B, 7) is not mediated by the inhi-
bition of cAMP-specific phosphodiesterase, PDE4.
Discussion
Catechins that have an anti-inflammatory effect
are known to have anti-platelet function by inhibiting
the liberation of TXA2 precursor 20:4 out of platelet
membrane phospholipids and TXA2 synthase activity
in collagen-activated platelets13, 27, 28). EGCG has been
reported to inhibit collagen-activated Ca2＋-dependent
PLC-γ2 and TXA2 production13). Since EGCG inhib-
ited [Ca2＋]I mobilization (Fig.2A) and TXA2 produc-
tion (Fig.2B), but increased cAMP production
(Fig.3B, Table 1), as in previous reports13, 27, 28), this
suggested that EGCG-elevated Ca2＋-antagonistic
cAMP (Fig.3B) may be involved in the inhibition of
20:4 liberation and TXA2 production by suppressing
[Ca2＋]I mobilization and Ca2＋-dependent PLC-γ2
activity. TXA2 is produced via cyclooxygenase-1
(COX-1) and the TXA2 synthase (TXAS) pathway
from 20:4. In our other experiments, EGCG inhibited
microsomal COX-1 activity to 91% as compared with
that (1.4 nmoL/protein-mg/min) of the control, and
inhibited microsomal TXAS activity to 20% as com-
pared with the control (0.17 nmoL/protein-mg/min)
(data not shown). It is thought that EGCG has multi-
ple actions that reduce [Ca2＋]i mobilization and TXA2
production-associated enzyme (COX-1, TXAS) activi-
ties to inhibit TXA2 production, and 20:4 is metabo-
lized to prostaglandin E2, an inflammatory mediator,
tion by cAMP is known to occur via the activation of
A-kinase which phosphorylates VASP (Ser157)12, 26) in
platelet aggregation; therefore, because the inhibition
of A-kinase activation and VASP (Ser157) phosphoryla-
tion induced platelet aggregation, we investigated
whether EGCG affects collagen-stimulated platelet
aggregation and VASP (Ser157) phosphorylation in the
presence of A-kinase inhibitor, Rp-8-Br-cAMPS.
As shown in Fig.6A, in the presence of both Rp-
8-Br-cAMPS and EGCG, the degree of collagen-
induced platelet aggregation (58.0±2.0%) was higher
than that (18.0±3.0%) in the presence of EGCG
alone. The band density (Fig.6B lane 2, Table 4) of
p-VASP (Ser157) phosphorylated by both collagen and
EGCG in the presence of Rp-8-Br-cAMPS was
decreased to 76.1 % as compared with that (Fig.6B
lane 1, Table 4) in the absence of Rp-8-Br-cAMPS.
These results reflect that the inhibitory effect of
EGCG on collagen-induced platelet aggregation is
involved in the phosphorylation of VASP (Ser157) by
the cAMP/A-kinase pathway.
Effects of EGCG on cAMP Production in the
Presence of PDE4 Inhibitor Etazolate
As shown in Fig.7, when washed platelets were
stimulated by collagen in the presence of etazolate
alone (20 μM), the cAMP level was increased signifi-
cantly from 3.5±0.3 pmoL/108 platelets to 5.3±0.5
pmoL/108 platelets. On the other hand, when washed
platelets were stimulated by collagen in the presence
Table 2. Changes of p-VASP (Ser157) by SQ22536
Blot (intensity/mm2)
Collagen
(10 μg/mL)
EGCG
(50 μM)
Collagen＋
EGCG
SQ22536
(10 μM)
Collagen＋EGCG
＋SQ22536
Forskolin
(10 μM)
VASP
50 kDa
46 kDa
19403±2941
14560±1471
17557±7353＊
28618±6470
22166±8235＊
18345±6764
15183±9117
32285±1765
24407±5882＊ ＊
23578±3235
31587±2941
23069±5882
p-VASP (Ser157)50 kDa 23605±14720436±176 25101±11815131±8819396±1471 27994±29
p-VASP (Ser157)-50 kDa
1.221.16 1.131.00.790.89
VASP (50 kDa)
Data were from Fig.5. ＊p＜0.05 vs collagen. ＊ ＊p＜0.05 vs collagen＋EGCG.
Table 3. Changes (%) of p-VASP (Ser157) by EGCG or SQ22536
Collagen Collagen＋EGCG Collagen＋EGCG＋SQ22536
0
＋6.31)
−22.72)
Data are calculated from Table 2. 1) change to collagen; 2) change to
collagen plus EGCG.

Page 9

345
Anti-Platelet Effects of EGCG
lated platelets (Table 1). The ratio of cAMP to cGMP
was increased up to 64.3% in the presence of both
collagen and EGCG as compared with that (2.8) in
the absence of EGCG (Table 1). This suggests that
EGCG directly produced cAMP and used it to
decrease [Ca2＋]i, an essential molecule for TXA2 pro-
duction. cAMP and cGMP are involved in the inhibi-
tion of platelet aggregation by phosphorylating IP3
receptor or VASP via the A-kinase or G-kinase path-
way. IP3 receptor phosphorylation is involved in the
inhibition of [Ca2＋]i mobilization30, 31), and VASP
phosphorylation is known to regulate VASP affinity
for platelet filamentous actin32). In the present study,
it is unknown whether the inhibitory effect of [Ca2＋]i
by EGCG is due to the phosphorylation of IP3 recep-
tor by cAMP or cGMP. In intact and collagen-acti-
vated platelets, EGCG phosphorylated A-kinase sub-
strate VASP (Ser157) markedly, but did not phosphory-
by cyclooxygenase-2 (COX-2). Because it was observed
in our other results that EGCG inhibits TXA2 pro-
duction (Fig.2B) and microsomal COX-1 (data not
shown), it is clear that EGCG has an anti-inflamma-
tory effect by inhibiting COX-2 activity, in the same
way as EGCG has an anti-prostate carcinogenic effect
by inhibiting COX-2 activity29). Because both platelet
aggregation and inflammation are the cause of athero-
sclerosis, it is thought that EGCG could contribute to
the treatment of cardiovascular disease.
Agonist-mobilized [Ca2＋]i is decreased by cyclic
nucleotides (cAMP and cGMP), and their receptors
A-kinase and G-kinase are key modulators controlling
platelet inhibition24, 26). EGCG (50 μM) significantly
increased cAMP rather than cGMP in intact and col-
lagen-induced platelet aggregation (Table 1). EGCG
itself increased up to 46.4% the ratio of cAMP to
cGMP from 2.8, the basal ratio, to 4.1 in unstimu-
Fig.6. Effect of EGCG on collagen-induced platelet aggregation and VASP (Ser157) phosphorylation in the presence of A-kinase
inhibitor, Rp-8-Br-cAMPS. (A) Effects of EGCG in the presence of Rp-8-Br-cAMPs on collagen-stimulated platelets.
Washed platelets (108/mL) were preincubated with EGCG (50 μM) with Rp-8-Br-cAMPS (20 μM), a A-kinase inhibitor,
in the presence of 2 mM CaCl2 for 3 min at 37℃, and then stimulated with collagen (10 μg/mL) for 5 min. Platelet aggre-
gation (%) was recorded as an increase in light transmission. Data are expressed as the means±S.E.M. (n=4). ＊ ＊p＜0.001.
(B) Effects of EGCG in the presence of Rp-8-Br-cAMPs on VASP (Ser157). Lane 1, collagen＋EGCG; lane 2, collagen＋
EGCG＋Rp-8-Br-cAMPS. Collagen-induced platelet aggregation was terminated by adding an equal volume of lysis buffer.
Proteins were extracted, separated by SDS-PAGE (8-10%), transferred to PVDF and immunoblotted with the indicated
corresponding antibodies, anti-phosphor VASP, or Ser157. Blots were visualized by the ECL plus kit (Amersham) and immu-
noblot panels are representative of 3-4 similar experiments. Blots were analyzed using Quantity One, Ver. 4.5 program (Bio-
rad), and units are expressed as intensity/mm2.
A
90
80
70
60
50
40
30
20
10
0
＊ ＊
＊ ＊
Light transmission (%)
Collagen (10μg/mL)
EGCG (50μM)
Rp-8-Br-cAMPS (20μM)
＋
－
＋
＋
＋
－
＋
＋
＋
＋
－
－
B
＋
＋
＋
＋
＋
－
p-VASP (Ser157)
p-VASP (Ser157)
Collagen (10μg/mL)
EGCG (50μM)
Rp-8-Br-cAMPS (20μM)
12
1
2
Table 4. Changes (%) of p-VASP (Ser157) by EGCG or Rp-8-Br-cAMPS
Band density of p-VASP (Ser157)
(Intensity/mm2)
Inhibition of p-VASP (Ser157)
(%)
Collagen＋EGCG
Collagen＋EGCG＋Rp-8-Br-cAMPS
31800
7600
0
76.1
Data were calculated from Fig.6B.

Page 10

346
Ok et al.
is 0.06 μM (60 pmoL/mL)35); therefore, EGCG-ele-
vated cAMP (Table 1, Fig.3B) is not due to the inhi-
bition of PDE3.
The next question therefore concerns the mecha-
nism by which EGCG up-regulated the intracellular
cAMP level. To solve this question, we used SQ22536
as an adenylate cyclase inhibitor and etazolate as a
PDE4 inhibitor. The EGCG-elevated cAMP level was
decreased by SQ22536 (Fig.5A), which results from
inhibition of adenylate cyclase by SQ22536. Because
SQ22536 decreased cAMP production, the phosphor-
ylation of VASP (Ser157) in the presence of EGCG was
inhibited by SQ22536 (Fig.5B). These results suggest
that EGCG produced cAMP from ATP by activating
adenylate cyclase, and phosphorylated VASP (Ser157)-
50 kDa by activating A-kinase to inhibit collagen-
induced platelet aggregation. Otherwise, p-VASP
(Ser157) phosphorylated by both EGCG and collagen
would not be inhibited in the presence of Rp-8-Br-
cAMPS, an A-kinase inhibitor (Fig.6, Table 4). On
the other hand, even though PDE4 inhibitor etazolate
increased cAMP in the presence of collagen, it did not
increase cAMP in the presence of both EGCG and
collagen (Fig.7). This means that EGCG-elevated
cAMP is not due to the inhibition of PDE4 by EGCG.
Nakagawa et al. reported that human plasma
EGCG concentrations before administration were all
below the detection limit (＜2 pmoL/mL), but 90
min after, they were significantly and dose-depend-
late G-kinase substrate VASP (Ser239) (Fig.4A). These
results are in accord with the result that EGCG mark-
edly enhanced cAMP rather than cGMP in intact and
collagen-stimulated platelets (Table 1). Eigenthaler et
al.33) reported that a small elevation in cAMP is
enough to activate most A-kinase, whereas even sev-
eral fold elevation of the cGMP level may stimulate
only a small fraction of total G-kinase. This previous
report33) reflects that even a little cAMP is enough to
phosphorylate VASP (Ser157), an A-kinase substrate,
and much cGMP is required to phosphorylate VASP
(Ser239), a G-kinase substrate. Accordingly, even
though EGCG increased cGMP to 18.2 % in colla-
gen-induced platelets as compared with that (1.21±
0.15 pmoL/108 platelets) by collagen only, it is
thought that the EGCG-elevated cGMP level (1.43±
0.04 pmoL/108 platelets) is not enough to phosphory-
late VASP (Ser239) (Table 1, Fig.4A, D). In addition,
EGCG-phosphorylated VASP-Ser157 was inhibited by
Rp-8-Br-cAMPS, an A-kinase inhibitor, which sug-
gests that EGCG phosphorylates VASP-Ser157 via
both cAMP production and A-kinase activation. Sudo
et al.34), using cilostazole, reported a similar result to
our finding, and suggested that its antiplatelet effect is
mediated through an increase of the cAMP level,
A-kinase activity, and VASP phosphorylation. The lev-
els of intracellular cAMP and cGMP are regulated by
the balance between cyclic nucleotide-producing
enzymes, adenylate/guanylate cyclases, and hydrolyz-
ing enzymes, cAMP/cGMP PDEs. If EGCG inhibited
the activity of PDE2 to produce cAMP and cGMP in
intact and collagen-stimulated platelets, because
PDE2 hydrolyzes both cAMP and cGMP26), EGCG
would increase the level of cAMP and cGMP in intact
and collagen-stimulated platelets; however, EGCG
markedly increased the cAMP level only, and hardly
increased the cGMP level (Table 1). In particular,
since EGCG did not increase but decreased cGMP
production in intact platelets (Table 1), even though
the cGMP level (1.43±0.04 pmoL/108 platelets) was
increased to 18.2% in the presence of both collagen
and EGCG as compared with that (1.21±0.15
pmoL/108 platelets) in the absence of EGCG (Table 1),
it is thought that EGCG-elevated cAMP (Fig.3B) is
not due to the regulation of cGMP-stimulated PDE2,
cGMP-inhibited PDE3, and cGMP-binding-cGMP-
specific PDE5. EGCG increased cGMP to about 0.22
pmoL/108 platelets as compared with that (1.21±0.15
pmoL/108 platelets) in the presence of collagen (10
μg/mL) (Table 1, Fig.3A). This increased cGMP
level (0.22 pmoL/108 platelets) is not sufficient to
inhibit PDE3 to increase cAMP, since it is known that
Ki for cGMP inhibition of cAMP hydrolysis by PDE3
Fig.7. Effects of EGCG with etazolate, cAMP-specific phos-
phodiesterase inhibitor, on cAMP production in colla-
gen-stimulated platelets. Washed platelets (108/mL)
were preincubated with or without etazolate or EGCG
for 3 min in the presence of 2 mM CaCl2 and then
stimulated with collagen (10 μg/mL) for 5 min at
37℃. The reactions were terminated by adding 80%
ice-cold ethanol. cAMP contents were measured using
EIA kits. Data are expressed as the means±S.E.M.
(n=4). ＊p＜0.05. ＊ ＊p＜0.001.
8
7
6
5
4
3
2
1
0
＊ ＊
＊ ＊
＊
cAMP (pmoL/108 platelets)
Collagen (10μg/mL)
EGCG (50μM)
Etazolate (20μM)
＋＋＋－＋
－
－
－
＋
＋
－
＋
＋
－
－

Page 11

347
Anti-Platelet Effects of EGCG
Gerzer R: Influence of the cGMP analog 8-PCPT-cGMP
on agonist-induced increases in cytosolic ionized Ca2＋ and
on aggregation of human platelets. Eur J Pharmacol,
1993; 245: 281-284
6) Pasqui AL, Capecci PL, Ceccatelli L, Mazza S, Gistri A,
Laghi Pasini F, Di Perri, T: Nitroprusside in vitro inhibits
platelet aggregation and intracellular calcium transloca-
tion. Effect of haemoglobin. Thromb Res, 1991; 61: 113-
122
7) Radomski MW, Palmer RMJ, Monacade S: An L-argi-
nine/nitric oxide pathway present in human platelets reg-
ulates aggregation. Proc Natl Acad Sci (USA); 1990; 87:
5193-5197
8) Halbrügge M, Walter U: Purification of a vasodilator-reg-
ulated phosphoprotein from human platelets. Eur J Bio-
chem, 1989; 185: 41-50
9) Halbrügge M, Friedrich C, Eigenthaler M, Schanzen-
bächer P, Walter U: Stoichiometric and reversible phos-
phorylation of a 46-kDa protein in human platelets in
response to cGMP- and cAMP-elevating vasodilators. J
Biol Chem, 1990; 265: 3088-3093
10) Butt E, Abel K, Krieger M, Palm D, Hoppe V, Hoppe J,
Walter U: cAMP- and cGMP-dependent protein kinase
phosphorylation sites of the focal adhesion vasodilator-
stimulated phosphoprotein (VASP) in vitro and in intact
human platelets. J Biol Chem, 1994; 269: 14509-14517
11) Smolenski A, Bachmann C, Reinhard K, Hönig-Liedl P,
Jarchau T, Hoschuetzky H, Walter U: Analysis and regu-
lation of vasodilator-stimulated phosphoprotein serine
239 phosphorylation in vitro and in intact cells using a
phosphospecific monoclonal antibody. J Biol Chem,
1998; 273: 20029-20035
12) Horstrup K, Jablonka B, Hönig-Liedl P, Just M, Kochsiek
K, Walter U: Phosphorylation of focal adhesion vasodila-
tor-stimulated phosphoprotein at Ser157 in intact human
platelets correlates with fibrinogen receptor inhibition.
Eur J Biochem, 1994; 225: 21-27
13) Lill G, Voit S, Schor K, Weber AA: Complex effects of
different green tea catechins on human platelets. FEBS
Lett, 2003; 546: 265-270
14) Deana R, Turetta L, Donella-Deana A, Dona M, Brunati
AM, Demichiel L, Garbisa S: Green tea epigallocatechin-
3-gallate inhibits platelet signaling pathways triggered by
both proteolytic and non-proteolytic agonists. Thrombo
Haemost, 2003; 89: 866-874
15) Jin YR, Im JH, Park ES, Cho MR, Han XH, Lee JJ, Lim
Y, Kim TJ, Yun YP: Anti-platelet activity of epigallocate-
chin gallate is mediated by the inhibition of PLCr2 phos-
phorylation, elevation of PGD2 production, and main-
taining calcium-ATPase activity. J Cardiovasc Pharmacol,
2008; 51: 45-54
16) Sagesaka-Mitane Y, Miwa M, Okada S: Platelet aggrega-
tion inhibitors in hot water extract of green tea. Chem
Pharm Bull (Tokyo), 1990; 38: 790-793
17) GUO Q, Zhao B, Shen S, Hou J, Hu JXW; ERS study
on the structure-antioxidant activity relationship of tea
catechins and their epimers. Biochem Biophys Acta,
1999; 1427: 13-23
18) Sehm J, Polster J, Pfaffl MW: Effects of varied EGCG and
(＋)-catechin concentrations on proinflammatory cyto-
ently increased to 657, 4300, and 4410 pmoL EGCG/
mL, in subjects who received 3, 5, and 7 capsules,
respectively (corresponding to 225, 375, and 525 mg
EGCG, respectively). EGCG levels detected in plasma
corresponded to 0.2-2.0% of the ingested amount36).
In the present study, although it is unknown whether
46-460 μg, corresponding to 0.2-2.0% of EGCG
(MW. 458.37) 50 μM (23 mg) having anti-platelet
activities, has clinic anti-platelet activity in vivo, which
should be studied in the future, and if it has a dietary
effect, it could be used to evaluate the anti-platelet
effects of EGCG.
In conclusion, the most important result of this
study is that EGCG significantly inhibits the level of
[Ca2＋]i and TXA2, platelet-aggregating molecules. The
inhibition of [Ca2＋]i by EGCG depends on the up-
regulation of Ca2＋-antagonistic intracellular cAMP
level through the activation of adenylate cyclase. Fur-
thermore, our findings showed that EGCG-elevated
cAMP phosphorylates VASP (Ser157) through A-kinase
activation to inhibit collagen-induced platelet aggrega-
tion. Therefore, these results suggest that EGCG is a
physiologically effective negative feedback regulator
during platelet aggregation, a cause of thrombosis,
atherosclerosis, and myocardial infarction.
Acknowledgments
This study was supported by a grant (2011-
0012143 to Hwa-Jin Park) from the basic Science
Research Program via the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Edu-
cation, Science and Technology, and supported in part
by a grant (2010-0024028) from the Basic Science
Research Program through the National Research
Foundation of Korea funded by the Ministry of Edu-
cation, Science and Technology.
References
1) Schwartz SM, Heinmark RL, Majesky MW: Develop-
mental mechanisms underlying pathology of arteries.
Physiol Rev, 1990; 70: 1177-1209
2) Cattaneo M, Tenconi PM, Lecchi A, Mannucci PM: In
vitro effects of picotamide on human platelet aggregation,
the release reaction and thromboxane B2 production.
Thromb Res, 1991; 62: 717-724
3) Nishikawa M, Tanaka T, Hidaka H: Ca2＋-calmodulin-
dependent phosphorylation and platelet secretion. Nature,
1980; 287: 863-865
4) Kaibuchi K, Sano K, Hoshijima M, Takai Y, Nishizuka Y:
Phosphatidylinositol turnover in platelet activation; cal-
cium mobilization and protein phosphorylation. Cell Cal-
cium, 1982; 3: 323-335
5) Menshikov MYu, Ivanova K, Schaefer M, Drummer C,

Page 12

348
Ok et al.
chins: a possible mechanism through arachidonic acid
pathway. Prostaglandins Leukotriens and Essential Fatty
acids, 2004; 71: 25-31
29) Liu FC, Liao CH, Chang YW, Liou JT, Day YJ: A new
insight of anti-platelet effects of sirtinol in platelets aggre-
gation via cyclic AMP phosphodiesterase. Biochem Phar-
macol, 2009; 77: 1364-1373
30) Homer KL, Wanstall JC: Inhibition of rat platelet aggre-
gation by the diazeniumdiolate nitric oxide donor
MAHMA NONOate. Br J Pharmacol, 2002; 137: 1071-
1081
31) Park WH, Kim HK, Nam KS, Shon YH, Jeon BH, Moon
SK, Kim MG, Kim CH: Inhibitory effect of GBH on
platelet aggregation through inhibition of intracellular
Ca2＋ mobilization in activated human platelets. Life Sci,
2004; 75: 3063-3076
32) Laurent V, Loisel TP, Harbeck B, Wehman A, Gröbe L,
Jockusch BM, Wehland J, Gertler FB, Carlier MF: Role
of proteins of the Ena/VASP family in actin-based motil-
ity of Listeria monocytogenes. J Cell Biol, 1999; 144:
1245-1258
33) Eigenthaler M, Nolte C, Halbrügge M, Walter U: Con-
centration and regulation of cyclic nucleotides, cyclic-
nucleotide-dependent protein kinases and one of their
major substrates in human platelets. Estimating the rate
of cAMP-regulated and cGMP-regulated protein phos-
phorylation in intact cells. Eur J Biochem, 1992; 205:
471-481
34) Sudo T, Ito H, Kimura Y: Phosphorylation of the vasodi-
lator-stimulated phosphoprotein (VASP) by the antiplate-
let drug, cilostazol, in platelets. Platelets, 2003; 14: 381-
390
35) Harrison SA, reifsnyder DH, Gallis B, Cadd GG, Beavo
JA: Isolation and characterization of bovine cardiac mus-
cle cGMP-inhibited phosphodiesterase: a receptor for new
cardiotonic drugs. Mol Pharmacol, 1986; 29: 506-514
36) Nakagawa K, OKuda S, Miyazawa T: Dose-dependent
incorporation of tea catechins, (−)-epigallocatechim-3-
gallate and (−)-epigallocatechim, into human plasma.
Biosci Biotech Biochem, 1997; 61: 1981-1985
kines mRNA expression in cona-stimulated primary white
blood cell cultures. J Agric Food Chem, 2005; 53: 6907-
6911
19) Hussain T, Gupta S, Adhami VM, Mukhtar H: Green tea
constituent epigallocatechin-3-gallate selectively inhibits
COX-2 without affecting COX-1 expression in human
prostate carcinnoma cells. Int J Cancer, 2005; 113: 660-
669
20) Negishi H, Xu Jw, Ikeda K: Black and green tea polyphe-
nols attenuate blood pressure increases in stroke-prone
spontaneously rat. J Nutr, 2004; 134: 38-42
21) Velayutham P, Babu A, Liu D: Green tea catechins and
cardiovascular health: An update. Curr Med Chem, 2008;
15: 1840-1850
22) Schaeffer J, Blaustein MP: Platelet free calcium concentra-
tions measured with fura-2 are influenced by the trans-
membrane sodium gradient. Cell Calcium, 1989; 10:
101-113
23) Cho HJ, Cho JY, Rhee MH, Park HJ: Cordycepin
(3’-deoxyadenosine) inhibits human platelet aggregation
in a cyclic cAMP-and cyclic GMP-dependent manner.
Eur J Pharmacol, 2007; 558: 43-51
24) Jang EK, Azzam JE, Dickinson NT, Davidson MM,
Haslam RJ: Roles for both cyclic GMP and cyclic AMP
in the inhibition of collagen-induced platelet aggregation
by nitroprusside. Br J Haematol, 2002; 117: 664-675
25) Gambaryan S, Kobsar A, Rukoyatkina N, Herterich S,
Geiger J, Smolenski A, Lohmann SM, Walter U: Throm-
bin and collagen induce a feedback inhibitory signaling
pathway in platelets involving dissociation of the catalytic
subunit of protein kinase A from an NF kappaB-IkappaB
complex. J Biol Chem, 2010; 285: 18352-18363
26) Schwarz UR, walter U, Eigenthaler M: Taming platelets
with cyclic nucleotides. Biochem Pharmacol, 2001; 62:
1153-1161
27) Jackson SP, Nesbitt WS, Kulkarni S: Signaling events
underlying thrombus formation. J Thromb Haemost,
2003; 1: 1602-1612
28) Son DJ, Cho MR, Jin YR, Kim SY, Park YH, Lee SH,
Akiba T, Yun YP : Antiplatelet effect of green tea cate-