JJK694, a synthesized obovatol derivative, inhibits platelet activation by suppressing cyclooxygenase and lipoxygenase activities

Article abstract of DOI:10.1271/bbb.120369

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, antiplatelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative

Full text of DOI:10.1271/bbb.120369

Biosci. Biotechnol. Biochem., 76 (11), 2038–2043, 2012
JJK694, a Synthesized Obovatol Derivative, Inhibits Platelet Activation
by Suppressing Cyclooxygenase and Lipoxygenase Activities
1;*
2;*
3
1
4
Ji-Yeon YU, Jung-Jin LEE, Jae-Kyung JUNG, Yong-Ki MIN, Tack-Joong KIM,
2
5
3;y
Jin Yeul MA, Mi-Yea LEE, and Yeo-Pyo YUN
1
Laboratory for Chemical Genomics, Korea Research Institute of Chemical Technology,
Daejeon 305-600, Republic of Korea
2
Korean Medicine (KM)-Based Herbal Drug Research Group, Korea Institute of Oriental Medicine,
Daejeon 305-811, Republic of Korea
3
College of Pharmacy, Research Center for Bioresource and Health, Chungbuk National University,
2 Gaesin-Dong, Heungduk-gu, Cheongju 361-763, Republic of Korea
1
4
Division of Biological Science and Technology, College of Science and Technology, Yonsei University,
Wonju 220-710, Republic of Korea
5
Department of Nursing, Kyungbok University, Pocheon 487-717, Republic of Korea
Received May 17, 2012; Accepted August 21, 2012; Online Publication, November 7, 2012
[doi:10.1271/bbb.120369]
Obovatol has various biological activities, including
atheroma, such as transient ischemic attack, myocardial
–3)
1
anti-proliferative, neurotrophic, anti-fibrillogenic, anti-
platelet, anti-fungal and anti-inflammatory activities. In
this study, we investigated the effects of JJK694, a
synthesized obovatol derivative, on rabbit platelet
activation and its molecular mechanisms. JJK694
significantly inhibited washed rabbit platelet aggrega-
tion and serotonin secretion induced by collagen and
arachidonic acid, but had little effect on thrombin- or
U46619-induced aggregation. These results suggest that
JJK694 selectively inhibits collagen- and arachidonic
acid-mediated signaling. JJK694 also showed a concen-
infarction, acute coronary syndrome, and stroke.
Cyclooxygenase (COX)-1 and lipoxygenase (LOX)
are two major enzyme families that catalyze the rate-
limiting step in the formation of prostanoids, prosta-
glandins (PGs), including PGD , and thromboxane A2
2
(TXA ) by the COX-1 pathway, and leukotrienes (LTs),
2
including 12-hydroxy-5,8,10,14-eicosatetraenoic acid
(12-HETE), by the LOX pathway, whose products are
4
)
significant mediators of pain, fever and inflammation.
TXA2, formed by platelets, has been reported to be a
potent constrictor of blood vessels and an aggregator of
platelets, and is activated by various agonists such as
collagen, arachidonic acid (AA), adenosine diphosphate
(ADP), epinephrine, and the platelet activating factor
tration-dependent decrease in cytosolic Ca2 mobiliza-
tion, but it had no effect on arachidonic acid liberation.
On the other hand, it significantly inhibited the for-
mation of arachidonic acid metabolites, including
thromboxane A2 (TXA2), prostaglandin D2, and 12-
hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by
suppression of cyclooxygenase (COX)-1 and lipoxyge-
nase (LOX) activities. These results indicate that
JJK694 hasanti-platelet activities through inhibition of
arachidonic acid metabolite production by suppression
of COX-1 and LOX activities.
þ
5
,6)
(PAF).
COX-1-TXA synthase from AA, which is increased
In platelets, TXA2 is produced mainly via
2
dramatically via the action of phospholipase (PL) C and
PLA2-mediated phospholipid hydrolysis when platelets
7
–11)
are activated by various inducers.
12-HETE, a parallel metabolite of AA via 12-LOX, has
On the other hand,
also been reported to act as a platelet activator
1
2,13)
promoting thrombus formation in vivo and to give
rise to platelet aggregation and aortic smooth muscle
1
4–16)
Key words: anti-platelet activity; obovatol derivative;
arachidonic acid metabolites; cyclooxyge-
nase; lipoxygenase
cell migration in vitro. It is likely that 12-HETE as
well as TXA is involved in the initiation and prop-
2
agation of thrombotic and atherosclerotic disorders.
Recently, anti-platelet agents that have inhibitory effects
on TXA2 production and 12-HETE formation have
proven beneficial for patients with cardiovascular
diseases.
Platelets play crucial roles in the development and
complications of coronary heart disease. Platelet adhe-
sion after response to injury by plaque rupture or erosion
of the atherosclerotic artery leads to the subsequent
cascades of thrombus formation, but it also contributes
to the formation of pathogenic thrombus associated with
acute clinical symptoms of thrombotic disease in
Magnolia extract contains various ingredients, includ-
ing flavonoids, alkaloids, coumarins, lignans, neoli-
1
7)
gnans, terpenoids, and phenylpropanoids. Among
Magnolia extracts, obovatol is known to have various
y
*
To whom correspondence should be addressed. Tel: +82-43-261-2821; Fax: +82-43-268-2732; E-mail: ypyun@chungbuk.ac.kr
These authors contributed equally to this work.
Abbreviations: AA, arachidonic acid; TXA2, thromboxane A2; PGD2, prostaglandin; COX, cyclooxygenase; LOX, lipoxygenase; PLC,
phospholipase C; 12-HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid; JY0695, 5-allyl-3-(4-allylphenoxy)-1,2-phenylene diacetate; U46619, 9,11-
dideoxy-9ꢀ,11ꢀ-methanoepoxy prostaglandin F2ꢀ; BSA, bovine serum albumin; Fura 2/AM, fura 2 acetoxymethyl ester; OPT, o-phthalaldehyse;
DMSO, dimethyl sulfoxide; ACD, anticoagulant citrate dextrose; TCA, trichloroacetic acid; PRP, platelet-rich plasma

Antiplatelet Activity of JJK694
2039
1
8–20)
biological activities, including anti-proliferative,
22)
of tetrazolium salt by cellular NADH or NADPH. Working solution
(10 mL) containing WST-8 reagent and JJK694 was added to the PRP
21)
23,24)
neurotrophic,
anti-fibrillogenic,
anti-platelet,
8
anti-fungal,2 and anti-inflammatory activities.26) Park
5)
(200 mL) containing the platelets (3 ꢁ 10 cells/mL) in a 96-well
ꢀ
microtiter plate (Disposable Products, Adelaide). Over 2 h at 37 C, the
et al. found that obovatol inhibited platelet activation in
absorbance of the colored product (formazan dye) was read on a
microplate reader (Well Reader SK601, Seikagaku, Tokyo), using a
test wavelength of 450 nm against a reference wavelength of 650 nm.
1
8)
vitro and thrombus formation in vivo. Hence, the
present study focused on the development of obovatol
derivatives as anti-platelet agents. Recently, we pre-
pared a synthetic obovatol derivative and named it
JJK694 (Fig. 1A), and investigated the effects of
obovatol derivative on platelet activation and possible
molecular mechanisms.
Serotonin secretion assay. Serotonin release was determined as
described previously.27,31) In brief, to prevent re-uptake of secreted
serotonin, imipramine (a serotonin re-uptake inhibitor, 5 mM) was
added to the platelet suspension. Washed rabbit platelets were treated
ꢀ
with various concentrations of JJK694 or DMSO at 37 C for 3 min
prior to the addition of the agonist (collagen 10 mg/mL or AA 100 mM)
over 5 min. An aliquot (0.35 mL) of the washed rabbit platelets was
mixed with 5 mM EDTA on ice, and the mixture centrifuged at
12;000 ꢁ g for 2 min. The supernatant was mixed with 6 M trichloro-
acetic acid (TCA) and centrifuged at 12;000 ꢁ g for 2 min. An aliquot
(0.3 mL) of the TCA supernatant was mixed with 1.2 mL of the
solution (0.5% OPT in ethanol diluted 1:10 with 8 N HCl), left in a
boiling water bath for 10 min, and then cooled on ice. The excess lipids
were extracted with chloroform, and the fluorophore was measured
at excitation and emission wavelengths of 360 nm and 475 nm
respectively.
Materials and Methods
Materials and animals. JJK694 (5-propyl-3-(4-propylphenoxy)
bezene-1,2-diol, Fig. 1A) was kindly provided by Professor Jae-Kyung
Jung of the College of Pharmacy, Chungbuk National University,
Korea. Collagen, arachidonic acid (AA), ADP, and thrombin were
purchased from Chrono-Log (Havertown, PA). U46619 (9,11-dideoxy-
9ꢀ,11ꢀ-methanoepoxy prostaglandin F2ꢀ), TXB2, PGD2, and 12-
HETE were from Cayman Chemical (Ann Arbor, MI). Indomethacin,
bovine serum albumin (BSA), ethylene glycol-bis (ꢁ-aminoethyl
0
0
ether)-N,N,N ,N -tetraacetic acid (EGTA), fura-2 acetoxymethyl ester
Fura-2/AM), serotonin creatinine sulfate, o-phthalaldehyse (OPT),
imipramine, and dimethyl sulfoxide (DMSO) were from Sigma
(
2þ
Cytosolic calcium mobilization assay. Cytosolic Ca mobiliza-
tion was measured by fluorescent dye fura-2/AM, as described
previously,27,31) which involved incubating the platelets with cell
permeant acetoxy methyl ester. Rabbit platelets were incubated with
3
Chemical (St. Louis, MO). [ H] AA (250 mCi/mmol) was from New
England Nuclear (Boston, MA). All other chemicals were of analytical
grade. New Zealand white rabbits were purchased from Sam-Tako
ꢀ
2
mM fura-2/AM at room temperature for 1 h on a rocking platform in
the loading buffer (137 mM NaCl, 27 mM KCl, 0.4 mM NaH2PO4,
0 mM HEPES, 12 mM NaHCO3, 5.5 mM dextrose, 0.35% BSA,
Animal (Osan, Korea). They acclimatized for 1 week at 24 C at 55%
humidity. They had free access to a commercial pellet diet from
Samyang (Wonju, Korea) and drinking water before the experiments.
1
pH 7.4). Excess fura-2/AM was removed by centrifugation (500 ꢁ g
for 10 min), and the platelets were suspended in fresh buffer without
added EGTA. Aliquots of the platelet suspension (2.5 mL) were
inserted into a 4 mL cuvette containing a teflon coated stirrer bar
(Chrono-Log, Havertown, PA). Just before [Ca2 ]i measurement was
done, Ca2 was added back to the buffer to a final concentration of
Synthesis and characterization of JJK694. 1HNMR spectra were
recorded on a Bruker DPS300 spectrometer in deuteriochloroform
þ
(CDCl3). Chemical shifts were expressed in parts per million (ppm, ꢂ)
þ
downfield of tetramethylsilane, and are referenced to the deutered
solvent (CHCl3). Reagents were purchased from Aldrich Chemical.
JJK694 (5-propyl-3-(4-propylphenoxy) bezene-1,2-diol) prepared by
saturation of obovatol with ethanol was added Pd/C (10 mg) by a
1 mM, and then JJK694 (25 mL at various concentration) and collagen
2þ
(10 mg/mL) and AA (100 mM) were added. Measurement of [Ca ]i
was done at room temperature in a MSIII fluorimeter (Photon
Technology International, South Brunswick, NJ) at excitation wave-
lengths of 340 and 380 and an emission wavelength of 505 nm. [Ca2 ]i
was calculated using the SPEX dM3000 software package.
1
method reported elsewhere. HNMR (CDCl3, 300 MHz): ꢂ 7.14 (d, 2H,
þ
J ¼ 8:4 Hz), 6.93 (d, 2H, J ¼ 8:4 Hz), 6.56 (s, 1H), 6.28 (s, 1H), 2.57
(
t, 2H, J ¼ 7:6 Hz), 2.41 (t, 2H, J ¼ 7:6 Hz), 1.70–1.42 (m, 4H), 0.95
t, 3H, J ¼ 7:3 Hz), 0.88 (t, 3H, J ¼ 7:3 Hz).
(
Arachidonic acid liberation assay. AA liberation was measured as
previously described.27,31) In brief, PRP was preincubated with [ H]
3
Washed rabbit platelet preparation and platelet aggregation assay.
ꢀ
Blood was withdrawn from the ear artery of male New Zealand white
rabbits and collected directly into 0.15 (v/v) of anticoagulant citrate
dextrose (ACD) solution that contained 0.8% citric acid, 2.2% sodium
citrate, and 2% dextrose (w/v). Washed platelets were prepared as
described previously.27) Briefly, platelet rich plasma (PRP) was
obtained by centrifugation of rabbit blood at 230 ꢁ g for 10 min. The
platelets were sedimented by centrifugation of the PRP at 800 ꢁ g for
AA (1 mCi/mL) at 37 C for 1.5 h, and then washed as described above.
3
8
[ H] AA labeled platelets (4 ꢁ 10 cells/mL) were pretreated with
100 mM BW755C, a COX-1 and LOX inhibitor, and various concen-
ꢀ
trations of JJK694 at 37 C for 3 min in the presence of 1 mM CaCl ,
2
and then stimulated with collagen (10 mg/mL). The reaction was
terminated by the addition of chloroform/methanol/HCl (200/200/1,
v/v/v). Lipids were extracted and separated by TLC on silica gel G
plates by the following development system: petroleum ether/diethyl
ether/acetic acid (40/40/1, v/v/v). The area corresponding to each of
the lipids was scraped off, and radioactivity was determined by liquid
scintillation counting.
15 min, and were washed with Hepes buffer (137 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2, 5.6 mM glucose, and 3.8 mM Hepes, pH 6.5)
containing 0.35% BSA and 0.4 mM EGTA (ethylene glycol bis (ꢁ-
0
0
aminoethyl ether) N,N,N ,N -tetraacetic acid). The washed platelets
were resuspended in Hepes buffer (pH 7.4), and were adjusted to
8
4
ꢁ 10 cells/mL. Platelet aggregation was measured by aggregometer
Measurement of arachidonic acid metabolites. TXB , PGD2 and
2
2
8)
27,31)
(Chrono-Log, Havertown, PA) by the turbidimetry method of Born,
as described previously. Briefly, the washed platelet suspension was
12-HETE generations were measured as previously described.
8
In
2
9)
brief, washed rabbit platelets (4 ꢁ 10 cells/mL) were preincubated
ꢀ
ꢀ
incubated at 37 C in the aggregometer with stirring at 1,000 rpm, and
then DMSO or various concentrations of JJK694 were added. After
with various concentrations of JJK694 at 37 C for 3 min and further
incubated with a mixture of [3H] AA and unlabeled AA (2 mM, 1 mCi/
3
min of preincubation, platelet aggregation was induced by the
mL) for 5 min. The reaction was terminated by the addition of stop
solution (2.6 mM EGTA, 130 mM BW755C). Lipids were extracted and
separated by TLC on silica gel G plates by the following development
system: ethyl acetate/isooctane/acetic acid/H2O (11/5/2/10, v/v/v/
v). The area corresponding to each of the lipids was scraped off and
radioactivity was measured by liquid scintillation counting.
addition of collagen (10 mg/mL), AA (100 mM), U46619 (1 mM), or
thrombin (0.05 U/mL).
Cell viability. Cell viability of platelets was determined as described
previously.30) In brief, cell death detection of platelets by JJK694 was
performed cell counting Kit 8 following the manufacturer’s instruc-
tions (Wako, Tokyo). In vitro viability was determined by measuring
reduced formazan, which is a colorimetric assay based on the reduction
Statistical analysis. Experimental results were expressed as
mean ꢂ SEM. One-way analysis of variance (ANOVA) was used for

2040
J.-Y. YU et al.
A
B
C
Fig. 1. Chemical Structure of JJK694 and Effects of JJK694 on Washed Rabbit Platelet Aggregation and Cell Viability.
Washed platelets were prepared and platelet aggregation and cell viability were measured as described in ‘‘Materials and Methods.’’ (A)
Chemical structure of JJK694; (B) Platelet aggregation induced by collagen (10 mg/mL), arachidonic acid (100 mM), U46619 (1 mM), and
thrombin (0.05 U/mL); (C) Cell viability under JJK694. The aggregation percentages are expressed as percentages of maximum aggregation by
the respective inducers. Data are expressed as mean ꢂ SEM (n ¼ 4).
A
B
Fig. 2. Effects of JJK694 on Serotonin Secretion.
ꢀ
Washed rabbit platelet suspension was incubated with imipramine (5 mM) and various concentrations of JJK694 at 37 C for 3 min prior to the
addition of collagen (10 mg/mL) (A), arachidonic acid (100 mM) (B). The serotonin concentration was determined by a fluorimetric method, as
described in ‘‘Materials and Methods.’’ Aspirin (ASA, 100 mM), an anti-platelet agent, was used as positive control. Data are expressed as
ꢃꢃ
mean ꢂ SEM (n ¼ 3). p < 0:01 vs. stimulus control.
multiple comparison, followed by Dunnett’s test (GraphPad, San
Diego, CA). Data were considered significant p < 0:05.
(Fig. 2A), and 32.6, 93.6, and 96.4% for AA (Fig. 2B)
at 10, 20, and 40 mM, respectively. In addition, total
serotonin content was expressed as lysis (Fig. 2).
Results
Effects of JJK694 on cytosolic calcium mobilization
Anti-platelet agents that block calcium signaling can
exert their beneficial action by inhibition of platelet
activation. Agonists, collagen and AA, added to induce
Effects of JJK694 on washed rabbit platelet aggre-
gation in vitro
To determine the selectivity of JJK694 on various
agonist-induced forms of platelet aggregation, agonist
concentrations were determined by a preliminary study
and set at 10 mg/mL for collagen, 100 mM for AA, 1 mM
for U46619, and 0.05 U/mL for thrombin. As shown in
Fig. 1B, JJK694 significantly inhibited collagen- and
AA-induced washed rabbit platelet aggregation, with
IC50 values of 13:14 ꢂ 1:25 and 11:85 ꢂ 5:50 mM,
respectively, but it had little effect on U46619- or
thrombin-induced platelet aggregation. These results
indicate that JJK694 selectively affects collagen- and
AA-mediated signal transduction in platelet activation.
In addition, we confirmed by cell viability assay that this
anti-platelet effect of JJK694 on platelets was not due to
cellular cytotoxicity (Fig. 1C).
2þ
[Ca ]i mobilization are shown in Fig. 3. Treatment of
the platelet suspension with JJK694 inhibited the
2
þ
elevation of [Ca ]i in response to collagen by 51.8,
69.2, and 85.0% (Fig. 3A) in response to AA by 53.0,
87.1, and 93.3% (Fig. 3B) at concentrations of 10, 20,
and 40 mM, respectively. Bar graphs of right panel in
Fig. 3 show the average of three separate experiments.
Effects of JJK694 on arachidonic acid and diacylgly-
cerol liberation
As shown in Fig. 4, pretreatment with JJK694 had no
effect on collagen-induced arachidonic acid (A) or
3
diacylglycerol liberation (B) in [ H] AA pre-labeled
rabbit platelets. U73122, a PLC inhibitor used as
positive control, significantly blocked arachidonic acid
liberation at a concentration of 20 mM.
Effects of JJK694 on serotonin secretion
JJK694 inhibited the platelet aggregation induced by
collagen and AA, and it reduced the release of serotonin.
In this result, JJK694 reduced the release of serotonin
in a concentration-dependent manner, with inhibition
percentages of 36.2, 70.1, and 78.4% for collagen
Effects of JJK694 on TXB , PGD , and 12-HETE
2
2
formation
JJK694 concentration dependently suppressed TXB2
(A), PGD2 (B), and 12-HETE (C) generation induced by

Antiplatelet Activity of JJK694
2041
A
B
Fig. 3. Effect of JJK694 on Agonist-Stimulated [Ca2 ]i.
Calcium (1 mM) was added to the platelet suspension 10 s before data collection (time 0). JJK694 was added to final concentrations of 10, 20,
þ
and 40 mM in the platelet suspension. Collagen (10 mg/mL) (A), or arachidonic acid (100 mM) (B) was added 3 min later. Taces are from a
ꢃꢃ
representative experiment, with similar results right panel as mean ꢂ SEM (n ¼ 3). p < 0:01 vs. stimulus control.
A
B
Fig. 4. Effect of JJK694 on Collagen-Induced Arachidonic Acid Liberation.
[3
H] Arachidonic acid-prelabeled platelets were incubated with various concentrations of JJK694 at 37 C for 3 min in the presence of 100 mM
ꢀ
3
BW755C, and then stimulated with 50 mg/mL of collagen for 2 min. Liberated [ H] arachidonic acid was measured as described in ‘‘Materials
ꢃꢃ
and Methods.’’ Data are expressed as mean ꢂ SEM (n ¼ 3). p < 0:01 vs. stimulus control.
3
addition of [ H] AA to intact rabbit platelets (Fig. 5).
These results suggest that JJK694 inhibits COX-1 and
LOX activities rather than TXA2 synthase, because
TXA2, PGD2, and 12-HETE are simultaneously pro-
duced from AA through the COX-1 and LOX pathways.
Aspirin (ASA), a COX-1 inhibitor used as positive
control, significantly blocked TXB2 and PGD2 at a
concentration of 100 mM.
gression of thrombosis and is therefore of importance in
the prevention of complications after an acute coro-
3
2)
nary. It is generally considered that platelet activation
is mediated mainly through adhesion of platelets to the
site of injury and through the action of endogenous
agonists such as collagen, ADP, and thrombin, followed
by the release of TXA2, which acts as an amplifying
3
3,34)
factor in the platelet aggregation.
We identified recently an anti-platelet effect of
obovatol, which appeared to be mainly mediated by
Discussion
1
8)
inhibition of PLC ꢃ2 phosphorylation. However, the
inhibitory effect of obovatol derivatives on platelet
aggregation and the mechanisms are not understood.
Hence, we are investigating the antiplatelet effect of
Platelet activation and aggregation play pivotal roles
in atherothrombosis. Inhibition of platelet aggregation
by clinical treatment impairs the formation and pro-

2042
J.-Y. YU et al.
A
B
C
Fig. 5. Effects of JJK694 on Arachidonic Acid Metabolites.
3
3
3
3
3
Arachidonic acid-mediated [ H] TXB2, [ H] PGD2, and [ H] 12-HETE formations were assayed using [ H] arachidonic acid. [ H] TXB2 (A),
3
[3
H] PGD2 (B), and [ H] 12-HETE (C) were extracted and separated by TLC on a silica gel G plate. The area corresponding to each lipid was
scraped off, and radioactivity was measured by liquid scintillation counting. Aspirin (ASA, 100 mM), an anti-platelet agent, was used as the
ꢃꢃ
positive control. Data are expressed as mean ꢂ SEM (n ¼ 3). p < 0:01 vs. stimulus control.
JJK694, an obovatol derivative, on washed rabbit
platelets. In the present study, JJK694 showed potential
as a potent anti-platelet agent in response to collagen
and AA. The anti-platelet activity of JJK694 might be
mediated by the suppression of COX-1 and LOX
activities.
LOX activities, and that the beneficial effect of JJK694
on thrombotic disease might be also due to its
modulation of platelet activity.
Acknowledgments
In this study, JJK694 dose-dependently inhibited
collagen- and AA-induced platelet aggregation but not
U46619- or thrombin-induced platelet aggregation. It
inhibited not only platelet aggregation induced by
collagen and AA, but also the selective cytosolic
calcium mobilization and serotonin secretion induced
by collagen and AA. Changes in cytosolic calcium
concentration play a central role in platelet activation as
an upstream event that further interacts with intracellular
targets to evoke functional and structural changes in
platelets,3 leading to the stimulation of enzymes that
are not fully functional at the low calcium concentra-
This work was supported by the National R&D
Program for Cancer Control, funded by the Ministry of
Health and Welfare of the Republic of Korea (1020090)
and by the National R&D Program funded by the
Ministry of Education, Science, and Technology of the
Republic of Korea (Chemical Genomics Research,
20100002073) and Grant K12050, awarded to the Korea
Institute of Oriental Medicine (KIOM) by Ministry of
Education, Science and Technology (MEST) of the
Republic of Korea.
5)
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2
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1
We suggest that JJK694 selectively inhibits platelet
aggregation, perhaps due to inhibition of COX-1 and
17) Lee YJ, Lee YM, Lee CK, Jung JK, Han SB, and Hong JT,

Antiplatelet Activity of JJK694
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