Antiplatelet and antithrombotic activities of CP201, a newly synthesized
Yong-Ri Jina, Kyung-Ae Hwanga, Mi-Ra Choa, Soo-Yeon Kima, Jin-Ho Kima,
Chung-Kyu Ryub, Dong-Ju Sonc, Young-Hyun Parkc, Yeo-Pyo Yuna,d,*
aCollege of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea
bCollege of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea
cCollege of Natural Sciences, Soonchunhyang University, Asan 336-745, South Korea
dResearch Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea
Received 5 December 2003; received in revised form 5 December 2003; accepted 13 April 2004
The antiplatelet and antithrombotic activities of a newly synthesized CP201, 2-(3,5-di-tert-butyl-4-hydroxyl)-3-chloro-1,4-naphthoqui-
none on human platelet aggregation in vitro and murine pulmonary thrombosis in vivo were examined. In addition, the antiplatelet activity of
CP201 involved in calcium-signaling cascade was also investigated. CP201 showed concentration-dependent inhibitory effects on platelet
aggregation induced by collagen and thrombin, with IC50values of 4.1F0.3 and 4.6F0.4 AM, respectively. Orally administered CP201
protected mice against the collagen plus epinephrine-induced thromboembolic death in a dose-dependent manner. On the other hand, CP201
did not alter such coagulation parameters as activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) in
human plasma in vitro. These results suggest that the antithrombotic activity of CP201 may be due to antiplatelet rather than anticoagulation
activity. CP201 potently inhibited platelet aggregation challenged by calcium ionophore A23187 and thapsigargin, which is a selective
inhibitor of the Ca2 +-ATPase pump, in a concentration-dependent manner, indicating that CP201 may have an inhibitory effect on calcium-
signaling cascade. This was supported by measuring [Ca2 +]iin platelets loaded with fura-3AM, where CP201 inhibited the rise in cytosolic
Ca2 +mediated by thrombin. Taken together, these results suggest that CP201 may be a promising antithrombotic agent, and the
antithrombotic effect of CP201 may be due to antiplatelet activity, which was mediated, at least partly, by the inhibition of cytosolic calcium
D 2004 Elsevier Inc. All rights reserved.
Keywords: Naphthoquinone; Antithrombosis; Antiplatelet; Calcium mobilization
Platelets are blood cells that participate in the human
primary hemostatic mechanism. Platelets need to be acti-
vated to perform all of their functions, and this activation
can be initially produced after an endothelial injury that
exposes subendothelial structures to the blood flow. Aggre-
gation (platelets–platelets interaction) has the final purpose
to produce a platelet thrombus that constitutes the primary
hemostatic plug. In addition, platelet aggregation plays a
critical role in the pathophysiology of thrombotic diseases,
and as a consequence, antiplatelet agents have been used
clinically in patients at risk for brain ischemia, unstable
angina, and acute myocardial infarction (Albers, 1995;
George, 2000). Because platelet inhibitors, such as aspirin,
clopidogrel, and abciximab, have been proven to be of
benefit to patients with these disorders (Antithrombotic
Trialists’ Collaboration, 2002; Kaul et al., 2002; Mazur et
al., 2002; Montalescot et al., 2001), there has been a
directed search for more effective and safe antiplatelet
Platelet aggregation involves a sequence of biochemical
reactions that are initiated by the contact of an agonistic
agent with receptors on the platelet membrane (Vizcaino-
Salazar, 1994). There is increasing evidence that internal
Ca2 +stores play a central role in the response of platelets to
activating agents (Rink and Sage, 1990; Sage, 1997; Siess,
1537-1891/$ – see front matter D 2004 Elsevier Inc. All rights reserved.
* Corresponding author. College of Pharmacy, Chungbuk National
University, 48 Gaeshin-Dong, Cheongju, 361-763, South Korea. Tel.: +82-
43-261-2821; fax: +82-43-268-2732.
E-mail address: firstname.lastname@example.org (Y.-P. Yun).
Vascular Pharmacology 41 (2004) 35–41
1989), such as the following that a rise in cytosolic Ca2 +
levels accompanies platelet activation through stimulation
of the enzymes, which are not fully functional at the low
Ca2 +concentration present in the resting platelets (Ber-
ridge, 1993; Heemskerk et al., 1993).
The compounds with the backbone of a 1,4-naphtho-
quinone chemical structure have shown a wide variety of
pharmacological effects such as antiviral, antifungal, anti-
cancer, and antiplatelet activities (Chen et al., 2002; Ko et
al., 2001; Lien et al., 2002; Rodriguez et al., 1995). Liao
et al. (1998) reported that PP1D-1 (2-chloro-3-methoxy-
carbonylpropionamido-1,4-naphthalenedione) exerts anti-
platelet effects mainly by inhibiting phosphoinositide
turnover. Shen et al. (1999) reported that NTP (2-p-
mercaptophenyl-1,4-naphthoquinone) showed an antiplate-
let activity on human platelets by suppressing the perfor-
mance of fibrinogen receptors and decreasing [Ca2 +]i
Among numerous 1,4-naphthoquinone derivatives so far
synthesized, CP201 [2-(3,5-di-tert-butyl-4-hydroxyl)-3-
chloro-1,4-naphthoquinone; Fig. 1] was one of the most
potent antithrombotic agents when administered to experi-
mental animals; whereas, it did not show any toxic effect. In
the present study, we examined a possibility of CP201 as a
novel antithrombotic agent by determining the effects of
CP201 on human platelet aggregation in vitro and murine
pulmonary thrombosis in vivo. Furthermore, the antiplatelet
activity of CP201 involved in calcium-signaling cascade
was also examined.
2. Materials and methods
CP201 was synthesized and characterized as previously
described (Ryu, 1988). In brief, a solution of 1,4-naphtho-
quinone (0.01 mol) and 4-acetophenyl amine (0.011 M) in
150 ml of 95% EtOH was refluxed for 5 h. After the
reaction mixture was kept overnight at 4 jC, precipitate
was collected by filtration. Crystallization of the precipitate
from MeOH afforded CP201. CP201 had color: bright red
crystal; m.p.: 184.0 jC; IR (KBr, cm? 1): 3500-3400 (OH),
3100 (w, aromatic ring), 2961, 1674 (s, C=O), 1436, 1363;
1H-NMR (DMSO-d6): y 1.5 (s, 18H, 2C(CH3)3), 5.5 (s, 1H,
OH), 7.2 (s, 2H, H-2,6), 7.9–8.2 (m, 4H, H5–H8, benzene);
MS (m/e): 396 (M+), 381, 340, 325, 284, 215, 173, 159,
151, 57; EA (%): Calcd. for C24H25O3Cl (396.91): C, 72.63;
H, 6.35; Found: C, 72.97; H, 6.40. 1,4-Naphthoquinone and
4-hydroxyphenyl amine were obtained from Aldrich Chem-
ical (St. Louis, MO, USA). Thrombin was purchased from
Chrono-Log (PA, USA). ADP, collagen, calcium ionophore
A23187, bovine serum albumin (BSA), thapsigargin, and
dimethylsulfoxide (DMSO) were purchased from Sigma (St.
Louis, MO, USA). Fluo-3, AM was from Molecular Probes
(Eugene, OR, USA). Other chemicals were of analytical
Male ICR mice (approximately 25–30 g) were purchased
from Samtako Biokorea (Osan, Korea) and acclimatized for
1 week at a temperature of 24F1 jC and a humidity of
55F5%. The animals had free access to a commercial
pellet diet and drinking water before experiments. The
animal studies have been carried out in accordance with
the Guide for the Care and Use of Laboratory Animals,
Chungbuk National University, Korea.
2.3. Washed human platelet preparation
Washed human platelets were prepared at room temper-
ature as previously described (Kang et al., 2001). In brief,
blood from healthy volunteers who had not taken any drug
for at least 15 days was collected by venipuncture in a
plastic flask containing 3.15% sodium citrate (1:9 v/v).
Platelet-rich plasma (PRP) was prepared by centrifugation
at 120 ?g for 15 min, and platelet-poor plasma (PPP) was
prepared by further centrifugation at 850 ?g for 15 min.
Washed platelets were prepared from PRP. The super-
natants were pooled and centrifuged at 600 ?g for 15
min. The platelet pellets were washed with modified
Tyrode–HEPES buffer (129 mM NaCl, 2.8 mM KCl,
8.9 mM NaHCO3, 0.8 mM MgCl2, 0.8 mM KH2PO4, 2
mM EGTA, 5.6 mM glucose, 10 mM HEPES, 0.35%
BSA, pH 7.4) and centrifuged at 600 ?g for 15 min.
Finally, platelets were gently resuspended in Tyrode–
HEPES buffer (129 mM NaCl, 2.8 mM KCl, 8.9 mM
NaHCO3, 0.8 mM MgCl2, 0.8 mM KH2PO4, 1 mM CaCl2,
5.6 mM glucose, 10 mM HEPES, 0.35% BSA, pH 7.4).
The platelets were counted using a Coulter Counter (Coul-
ter Electronics, Hialeah, FL, USA) and adjusted to a
concentration of 3?108platelets/ml.
2.4. Antiplatelet aggregation assay
Platelet aggregation was measured by using an aggreg-
ometer (470-VS, Chrono-Log, Havertown, PA, USA) as
Fig. 1. Chemical structure of CP201 (2-(3,5-di-tert-butyl-4-hydroxyl)-3-
Y.-R. Jin et al. / Vascular Pharmacology 41 (2004) 35–41
Born and Cross (1963) described. Briefly, washed human
platelet suspension was incubated at 37 jC for 4 min in
the aggregometer with stirring at 1000 rpm before aggre-
gation was challenged by the addition of collagen (50 Ag/
ml), thrombin (0.5 U/ml), calcium ionophore A23187 (1
AM), or thapsigargin (1 AM). The resulting aggregation,
measured as the change in light transmission, was
recorded for 8 min. The inhibition extent of platelet
aggregation is expressed as % inhibition (X) using the
following equation: X=[(A?B)/A]?100, where A is the
maximal aggregation rate of vehicle-treated platelets, and
B is the maximal aggregation rate of sample-treated
2.5. In vivo antithrombosis assay
The antithrombotic effect of CP201 was performed by
mouse pulmonary thrombosis test as DiMinno and Silver
(1983) described. In brief, male ICR mice, weighing about
25–30 g, were used after overnight fasting. CP201 (50, 100
mg/kg), aspirin (50 mg/kg) as a positive control, or 0.5%
CMC solution as a vehicle were administered orally. Ninety
minutes after oral administration of sample, a mixture
solution of collagen (114 Ag/mouse) and epinephrine (1.83
Ag/mouse) was injected into the tail vein to induce pulmo-
nary thrombosis. The number of dead or paralyzed mice was
recorded up to 15 min, and the percentage of protection was
2.6. In vitro anticoagulation assay
Measurements of plasma activated partial thromboplas-
tin time (APTT), prothrombin time (PT), and thrombin
time (TT) were performed by using Automated Coagula-
tion Laboratory 100 Instrument and an ACL-assay reagent
kit (Instrumentation Laboratory, Milano, Italy), as previ-
ously described (Yuk et al., 2000). In brief, the PPP was
incubated with CP201 or heparin for 7 min at 37 jC. One
hundred microliters of the incubated plasma was mixed
with 50 Al of cephalin in the process plate, and the
coagulation was started by addition of CaCl2 (1 mM),
100 Al of thromboplastin, and 100 Al of bovine thrombin
into the 100 Al of incubated plasma for APTT, PT, and TT
2.7. Cytosolic calcium mobilization measurement
Cytosolic calcium mobilization was measured by using
a laser-scanning confocal microscope (Carl Zeiss LSM
410, Germany), as Lee et al. (1998) described. In brief,
washed human platelets were incubated with 4 AM fluo-3,
AM in washing buffer for 40 min. Excess fluo-3 was
removed by centrifugation (500 ?g for 10 min) and
washed two times with calcium-free washing buffer con-
taining 2 mM EGTA. Each coverslip containing stained
platelets was mounted on a perfusion chamber, subjected
to a confocal laser-scanning microscope, and then scanned
every second with a 488-nm excitation argon laser and a
515-nm-long pass emission filter. After treatment with
CP201 for 3 min, thrombin was added to the platelets
by using an automatic pumping system. All images from
the scanning were processed to analyze changes of calcium
in a single-cell level. The results were expressed as the
relative fluorescence intensity.
2.8. Lactate dehydrogenase (LDH) assay
The released LDH activity was measured spectropho-
tometrically by recording the decrease in the optical
density of h-NADH at 340 nm, as previously described
(Yuk et al., 2000). Samples (20 AM) were incubated with
washed human platelet for 120 min and then centrifuged
for 4 min at 1312 ?g. The LDH activity in the platelet
suspension and cellular LDH activity from platelets,
which was lysed with 1% Triton-X 100, were deter-
mined. Total LDH activity was the summation of both
released and cellular LDH activities. The released LDH
activity was expressed as a percentage of total LDH
2.9. Statistical analysis
The experimental results were expressed as the meanF
S.E.M. Fisher’s Exact Test for the pulmonary thrombosis
model, and unpaired Student’s t test for the sample-
treated groups and control groups were performed. The
data were considered significant with a probability less
3.1. Effect of CP201 on human platelet aggregation
As shown in Fig. 2, CP201 inhibited collagen (50 Ag/
ml)-, thrombin (0.5 U/ml)-, thapsigargin (1 AM)-, and
A23187 (1 AM)-induced platelet aggregation in a concen-
tration-dependent manner, with IC50 values of 4.1F0.3,
4.6F0.4, 2.8F0.3, and 2.6F0.1 AM, respectively.
3.2. Effect of CP201 on mouse pulmonary thrombosis
Intravenous injection of a mixture of collagen (114 Ag/
mouse) and epinephrine (1.83 Ag/mouse) into the tail vein of
mice caused pulmonary thrombosis, resulting in death and
giving a survival rate of 14.3% in the control group.
Previous oral administration of CP201 (50, 100 mg/kg)
exhibited a significant dose-dependent preventive effect,
giving a survival rate of 54.5% and 73.7%, respectively
(Table 1); while aspirin, as a reference drug, showed a
protective effect by 47.4% at dose of 50 mg/kg.
Y.-R. Jin et al. / Vascular Pharmacology 41 (2004) 35–41
3.3. Effect of CP201 on coagulation parameters in vitro
The effects of CP201 on coagulation time were evaluated
by APTT, PT, and TT assay using human PPP. APTT, PT,
and TT were not affected by CP201 treatment at the
concentration up to 100 AM (Table 2).
3.4. Effect of CP201 on thrombin-induced calcium mobili-
zation in human platelet
To examine the effect of CP201 on the calcium
mobilization in activated platelets, the calcium level was
measured in washed human platelets in the absence of
extracellular calcium. After treatment with thrombin (0.5
U/ml), it caused a rapid, but transient, increase in
[Ca2 +]i. Whereas, treatment of CP201 (10 AM) complete-
ly blocked this increase of [Ca2 +]i in human platelets
3.5. Platelet cytotoxicity
The released LDH activity was assayed to study the
cytotoxic effect of CP201 on human platelets. The LDH
activities were approximately 5.1% for the vehicle- and
5.2% for the sample (20 AM)-treated platelets for 120
min. No significant increase in LDH release was ob-
Effect of CP201 on pulmonary thrombosis in mice
DrugDose (mg/kg) No. dead or paralyzed/
CP201 was orally administered 90 min before the intravenous injection of
epinephrine (1.83 Ag/mouse) plus collagen (114 Ag/mouse).
* P<.01 compared with control.
** P<.001 compared with control.
Effect of CP201 on human plasma coagulation time
DrugDose (AM)APTT (s)PT (s)TT (s)
The results are expressed as meanFS.E.M. (n=5). N.C.—no coagulation.
*P<.01 compared with control.
**P<.001 compared with control.
Fig. 2. Effect of CP201 on washed human platelet aggregation. Washed
human platelet suspension was incubated at 37 jC in an aggregometer
with stirring at 1000 rpm, and CP201 was then added. After 3 min of
preincubation, the platelet aggregation was induced by the addition of
collagen (50 Ag/ml), thrombin (0.5 U/ml), A23187 (1 AM), or
thapsigargin (1 AM), respectively. Data were expressed as meanF
Fig. 3. Effect of CP201 on thrombin-induced calcium mobilization in
human platelet. Washed human platelet was incubated with 4 AM fluo-3,
AM in washing buffer for 40 min and was treated with 10 AM CP201 for 3
min. Then, the thrombin (0.5 U/ml) was added at the indicated time. The
[Ca2 +]iwas monitored using a confocal laser-scanning microscope. The
results were expressed as the relative fluorescence intensity (RFI). Each
trace is a single-cell representative from at least three separate experiments.
Y.-R. Jin et al. / Vascular Pharmacology 41 (2004) 35–41
served between CP201- and vehicle-treated platelets
The results of the present study demonstrate that CP201
none) displays a potent protective effect on pulmonary
thrombosis in mice in vivo and strong inhibitory effect on
human platelet aggregation in vitro, and the antiplatelet
activity of CP201 may be due, at least partly, to the
inhibition of cytosolic calcium mobilization.
CP201 inhibited washed human platelet aggregation
induced by thrombin, collagen, calcium ionophore
A23187, and thapsigargin in a concentration-dependent
manner (Fig. 2). Thrombin and collagen, both of which
are strong agonists, have different platelet aggregation
mechanisms (Colman et al., 1994). Thrombin interacts with
platelet through a specific receptor belonging to the super-
family of receptors that are coupled to G proteins and
phospholipase Ch, producing diacylglycerol, which stimu-
lates protein kinase C that is closely linked to secretion.
Inositol trisphosphate is also produced and plays a role in
increasing intracellular calcium (Lapetina, 1990). Whereas,
collagen induces platelet activation through a tyrosine
kinase-based signaling pathway that involves the kinase
Syk and phospholipase Cg2, which results into calcium
increase, shape change, and granule release; adhesion is
partly and aggregation is largely dependent on ADP and
prostaglandin (PG) H2/TXA2release (Cowan, 1981; Cowan
et al., 1981). As our aggregation data shown in Fig. 2,
CP201 displayed almost the same half-maximal inhibitory
concentrations (IC50) against platelet aggregation induced
by both agonists in an unselective pattern, indicating that
CP201 does not interfere with the interactions between
aggregation inducers and their receptors on the platelet
membrane surface. Judging from the chemical structure,
CP201 may not be a disintegrin. It seems that CP201 may
interfere with any signal transductions that are commonly
activated by both inducers. It is thus suggested that CP201
may inhibit both agonists-induced platelet aggregation by
inhibiting calcium mobilization, because calcium mobiliza-
tion is involved in the major cellular processes of platelet
activation (Godfraind, 1994; Niiya et al., 1987; Oates,
1996), and it has been widely accepted that the intracellular
calcium mobilization plays a central role in platelet aggre-
gation challenged by both agonists (Kucera and Ritten-
house, 1990; Nolan and Lapetina, 1990; Smith et al., 1992).
To determine the effect of CP201 on the calcium mobi-
lization, we measured [Ca2 +]iin platelets loaded with fluo-
3, AM. At a concentration of 10 AM, CP201 completely
inhibited the transient increase of cytosolic calcium mobi-
lization induced by thrombin, which activates phospholipase
C pathway and elevates cytosolic calcium by an inositol
1,4,5-triphosphate-dependent release from internal stores
(Kucera and Rittenhouse, 1990; Nolan and Lapetina,
1990; Fig. 3). It has been reported that several 1,4-naph-
thoquinone derivatives inhibited platelet aggregation by
suppression of intracellular calcium mobilization, which
were mediated by the inhibition of IP3 production or
elevation of cAMP level (Chang et al., 2001; Ko et al.,
1995; Liao et al., 1998; Zhang et al., 2001). Considering
that CP201 also inhibited platelet aggregation induced by
calcium ionophore A23187 and tharpsigargin (Fig. 2), it
seems that the inhibitory effect of CP201 on calcium
mobilization may be due to the elevation of cAMP level
rather than IP3 inhibition. Because calcium ionophore
A23187 can penetrate membranes and directly mobilize
the intracellular calcium from intracellular stores and also
influx from extracellular medium, resulting platelet aggre-
gation independent of IP3formation (Fuse et al., 2001);
whereas, thapsigargin, which can selectively inhibit the
endoplasmic reticulum Ca2 +-ATPase pump, may also cause
IP3-independent increase of Ca2 +levels from intracellular
stores and influx from outside, and induce platelet aggrega-
tion in an extracellular Ca2 +-dependent way (Huang and
Kwan, 1998; Sun and Kambayashi, 2000). It has been well
reported that drugs which increase cAMP level can inhibit
both calcium ionophore A23187- and thapsigargin-induced
platelet aggregation (Siess, 1989). It was not unreasonable
to speculate that CP201 may be able to increase cAMP
level, although further study on this direction is still needed.
In the present in vivo antithrombotic study, the lethal
effect of aggregating agonists on mice is caused by
massive occlusion of the microcirculation of lung by
platelet thrombosis or by vasoconstriction due to the
increase of TXA2and PGF2ain platelets (Cerskus et al.,
1978; Nishizawa et al., 1972; Silver et al., 1974). CP201
significantly prevented the death due to mouse pulmonary
thrombosis induced by platelet aggregation in a dose-
dependent manner (Table 1). The present result is consis-
tent with reports that drugs having calcium antagonistic
activity showed potent protective effect on the present
pulmonary thrombosis model (Kim et al., 1999; Odawara
et al., 1996). On the other hand, CP201 did not affect the
Effect of CP201 on platelet LDH release
Time (min) Released LDH (%)
CP201 (20 AM) or vehicle (DMSO 0.5%) was incubated with washed
human platelet for 120 min, and LDH activity was determined as the
indicated time point. The extent of LDH release was expressed as the
percent of total enzyme activity lysed with 0.1% Triton X-100. Results are
expressed as meanFS.E.M. (n=3).
Y.-R. Jin et al. / Vascular Pharmacology 41 (2004) 35–41
clotting parameters, whereas heparin, a reference drug,
prolonged the clotting time (Table 2).
Possible toxicity of CP201 was examined by in vitro
released LDH and in vivo administration of CP201 to mice.
CP201 showed neither any significant increase in released
LDH compared with the vehicle (Table 3) nor any toxic
effects when administered orally to mice (data not shown).
In conclusion, these results suggest that CP201 may be a
promising antithrombotic agent and that the antithrombotic
effect of CP201 was due to antiplatelet activity, which may
be mediated by inhibition of cytosolic calcium mobilization.
This work was supported by the Program of Research
Centre for Bioresource and Health from MOSTand KOSEF.
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