Fitoterapia 91 (2013) 1–8

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Antithrombotic activities of pellitorine in vitro and in vivo

Sae-Kwang Ku a,1, In-Chul Lee b,1, Jeong Ah Kim c, Jong-Sup Bae c,⁎
a
Department of Anatomy and Histology, College of Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea
b
Department of Cosmetic Science and Technology, Seowon University, Cheongju 361-742, Republic of Korea
c
College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Pellitorine (PLT), an active amide compound, is well known to possess insecticidal, anti-
Received 10 July 2013 bacterial and anticancer properties. However, the anti-coagulant functions of PLT are not
Accepted in revised form 9 August 2013 studied yet. Here, the anticoagulant activities of PLT were examined by monitoring activated
Available online 22 August 2013 partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of cell-based
thrombin and activated factor X (FXa). Furthermore, the effects of PLT on the expressions of
Keywords: plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA)
Pellitorine were tested in tumor necrosis factor (TNF)-α activated human umbilical vein endothelial cells
Coagulation cascade (HUVECs). Treatment with PLT resulted in prolonged aPTT and PT and inhibition of the
Fibrinolysis
activities of thrombin and FXa, and PLT inhibited production of thrombin and FXa in HUVECs.
Endothelium
And PLT inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. In
accordance with these anticoagulant activities, PLT elicited anticoagulant effects in mouse. In
addition, treatment with PLT resulted in the inhibition of TNF-α-induced production of PAI-1
and in the significant reduction of the PAI-1 to t-PA ratio. Collectively, PLT possesses anti-
thrombotic activities and offers bases for development of a novel anticoagulant.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction precise and balanced generation of thrombin at sites of

The leading causes of death (30% of total) in the world are
diseases involving the heart and blood vessels and, conse-
quently, thrombosis [1]. Most thromboembolic processes
require anticoagulant therapy and this explains the current
efforts to develop specific and potent anticoagulant and
antithrombotic agents [2]. Primary haemostatic events are
triggered in response to damage of the vascular wall by the
exposure of blood to the subendothelial extracellular matrix
[3,4]. Thrombin is the key effector enzyme of the coagulation
system, having many biologically important functions such as
the activation of platelets, conversion of fibrinogen to a fibrin
network, and feedback amplification of coagulation [4,5]. The
⁎ Corresponding author at: College of Pharmacy, CMRI, Research Institute
of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro,
Buk-gu, Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82
53 950 8557.
E-mail address: baejs@knu.ac.kr (J.-S. Bae).
1
First two authors contributed equally to this work.
vascular injury is the result of an ordered series of reactions
collectively referred to as blood coagulation [4,5]. Clots are
eventually broken down by plasmin, which is activated by
tissue-type plasminogen activator (t-PA) from plasminogen.
Thrombin is also an activator of inflammation and an
inhibitor of fibrinolysis [6]. The hemostatic plug that forms
within blood vessels, often within the veins or arteries of the
heart, in pathological conditions associated with arterial
disease, referred to as a thrombus [6], is a major cause of
morbidity and death. Clotting time assays measure the time
required to generate thrombin [7] and activated partial
thromboplastin time (aPTT) measures the efficacy of the
contact activation and common coagulation pathways [7].
Furthermore, the aPTT or prothrombin time (PT) mainly
serves to aid the diagnosis of deficiencies in certain factors
[8].
Asarum sieboldii Miq. (Aristolochiaceae) is a wide-ranging
species found through North America, Europe, and Asia [9].
The radix of A. sieboldii is a traditional herb medicine used as

0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2013.08.004
2 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8
remedies for aphthous stomatitis, toothache, and gingivitis
and as a local anesthetic agent in Korea and China [10]. Previous
studies on the constituents of A. sieboldii have revealed isolation
of various types of essential oils, amide, alkaloids, lignans, and
flavonoids [9,11,12]. Pellitorine (PLT) was isolated from the
methylene chloride soluble fraction of the roots of A. sieboldii
using a combination of silica gel column chromatography and
recrystallization. A conjugated alkaldienamide, pellitorine was
isolated mainly from the Piper species [13]. Pellitorine showed
various biological properties including insecticidal [14], lavicidal
[14], antibacterial [15], and anti-cancer [16] activities. However,
no studies on the anticoagulant activities of PLT have been
reported. Therefore, in the current study, we examined the
anticoagulant activities of PLT in the production of FXa and
thrombin, and their effects on PT and aPTT and on fibrinolytic
activity.
2. Materials and methods
2.1. Reagents
TNF-α was purchased from Abnova (Taiwan). Anti-tissue
factor antibody was purchased from Santa Cruz Biologics (Santa
Cruz, CA). c-Jun N-terminal kinase (JNK) inhibitor (SP600125),
Nuclear factor (NF)-κB inhibitor (Emodin), and extracellular
signal regulated kinase (ERK) inhibitor (PD98059) were pur-
chased from R&D Systems (Minneapolis, MN). Factors V, Vll,
Vlla, FX, and FXa, antithrombin III (AT III), prothrombin, and
thrombin were obtained from Haematologic Technologies
(Essex Junction, VT, USA). aPTT assay reagent and PT reagents
were purchased from Fisher Diagnostics (Middletown, Virginia,
USA), and the chromogenic substrates, S-2222 and S-2238, were
purchased from Chromogenix AB (Sweden). The PAI-1 and t-PA
ELISA kits were purchased from American Diagnostica Inc.
(Stamford, CT, USA). Other reagents were of the highest
commercially available grades. Oleanolic acid (OA) was pre-
pared as described previously [17].
2.2. Plant material, extraction, and purification
Melting points were obtained with an Electrothermal 9100
melting point apparatus (Electrothermal Ltd.). 1H and 13C NMR
spectra were obtained on a Bruker ARX spectrometer (250 MHz)
and chemical shifts given in δ (ppm) from tetramethylsilane
(TMS) as an internal standard. Column chromatography was
carried out on silica gel (70–230 mesh, Merck). Thin layer
chromatography (TLC) analysis was performed on Kieselgel 60
F254 (Merck 1.05715) aluminum plate; spots were visualized by
spraying with 10% aqueous H2SO4 followed by heating. All other
chemicals and solvents were of analytical grade and used
without further purification.
The roots of A. sieboldii were purchased from herbal market at
Daegu, Korea, in February 2006. The plant material was identified
by Dr. Seung Ho Lee at the College of Pharmacy, Yeungnam
University. A voucher specimen (SH0602) was deposited at the
herbarium, College of Pharmacy, Yeungnam University. The
dried roots of A. sieboldii (6.0 kg) were extracted with MeOH at
room temperature for 5 days. After being concentrated, the
MeOH extract (564.0 g) was suspended in water and then
partitioned successively with methylene chloride (MC) and ethyl
acetate (EtOAc) to give MC (298.0 g), EtOAc (15.0 g), and water
(251.0 g) extractions, respectively. The MC extraction was
fractioned by silica gel column chromatography eluting with
EtOAc in n-hexane (0–100%, step-wise), to yield twenty fractions
(MC1–MC20). MC15 (8.2 g) was chromatographed on silica gel
column using a solvent system of EtOAc in n-hexane (15%) to
give seven fractions (MC15-1–MC15-7). MC15-4 (16.6 g) was
purified by recrystallization from chloroform to afford a
needle-type solid, compound 1 [675.0 mg, 0.12% (w/w) of
MeOH extract]. The structure of compound 1 (Fig. 1) was
identified by a combination of spectroscopic methods and
comparisons with the literature data [18].
2.3. Pellitorine (1)
Needles, mp 69 °C; 1H NMR (250 MHz, CDCl3): δ 0.91 (3H,
s, H-10), 0.93 (3H, s, H-3′), 0.96 (3H, s, H-4′), 1.32 (4H, m, H-8,
H-9), 1.44 (2H, m, H-7), 1.82 (1H, m, H-2′), 2.18 (2H, dd, J =
12.5, 6.2 Hz, H-6), 3.18 (2H, t, J = 12.9, 6.6 Hz, H-1′), 5.18 (1H,
d, J = 15.0 Hz, H-2), 5.58 (NH, br s), 6.07 (1H, m, H-5), 6.17
(1H, m, H-4), 7.20 (1H, m, H-3); 13C NMR (63 MHz, CDCl3): δ
14.4 (C-10), 20.5 (C-3′, C-4′), 22.9 (C-9), 28.9 (C-7), 29.0 (C-2′),
31.8 (C-8), 33.3 (C-6), 47.3 (C-1′), 122.0 (C-2), 128.6 (C-4),
141.8 (C-3), 143.8 (C-5), 166.9 (C-1).
2.4. Isolation of plasma
Blood samples were taken in the morning from 10 healthy
volunteers in fasting status (aged between 24 and 28 years, 4
males and 6 females) without cardiovascular disorders, allergy
and lipid or carbohydrate metabolism disorders, untreated with
drugs. All subjects gave written informed consent before
participation. Healthy subjects did not use addictive substances
and antioxidant supplementation, and their diet was balanced
(meat and vegetables). Human blood was collected into sodium
citrate (0.32% final concentration) and immediately centrifuged
(2000 ×g 15 min) to obtain plasma.
2.5. Anticoagulation assay
aPTT and PT were determined using a Thrombotimer
(Behnk Elektronik, Germany), according to the manufacturer's
instructions as described previously [19]. In brief, citrated
normal human plasma (90 μl) was mixed with 10 μl of PLT or
OA and incubated for 1 min at 37 °C. aPTT assay reagent
(100 μl) was added and incubated for 1 min at 37 °C, and then
20 mM CaCl2 (100 μl) was added. Clotting times were
recorded. For PT assays, citrated normal human plasma
(90 μl) was mixed with 10 μl of PLT or OA stock and incubated
for 1 min at 37 °C. PT assay reagent (200 μl), which has been
preincubated for 10 min at 37 °C, was then added and clotting
time was recorded. PT results are expressed in seconds and as
International Normalized Ratios (INR), and aPTT results are
expressed in seconds. INR = (PT sample / PT control)ISI. ISI =
international sensitivity index.
Fig. 1. The chemical structure of PLT.
 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8
2.6. Platelet aggregation assay
Mouse platelets from platelet-rich plasma (PRP) were
washed once with HEPES buffer (5 mM HEPES, 136 mM
NaCl, 2.7 mM KCl, 0.42 mM NaH2PO4, 2 mM MgCl2, 5.6 mM
glucose, 0.1% BSA (w/v), pH to 7.45). The platelet aggregation
study was carried out according to a method previously
reported [20]. Washed platelets were incubated with indi-
cated PLT or OA for 3 min, and then stimulated by thrombin
(3 U/ml, Sigma) in 0.9% saline solution at 37 °C for 5 min.
Platelet aggregation was recorded using an aggregometer
(CHRONO-LOG, Havertown, PA, USA).
2.7. Thrombin-catalyzed fibrin polymerization
Thrombin-catalyzed polymerization was determined
every 6 s for 20 min by monitoring turbidity at 360 nm using
a spectrophotometer (TECAN, Switzerland) at ambient tem-
perature. Control plasma and plasma incubated with PLT or OA
were trebly diluted TBS (50 mM Tris-buffered physiological
saline solution pH 7.4) and clotted with thrombin (final
concentration — 0.5 U/ml). The maximum polymerization
rate (Vmax, ΔmOD/min) of each absorbance curve was
recorded [21]. All experiments were performed three times.
2.8. Cell culture
Primary HUVECs were obtained from Cambrex Bio Science
(Charles City, IA) and were maintained using a previously
described method [22,23]. Briefly, the cells were cultured until
confluent at 37 °C at 5% CO2 in EBM-2 basal media supplemented
with growth supplements (Cambrex Bio Science).
2.9. Cell viability assay
MTT was used as an indicator of cell viability. The cells were
grown in 96-well plates at a density of 5 × 103/well. After 24 h,
the cells were washed with fresh medium, followed by
treatment with PLT. After a 48-h incubation period, the cells
were washed, and 100 μl of 1 mg/ml MTT was added, followed
by incubation for 4 h. Finally, 150-μl DMSO was added to
solubilize the formazan salt formed, the amount of which was
determined by measuring the absorbance at 540 nm using a
microplate reader (Tecan Austria GmbH, Austria).
2.10. Factor Xa production on the surfaces of HUVECs
TNF-α (10 ng/ml for 6 h in serum-free medium) stimu-
lated confluent monolayers of HUVECs (preincubated with
the indicated concentrations of PLT or OA for 10 min) in a
96-well culture plate were incubated with FVIIa (10 nM) in
buffer B (buffer A supplemented with 5 mg/ml bovine serum
albumin [BSA] and 5 mM CaCl2) for 5 min at 37 °C in the
presence or absence of anti-TF IgG (25 μg/ml). FX (175 nM)
was then added to the cells (final reaction mixture volume,
100 μl) and incubated for 15 min. The reaction was stopped
by adding buffer A (10 mM HEPES, pH 7.45, 150 mM NaCl,
4 mM KCl, and 11 mM glucose) containing 10 mM EDTA and
the amounts of FXa generated were measured by using a
chromogenic substrate. Changes in absorbance at 405 nm
over 2 min were monitored using a microplate reader. Initial
3
rates of color development were converted into FXa concen-
trations using a standard curve prepared with known dilutions
of purified human FXa.
2.11. Thrombin production on the surfaces of HUVECs
Measurement of thrombin production by HUVECs was
quantitated as previously described [19,23]. Briefly, HUVECs
were pre-incubated in 300 μl containing PLT or OA in 50 mM
Tris–HCl buffer, 100 pM FVa, and 1 nM FXa for 10 min,
followed by addition of prothrombin to a final concentration
of 1 μM. After 10 min, duplicate samples (10 μl each) were
transferred to a 96-well plate containing 40 μl of 0.5 M EDTA
in Tris-buffered saline per well to terminate prothrombin
activation. Activated prothrombin was determined by mea-
suring the rate of hydrolysis of S2238 at 405 nm. Standard
curves were prepared using the amounts of purified thrombin.
2.12. Thrombin or factor Xa (FXa) activity assay
PLT or OA in 50 mM Tris–HCl buffer (pH 7.4) containing
7.5 mM EDTA and 150 mM NaCl was mixed in the presence
of 150 μl of AT III (200 nM). The heparins with AT III
(200 nM) were dissolved in physiological saline and placed
in the sample wells. After incubation at 37 °C for 2 min,
thrombin solution (150 μl; 10 U/ml) was added, followed by
incubation at 37 °C for 1 min. S-2238 (a thrombin substrate;
150 μl; 1.5 mM) solution was then added and absorbance at
405 nm was monitored for 120 s using a spectrophotometer
(TECAN, Switzerland). And FXa activity assay was performed
in the same manner as the thrombin activity assay, but using
factor Xa (1 U ml/1) and S-2222 as substrates.
2.13. In vivo bleeding time
Tail bleeding times were measured using the method
described by Dejana et al. [19,24]. Briefly, ICR mice were
fasted overnight before experiments. One hour after intrave-
nous administration of PLT or OA, tails of mice were
transected at 2 mm from their tips. Bleeding time was
defined as the time elapsed until bleeding stopped. When
the bleeding time exceeded 15 min, bleeding time was
recorded as 15 min for the analysis. All animals were treated
in accordance with the Guidelines for the Care and Use of
Laboratory Animals issued by Kyungpook National University.
2.14. ELISA for PAI-1 and t-PA
The concentrations of PAI-1 and t-PA in HUVEC cultured
supernatants were determined using ELISA kits (American
Diagnostica Inc. CT, USA).
2.15. Statistical analysis
Data are expressed as mean ± SEM (standard error of the
mean) of at least three independent experiments. Statistical
significance between two groups was determined using the
Student's t-test. Statistical significance was accepted for
p b 0.05.
4 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8

Table 1 recently we published that OA has anticoagulant activities in

Anticoagulant activity of PLTa. vitro and in vivo [17].
In vitro coagulant assay
3.1. Effects of PLT on aPTT and PT
Sample Dose aPTT (s) PT (s) PT (INR)

Control Saline 30.6 ± 0.6 14.0 ± 0.2 1.00 Incubation with PLT resulted in changes in the coagulation
PLT 1 μM 31.2 ± 0.4 14.2 ± 0.4 1.03
properties of human plasma. The anticoagulant properties of PLT
2 μM 30.8 ± 0.6 14.6 ± 0.6 1.10
3 μM 31.4 ± 0.8 15.0 ± 0.4 1.16 in human plasma were tested using aPTT and PT assays; a
5 μM 32.4 ± 1.2 15.2 ± 0.2 1.20 summary of the results is shown in Table 1. Although the
10 μM 40.6 ± 0.4⁎⁎ 23.2 ± 0.2 3.04⁎⁎ anticoagulant activities of PLT were weaker than those of
20 μM 48.2 ± 0.6⁎⁎ 27.4 ± 0.4 4.38⁎⁎ heparin, aPTT and PT were significantly prolonged by treatment
OA 40 μM 63.4 ± 1.0⁎⁎ 28.2 ± 0.6 4.67⁎⁎
Heparin 0.5 μg/ml 10 μg/ml 7.32⁎⁎
with PLT at concentrations greater than 10 μM. The result
114.8 ± 1.2⁎⁎ 34.6 ± 0.8⁎⁎ showing prolongation of aPTT suggests inhibition of the intrinsic
and/or the common pathway, whereas prolongation of PT
In vivo bleeding time
indicates that PLT could also inhibit the extrinsic coagulation
Sample Dose Tail bleeding time (s) n pathway. To confirm these data in vivo, PLT was administered
Control Saline 41.6 ± 1.2 10
into mouse via intravenous injection. As shown in Table 1, tail
PLT 4.5 μg/mouse 57.6 ± 0.8⁎⁎ 10 bleeding times were significantly prolonged by treatment with
9.0 μg/mouse 71.8 ± 1.2⁎⁎ 10 PLT. Assuming that the average weight of a mouse was 20 g, and
OA 36.5 μg/mouse 74.6 ± 1.4⁎⁎ 10 the average blood volume was 2 ml, the amount of PLT injected
Heparin 1 mg/mouse 121.5 ± 1.2⁎⁎ 10
(4.5 or 9.0 μg per mouse) or OA injected (36.5 μg per mouse)
a
Each value represents the means ± SEM (n = 10). was equivalent to PLT 10, 20 μM or OA 40 μM in peripheral
⁎⁎ p b 0.01 as compared to control.
blood.

3.2. Effects of PLT on thrombin-catalyzed platelet aggregation

3. Results and discussion and fibrin polymerization and cellular viability

In this study, we examined the anticoagulant effects of The effects of PLT on thrombin-catalyzed fibrin polymer-
pellitorine (PLT, Fig. 1) for the first time and sought to ization in human plasma were monitored as changes in
identify the mechanisms responsible for these effects. absorbance at 360 nm, as described in the Materials and
Oleanolic acid (OA) was used as a positive control because methods section. The results, shown in Fig. 2A, demonstrate

Fig. 2. Effects of PLT on fibrin polymerization in human plasma and cytotoxicity. (A) Thrombin-catalyzed fibrin polymerization at the indicated concentrations of
PLT or OA (40 μM, positive control) was monitored using a catalytic assay, as described in the “Materials and methods” section. The results are Vmax values
expressed as percentages versus controls. (B) Effect of PLT or OA (40 μM) on mouse platelet aggregation induced by 3 U/ml thrombin. (C) Effect of PLT on cellular
viability was measured by MTT assay. Data represent the mean ± SEM of three independent experiments performed in triplicate. **p b 0.01 vs. Th alone.
 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8 5

Fig. 3. Effects of PLT on inactivation of thrombin and factor Xa. (A) Inhibition of thrombin (Th) by PLT (○) or OA (40 μM, ■) was measured using a chromogenic
assay, as described in the “Materials and methods” section. (B) Inhibition of factor Xa (FXa) by PLT (○) or OA (40 μM, ■) was monitored using a chromogenic
assay, as described in the “Materials and methods” section. Heparin (●) was used as a positive control. **p b 0.01 vs. 0.

that incubation of human plasma with PLT resulted in a
significant decrease in the maximal rate of fibrin polymeri-
zation. To eliminate the effect of sample pH, all dilutions
were performed using 50 mM TBS (pH 7.4). We also
evaluated the effect of the same volume of DMSO on human
plasma; however, coagulation properties were unaffected. To
confirm the anticoagulant activities of PLT, thrombin-
catalyzed platelet aggregation assay was conducted. As
shown in Fig. 2B, PLT significantly inhibited mouse platelet
aggregation induced by thrombin (final concentration: 3 U/
ml) in a concentration dependent manner. To exclude the
possibility that the decrease of the polymerization could be
due to direct effect on thrombin leading to decrease in fibrin
production rather than polymerization of fibrin formed,
reptilase-catalyzed polymerization assay was introduced.
Results showed that PLT significantly decreased reptilase-
catalyzed polymerization (data not shown). To determine the
cellular viability of PLT, cellular viability assay (MTT assay)
was performed in HUVECs treated with PLT for 24 h. At
concentrations up to 30 μM, PLT did not affect cell viability
(Fig. 2C).
3.3. Effects of PLT on the activities of thrombin and FXa
To elucidate the mechanism responsible for inhibition of
coagulation by PLT, the inhibitory effects of PLT on the
activities of thrombin and FXa were measured using
chromogenic substrates. In the results presented in Fig. 3A,
treatment with PLT resulted in dose-dependent inhibition of
the amidolytic activity of thrombin, indicating direct inhibi-
tion of thrombin activity by the anticoagulant. In addition, we
also investigated the effects of PLT on FXa activity. PLT
inhibited the effects on FXa activities (Fig. 3B). These results
are consistent with the results of our antithrombin assay, and
therefore suggest that the antithrombotic mechanisms of PLT
appear to be due to inhibition of fibrin polymerization and/or
the intrinsic/extrinsic pathway.
3.4. Effects of PLT on production of thrombin and FXa
Previously, Sugo et al. reported that endothelial cells are
able to support prothrombin activation by FXa [25]. In the
current study, pre-incubation of HUVECs with FVa and FXa in

Fig. 4. Inhibition of thrombin and FXa production by PLT in HUVECs. (A) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10 min
with the indicated concentrations of PLT or OA (40 μM). Prothrombin was added to a final concentration of 1 μM and prothrombin activation was determined
30 min later, as described in the “Materials and methods” section. (B) HUVECs were pre-incubated with indicated concentrations of PLT or OA (40 μM) for
10 min. TNF-α- (10 ng/ml for 6 h) stimulated HUVECs were incubated with FVIIa (10 nM) and FX (175 nM) in the absence or presence of anti-TF IgG (25 μg/ml)
and FXa production was determined as described in the “Materials and methods” section. *p b 0.05 or **p b 0.01 vs. 0 (A) or TNF-α alone (B).
6 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8
Fig. 5. Effects of PLT on secretion of PAI-1 by HUVECs stimulated with TNF-α.
HUVECs were cultured with PLT or OA (40 μM) in the absence or presence of
TNF-α (10 ng/ml) for 18 h and PAI-1 concentrations in culture media were
determined as described in the “Materials and methods” section. *p b 0.05
or **p b 0.01 vs. TNF-α alone; n.s., not significant.
the presence of CaCl2 prior to addition of prothrombin
resulted in production of thrombin (Fig. 4A). In addition,
treatment with PLT resulted in dose-dependent inhibition of
production of thrombin from prothrombin (Fig. 4A).
According to findings reported by Rao et al., the endothelium
provides the functional equivalent of procoagulant phospho-
lipids and supports activation of FX [26], and, in TNF-α
stimulated HUVECs, activation of FX by FVIIa occurred in a TF
expression-dependent manner [27]. Thus, we investigated
the effects of PLT on activation of FX by FVIIa. HUVECs were
stimulated with TNF-α for induction of TF expression, and, as
shown in Fig. 4B, the rate of FX activation by FVIIa was
18-fold higher in stimulated HUVECs (95.2 ± 8.2 nM) than
in non-stimulated HUVECs (5.3 ± 1.2 nM), and this increase
in activation was abrogated by anti-TF IgG (13.8 ± 2.8 nM).
In addition, pre-incubation with PLT resulted in dose-
dependent inhibition of FX activation by FVIIa (Fig. 4B).
Therefore, these results suggest that PLT can inhibit produc-
tion of thrombin and FXa.
3.5. Effects of PLT on secretion of PAI-1 or t-PA protein
It is well known that TNF-α appears to inhibit the fibrinolytic
system in HUVECs by inducing production of PAI-1 and altering
the balance between t-PA and PAI-1 is known to modulate
coagulation and fibrinolysis [28,29]. To determine the direct
Fig. 6. Effects of PLT on secretion of t-PA by HUVECs stimulated with TNF-α. (A) HUVECs were cultured with PLT or OA (40 μM) in the absence or presence of
TNF-α (10 ng/ml) for 18 h and t-PA concentrations in culture media were determined as described in the “Materials and methods” section. PAI-1/t-PA ratio by
PLT in TNF-α activated HUVECs by ELISA was shown in (B). *p b 0.05; n.s., not significant.
effects of PLT on TNF-α-stimulated secretion of PAI-1, HUVECs
were cultured in media with or without PLT in the absence or
presence of TNF-α for 18 h. As shown in Fig. 5, treatment with
PLT resulted in dose-dependent inhibition of TNF-α-induced
secretion of PAI-1 from HUVECs, and these decreases became
significant at a PLT dose of 20 μM.
TNF-α does not have a significant effect on t-PA production
[30] and the balance between plasminogen activators and their
inhibitors reflects net plasminogen-activating capacity [4,5,7];
therefore, we investigated the effect of TNF-α with PLT on
secretion of t-PA from HUVECs. The results obtained were
consistent with those of a prior study reporting a modest
decrease in production of t-PA by TNF-α in HUVECs [31]. This
decrease was not significantly altered by treatment with PLT
(Fig. 6A). Therefore, collectively, these results indicate that the
PAI-1/t-PA ratio was increased by TNF-α and that PLT
prevented this increase (Fig. 6B).
3.6. Effects of signal pathway inhibitors on basal and TNF-α
induced PAI-1 secretion in the presence or absence of PLT
It is well known that both NF-κB and ERK dependent
pathways are involved in TNF-α induced PAI-1 production
[30]. To define the targets of PLT in the signal transduction
pathways leading to TNF-α induced PAI-1 expression, we
investigated the effects of three signal transduction inhibi-
tors, Emodin (NF-κB inhibitor), PD98059 (ERK inhibitor), and
SP600125 (JNK inhibitor) on TNF-α induced PAI-1 expression
in the presence or absence of PLT. We first demonstrated the
effect of increasing doses of each inhibitor on HUVECs
viability, and selected appropriate non-cytotoxic doses. To
confirm the specificity of the inhibitors, we checked the cell
viability for all agents by MTT assay. We found that the
inhibitors combined with TNF-α and/or PLT as used in this
study has no effect on HUVECs viability (data not shown).
Experiments performed showed that PD98059 and Emodin
inhibited TNF-α induced PAI-1 accumulation, which is
consistent with the previous study (Fig. 7A) [30]. In addition,
we observed that SP600125 did inhibit TNF-α induced PAI-1
secretion (Fig. 7A). Furthermore, neither PD98059 nor
Emodin showed any additional inhibitory effects in the
presence of PLT (Fig. 7B). However, the inhibitory effects of
 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8
Fig. 7. Effect of signal transduction inhibitors on TNF-α induced PAI-1 secretion. (A) HUVECs were cultured with SP600125 (2 μM), Emodin (2 μg/ml), and
PD98059 (10 μM) in the absence or presence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentration in the culture mediums was examined as described in the
“Materials and methods” section. (B) the same as (A) except that cells were preincubated with PLT 20 μM. *p b 0.05 as compared to TNF-α alone (A) or
TNF-α + PLT (B).
SP600125 were essentially additive with those of PLT
(Fig. 7B). These results suggest that the ERK and NF-κB
pathways are involved in PLT-mediated inhibition of TNF-α
induced PAI-1 expression in HUVECs. No significant effects of
the three inhibitors on basal levels of PAI-1 production could
be explained by the fact that the activities of ERK, JNK, and
NF-κB are relatively low in the unstimulated cells [32,33].
Thus, these results seem to indicate that PLT decreases PAI-1
levels via inhibition of the ERK and NF-κB pathways. However,
additional work will be required to elucidate whether PLT has
beneficial effects on fibrinolytic systems in vivo.
The pre-clinical evaluation of the antithrombotic poten-
tial of novel molecules requires the use of reliable and
reproducible experimental models. PT, aPTT, fibrin polymer-
ization, and platelet aggregation are the most established and
commonly used preparations to determine the efficacy of novel
antithrombotic drugs [34,35]. In our experiment, PT and aPTT
using human plasma were used to evaluate the antithrombotic
effect of PLT. Fibrin polymerization and platelet aggregation are
also used to determine the anti-platelet functions of PLT. Data
showed that PLT prolonged PT and aPTT values and PLT
resulted in a significant decrease in the maximal rate of fibrin
polymerization and inhibited platelet aggregation without
having cytotoxicity on human endothelial cells.
There is ample evidence that inflammation and coagula-
tion are intricately related processes that may considerably
affect each other [36,37]. This cross-talk occurs at the levels
of platelet activation, fibrin formation, and resolution as well
as physiological anticoagulant pathways [36,37]. On the basis
of our current experimental studies, it can be hypothesized
that inhibitory modulation of coagulation by PLT could give
promising anti-inflammatory mediators. Well-designed pro-
spective studies are needed to prove this hypothesis.
In conclusion, results of this study demonstrate that PLT
inhibited the extrinsic and intrinsic blood coagulation
pathways through inhibition of FXa and thrombin produc-
tion in HUVECs, and that PLT inhibits TNF-α-induced
secretion of PAI-1. These results add to previous work on
the topic, and should be of interest to those designing
pharmacological strategies for the treatment or prevention
of vascular diseases.
7
Conflict of interest statement
The authors have no conflict of interest to declare.
Acknowledgments
This study was supported by the National Research
Foundation of Korea (NRF) funded by the Korean govern-
ment [MSIP] (Grant No. 2012-000940).
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