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

vascular injury is the result of an ordered series of reactions
The leading causes of death (30% of total) in the world are collectively referred to as blood coagulation [4,5]. Clots are
diseases involving the heart and blood vessels and, conse- eventually broken down by plasmin, which is activated by
quently, thrombosis [1]. Most thromboembolic processes tissue-type plasminogen activator (t-PA) from plasminogen.
require anticoagulant therapy and this explains the current Thrombin is also an activator of inflammation and an
efforts to develop specific and potent anticoagulant and inhibitor of fibrinolysis [6]. The hemostatic plug that forms
antithrombotic agents [2]. Primary haemostatic events are within blood vessels, often within the veins or arteries of the
triggered in response to damage of the vascular wall by the heart, in pathological conditions associated with arterial
exposure of blood to the subendothelial extracellular matrix disease, referred to as a thrombus [6], is a major cause of
[3,4]. Thrombin is the key effector enzyme of the coagulation morbidity and death. Clotting time assays measure the time
system, having many biologically important functions such as required to generate thrombin [7] and activated partial
the activation of platelets, conversion of fibrinogen to a fibrin thromboplastin time (aPTT) measures the efficacy of the
network, and feedback amplification of coagulation [4,5]. The contact activation and common coagulation pathways [7].
Furthermore, the aPTT or prothrombin time (PT) mainly
⁎ Corresponding author at: College of Pharmacy, CMRI, Research Institute serves to aid the diagnosis of deficiencies in certain factors
of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, [8].
Buk-gu, Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82
53 950 8557.
Asarum sieboldii Miq. (Aristolochiaceae) is a wide-ranging
E-mail address: [email protected] (J.-S. Bae). species found through North America, Europe, and Asia [9].
1
First two authors contributed equally to this work. 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 (251.0 g) extractions, respectively. The MC extraction was
and as a local anesthetic agent in Korea and China [10]. Previous fractioned by silica gel column chromatography eluting with
studies on the constituents of A. sieboldii have revealed isolation EtOAc in n-hexane (0–100%, step-wise), to yield twenty fractions
of various types of essential oils, amide, alkaloids, lignans, and (MC1–MC20). MC15 (8.2 g) was chromatographed on silica gel
flavonoids [9,11,12]. Pellitorine (PLT) was isolated from the column using a solvent system of EtOAc in n-hexane (15%) to
methylene chloride soluble fraction of the roots of A. sieboldii give seven fractions (MC15-1–MC15-7). MC15-4 (16.6 g) was
using a combination of silica gel column chromatography and purified by recrystallization from chloroform to afford a
recrystallization. A conjugated alkaldienamide, pellitorine was needle-type solid, compound 1 [675.0 mg, 0.12% (w/w) of
isolated mainly from the Piper species [13]. Pellitorine showed MeOH extract]. The structure of compound 1 (Fig. 1) was
various biological properties including insecticidal [14], lavicidal identified by a combination of spectroscopic methods and
[14], antibacterial [15], and anti-cancer [16] activities. However, comparisons with the literature data [18].
no studies on the anticoagulant activities of PLT have been
reported. Therefore, in the current study, we examined the 2.3. Pellitorine (1)
anticoagulant activities of PLT in the production of FXa and
thrombin, and their effects on PT and aPTT and on fibrinolytic Needles, mp 69 °C; 1H NMR (250 MHz, CDCl3): δ 0.91 (3H,
activity. 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 =
2. Materials and methods 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
2.1. Reagents (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′),
TNF-α was purchased from Abnova (Taiwan). Anti-tissue 31.8 (C-8), 33.3 (C-6), 47.3 (C-1′), 122.0 (C-2), 128.6 (C-4),
factor antibody was purchased from Santa Cruz Biologics (Santa 141.8 (C-3), 143.8 (C-5), 166.9 (C-1).
Cruz, CA). c-Jun N-terminal kinase (JNK) inhibitor (SP600125),
Nuclear factor (NF)-κB inhibitor (Emodin), and extracellular 2.4. Isolation of plasma
signal regulated kinase (ERK) inhibitor (PD98059) were pur-
chased from R&D Systems (Minneapolis, MN). Factors V, Vll, Blood samples were taken in the morning from 10 healthy
Vlla, FX, and FXa, antithrombin III (AT III), prothrombin, and volunteers in fasting status (aged between 24 and 28 years, 4
thrombin were obtained from Haematologic Technologies males and 6 females) without cardiovascular disorders, allergy
(Essex Junction, VT, USA). aPTT assay reagent and PT reagents and lipid or carbohydrate metabolism disorders, untreated with
were purchased from Fisher Diagnostics (Middletown, Virginia, drugs. All subjects gave written informed consent before
USA), and the chromogenic substrates, S-2222 and S-2238, were participation. Healthy subjects did not use addictive substances
purchased from Chromogenix AB (Sweden). The PAI-1 and t-PA and antioxidant supplementation, and their diet was balanced
ELISA kits were purchased from American Diagnostica Inc. (meat and vegetables). Human blood was collected into sodium
(Stamford, CT, USA). Other reagents were of the highest citrate (0.32% final concentration) and immediately centrifuged
commercially available grades. Oleanolic acid (OA) was pre- (2000 ×g 15 min) to obtain plasma.
pared as described previously [17].
2.5. Anticoagulation assay
2.2. Plant material, extraction, and purification
aPTT and PT were determined using a Thrombotimer
Melting points were obtained with an Electrothermal 9100 (Behnk Elektronik, Germany), according to the manufacturer's
melting point apparatus (Electrothermal Ltd.). 1H and 13C NMR instructions as described previously [19]. In brief, citrated
spectra were obtained on a Bruker ARX spectrometer (250 MHz) normal human plasma (90 μl) was mixed with 10 μl of PLT or
and chemical shifts given in δ (ppm) from tetramethylsilane OA and incubated for 1 min at 37 °C. aPTT assay reagent
(TMS) as an internal standard. Column chromatography was (100 μl) was added and incubated for 1 min at 37 °C, and then
carried out on silica gel (70–230 mesh, Merck). Thin layer 20 mM CaCl2 (100 μl) was added. Clotting times were
chromatography (TLC) analysis was performed on Kieselgel 60 recorded. For PT assays, citrated normal human plasma
F254 (Merck 1.05715) aluminum plate; spots were visualized by (90 μl) was mixed with 10 μl of PLT or OA stock and incubated
spraying with 10% aqueous H2SO4 followed by heating. All other for 1 min at 37 °C. PT assay reagent (200 μl), which has been
chemicals and solvents were of analytical grade and used preincubated for 10 min at 37 °C, was then added and clotting
without further purification. time was recorded. PT results are expressed in seconds and as
The roots of A. sieboldii were purchased from herbal market at International Normalized Ratios (INR), and aPTT results are
Daegu, Korea, in February 2006. The plant material was identified expressed in seconds. INR = (PT sample / PT control)ISI. ISI =
by Dr. Seung Ho Lee at the College of Pharmacy, Yeungnam international sensitivity index.
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 Fig. 1. The chemical structure of PLT.
 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8 3

2.6. Platelet aggregation assay rates of color development were converted into FXa concen-
trations using a standard curve prepared with known dilutions
Mouse platelets from platelet-rich plasma (PRP) were of purified human FXa.
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
2.11. Thrombin production on the surfaces of HUVECs
glucose, 0.1% BSA (w/v), pH to 7.45). The platelet aggregation
study was carried out according to a method previously
Measurement of thrombin production by HUVECs was
reported [20]. Washed platelets were incubated with indi-
quantitated as previously described [19,23]. Briefly, HUVECs
cated PLT or OA for 3 min, and then stimulated by thrombin
were pre-incubated in 300 μl containing PLT or OA in 50 mM
(3 U/ml, Sigma) in 0.9% saline solution at 37 °C for 5 min.
Tris–HCl buffer, 100 pM FVa, and 1 nM FXa for 10 min,
Platelet aggregation was recorded using an aggregometer
followed by addition of prothrombin to a final concentration
(CHRONO-LOG, Havertown, PA, USA).
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
2.7. Thrombin-catalyzed fibrin polymerization
in Tris-buffered saline per well to terminate prothrombin
activation. Activated prothrombin was determined by mea-
Thrombin-catalyzed polymerization was determined
suring the rate of hydrolysis of S2238 at 405 nm. Standard
every 6 s for 20 min by monitoring turbidity at 360 nm using
curves were prepared using the amounts of purified thrombin.
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 2.12. Thrombin or factor Xa (FXa) activity assay
saline solution pH 7.4) and clotted with thrombin (final
concentration — 0.5 U/ml). The maximum polymerization PLT or OA in 50 mM Tris–HCl buffer (pH 7.4) containing
rate (Vmax, ΔmOD/min) of each absorbance curve was 7.5 mM EDTA and 150 mM NaCl was mixed in the presence
recorded [21]. All experiments were performed three times. of 150 μl of AT III (200 nM). The heparins with AT III
(200 nM) were dissolved in physiological saline and placed
2.8. Cell culture in the sample wells. After incubation at 37 °C for 2 min,
thrombin solution (150 μl; 10 U/ml) was added, followed by
Primary HUVECs were obtained from Cambrex Bio Science incubation at 37 °C for 1 min. S-2238 (a thrombin substrate;
(Charles City, IA) and were maintained using a previously 150 μl; 1.5 mM) solution was then added and absorbance at
described method [22,23]. Briefly, the cells were cultured until 405 nm was monitored for 120 s using a spectrophotometer
confluent at 37 °C at 5% CO2 in EBM-2 basal media supplemented (TECAN, Switzerland). And FXa activity assay was performed
with growth supplements (Cambrex Bio Science). in the same manner as the thrombin activity assay, but using
factor Xa (1 U ml/1) and S-2222 as substrates.
2.9. Cell viability assay
2.13. In vivo bleeding time
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, Tail bleeding times were measured using the method
the cells were washed with fresh medium, followed by described by Dejana et al. [19,24]. Briefly, ICR mice were
treatment with PLT. After a 48-h incubation period, the cells fasted overnight before experiments. One hour after intrave-
were washed, and 100 μl of 1 mg/ml MTT was added, followed nous administration of PLT or OA, tails of mice were
by incubation for 4 h. Finally, 150-μl DMSO was added to transected at 2 mm from their tips. Bleeding time was
solubilize the formazan salt formed, the amount of which was defined as the time elapsed until bleeding stopped. When
determined by measuring the absorbance at 540 nm using a the bleeding time exceeded 15 min, bleeding time was
microplate reader (Tecan Austria GmbH, Austria). recorded as 15 min for the analysis. All animals were treated
in accordance with the Guidelines for the Care and Use of
2.10. Factor Xa production on the surfaces of HUVECs Laboratory Animals issued by Kyungpook National University.

TNF-α (10 ng/ml for 6 h in serum-free medium) stimu-

2.14. ELISA for PAI-1 and t-PA
lated confluent monolayers of HUVECs (preincubated with
the indicated concentrations of PLT or OA for 10 min) in a
The concentrations of PAI-1 and t-PA in HUVEC cultured
96-well culture plate were incubated with FVIIa (10 nM) in
supernatants were determined using ELISA kits (American
buffer B (buffer A supplemented with 5 mg/ml bovine serum
Diagnostica Inc. CT, USA).
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, 2.15. Statistical analysis
100 μl) and incubated for 15 min. The reaction was stopped
by adding buffer A (10 mM HEPES, pH 7.45, 150 mM NaCl, Data are expressed as mean ± SEM (standard error of the
4 mM KCl, and 11 mM glucose) containing 10 mM EDTA and mean) of at least three independent experiments. Statistical
the amounts of FXa generated were measured by using a significance between two groups was determined using the
chromogenic substrate. Changes in absorbance at 405 nm Student's t-test. Statistical significance was accepted for
over 2 min were monitored using a microplate reader. Initial 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 3.3. Effects of PLT on the activities of thrombin and FXa
significant decrease in the maximal rate of fibrin polymeri-
zation. To eliminate the effect of sample pH, all dilutions To elucidate the mechanism responsible for inhibition of
were performed using 50 mM TBS (pH 7.4). We also coagulation by PLT, the inhibitory effects of PLT on the
evaluated the effect of the same volume of DMSO on human activities of thrombin and FXa were measured using
plasma; however, coagulation properties were unaffected. To chromogenic substrates. In the results presented in Fig. 3A,
confirm the anticoagulant activities of PLT, thrombin- treatment with PLT resulted in dose-dependent inhibition of
catalyzed platelet aggregation assay was conducted. As the amidolytic activity of thrombin, indicating direct inhibi-
shown in Fig. 2B, PLT significantly inhibited mouse platelet tion of thrombin activity by the anticoagulant. In addition, we
aggregation induced by thrombin (final concentration: 3 U/ also investigated the effects of PLT on FXa activity. PLT
ml) in a concentration dependent manner. To exclude the inhibited the effects on FXa activities (Fig. 3B). These results
possibility that the decrease of the polymerization could be are consistent with the results of our antithrombin assay, and
due to direct effect on thrombin leading to decrease in fibrin therefore suggest that the antithrombotic mechanisms of PLT
production rather than polymerization of fibrin formed, appear to be due to inhibition of fibrin polymerization and/or
reptilase-catalyzed polymerization assay was introduced. the intrinsic/extrinsic pathway.
Results showed that PLT significantly decreased reptilase-
catalyzed polymerization (data not shown). To determine the 3.4. Effects of PLT on production of thrombin and FXa
cellular viability of PLT, cellular viability assay (MTT assay)
was performed in HUVECs treated with PLT for 24 h. At Previously, Sugo et al. reported that endothelial cells are
concentrations up to 30 μM, PLT did not affect cell viability able to support prothrombin activation by FXa [25]. In the
(Fig. 2C). 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

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
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
consistent with those of a prior study reporting a modest
TNF-α (10 ng/ml) for 18 h and PAI-1 concentrations in culture media were decrease in production of t-PA by TNF-α in HUVECs [31]. This
determined as described in the “Materials and methods” section. *p b 0.05 decrease was not significantly altered by treatment with PLT
or **p b 0.01 vs. TNF-α alone; n.s., not significant. (Fig. 6A). Therefore, collectively, these results indicate that the
PAI-1/t-PA ratio was increased by TNF-α and that PLT
the presence of CaCl2 prior to addition of prothrombin prevented this increase (Fig. 6B).
resulted in production of thrombin (Fig. 4A). In addition,
treatment with PLT resulted in dose-dependent inhibition of 3.6. Effects of signal pathway inhibitors on basal and TNF-α
production of thrombin from prothrombin (Fig. 4A). induced PAI-1 secretion in the presence or absence of PLT
According to findings reported by Rao et al., the endothelium
provides the functional equivalent of procoagulant phospho- It is well known that both NF-κB and ERK dependent
lipids and supports activation of FX [26], and, in TNF-α pathways are involved in TNF-α induced PAI-1 production
stimulated HUVECs, activation of FX by FVIIa occurred in a TF [30]. To define the targets of PLT in the signal transduction
expression-dependent manner [27]. Thus, we investigated pathways leading to TNF-α induced PAI-1 expression, we
the effects of PLT on activation of FX by FVIIa. HUVECs were investigated the effects of three signal transduction inhibi-
stimulated with TNF-α for induction of TF expression, and, as tors, Emodin (NF-κB inhibitor), PD98059 (ERK inhibitor), and
shown in Fig. 4B, the rate of FX activation by FVIIa was SP600125 (JNK inhibitor) on TNF-α induced PAI-1 expression
18-fold higher in stimulated HUVECs (95.2 ± 8.2 nM) than in the presence or absence of PLT. We first demonstrated the
in non-stimulated HUVECs (5.3 ± 1.2 nM), and this increase effect of increasing doses of each inhibitor on HUVECs
in activation was abrogated by anti-TF IgG (13.8 ± 2.8 nM). viability, and selected appropriate non-cytotoxic doses. To
In addition, pre-incubation with PLT resulted in dose- confirm the specificity of the inhibitors, we checked the cell
dependent inhibition of FX activation by FVIIa (Fig. 4B). viability for all agents by MTT assay. We found that the
Therefore, these results suggest that PLT can inhibit produc- inhibitors combined with TNF-α and/or PLT as used in this
tion of thrombin and FXa. study has no effect on HUVECs viability (data not shown).
Experiments performed showed that PD98059 and Emodin
3.5. Effects of PLT on secretion of PAI-1 or t-PA protein inhibited TNF-α induced PAI-1 accumulation, which is
consistent with the previous study (Fig. 7A) [30]. In addition,
It is well known that TNF-α appears to inhibit the fibrinolytic we observed that SP600125 did inhibit TNF-α induced PAI-1
system in HUVECs by inducing production of PAI-1 and altering secretion (Fig. 7A). Furthermore, neither PD98059 nor
the balance between t-PA and PAI-1 is known to modulate Emodin showed any additional inhibitory effects in the
coagulation and fibrinolysis [28,29]. To determine the direct presence of PLT (Fig. 7B). However, the inhibitory effects of

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.
 S.-K. Ku et al. / Fitoterapia 91 (2013) 1–8 7

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 Conflict of interest statement
(Fig. 7B). These results suggest that the ERK and NF-κB
pathways are involved in PLT-mediated inhibition of TNF-α The authors have no conflict of interest to declare.
induced PAI-1 expression in HUVECs. No significant effects of
the three inhibitors on basal levels of PAI-1 production could
Acknowledgments
be explained by the fact that the activities of ERK, JNK, and
NF-κB are relatively low in the unstimulated cells [32,33].
This study was supported by the National Research
Thus, these results seem to indicate that PLT decreases PAI-1
Foundation of Korea (NRF) funded by the Korean govern-
levels via inhibition of the ERK and NF-κB pathways. However,
ment [MSIP] (Grant No. 2012-000940).
additional work will be required to elucidate whether PLT has
beneficial effects on fibrinolytic systems in vivo.
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