The American Journal of Chinese Medicine, Vol. 41, No. 6, 1313–1327 © 2013 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X13500882
Am. J. Chin. Med. 2013.41:1313-1327. Downloaded from www.worldscientific.com by 222.80.15.134 on 01/22/15. For personal use only.
Antithrombotic Activities of Epi-Sesamin in vitro and in vivo Sae-Kwang Ku,* Jeong Ah Kim,† Chang-Kyun Han‡ and Jong-Sup Bae† *Department
of Anatomy and Histology College of Oriental Medicine Daegu Haany University Gyeongsan 712–715, Republic of Korea † College of Pharmacy CMRI, Research Institute of Pharmaceutical Sciences Kyungpook National University Daegu 702–701, Republic of Korea ‡
Department of Oriental Medicinal Materials and Processing College of Life Science, Kyung Hee University Youngin 446–701, Republic of Korea
Abstract: Sesamin (SM) and epi-sesamin (ESM) were isolated from Asarum sieboldii and their anticoagulant activities were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of cell-based thrombin and activated factor X (FXa). In addition, the effects of SM and ESM on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor- (TNF-) activated human umbilical vein endothelial cells (HUVECs). Treatment with ESM, but not SM, resulted in significantly prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, and ESM inhibited production of thrombin and FXa in HUVECs; and ESM inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. In accordance with these anticoagulant activities, ESM elicited anticoagulant effects in mice. In addition, treatment with ESM, but not SM, resulted in the inhibition of TNF--induced production of PAI-1, and treatment with ESM resulted in a significant reduction of the PAI-1 to t-PA ratio. Of particular interest, inhibition of the anticoagulant activity by ESM was more potent than that by SM, likely due to differences between their three-dimensional structures. Collectively, ESM possesses antithrombotic activities and offers a basis for the development of a novel anticoagulant. Keywords: Epi-sesamin; Coagulation Cascade; Fibrinolysis; Endothelium.
Correspondence to: Dr. Jong-Sup Bae, College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702–701, Republic of Korea. Tel: (þ82) 53-950-8570, Fax: (þ82) 53-950-8557, E-mail: [email protected]
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Introduction Blood coagulation involves the conversion of fluid blood to a solid gel or a clot, and clot formation contributes to hemostasis (Davie, 1995). Hemostasis is a dynamic process in which blood coagulation is initiated and terminated in a rapid and tightly regulated fashion, which requires coordinated interactions between tissue components, platelets, and plasma proteins (Davie et al., 1991). Primary hemostatic events are triggered in response to damage of the vascular wall by the exposure of blood to the subendothelial extracellular matrix (Davie et al., 1991). Thrombin, an important contributor to all major thrombotic processes, including physiologic hemostasis and pathologic thrombosis, is required for the conversion of fibrinogen to fibrin (Davie, 1995). 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 (Esmon, 2001). 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 (Esmon, 2001), is a major cause of morbidity and death. Clotting time assays measure the time required for the generation of thrombin (Quinn et al., 2000) and activated partial thromboplastin time (aPTT) measures the efficacy of the contact activation and common coagulation pathways (Quinn et al., 2000). In addition, the aPTT or prothrombin time (PT) mainly serves as an aid in diagnosis of deficiencies in certain factors (Levi et al., 2011). The Asarum species of the family Aristolochiaceae is a perennial plant with a wide distribution in east Asia (Kelly, 1998). The roots of A. sieboldii have traditionally been used as antiallergic (Hashimoto et al., 1994), antinociceptive (Kim et al., 2003), and antifungal agents (Lee et al., 2005; Yu et al., 2006). The lignan sesamin (SM) is abundant in sesame seeds and oils (Kamal-Eldin et al., 2011), as well as in Asarum species (Han et al., 2008), and has been reported to influence lipid metabolism in experimental animals (Ashakumary et al., 1999). Sesame is a well-known health food, and the association of sesame oil with lower blood pressure and lipid peroxidation and increased antioxidant status in hypertensive patients has been reported (Alipoor et al., 2012). The search for anticoagulant agents in natural herbal medicines represents an area of considerable interest (Li et al., 2009). During sesame oil refinement, sesamin is epimerized to a mixture of sesamin and epi-sesamin (ESM), which accounts for some of the different physiological effects of sesame oils. (Fukuda et al., 1986). In our continued search for natural products with the capacity for modulation of anticoagulant activities, we found that the methanol extract of A. sieboldii roots exhibited potent anticoagulant activities in both cell and animal models. Activity-guided purification of this root extract resulted in the isolation of one active principle compound, which was subsequently identified as epi-sesamin. In addition, no studies on the anticoagulant activities of SM or ESM have been reported. Therefore, in the current study, we examined the anticoagulant activities of SM and ESM in the production of FXa and thrombin, and their effects on PT and aPTT and on fibrinolytic activity.
ANTICOAGULANT ACTIVITIES OF ESM
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Materials and Methods
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Reagents TNF- was purchased from Abnova (Twawan). Anti-tissue factor antibody was purchased from Santa Cruz Biologics (Santa Cruz, CA). Factor V, Vll, Vlla, FX, 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). 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. Plant Materials, Extraction, and Isolation of Sesamin (SM) and Epi-sesamin (ESM) Roots of A. sieboldii were purchased at an herbal market in Daegu, Korea in February 2006. The plant material was identified by Dr. Seung Ho Lee at the College of Pharmacy, Yeungnam University, and 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 methanol (MeOH) at room temperature for five days. After concentration, the MeOH extract (564.0 g) was suspended in water and partitioned sequentially with Asarum sieboldii (6 kg) MeOH (3 × 20 L) MC EtOAc
MC ext. (298 g) EtOAc ext. (15 g)
H2O ext. (251 g)
(A)
Sesamin
epi-Sesamin
(B) Figure 1. Procedure for extraction of SM and ESM, HPLC and structures of SM and ESM. (A) Solvent extraction scheme used for SM and ESM was performed as described in the “Materials and Methods” section. HPLC of isolated SM and ESM is shown in (B) and chemical structures of SM and ESM are shown in (C).
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O O
O H
H O
O
O
O H
O
O
Sesamin (1)
epi-sesamin (2) (C)
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Figure 1. (Continued )
methylene chloride (MC) and ethyl acetate (EtOAc) to produce MC (298.0 g), EtOAc (15.0 g), and water (251.0 g) fractions. Chromatography of the MC fraction was performed using a silica gel column, with a stepwise gradient of EtOAc in n-hexane (0–100 %), to yield 20 fractions (MC1-MC20). Compounds 1 [2.1 g, 0.37% (w/w) of the MeOH extract] and 2 [8.5 g, 1.5% (w/w) of the MeOH extract] were obtained from MC10 and MC13 by precipitation in cold MeOH. A combination of spectroscopic methods was used for structural identification of compounds 1 (SM) and 2 (ESM, Fig. 1), as reported in a previous study (Venkataraman and Gopalakrishnan, 2002; Lee et al., 2013). Values were compared with those in the literature ( Venkataraman and Gopalakrishnan, 2002). Isolation of Plasma Blood samples were taken in the morning from 10 healthy volunteers in fasting status (aged between 24 and 28 years, four males and six females) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, and untreated with drugs. All subjects gave written informed consent prior to 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 15 min) in order to obtain plasma. Anticoagulation Assay aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Germany), according to the manufacturer’s instructions, as described previously (Kim et al., 2012). In brief, citrated normal human plasma (90 l) was mixed with 10 l of SM or ESM and incubated for 1 min at 37 C. aPTT assay reagent (100 l) was added, followed by incubation for 1 min at 37 C; 20 mM CaCl2 (100 l) was then added. Clotting times were recorded. For PT assays, citrated normal human plasma (90 l) was mixed with 10 l of SM or ESM stock and incubated for 1 min at 37 C. PT assay reagent (200 l), which had 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 ¼ (PT sample / PT control) ISI . ISI ¼ international sensitivity index.
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Platelet Aggregation Assay
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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 performed according to a previously reported method (Kim et al., 2011). Washed platelets were incubated with indicated (E)SM for 3 min, followed by stimulation with thrombin (3 U/ml, Sigma) in 0.9% saline solution at 37 C for 5 min. Platelet aggregation was recorded using an aggregometer (Chronolog, Havertown, PA, USA). 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 temperature. Control plasma and plasma incubated with SM or ESM were diluted three times in 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, mOD/min) of each absorbance curve was recorded (Nowak et al., 2007). All experiments were performed three times. Cell Culture Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA) and were maintained using a previously described method (Bae and Rezaie, 2008; Bae, 2011). Briefly, cells were cultured until confluent at 37 C at 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). Cell Viability Assay MTT was used as an indicator of cell viability. Cells were grown in 96-well plates at a density of 5 10 3 /well. After 24 h, cells were washed with fresh medium, followed by treatment with SM or ESM. After a 48-h incubation period, cells were washed and 100 l of 1 mg/ml MTT was added, followed by incubation for 4 h. Finally, 150 l DMSO was added in order 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). Data were expressed as meanSD of at least three independent experiments. Factor Xa Production on the Surfaces of HUVECs TNF- (10 ng/ml for 6 h in serum-free medium) stimulated confluent monolayers of HUVECs (preincubated with the indicated concentrations of SM or ESM for 10 min) in a 96well 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
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presence or absence of anti-TF IgG (25 g/ml). FX (175 nM) was then added to the cells (final reaction mixture volume, 100 l) and incubated for 15 min. The reaction was stopped by the addition of 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 using a chromogenic substrate. Changes in absorbance at 405 nm over 2 min were monitored using a microplate reader. Initial rates of color development were converted to FXa concentrations using a standard curve prepared with known dilutions of purified human FXa.
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Thrombin Production on the Surfaces of HUVECs Measurement of thrombin production by HUVECs was quantitated as previously described (Bae, 2011; Kim et al., 2012). HUVECs were brieflt pre-incubated in 300 l containing SM or ESM 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 M. After 10 min, duplicate samples (10 l each) were transferred to a 96-well plate containing 40 l of 0.5 M EDTA in Tris-buffered saline per well for termination of prothrombin activation. Activated prothrombin was determined by measuring the rate of hydrolysis of S2238 at 405 nm. Standard curves were prepared using amounts of purified thrombin. Thrombin Activity Assay SM or ESM in 50 mM Tris–HCl buffer (pH 7.4) containing 7.5 mM EDTA and 150 mM NaCl was mixed in the absence or presence of 150 l of AT III (200 nM). 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, the thrombin solution (150 l; 10 U/ml) was added, followed by incubation at 37 C for 1 min. S-2238 (a thrombin substrate; 150 l; 1.5 mM) solution was then added and its absorbance at 405 nm was monitored for 120 s using a spectrophotometer (TECAN, Switzerland). Factor Xa (FXa) Activity Assay These assays were performed in the same manner as the thrombin activity assay, but using factor Xa (1 U ml/1) and S-2222 as substrates. In Vivo Bleeding Time Tail bleeding times were measured using the method described by (Dejana et al., 1979; Kim et al., 2012). Briefly, ICR mice were fasted overnight before the experiments. One hour after intravenous administration of SM or ESM, the 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.
ANTICOAGULANT ACTIVITIES OF ESM
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ELISA for PAI-1 and t-PA Concentrations of PAI-1 and t-PA in HUVEC cultured supernatants were determined using ELISA kits (American Diagnostica Inc. CT, USA). Statistical Analysis
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Data are expressed as meanS.E.M. (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 < 0:05. Results and Discussion Sesamin (SM) and epi-sesamin (ESM) have been reported to contain antiallergic (Hashimoto et al., 1994), antinociceptive (Kim et al., 2003), and antifungal agents (Lee et al., 2005; Yu et al., 2006). In this study, we evaluated the anticoagulant effects of SM or ESM from Asarum species for the first time and sought to identify the mechanisms responsible for these effects. Effects of SM or ESM on aPTT and PT Incubation with ESM, but not SM, resulted in changes in the coagulation properties of human plasma. The anticoagulant properties of SM or ESM in human plasma were tested Table 1. Anticoagulant Activity of Sesamin (SM) a In Vitro Coagulant Assay Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control
Saline
32.1 0.5
14.2 0.4
1.00
SM
0.5 M 1 M 2 M 5 M 10 M 20 M
33.7 33.1 32.9 34.2 33.6 32.4
1.05 0.98 1.08 1.05 0.95 1.02
Heparin
0.5 0.3 0.5 0.3 1.2 0.4
0.5 (g/ml) 98.6 1.2**
14.5 14.1 14.7 14.5 13.9 14.3
0.3 0.7 0.2 0.5 0.7 0.3
10 (g/ml) 38.4 0.6**
8.92**
In Vivo Bleeding Time Sample
Dose
Tail Bleeding Time (s)
n
Control SM Heparin
Saline 7 g/mouse 1 mg/mouse
45.9 1.2 46.5 1.7 137.4 2.3**
10 10 10
a Each value represents the means S.D. (n ¼ 5). *p < 0:05 as compared to control. **p < 0:01 as compared to control.
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S.-K. KU et al. Table 2. Anticoagulant Activity of Epi-Sesamin (ESM) a
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In Vitro Coagulant Assay Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control
Saline
32.1 0.5
14.2 0.4
1.00
ESM
0.5 M 1 M 2 M 5 M 10 M 20 M
32.1 33.5 38.1 45.2 64.2 71.7
Heparin
0.6 1.1 0.4* 0.6** 0.5** 0.4**
0.5 (g/ml) 98.6 1.2**
14.3 14.9 18.3 21.3 24.3 28.7
0.6 0.8 0.6 0.5 0.4 0.5
10 (g/ml) 38.4 0.6**
1.02 1.11 1.75* 2.44** 3.26** 4.70** 8.92**
In vivo Bleeding Time Sample
Dose
Tail Bleeding time (s)
n
Control ESM Heparin
Saline 7 g/mouse 1 mg/mouse
45.9 1.2 106.7 1.7** 137.4 2.3**
10 10 10
value represents the means S.D. (n ¼ 5). *p < 0:05 as compared to control. **p < 0:01 as compared to control. a Each
using aPTT and PT assays. A summary of the results is shown in Tables 1 and 2. Although the anticoagulant activities of ESM were weaker than those of heparin, treatment with ESM at concentrations greater than 2 M resulted in significantly prolonged aPTT and PT. The result showing prolongation of aPTT suggests the inhibition of the intrinsic and/or the common pathway, whereas prolongation of PT indicates that ESM could also inhibit the extrinsic coagulation pathway. Assuming an average body weight of 20 g and an average blood volume of 2 ml, the amounts of both of the test compounds (SM or ESM, M:W ¼ 354, 7g per mouse) produced a concentration of approximately 10 M in peripheral blood. As shown in Tables 1 and 2, treatment with ESM, but not SM, resulted in significantly prolonged tail bleeding times. Effects of SM or ESM on Production of Thrombin and FXa and Cytotoxicity Based on the current finding that clotting times were prolonged by treatment with ESM, the effects of SM or ESM on the production of thrombin and FXa were determined because FXa alone can convert prothrombin to thrombin and thrombin is required for conversion of fibrinogen to fibrin (Davie, 1995). In a previous study, Sugo et al. reported that endothelial cells are able to support prothrombin activation by FXa (Sugo et al., 1995). In the current study, pre-incubation of HUVECs with FVa and FXa in the presence of CaCl2 prior to the addition of prothrombin resulted in production of thrombin (Fig. 2A). In addition,
*
300
**
**
200 100 0 0
0.5 1 2 5 SM or ESM, [µM]
10
80 **
60
**
40
**
20
****
0 (E)SM [µM] 0
10
0
1
2
5
10
0
-
-
+
+
+
+
+
+
TNF-α
(B)
Relative Cell Viability (%)
(A)
α-TF IgG
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100
400
FXa Generation (nM)
Th Generation (nM)
ANTICOAGULANT ACTIVITIES OF ESM
120 100 80 60 40 20 0 Cont 1
2
5
10
20
50
SM or ESM, [µM] (C) Figure 2. Inhibition of thrombin and FXa production by SM or ESM in HUVECs and cytotoxicity. (A) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10 min with the indicated concentrations of SM (WÞ or ESM (¥). Prothrombin was added to a final concentration of 1 M 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 SM (W) or ESM (¥) for 10 min. TNF- (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 g/ml) and FXa production was determined as described in the “Materials and Methods” section. (C) Effect of SM or ESM on cellular viability was measured using the MTT assay. *p < 0:05 or **p < 0:05vs. 0 (A) or TNF- alone (B).
treatment with ESM, but not SM, resulted in dose-dependent inhibition of the production of thrombin from prothrombin (Fig. 2A). According to findings reported by Rao et al., the endothelium provides the functional equivalent of procoagulant phospholipids and supports the activation of FX (Rao et al., 1988), and in TNF- stimulated HUVECs, the activation of FX by FVIIa occurred in a TF expression-dependent manner (Ghosh et al., 2007). Thus, we investigated the effects of SM or ESM on the activation of FX by FVIIa. HUVECs were stimulated with TNF- for induction of TF expression, and as shown in Fig. 2B, the rate of FX activation by FVIIa was 40-fold higher in stimulated HUVECs (82.3 6.1 nM) than in non-stimulated HUVECs (2.8 0.07 nM); this increase in
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activation was abrogated by anti-TF IgG (8.2 1.5 nM). In addition, pre-incubation with ESM resulted in dose-dependent inhibition of FX activation by FVIIa (Fig. 2B). Therefore, these results suggest that ESM can inhibit the production of thrombin and FXa. For determination of the cytotoxicity of SM or ESM, a cellular viability assay (MTT assay) was performed in HUVECs treated with SM or ESM for 24 h. At concentrations up to 50 M, neither SM nor ESM had an effect on cell viability (Fig. 2C).
The effects of SM or ESM on thrombin-catalyzed fibrin polymerization in human plasma were monitored as changes in absorbance at 360 nm, as described in the “Materials and Methods” section. As shown in Fig. 3A, the results demonstrate that the incubation of human plasma with ESM, but not SM, resulted in a significant decrease in the maximal rate of fibrin polymerization. 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. A thrombin-catalyzed platelet aggregation assay was performed in order to confirm the anticoagulant activities of ESM. As shown in Fig. 3B, ESM significantly inhibited mouse platelet aggregation induced by thrombin (final concentration: 3 U/ml) in a concentration dependent manner. The reptilase-catalyzed polymerization assay was performed in order to exclude the possibility that the decrease of polymerization could be due to a direct effect on thrombin, leading to a decrease in fibrin production rather than in the polymerization of fibrin formed. Results showed that treatment with ESM resulted in a significant decrease in reptilasecatalyzed polymerization (data not shown).
100
100 *
80
**
60
**
40 20
Aggregation (%)
120 Vmax (% Control 0)
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Effects of SM or ESM on Thrombin-catalyzed Platelet Aggregation and Fibrin Polymerization
80
*
60
**
40
**
20
0 (E)SM [µM]
0
10
0
1
2
5
10
0 (E)SM [µM]
0
10
0
1
2
5
10
Th
-
-
+
+
+
+
+
Th
-
-
+
+
+
+
+
(A)
(B)
Figure 3. Effects of SM or ESM on fibrin polymerization in human plasma. (A) Thrombin-catalyzed fibrin polymerization at the indicated concentrations of SM (W) or ESM (¥) 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 SM (W) or ESM (¥) on mouse platelet aggregation induced by 3U/ml thrombin. *p < 0:05 or **p < 0:05 vs. Th alone.
ANTICOAGULANT ACTIVITIES OF ESM
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120
Relative Th Activity (%)
Relative Th Activity (%)
To elucidate the mechanism responsible for inhibition of coagulation by ESM, a measurement of the inhibitory effects of ESM on the activities of thrombin and FXa was performed using chromogenic substrates. According to results shown in Figs. 4A and 4B, treatment with ESM, but not SM, resulted in dose-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by the anticoagulant. In addition, we also investigated the effects of SM or ESM on FXa activity. ESM, but not SM, inhibited the effects on FXa activities (Figs. 4C and 4D). These results are consistent with those of our antithrombin assay, and therefore suggest that the antithrombotic mechanisms of ESM appear to be due to the inhibition of fibrin polymerization and/or the intrinsic/extrinsic pathway.
SM
100 80 60 40 20
Heparin
0
120 100
*
80
20
Heparin
0 100
10
1
0.1
0.01
0.001
0
ESM, [µM] or Heparin, [µg/ml] (B)
120
SM
100 80 60 40 20
Heparin
0
120 100
*
80 60
** ESM ** **
40 20
Heparin
0 100
10
1
0.1
0.01
0.001
0
100
10
1
0.1
0.01
0.001
0
SM, [µM] or Heparin, [µg/ml]
Relative FXa Activity (%)
(A)
Relative FXa Activity (%)
**
40
SM, [µM] or Heparin, [µg/ml]
(C)
ESM
** **
60
100
10
1
0.1
0.01
0.001
0
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Effects of SM or ESM on the Activities of Thrombin and FXa
ESM, [µM] or Heparin, [µg/ml] (D)
Figure 4. Effects of SM or ESM on inactivation of thrombin and factor Xa. Inhibition of thrombin (Th) by SM (A) or ESM (B) was measured using a chromogenic assay, as described in the “Materials and Methods” section. Inhibition of factor Xa (FXa) by SM (C) or ESM (D) was monitored using a chromogenic assay, as described in the “Materials and Methods” section. Heparin (W) was used as a positive control. *p < 0:05 or **p < 0:05 vs. 0.
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TNF- appears to inhibit the fibrinolytic system in HUVECs by inducing the production of PAI-I and altering the balance between t-PA and PAI-1 is known to result in modulation of coagulation and fibrinolysis (Schleef et al., 1988; Philip-Joet et al., 1995). To determine the direct effects of SM or ESM on TNF--stimulated secretion of PAI-1, HUVECs were cultured in media with or without SM or ESM in the absence or presence of TNF- for 18 h. As shown in Fig. 5, treatment with ESM, but not SM, resulted in dose-dependent inhibition of TNF--induced secretion of PAI-1 from HUVECs, and these decreases became significant at an ESM dose of 2 M. TNF- does not have a significant effect on t-PA production (Hamaguchi et al., 2003) and the balance between plasminogen activators and their inhibitors reflects net plasminogen-activating capacity (Davie et al., 1991; Davie, 1995; Quinn et al., 2000); therefore, we investigated the effect of TNF- with ESM on the secretion of t-PA from HUVECs. The results were consistent with those of a previous study reporting a modest decrease in the production of t-PA by TNF- in HUVECs (Lopez et al., 2000). This decrease was not significantly altered by treatment with ESM (Fig. 6). Therefore, collectively, these results indicate that the PAI-1/t-PA ratio was increased by TNF- and that ESM, but not SM, prevented this increase (Table 3). ESM, which possesses anti-aryl side chains, exhibited anticoagulant activity both in vitro and in vivo, however, SM, which has a similar structure, did not exhibit such activity. Molecular modeling studies performed in order to determine the reason for this difference showed the difference in the three-dimensional (3D) structures of SM and ESM. The two aryl side chains of SM were found to adopt the syn orientation, whereas those of ESM adopted the anti orientation, suggesting that SM or ESM interacts with coagulation PAI-1 Concentration (ng/ml)
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Effects of SM or ESM on the Secretion of PAI-1 or t-PA Protein
400 300 200
*
**
**
n.s.
100 0 0 0.5 1
2
5
10
0 0.5 1
2
5
10
SM or ESM, [µM] TNF-α (10 ng/ml) Figure 5. Effects of SM or ESM on secretion of PAI-1 by HUVECs stimulated with TNF-. HUVECs were cultured with SM (W) or ESM (¥) in the absence or presence of TNF- (10 ng/ml) for 18 h and PAI-1 concentrations in culture media were determined as described in the “Materials and Methods” section. *p < 0:05 or **p < 0:05 vs. TNF- alone; n.s., not significant.
t-PA Concentration (ng/ml)
ANTICOAGULANT ACTIVITIES OF ESM 14 12 10 8 6 4 2 0
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* n.s.
0
0
0.5
1
2
5
10
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SM or ESM, [µM] TNF-α (10 ng/ml) Figure 6. Effects of SM or ESM on secretion of t-PA by HUVECs stimulated with TNF-. HUVECs were cultured with SM (W) or ESM (¥) in the absence or presence of TNF- (10 ng/ml) for 18 h and t-PA concentrations in culture media were determined as described in the “Materials and Methods” section. *p < 0:05; n.s., not significant.
factors and with signaling molecules downstream of coagulation factors in different ways. This finding suggests that novel structures should be investigated through face-to-face study of coagulation factor activities. In conclusion, results of this study demonstrate that ESM, but not SM, inhibited the extrinsic and intrinsic blood coagulation pathways through the inhibition of FXa and thrombin production in HUVECs, and that ESM inhibits TNF--induced secretion of PAI-1. Of particular interest, consistently greater anticoagulant and profibrinolytic effects were observed for ESM, compared with those of SM, suggesting that the different 3D structure of ESM enhances its anticoagulant effects, compared to SM. These results contribute to previous work on the topic, and should be of interest to those designing pharmacological strategies for the treatment or prevention of vascular diseases.
Table 3. PAI-1/t-PA ratio by SM or ESM in TNF-® activated HUVECs by ELISA a Dose
PBS
10 ng/ml
1.4 TNF- 43.7
0.5 M 1 M 2 M 5 M 10 M
SM 44.2 44.4 43.2 45.8 47.3
ESM 43.3 45.0 40.3 * 36.7 ** 35.2 **
value represents the means S.D. (n ¼ 3). < 0:05 as compared to TNF- alone. ** p < 0:01 as compared to TNF- alone.
a Each *p
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Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MEST] (Grant Nos. 2012028835 and 2012-0009400).
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References Alipoor, B., M.K. Haghighian, B.E. Sadat and M. Asghari. Effect of sesame seed on lipid profile and redox status in hyperlipidemic patients. Int. J. Food. Sci. Nutr. 63: 674–678, 2012. Ashakumary, L., I. Rouyer, Y. Takahashi, T. Ide, N. Fukuda, T. Aoyama, T. Hashimoto, M. Mizugaki and M. Sugano. Sesamin, a sesame lignan, is a potent inducer of hepatic fatty acid oxidation in the rat. Metabolism 48: 1303–1313, 1999. Bae, J.S. Antithrombotic and profibrinolytic activities of phloroglucinol. Food. Chem. Toxicol. 49: 1572–1577, 2011. Bae, J.S. and A.R. Rezaie. Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand. Thromb. Haemost. 100: 101–109, 2008. Davie, E.W. Biochemical and molecular aspects of the coagulation cascade. Thromb. Haemost. 74: 1–6, 1995. Davie, E.W., K. Fujikawa and W. Kisiel. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30: 10363–10370, 1991. Dejana, E., A. Callioni, A. Quintana and G. de Gaetano. Bleeding time in laboratory animals. II — A comparison of different assay conditions in rats. Thromb. Res. 15: 191–197, 1979. Esmon, C.T. Role of coagulation inhibitors in inflammation. Thromb. Haemost. 86: 51–56, 2001. Fukuda, Y., M. Isobe, M. Nagata, T. Owaea and M. Namiki. Acidic transformation of sesamolin, the sesami-oil constituent, into an antioxidant bisepoxylignan, Sesaminol. Heterocycles 24: 923– 926, 1986. Ghosh, S., M. Ezban, E. Persson, U. Pendurthi, U. Hedner and L.V. Rao. Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface. J. Thromb. Haemost. 5: 336–346, 2007. Hamaguchi, E., T. Takamura, A. Shimizu and Y. Nagai. Tumor necrosis factor-alpha and troglitazone regulate plasminogen activator inhibitor type 1 production through extracellular signal-regulated kinase- and nuclear factor-kappaB-dependent pathways in cultured human umbilical vein endothelial cells. J. Pharmacol. Exp. Ther. 307: 987–994, 2003. Han, A.R., H.J. Kim, M. Shin, M. Hong, Y.S. Kim and H. Bae. Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells. Chem. Biodivers. 5: 346–351, 2008. Hashimoto, K., T. Yanagisawa, Y. Okui, Y. Ikeya, M. Maruno and T. Fujita. Studies on anti-allergic components in the roots of Asiasarum sieboldi. Planta Med. 60: 124–127, 1994. Kamal-Eldin, A., A. Moazzami and S. Washi. Sesame seed lignans: potent physiological modulators and possible ingredients in functional foods and nutraceuticals. Recent Pat. Food Nutr. Agric. 3: 17–29, 2011. Kelly, L. Phylogenetic relationships in Asarum (Aristolochiaceae) based on morphology and ITS sequences. Am. J. Bot. 85: 1454–1467, 1998. Kim, S.J., C. Gao Zhang and J. Taek Lim. Mechanism of anti-nociceptive effects of Asarum sieboldii Miq. radix: potential role of bradykinin, histamine and opioid receptor-mediated pathways. J. Ethnopharmacol. 88: 5–9, 2003.
Am. J. Chin. Med. 2013.41:1313-1327. Downloaded from www.worldscientific.com by 222.80.15.134 on 01/22/15. For personal use only.
ANTICOAGULANT ACTIVITIES OF ESM
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Kim, S.Y., S. Kim, J.M. Kim, E.H. Jho, S. Park, D. Oh and H.S. Yun-Choi. PKC inhibitors RO 318220 and Go 6983 enhance epinephrine-induced platelet aggregation in catecholamine hyporesponsive platelets by enhancing Akt phosphorylation. BMB Rep. 44: 140–145, 2011. Kim, T.H., S.K. Ku and J.S. Bae. Antithrombotic and profibrinolytic activities of eckol and dieckol. J. Cell Biochem. 113: 2877–2883, 2012. Lee, J.Y., S.S. Moon and B.K. Hwang. Isolation and antifungal activity of kakuol, a propiophenone derivative from Asarum sieboldii rhizome. Pest Manag. Sci. 61: 821–825, 2005. Lee, W., S.K. Ku, J.A. Kim, T. Lee and J.S. Bae. Inhibitory effects of epi-sesamin on HMGB1induced vascular barrier disruptive responses in vitro and in vivo. Toxicol. Appl. Pharmacol. 267: 201–208, 2013. Levi, M., M. Schultz and T. van der Poll. Coagulation biomarkers in critically ill patients. Crit. Care Clin. 27: 281–297, 2011. Li, Y., Z.J. Qian, B. Ryu, S.H. Lee, M.M. Kim and S.K. Kim. Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia cava. Bioorg. Med. Chem. 17: 1963–1973, 2009. Lopez, S., F. Peiretti, B. Bonardo, I. Juhan-Vague and G. Nalbone. Effect of atorvastatin and fluvastatin on the expression of plasminogen activator inhibitor type-1 in cultured human endothelial cells. Atherosclerosis 152: 359–366, 2000. Nowak, P., H.M. Zbikowska, M. Ponczek, J. Kolodziejczyk and B. Wachowicz. Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications induced by peroxynitrite: functional consequences. Thromb. Res. 121: 163–174, 2007. Philip-Joet, F., M.C. Alessi, C. Philip-Joet, M. Aillaud, J.R. Barriere, A. Arnaud and I. Juhan-Vague. Fibrinolytic and inflammatory processes in pleural effusions. Eur. Respir. J 8: 1352–1356, 1995. Quinn, C., J. Hill and H. Hassouna. A guide for diagnosis of patients with arterial and venous thrombosis. Clin. Lab. Sci. 13: 229–238, 2000. Rao, L.V., S.I. Rapaport and M. Lorenzi. Enhancement by human umbilical vein endothelial cells of factor Xa-catalyzed activation of factor VII. Blood 71: 791–796, 1988. Schleef, R.R., M.P. Bevilacqua, M. Sawdey, M.A. Gimbrone, Jr. and D.J. Loskutoff. Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor. J. Biol. Chem. 263: 5797–5803, 1988. Sugo, T., C. Nakamikawa, S. Tanabe and M. Matsuda. Activation of prothrombin by factor Xa bound to the membrane surface of human umbilical vein endothelial cells: its catalytic efficiency is similar to that of prothrombinase complex on platelets. J. Biochem. 117: 244–250, 1995. Venkataraman, R. and S. Gopalakrishnan. A lignan from the root of Ecbolium Linneanum Kurz. Phytochemistry 61: 963–966, 2002. Yu, H.H., S.J. Seo, J.M. Hur, H.S. Lee, Y.E. Lee and Y.O. You. Asarum Sieboldii extracts attenuate growth, acid production, adhesion, and water-insoluble glucan synthesis of Streptococcus mutans. J. Med. Food 9: 505–509, 2006.
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Antithrombotic Activities of Epi-Sesamin in vitro and in vivo Sae-Kwang Ku,* Jeong Ah Kim,† Chang-Kyun Han‡ and Jong-Sup Bae† *Department
of Anatomy and Histology College of Oriental Medicine Daegu Haany University Gyeongsan 712–715, Republic of Korea † College of Pharmacy CMRI, Research Institute of Pharmaceutical Sciences Kyungpook National University Daegu 702–701, Republic of Korea ‡
Department of Oriental Medicinal Materials and Processing College of Life Science, Kyung Hee University Youngin 446–701, Republic of Korea
Abstract: Sesamin (SM) and epi-sesamin (ESM) were isolated from Asarum sieboldii and their anticoagulant activities were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of cell-based thrombin and activated factor X (FXa). In addition, the effects of SM and ESM on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor- (TNF-) activated human umbilical vein endothelial cells (HUVECs). Treatment with ESM, but not SM, resulted in significantly prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, and ESM inhibited production of thrombin and FXa in HUVECs; and ESM inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. In accordance with these anticoagulant activities, ESM elicited anticoagulant effects in mice. In addition, treatment with ESM, but not SM, resulted in the inhibition of TNF--induced production of PAI-1, and treatment with ESM resulted in a significant reduction of the PAI-1 to t-PA ratio. Of particular interest, inhibition of the anticoagulant activity by ESM was more potent than that by SM, likely due to differences between their three-dimensional structures. Collectively, ESM possesses antithrombotic activities and offers a basis for the development of a novel anticoagulant. Keywords: Epi-sesamin; Coagulation Cascade; Fibrinolysis; Endothelium.
Correspondence to: Dr. Jong-Sup Bae, College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702–701, Republic of Korea. Tel: (þ82) 53-950-8570, Fax: (þ82) 53-950-8557, E-mail: [email protected]
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Introduction Blood coagulation involves the conversion of fluid blood to a solid gel or a clot, and clot formation contributes to hemostasis (Davie, 1995). Hemostasis is a dynamic process in which blood coagulation is initiated and terminated in a rapid and tightly regulated fashion, which requires coordinated interactions between tissue components, platelets, and plasma proteins (Davie et al., 1991). Primary hemostatic events are triggered in response to damage of the vascular wall by the exposure of blood to the subendothelial extracellular matrix (Davie et al., 1991). Thrombin, an important contributor to all major thrombotic processes, including physiologic hemostasis and pathologic thrombosis, is required for the conversion of fibrinogen to fibrin (Davie, 1995). 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 (Esmon, 2001). 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 (Esmon, 2001), is a major cause of morbidity and death. Clotting time assays measure the time required for the generation of thrombin (Quinn et al., 2000) and activated partial thromboplastin time (aPTT) measures the efficacy of the contact activation and common coagulation pathways (Quinn et al., 2000). In addition, the aPTT or prothrombin time (PT) mainly serves as an aid in diagnosis of deficiencies in certain factors (Levi et al., 2011). The Asarum species of the family Aristolochiaceae is a perennial plant with a wide distribution in east Asia (Kelly, 1998). The roots of A. sieboldii have traditionally been used as antiallergic (Hashimoto et al., 1994), antinociceptive (Kim et al., 2003), and antifungal agents (Lee et al., 2005; Yu et al., 2006). The lignan sesamin (SM) is abundant in sesame seeds and oils (Kamal-Eldin et al., 2011), as well as in Asarum species (Han et al., 2008), and has been reported to influence lipid metabolism in experimental animals (Ashakumary et al., 1999). Sesame is a well-known health food, and the association of sesame oil with lower blood pressure and lipid peroxidation and increased antioxidant status in hypertensive patients has been reported (Alipoor et al., 2012). The search for anticoagulant agents in natural herbal medicines represents an area of considerable interest (Li et al., 2009). During sesame oil refinement, sesamin is epimerized to a mixture of sesamin and epi-sesamin (ESM), which accounts for some of the different physiological effects of sesame oils. (Fukuda et al., 1986). In our continued search for natural products with the capacity for modulation of anticoagulant activities, we found that the methanol extract of A. sieboldii roots exhibited potent anticoagulant activities in both cell and animal models. Activity-guided purification of this root extract resulted in the isolation of one active principle compound, which was subsequently identified as epi-sesamin. In addition, no studies on the anticoagulant activities of SM or ESM have been reported. Therefore, in the current study, we examined the anticoagulant activities of SM and ESM in the production of FXa and thrombin, and their effects on PT and aPTT and on fibrinolytic activity.
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Materials and Methods
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Reagents TNF- was purchased from Abnova (Twawan). Anti-tissue factor antibody was purchased from Santa Cruz Biologics (Santa Cruz, CA). Factor V, Vll, Vlla, FX, 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). 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. Plant Materials, Extraction, and Isolation of Sesamin (SM) and Epi-sesamin (ESM) Roots of A. sieboldii were purchased at an herbal market in Daegu, Korea in February 2006. The plant material was identified by Dr. Seung Ho Lee at the College of Pharmacy, Yeungnam University, and 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 methanol (MeOH) at room temperature for five days. After concentration, the MeOH extract (564.0 g) was suspended in water and partitioned sequentially with Asarum sieboldii (6 kg) MeOH (3 × 20 L) MC EtOAc
MC ext. (298 g) EtOAc ext. (15 g)
H2O ext. (251 g)
(A)
Sesamin
epi-Sesamin
(B) Figure 1. Procedure for extraction of SM and ESM, HPLC and structures of SM and ESM. (A) Solvent extraction scheme used for SM and ESM was performed as described in the “Materials and Methods” section. HPLC of isolated SM and ESM is shown in (B) and chemical structures of SM and ESM are shown in (C).
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O O
O H
H O
O
O
O H
O
O
Sesamin (1)
epi-sesamin (2) (C)
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Figure 1. (Continued )
methylene chloride (MC) and ethyl acetate (EtOAc) to produce MC (298.0 g), EtOAc (15.0 g), and water (251.0 g) fractions. Chromatography of the MC fraction was performed using a silica gel column, with a stepwise gradient of EtOAc in n-hexane (0–100 %), to yield 20 fractions (MC1-MC20). Compounds 1 [2.1 g, 0.37% (w/w) of the MeOH extract] and 2 [8.5 g, 1.5% (w/w) of the MeOH extract] were obtained from MC10 and MC13 by precipitation in cold MeOH. A combination of spectroscopic methods was used for structural identification of compounds 1 (SM) and 2 (ESM, Fig. 1), as reported in a previous study (Venkataraman and Gopalakrishnan, 2002; Lee et al., 2013). Values were compared with those in the literature ( Venkataraman and Gopalakrishnan, 2002). Isolation of Plasma Blood samples were taken in the morning from 10 healthy volunteers in fasting status (aged between 24 and 28 years, four males and six females) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, and untreated with drugs. All subjects gave written informed consent prior to 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 15 min) in order to obtain plasma. Anticoagulation Assay aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Germany), according to the manufacturer’s instructions, as described previously (Kim et al., 2012). In brief, citrated normal human plasma (90 l) was mixed with 10 l of SM or ESM and incubated for 1 min at 37 C. aPTT assay reagent (100 l) was added, followed by incubation for 1 min at 37 C; 20 mM CaCl2 (100 l) was then added. Clotting times were recorded. For PT assays, citrated normal human plasma (90 l) was mixed with 10 l of SM or ESM stock and incubated for 1 min at 37 C. PT assay reagent (200 l), which had 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 ¼ (PT sample / PT control) ISI . ISI ¼ international sensitivity index.
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Platelet Aggregation Assay
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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 performed according to a previously reported method (Kim et al., 2011). Washed platelets were incubated with indicated (E)SM for 3 min, followed by stimulation with thrombin (3 U/ml, Sigma) in 0.9% saline solution at 37 C for 5 min. Platelet aggregation was recorded using an aggregometer (Chronolog, Havertown, PA, USA). 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 temperature. Control plasma and plasma incubated with SM or ESM were diluted three times in 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, mOD/min) of each absorbance curve was recorded (Nowak et al., 2007). All experiments were performed three times. Cell Culture Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA) and were maintained using a previously described method (Bae and Rezaie, 2008; Bae, 2011). Briefly, cells were cultured until confluent at 37 C at 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). Cell Viability Assay MTT was used as an indicator of cell viability. Cells were grown in 96-well plates at a density of 5 10 3 /well. After 24 h, cells were washed with fresh medium, followed by treatment with SM or ESM. After a 48-h incubation period, cells were washed and 100 l of 1 mg/ml MTT was added, followed by incubation for 4 h. Finally, 150 l DMSO was added in order 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). Data were expressed as meanSD of at least three independent experiments. Factor Xa Production on the Surfaces of HUVECs TNF- (10 ng/ml for 6 h in serum-free medium) stimulated confluent monolayers of HUVECs (preincubated with the indicated concentrations of SM or ESM for 10 min) in a 96well 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
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presence or absence of anti-TF IgG (25 g/ml). FX (175 nM) was then added to the cells (final reaction mixture volume, 100 l) and incubated for 15 min. The reaction was stopped by the addition of 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 using a chromogenic substrate. Changes in absorbance at 405 nm over 2 min were monitored using a microplate reader. Initial rates of color development were converted to FXa concentrations using a standard curve prepared with known dilutions of purified human FXa.
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Thrombin Production on the Surfaces of HUVECs Measurement of thrombin production by HUVECs was quantitated as previously described (Bae, 2011; Kim et al., 2012). HUVECs were brieflt pre-incubated in 300 l containing SM or ESM 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 M. After 10 min, duplicate samples (10 l each) were transferred to a 96-well plate containing 40 l of 0.5 M EDTA in Tris-buffered saline per well for termination of prothrombin activation. Activated prothrombin was determined by measuring the rate of hydrolysis of S2238 at 405 nm. Standard curves were prepared using amounts of purified thrombin. Thrombin Activity Assay SM or ESM in 50 mM Tris–HCl buffer (pH 7.4) containing 7.5 mM EDTA and 150 mM NaCl was mixed in the absence or presence of 150 l of AT III (200 nM). 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, the thrombin solution (150 l; 10 U/ml) was added, followed by incubation at 37 C for 1 min. S-2238 (a thrombin substrate; 150 l; 1.5 mM) solution was then added and its absorbance at 405 nm was monitored for 120 s using a spectrophotometer (TECAN, Switzerland). Factor Xa (FXa) Activity Assay These assays were performed in the same manner as the thrombin activity assay, but using factor Xa (1 U ml/1) and S-2222 as substrates. In Vivo Bleeding Time Tail bleeding times were measured using the method described by (Dejana et al., 1979; Kim et al., 2012). Briefly, ICR mice were fasted overnight before the experiments. One hour after intravenous administration of SM or ESM, the 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.
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ELISA for PAI-1 and t-PA Concentrations of PAI-1 and t-PA in HUVEC cultured supernatants were determined using ELISA kits (American Diagnostica Inc. CT, USA). Statistical Analysis
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Data are expressed as meanS.E.M. (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 < 0:05. Results and Discussion Sesamin (SM) and epi-sesamin (ESM) have been reported to contain antiallergic (Hashimoto et al., 1994), antinociceptive (Kim et al., 2003), and antifungal agents (Lee et al., 2005; Yu et al., 2006). In this study, we evaluated the anticoagulant effects of SM or ESM from Asarum species for the first time and sought to identify the mechanisms responsible for these effects. Effects of SM or ESM on aPTT and PT Incubation with ESM, but not SM, resulted in changes in the coagulation properties of human plasma. The anticoagulant properties of SM or ESM in human plasma were tested Table 1. Anticoagulant Activity of Sesamin (SM) a In Vitro Coagulant Assay Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control
Saline
32.1 0.5
14.2 0.4
1.00
SM
0.5 M 1 M 2 M 5 M 10 M 20 M
33.7 33.1 32.9 34.2 33.6 32.4
1.05 0.98 1.08 1.05 0.95 1.02
Heparin
0.5 0.3 0.5 0.3 1.2 0.4
0.5 (g/ml) 98.6 1.2**
14.5 14.1 14.7 14.5 13.9 14.3
0.3 0.7 0.2 0.5 0.7 0.3
10 (g/ml) 38.4 0.6**
8.92**
In Vivo Bleeding Time Sample
Dose
Tail Bleeding Time (s)
n
Control SM Heparin
Saline 7 g/mouse 1 mg/mouse
45.9 1.2 46.5 1.7 137.4 2.3**
10 10 10
a Each value represents the means S.D. (n ¼ 5). *p < 0:05 as compared to control. **p < 0:01 as compared to control.
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In Vitro Coagulant Assay Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control
Saline
32.1 0.5
14.2 0.4
1.00
ESM
0.5 M 1 M 2 M 5 M 10 M 20 M
32.1 33.5 38.1 45.2 64.2 71.7
Heparin
0.6 1.1 0.4* 0.6** 0.5** 0.4**
0.5 (g/ml) 98.6 1.2**
14.3 14.9 18.3 21.3 24.3 28.7
0.6 0.8 0.6 0.5 0.4 0.5
10 (g/ml) 38.4 0.6**
1.02 1.11 1.75* 2.44** 3.26** 4.70** 8.92**
In vivo Bleeding Time Sample
Dose
Tail Bleeding time (s)
n
Control ESM Heparin
Saline 7 g/mouse 1 mg/mouse
45.9 1.2 106.7 1.7** 137.4 2.3**
10 10 10
value represents the means S.D. (n ¼ 5). *p < 0:05 as compared to control. **p < 0:01 as compared to control. a Each
using aPTT and PT assays. A summary of the results is shown in Tables 1 and 2. Although the anticoagulant activities of ESM were weaker than those of heparin, treatment with ESM at concentrations greater than 2 M resulted in significantly prolonged aPTT and PT. The result showing prolongation of aPTT suggests the inhibition of the intrinsic and/or the common pathway, whereas prolongation of PT indicates that ESM could also inhibit the extrinsic coagulation pathway. Assuming an average body weight of 20 g and an average blood volume of 2 ml, the amounts of both of the test compounds (SM or ESM, M:W ¼ 354, 7g per mouse) produced a concentration of approximately 10 M in peripheral blood. As shown in Tables 1 and 2, treatment with ESM, but not SM, resulted in significantly prolonged tail bleeding times. Effects of SM or ESM on Production of Thrombin and FXa and Cytotoxicity Based on the current finding that clotting times were prolonged by treatment with ESM, the effects of SM or ESM on the production of thrombin and FXa were determined because FXa alone can convert prothrombin to thrombin and thrombin is required for conversion of fibrinogen to fibrin (Davie, 1995). In a previous study, Sugo et al. reported that endothelial cells are able to support prothrombin activation by FXa (Sugo et al., 1995). In the current study, pre-incubation of HUVECs with FVa and FXa in the presence of CaCl2 prior to the addition of prothrombin resulted in production of thrombin (Fig. 2A). In addition,
*
300
**
**
200 100 0 0
0.5 1 2 5 SM or ESM, [µM]
10
80 **
60
**
40
**
20
****
0 (E)SM [µM] 0
10
0
1
2
5
10
0
-
-
+
+
+
+
+
+
TNF-α
(B)
Relative Cell Viability (%)
(A)
α-TF IgG
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1321
100
400
FXa Generation (nM)
Th Generation (nM)
ANTICOAGULANT ACTIVITIES OF ESM
120 100 80 60 40 20 0 Cont 1
2
5
10
20
50
SM or ESM, [µM] (C) Figure 2. Inhibition of thrombin and FXa production by SM or ESM in HUVECs and cytotoxicity. (A) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10 min with the indicated concentrations of SM (WÞ or ESM (¥). Prothrombin was added to a final concentration of 1 M 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 SM (W) or ESM (¥) for 10 min. TNF- (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 g/ml) and FXa production was determined as described in the “Materials and Methods” section. (C) Effect of SM or ESM on cellular viability was measured using the MTT assay. *p < 0:05 or **p < 0:05vs. 0 (A) or TNF- alone (B).
treatment with ESM, but not SM, resulted in dose-dependent inhibition of the production of thrombin from prothrombin (Fig. 2A). According to findings reported by Rao et al., the endothelium provides the functional equivalent of procoagulant phospholipids and supports the activation of FX (Rao et al., 1988), and in TNF- stimulated HUVECs, the activation of FX by FVIIa occurred in a TF expression-dependent manner (Ghosh et al., 2007). Thus, we investigated the effects of SM or ESM on the activation of FX by FVIIa. HUVECs were stimulated with TNF- for induction of TF expression, and as shown in Fig. 2B, the rate of FX activation by FVIIa was 40-fold higher in stimulated HUVECs (82.3 6.1 nM) than in non-stimulated HUVECs (2.8 0.07 nM); this increase in
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activation was abrogated by anti-TF IgG (8.2 1.5 nM). In addition, pre-incubation with ESM resulted in dose-dependent inhibition of FX activation by FVIIa (Fig. 2B). Therefore, these results suggest that ESM can inhibit the production of thrombin and FXa. For determination of the cytotoxicity of SM or ESM, a cellular viability assay (MTT assay) was performed in HUVECs treated with SM or ESM for 24 h. At concentrations up to 50 M, neither SM nor ESM had an effect on cell viability (Fig. 2C).
The effects of SM or ESM on thrombin-catalyzed fibrin polymerization in human plasma were monitored as changes in absorbance at 360 nm, as described in the “Materials and Methods” section. As shown in Fig. 3A, the results demonstrate that the incubation of human plasma with ESM, but not SM, resulted in a significant decrease in the maximal rate of fibrin polymerization. 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. A thrombin-catalyzed platelet aggregation assay was performed in order to confirm the anticoagulant activities of ESM. As shown in Fig. 3B, ESM significantly inhibited mouse platelet aggregation induced by thrombin (final concentration: 3 U/ml) in a concentration dependent manner. The reptilase-catalyzed polymerization assay was performed in order to exclude the possibility that the decrease of polymerization could be due to a direct effect on thrombin, leading to a decrease in fibrin production rather than in the polymerization of fibrin formed. Results showed that treatment with ESM resulted in a significant decrease in reptilasecatalyzed polymerization (data not shown).
100
100 *
80
**
60
**
40 20
Aggregation (%)
120 Vmax (% Control 0)
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Effects of SM or ESM on Thrombin-catalyzed Platelet Aggregation and Fibrin Polymerization
80
*
60
**
40
**
20
0 (E)SM [µM]
0
10
0
1
2
5
10
0 (E)SM [µM]
0
10
0
1
2
5
10
Th
-
-
+
+
+
+
+
Th
-
-
+
+
+
+
+
(A)
(B)
Figure 3. Effects of SM or ESM on fibrin polymerization in human plasma. (A) Thrombin-catalyzed fibrin polymerization at the indicated concentrations of SM (W) or ESM (¥) 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 SM (W) or ESM (¥) on mouse platelet aggregation induced by 3U/ml thrombin. *p < 0:05 or **p < 0:05 vs. Th alone.
ANTICOAGULANT ACTIVITIES OF ESM
1323
120
Relative Th Activity (%)
Relative Th Activity (%)
To elucidate the mechanism responsible for inhibition of coagulation by ESM, a measurement of the inhibitory effects of ESM on the activities of thrombin and FXa was performed using chromogenic substrates. According to results shown in Figs. 4A and 4B, treatment with ESM, but not SM, resulted in dose-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by the anticoagulant. In addition, we also investigated the effects of SM or ESM on FXa activity. ESM, but not SM, inhibited the effects on FXa activities (Figs. 4C and 4D). These results are consistent with those of our antithrombin assay, and therefore suggest that the antithrombotic mechanisms of ESM appear to be due to the inhibition of fibrin polymerization and/or the intrinsic/extrinsic pathway.
SM
100 80 60 40 20
Heparin
0
120 100
*
80
20
Heparin
0 100
10
1
0.1
0.01
0.001
0
ESM, [µM] or Heparin, [µg/ml] (B)
120
SM
100 80 60 40 20
Heparin
0
120 100
*
80 60
** ESM ** **
40 20
Heparin
0 100
10
1
0.1
0.01
0.001
0
100
10
1
0.1
0.01
0.001
0
SM, [µM] or Heparin, [µg/ml]
Relative FXa Activity (%)
(A)
Relative FXa Activity (%)
**
40
SM, [µM] or Heparin, [µg/ml]
(C)
ESM
** **
60
100
10
1
0.1
0.01
0.001
0
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Effects of SM or ESM on the Activities of Thrombin and FXa
ESM, [µM] or Heparin, [µg/ml] (D)
Figure 4. Effects of SM or ESM on inactivation of thrombin and factor Xa. Inhibition of thrombin (Th) by SM (A) or ESM (B) was measured using a chromogenic assay, as described in the “Materials and Methods” section. Inhibition of factor Xa (FXa) by SM (C) or ESM (D) was monitored using a chromogenic assay, as described in the “Materials and Methods” section. Heparin (W) was used as a positive control. *p < 0:05 or **p < 0:05 vs. 0.
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TNF- appears to inhibit the fibrinolytic system in HUVECs by inducing the production of PAI-I and altering the balance between t-PA and PAI-1 is known to result in modulation of coagulation and fibrinolysis (Schleef et al., 1988; Philip-Joet et al., 1995). To determine the direct effects of SM or ESM on TNF--stimulated secretion of PAI-1, HUVECs were cultured in media with or without SM or ESM in the absence or presence of TNF- for 18 h. As shown in Fig. 5, treatment with ESM, but not SM, resulted in dose-dependent inhibition of TNF--induced secretion of PAI-1 from HUVECs, and these decreases became significant at an ESM dose of 2 M. TNF- does not have a significant effect on t-PA production (Hamaguchi et al., 2003) and the balance between plasminogen activators and their inhibitors reflects net plasminogen-activating capacity (Davie et al., 1991; Davie, 1995; Quinn et al., 2000); therefore, we investigated the effect of TNF- with ESM on the secretion of t-PA from HUVECs. The results were consistent with those of a previous study reporting a modest decrease in the production of t-PA by TNF- in HUVECs (Lopez et al., 2000). This decrease was not significantly altered by treatment with ESM (Fig. 6). Therefore, collectively, these results indicate that the PAI-1/t-PA ratio was increased by TNF- and that ESM, but not SM, prevented this increase (Table 3). ESM, which possesses anti-aryl side chains, exhibited anticoagulant activity both in vitro and in vivo, however, SM, which has a similar structure, did not exhibit such activity. Molecular modeling studies performed in order to determine the reason for this difference showed the difference in the three-dimensional (3D) structures of SM and ESM. The two aryl side chains of SM were found to adopt the syn orientation, whereas those of ESM adopted the anti orientation, suggesting that SM or ESM interacts with coagulation PAI-1 Concentration (ng/ml)
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Effects of SM or ESM on the Secretion of PAI-1 or t-PA Protein
400 300 200
*
**
**
n.s.
100 0 0 0.5 1
2
5
10
0 0.5 1
2
5
10
SM or ESM, [µM] TNF-α (10 ng/ml) Figure 5. Effects of SM or ESM on secretion of PAI-1 by HUVECs stimulated with TNF-. HUVECs were cultured with SM (W) or ESM (¥) in the absence or presence of TNF- (10 ng/ml) for 18 h and PAI-1 concentrations in culture media were determined as described in the “Materials and Methods” section. *p < 0:05 or **p < 0:05 vs. TNF- alone; n.s., not significant.
t-PA Concentration (ng/ml)
ANTICOAGULANT ACTIVITIES OF ESM 14 12 10 8 6 4 2 0
1325
* n.s.
0
0
0.5
1
2
5
10
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SM or ESM, [µM] TNF-α (10 ng/ml) Figure 6. Effects of SM or ESM on secretion of t-PA by HUVECs stimulated with TNF-. HUVECs were cultured with SM (W) or ESM (¥) in the absence or presence of TNF- (10 ng/ml) for 18 h and t-PA concentrations in culture media were determined as described in the “Materials and Methods” section. *p < 0:05; n.s., not significant.
factors and with signaling molecules downstream of coagulation factors in different ways. This finding suggests that novel structures should be investigated through face-to-face study of coagulation factor activities. In conclusion, results of this study demonstrate that ESM, but not SM, inhibited the extrinsic and intrinsic blood coagulation pathways through the inhibition of FXa and thrombin production in HUVECs, and that ESM inhibits TNF--induced secretion of PAI-1. Of particular interest, consistently greater anticoagulant and profibrinolytic effects were observed for ESM, compared with those of SM, suggesting that the different 3D structure of ESM enhances its anticoagulant effects, compared to SM. These results contribute to previous work on the topic, and should be of interest to those designing pharmacological strategies for the treatment or prevention of vascular diseases.
Table 3. PAI-1/t-PA ratio by SM or ESM in TNF-® activated HUVECs by ELISA a Dose
PBS
10 ng/ml
1.4 TNF- 43.7
0.5 M 1 M 2 M 5 M 10 M
SM 44.2 44.4 43.2 45.8 47.3
ESM 43.3 45.0 40.3 * 36.7 ** 35.2 **
value represents the means S.D. (n ¼ 3). < 0:05 as compared to TNF- alone. ** p < 0:01 as compared to TNF- alone.
a Each *p
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Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MEST] (Grant Nos. 2012028835 and 2012-0009400).
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References Alipoor, B., M.K. Haghighian, B.E. Sadat and M. Asghari. Effect of sesame seed on lipid profile and redox status in hyperlipidemic patients. Int. J. Food. Sci. Nutr. 63: 674–678, 2012. Ashakumary, L., I. Rouyer, Y. Takahashi, T. Ide, N. Fukuda, T. Aoyama, T. Hashimoto, M. Mizugaki and M. Sugano. Sesamin, a sesame lignan, is a potent inducer of hepatic fatty acid oxidation in the rat. Metabolism 48: 1303–1313, 1999. Bae, J.S. Antithrombotic and profibrinolytic activities of phloroglucinol. Food. Chem. Toxicol. 49: 1572–1577, 2011. Bae, J.S. and A.R. Rezaie. Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand. Thromb. Haemost. 100: 101–109, 2008. Davie, E.W. Biochemical and molecular aspects of the coagulation cascade. Thromb. Haemost. 74: 1–6, 1995. Davie, E.W., K. Fujikawa and W. Kisiel. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30: 10363–10370, 1991. Dejana, E., A. Callioni, A. Quintana and G. de Gaetano. Bleeding time in laboratory animals. II — A comparison of different assay conditions in rats. Thromb. Res. 15: 191–197, 1979. Esmon, C.T. Role of coagulation inhibitors in inflammation. Thromb. Haemost. 86: 51–56, 2001. Fukuda, Y., M. Isobe, M. Nagata, T. Owaea and M. Namiki. Acidic transformation of sesamolin, the sesami-oil constituent, into an antioxidant bisepoxylignan, Sesaminol. Heterocycles 24: 923– 926, 1986. Ghosh, S., M. Ezban, E. Persson, U. Pendurthi, U. Hedner and L.V. Rao. Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface. J. Thromb. Haemost. 5: 336–346, 2007. Hamaguchi, E., T. Takamura, A. Shimizu and Y. Nagai. Tumor necrosis factor-alpha and troglitazone regulate plasminogen activator inhibitor type 1 production through extracellular signal-regulated kinase- and nuclear factor-kappaB-dependent pathways in cultured human umbilical vein endothelial cells. J. Pharmacol. Exp. Ther. 307: 987–994, 2003. Han, A.R., H.J. Kim, M. Shin, M. Hong, Y.S. Kim and H. Bae. Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells. Chem. Biodivers. 5: 346–351, 2008. Hashimoto, K., T. Yanagisawa, Y. Okui, Y. Ikeya, M. Maruno and T. Fujita. Studies on anti-allergic components in the roots of Asiasarum sieboldi. Planta Med. 60: 124–127, 1994. Kamal-Eldin, A., A. Moazzami and S. Washi. Sesame seed lignans: potent physiological modulators and possible ingredients in functional foods and nutraceuticals. Recent Pat. Food Nutr. Agric. 3: 17–29, 2011. Kelly, L. Phylogenetic relationships in Asarum (Aristolochiaceae) based on morphology and ITS sequences. Am. J. Bot. 85: 1454–1467, 1998. Kim, S.J., C. Gao Zhang and J. Taek Lim. Mechanism of anti-nociceptive effects of Asarum sieboldii Miq. radix: potential role of bradykinin, histamine and opioid receptor-mediated pathways. J. Ethnopharmacol. 88: 5–9, 2003.
Am. J. Chin. Med. 2013.41:1313-1327. Downloaded from www.worldscientific.com by 222.80.15.134 on 01/22/15. For personal use only.
ANTICOAGULANT ACTIVITIES OF ESM
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Kim, S.Y., S. Kim, J.M. Kim, E.H. Jho, S. Park, D. Oh and H.S. Yun-Choi. PKC inhibitors RO 318220 and Go 6983 enhance epinephrine-induced platelet aggregation in catecholamine hyporesponsive platelets by enhancing Akt phosphorylation. BMB Rep. 44: 140–145, 2011. Kim, T.H., S.K. Ku and J.S. Bae. Antithrombotic and profibrinolytic activities of eckol and dieckol. J. Cell Biochem. 113: 2877–2883, 2012. Lee, J.Y., S.S. Moon and B.K. Hwang. Isolation and antifungal activity of kakuol, a propiophenone derivative from Asarum sieboldii rhizome. Pest Manag. Sci. 61: 821–825, 2005. Lee, W., S.K. Ku, J.A. Kim, T. Lee and J.S. Bae. Inhibitory effects of epi-sesamin on HMGB1induced vascular barrier disruptive responses in vitro and in vivo. Toxicol. Appl. Pharmacol. 267: 201–208, 2013. Levi, M., M. Schultz and T. van der Poll. Coagulation biomarkers in critically ill patients. Crit. Care Clin. 27: 281–297, 2011. Li, Y., Z.J. Qian, B. Ryu, S.H. Lee, M.M. Kim and S.K. Kim. Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia cava. Bioorg. Med. Chem. 17: 1963–1973, 2009. Lopez, S., F. Peiretti, B. Bonardo, I. Juhan-Vague and G. Nalbone. Effect of atorvastatin and fluvastatin on the expression of plasminogen activator inhibitor type-1 in cultured human endothelial cells. Atherosclerosis 152: 359–366, 2000. Nowak, P., H.M. Zbikowska, M. Ponczek, J. Kolodziejczyk and B. Wachowicz. Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications induced by peroxynitrite: functional consequences. Thromb. Res. 121: 163–174, 2007. Philip-Joet, F., M.C. Alessi, C. Philip-Joet, M. Aillaud, J.R. Barriere, A. Arnaud and I. Juhan-Vague. Fibrinolytic and inflammatory processes in pleural effusions. Eur. Respir. J 8: 1352–1356, 1995. Quinn, C., J. Hill and H. Hassouna. A guide for diagnosis of patients with arterial and venous thrombosis. Clin. Lab. Sci. 13: 229–238, 2000. Rao, L.V., S.I. Rapaport and M. Lorenzi. Enhancement by human umbilical vein endothelial cells of factor Xa-catalyzed activation of factor VII. Blood 71: 791–796, 1988. Schleef, R.R., M.P. Bevilacqua, M. Sawdey, M.A. Gimbrone, Jr. and D.J. Loskutoff. Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor. J. Biol. Chem. 263: 5797–5803, 1988. Sugo, T., C. Nakamikawa, S. Tanabe and M. Matsuda. Activation of prothrombin by factor Xa bound to the membrane surface of human umbilical vein endothelial cells: its catalytic efficiency is similar to that of prothrombinase complex on platelets. J. Biochem. 117: 244–250, 1995. Venkataraman, R. and S. Gopalakrishnan. A lignan from the root of Ecbolium Linneanum Kurz. Phytochemistry 61: 963–966, 2002. Yu, H.H., S.J. Seo, J.M. Hur, H.S. Lee, Y.E. Lee and Y.O. You. Asarum Sieboldii extracts attenuate growth, acid production, adhesion, and water-insoluble glucan synthesis of Streptococcus mutans. J. Med. Food 9: 505–509, 2006.
