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Antithrombotic activities of pellitorine in vitro and in vivo
Fitoterapia 91 (2013) 1–8
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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 b c
Department of Anatomy and Histology, College of Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea Department of Cosmetic Science and Technology, Seowon University, Cheongju 361-742, Republic of Korea 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
Article history: Received 10 July 2013 Accepted in revised form 9 August 2013 Available online 22 August 2013 Keywords: Pellitorine Coagulation cascade Fibrinolysis Endothelium
a b s t r a c t Pellitorine (PLT), an active amide compound, is well known to possess insecticidal, antibacterial and anticancer properties. However, the anti-coagulant functions of PLT are not studied yet. Here, the anticoagulant activities of PLT were examined by monitoring activated 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 plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor (TNF)-α activated human umbilical vein endothelial cells (HUVECs). Treatment with PLT resulted in prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, and PLT inhibited production of thrombin and FXa in HUVECs. 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 antithrombotic activities and offers bases for development of a novel anticoagulant. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The leading causes of death (30% of total) in the world are diseases involving the heart and blood vessels and, consequently, thrombosis [1]. Most thromboembolic processes require anticoagulant therapy and this explains the current efforts to develop specific and potent anticoagulant and antithrombotic agents [2]. Primary haemostatic events are triggered in response to damage of the vascular wall by the exposure of blood to the subendothelial extracellular matrix [3,4]. Thrombin is the key effector enzyme of the coagulation system, having many biologically important functions such as the activation of platelets, conversion of fibrinogen to a fibrin network, and feedback amplification of coagulation [4,5]. The ⁎ Corresponding author at: College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82 53 950 8557. E-mail address: [email protected] (J.-S. Bae). 1 First two authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.08.004
precise and balanced generation of thrombin at sites of vascular injury is the result of an ordered series of reactions collectively referred to as blood coagulation [4,5]. Clots are eventually broken down by plasmin, which is activated by tissue-type plasminogen activator (t-PA) from plasminogen. Thrombin is also an activator of inflammation and an inhibitor of fibrinolysis [6]. The hemostatic plug that forms within blood vessels, often within the veins or arteries of the heart, in pathological conditions associated with arterial disease, referred to as a thrombus [6], is a major cause of morbidity and death. Clotting time assays measure the time required to generate thrombin [7] and activated partial thromboplastin time (aPTT) measures the efficacy of the contact activation and common coagulation pathways [7]. Furthermore, the aPTT or prothrombin time (PT) mainly serves to aid the diagnosis of deficiencies in certain factors [8]. Asarum sieboldii Miq. (Aristolochiaceae) is a wide-ranging species found through North America, Europe, and Asia [9]. The radix of A. sieboldii is a traditional herb medicine used as
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remedies for aphthous stomatitis, toothache, and gingivitis and as a local anesthetic agent in Korea and China [10]. Previous studies on the constituents of A. sieboldii have revealed isolation of various types of essential oils, amide, alkaloids, lignans, and flavonoids [9,11,12]. Pellitorine (PLT) was isolated from the methylene chloride soluble fraction of the roots of A. sieboldii using a combination of silica gel column chromatography and recrystallization. A conjugated alkaldienamide, pellitorine was isolated mainly from the Piper species [13]. Pellitorine showed various biological properties including insecticidal [14], lavicidal [14], antibacterial [15], and anti-cancer [16] activities. However, no studies on the anticoagulant activities of PLT have been reported. Therefore, in the current study, we examined the anticoagulant activities of PLT in the production of FXa and thrombin, and their effects on PT and aPTT and on fibrinolytic activity. 2. Materials and methods 2.1. Reagents TNF-α was purchased from Abnova (Taiwan). Anti-tissue factor antibody was purchased from Santa Cruz Biologics (Santa Cruz, CA). c-Jun N-terminal kinase (JNK) inhibitor (SP600125), Nuclear factor (NF)-κB inhibitor (Emodin), and extracellular signal regulated kinase (ERK) inhibitor (PD98059) were purchased from R&D Systems (Minneapolis, MN). Factors V, Vll, Vlla, FX, and FXa, antithrombin III (AT III), prothrombin, and thrombin were obtained from Haematologic Technologies (Essex Junction, VT, USA). aPTT assay reagent and PT reagents were purchased from Fisher Diagnostics (Middletown, Virginia, USA), and the chromogenic substrates, S-2222 and S-2238, were purchased from Chromogenix AB (Sweden). The PAI-1 and t-PA ELISA kits were purchased from American Diagnostica Inc. (Stamford, CT, USA). Other reagents were of the highest commercially available grades. Oleanolic acid (OA) was prepared as described previously [17].
(251.0 g) extractions, respectively. The MC extraction was fractioned by silica gel column chromatography eluting with EtOAc in n-hexane (0–100%, step-wise), to yield twenty fractions (MC1–MC20). MC15 (8.2 g) was chromatographed on silica gel column using a solvent system of EtOAc in n-hexane (15%) to give seven fractions (MC15-1–MC15-7). MC15-4 (16.6 g) was purified by recrystallization from chloroform to afford a needle-type solid, compound 1 [675.0 mg, 0.12% (w/w) of MeOH extract]. The structure of compound 1 (Fig. 1) was identified by a combination of spectroscopic methods and comparisons with the literature data [18]. 2.3. Pellitorine (1) Needles, mp 69 °C; 1H NMR (250 MHz, CDCl3): δ 0.91 (3H, s, H-10), 0.93 (3H, s, H-3′), 0.96 (3H, s, H-4′), 1.32 (4H, m, H-8, H-9), 1.44 (2H, m, H-7), 1.82 (1H, m, H-2′), 2.18 (2H, dd, J = 12.5, 6.2 Hz, H-6), 3.18 (2H, t, J = 12.9, 6.6 Hz, H-1′), 5.18 (1H, d, J = 15.0 Hz, H-2), 5.58 (NH, br s), 6.07 (1H, m, H-5), 6.17 (1H, m, H-4), 7.20 (1H, m, H-3); 13C NMR (63 MHz, CDCl3): δ 14.4 (C-10), 20.5 (C-3′, C-4′), 22.9 (C-9), 28.9 (C-7), 29.0 (C-2′), 31.8 (C-8), 33.3 (C-6), 47.3 (C-1′), 122.0 (C-2), 128.6 (C-4), 141.8 (C-3), 143.8 (C-5), 166.9 (C-1). 2.4. Isolation of plasma Blood samples were taken in the morning from 10 healthy volunteers in fasting status (aged between 24 and 28 years, 4 males and 6 females) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. All subjects gave written informed consent before participation. Healthy subjects did not use addictive substances and antioxidant supplementation, and their diet was balanced (meat and vegetables). Human blood was collected into sodium citrate (0.32% final concentration) and immediately centrifuged (2000 ×g 15 min) to obtain plasma. 2.5. Anticoagulation assay
2.2. Plant material, extraction, and purification Melting points were obtained with an Electrothermal 9100 melting point apparatus (Electrothermal Ltd.). 1H and 13C NMR spectra were obtained on a Bruker ARX spectrometer (250 MHz) and chemical shifts given in δ (ppm) from tetramethylsilane (TMS) as an internal standard. Column chromatography was carried out on silica gel (70–230 mesh, Merck). Thin layer chromatography (TLC) analysis was performed on Kieselgel 60 F254 (Merck 1.05715) aluminum plate; spots were visualized by spraying with 10% aqueous H2SO4 followed by heating. All other chemicals and solvents were of analytical grade and used without further purification. The roots of A. sieboldii were purchased from herbal market at Daegu, Korea, in February 2006. The plant material was identified by Dr. Seung Ho Lee at the College of Pharmacy, Yeungnam University. A voucher specimen (SH0602) was deposited at the herbarium, College of Pharmacy, Yeungnam University. The dried roots of A. sieboldii (6.0 kg) were extracted with MeOH at room temperature for 5 days. After being concentrated, the MeOH extract (564.0 g) was suspended in water and then partitioned successively with methylene chloride (MC) and ethyl acetate (EtOAc) to give MC (298.0 g), EtOAc (15.0 g), and water
aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Germany), according to the manufacturer's instructions as described previously [19]. In brief, citrated normal human plasma (90 μl) was mixed with 10 μl of PLT or OA and incubated for 1 min at 37 °C. aPTT assay reagent (100 μl) was added and incubated for 1 min at 37 °C, and then 20 mM CaCl2 (100 μl) was added. Clotting times were recorded. For PT assays, citrated normal human plasma (90 μl) was mixed with 10 μl of PLT or OA stock and incubated for 1 min at 37 °C. PT assay reagent (200 μl), which has been preincubated for 10 min at 37 °C, was then added and clotting time was recorded. PT results are expressed in seconds and as International Normalized Ratios (INR), and aPTT results are expressed in seconds. INR = (PT sample / PT control)ISI. ISI = international sensitivity index.
Fig. 1. The chemical structure of PLT.
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2.6. Platelet aggregation assay Mouse platelets from platelet-rich plasma (PRP) were washed once with HEPES buffer (5 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH2PO4, 2 mM MgCl2, 5.6 mM glucose, 0.1% BSA (w/v), pH to 7.45). The platelet aggregation study was carried out according to a method previously reported [20]. Washed platelets were incubated with indicated PLT or OA for 3 min, and then stimulated by thrombin (3 U/ml, Sigma) in 0.9% saline solution at 37 °C for 5 min. Platelet aggregation was recorded using an aggregometer (CHRONO-LOG, Havertown, PA, USA). 2.7. Thrombin-catalyzed fibrin polymerization Thrombin-catalyzed polymerization was determined every 6 s for 20 min by monitoring turbidity at 360 nm using a spectrophotometer (TECAN, Switzerland) at ambient temperature. Control plasma and plasma incubated with PLT or OA were trebly diluted TBS (50 mM Tris-buffered physiological saline solution pH 7.4) and clotted with thrombin (final concentration — 0.5 U/ml). The maximum polymerization rate (Vmax, ΔmOD/min) of each absorbance curve was recorded [21]. All experiments were performed three times. 2.8. Cell culture Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA) and were maintained using a previously described method [22,23]. Briefly, the cells were cultured until confluent at 37 °C at 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science).
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rates of color development were converted into FXa concentrations using a standard curve prepared with known dilutions of purified human FXa. 2.11. Thrombin production on the surfaces of HUVECs Measurement of thrombin production by HUVECs was quantitated as previously described [19,23]. Briefly, HUVECs were pre-incubated in 300 μl containing PLT or OA in 50 mM Tris–HCl buffer, 100 pM FVa, and 1 nM FXa for 10 min, followed by addition of prothrombin to a final concentration of 1 μM. After 10 min, duplicate samples (10 μl each) were transferred to a 96-well plate containing 40 μl of 0.5 M EDTA in Tris-buffered saline per well to terminate prothrombin activation. Activated prothrombin was determined by measuring the rate of hydrolysis of S2238 at 405 nm. Standard curves were prepared using the amounts of purified thrombin. 2.12. Thrombin or factor Xa (FXa) activity assay PLT or OA in 50 mM Tris–HCl buffer (pH 7.4) containing 7.5 mM EDTA and 150 mM NaCl was mixed in the presence of 150 μl of AT III (200 nM). The heparins with AT III (200 nM) were dissolved in physiological saline and placed in the sample wells. After incubation at 37 °C for 2 min, thrombin solution (150 μl; 10 U/ml) was added, followed by incubation at 37 °C for 1 min. S-2238 (a thrombin substrate; 150 μl; 1.5 mM) solution was then added and absorbance at 405 nm was monitored for 120 s using a spectrophotometer (TECAN, Switzerland). And FXa activity assay was performed in the same manner as the thrombin activity assay, but using factor Xa (1 U ml/1) and S-2222 as substrates.
2.9. 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, the cells were washed with fresh medium, followed by treatment with PLT. After a 48-h incubation period, the cells were washed, and 100 μl of 1 mg/ml MTT was added, followed by incubation for 4 h. Finally, 150-μl DMSO was added to solubilize the formazan salt formed, the amount of which was determined by measuring the absorbance at 540 nm using a microplate reader (Tecan Austria GmbH, Austria). 2.10. Factor Xa production on the surfaces of HUVECs TNF-α (10 ng/ml for 6 h in serum-free medium) stimulated confluent monolayers of HUVECs (preincubated with the indicated concentrations of PLT or OA for 10 min) in a 96-well culture plate were incubated with FVIIa (10 nM) in buffer B (buffer A supplemented with 5 mg/ml bovine serum albumin [BSA] and 5 mM CaCl2) for 5 min at 37 °C in the presence or absence of anti-TF IgG (25 μg/ml). FX (175 nM) was then added to the cells (final reaction mixture volume, 100 μl) and incubated for 15 min. The reaction was stopped by adding buffer A (10 mM HEPES, pH 7.45, 150 mM NaCl, 4 mM KCl, and 11 mM glucose) containing 10 mM EDTA and the amounts of FXa generated were measured by using a chromogenic substrate. Changes in absorbance at 405 nm over 2 min were monitored using a microplate reader. Initial
Tail bleeding times were measured using the method described by Dejana et al. [19,24]. Briefly, ICR mice were fasted overnight before experiments. One hour after intravenous administration of PLT or OA, tails of mice were transected at 2 mm from their tips. Bleeding time was defined as the time elapsed until bleeding stopped. When the bleeding time exceeded 15 min, bleeding time was recorded as 15 min for the analysis. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University. 2.14. ELISA for PAI-1 and t-PA The concentrations of PAI-1 and t-PA in HUVEC cultured supernatants were determined using ELISA kits (American Diagnostica Inc. CT, USA). 2.15. Statistical analysis Data are expressed as mean ± SEM (standard error of the mean) of at least three independent experiments. Statistical significance between two groups was determined using the Student's t-test. Statistical significance was accepted for p b 0.05.
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recently we published that OA has anticoagulant activities in vitro and in vivo [17].
Table 1 Anticoagulant activity of PLTa. In vitro coagulant assay Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control PLT
Saline 1 μM 2 μM 3 μM 5 μM 10 μM 20 μM 40 μM
30.6 ± 0.6 31.2 ± 0.4 30.8 ± 0.6 31.4 ± 0.8 32.4 ± 1.2 40.6 ± 0.4⁎⁎ 48.2 ± 0.6⁎⁎ 63.4 ± 1.0⁎⁎ 0.5 μg/ml 114.8 ± 1.2⁎⁎
14.0 ± 0.2 14.2 ± 0.4 14.6 ± 0.6 15.0 ± 0.4 15.2 ± 0.2 23.2 ± 0.2 27.4 ± 0.4 28.2 ± 0.6 10 μg/ml 34.6 ± 0.8⁎⁎
1.00 1.03 1.10 1.16 1.20 3.04⁎⁎ 4.38⁎⁎ 4.67⁎⁎ 7.32⁎⁎
OA Heparin
In vivo bleeding time Sample
Dose
Tail bleeding time (s)
n
Control PLT
Saline
41.6 57.6 71.8 74.6 121.5
10 10 10 10 10
OA Heparin
4.5 μg/mouse 9.0 μg/mouse 36.5 μg/mouse 1 mg/mouse
± ± ± ± ±
1.2 0.8⁎⁎ 1.2⁎⁎ 1.4⁎⁎ 1.2⁎⁎
a Each value represents the means ± SEM (n = 10). ⁎⁎ p b 0.01 as compared to control.
3.1. Effects of PLT on aPTT and PT Incubation with PLT resulted in changes in the coagulation properties of human plasma. The anticoagulant properties of PLT in human plasma were tested using aPTT and PT assays; a summary of the results is shown in Table 1. Although the anticoagulant activities of PLT were weaker than those of heparin, aPTT and PT were significantly prolonged by treatment with PLT at concentrations greater than 10 μM. The result showing prolongation of aPTT suggests inhibition of the intrinsic and/or the common pathway, whereas prolongation of PT indicates that PLT could also inhibit the extrinsic coagulation pathway. To confirm these data in vivo, PLT was administered into mouse via intravenous injection. As shown in Table 1, tail bleeding times were significantly prolonged by treatment with PLT. Assuming that the average weight of a mouse was 20 g, and the average blood volume was 2 ml, the amount of PLT injected (4.5 or 9.0 μg per mouse) or OA injected (36.5 μg per mouse) was equivalent to PLT 10, 20 μM or OA 40 μM in peripheral blood.
3. Results and discussion
3.2. Effects of PLT on thrombin-catalyzed platelet aggregation and fibrin polymerization and cellular viability
In this study, we examined the anticoagulant effects of pellitorine (PLT, Fig. 1) for the first time and sought to identify the mechanisms responsible for these effects. Oleanolic acid (OA) was used as a positive control because
The effects of PLT 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. 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.
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Fig. 3. Effects of PLT on inactivation of thrombin and factor Xa. (A) Inhibition of thrombin (Th) by PLT (○) or OA (40 μM, ■) was measured using a chromogenic assay, as described in the “Materials and methods” section. (B) Inhibition of factor Xa (FXa) by PLT (○) or OA (40 μM, ■) was monitored using a chromogenic assay, as described in the “Materials and methods” section. Heparin (●) was used as a positive control. **p b 0.01 vs. 0.
that incubation of human plasma with PLT resulted in a significant decrease in the maximal rate of fibrin 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. To confirm the anticoagulant activities of PLT, thrombincatalyzed platelet aggregation assay was conducted. As shown in Fig. 2B, PLT significantly inhibited mouse platelet aggregation induced by thrombin (final concentration: 3 U/ ml) in a concentration dependent manner. To exclude the possibility that the decrease of the polymerization could be due to direct effect on thrombin leading to decrease in fibrin production rather than polymerization of fibrin formed, reptilase-catalyzed polymerization assay was introduced. Results showed that PLT significantly decreased reptilasecatalyzed polymerization (data not shown). To determine the cellular viability of PLT, cellular viability assay (MTT assay) was performed in HUVECs treated with PLT for 24 h. At concentrations up to 30 μM, PLT did not affect cell viability (Fig. 2C).
3.3. Effects of PLT on the activities of thrombin and FXa To elucidate the mechanism responsible for inhibition of coagulation by PLT, the inhibitory effects of PLT on the activities of thrombin and FXa were measured using chromogenic substrates. In the results presented in Fig. 3A, treatment with PLT resulted in dose-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by the anticoagulant. In addition, we also investigated the effects of PLT on FXa activity. PLT inhibited the effects on FXa activities (Fig. 3B). These results are consistent with the results of our antithrombin assay, and therefore suggest that the antithrombotic mechanisms of PLT appear to be due to inhibition of fibrin polymerization and/or the intrinsic/extrinsic pathway. 3.4. Effects of PLT on production of thrombin and FXa Previously, Sugo et al. reported that endothelial cells are able to support prothrombin activation by FXa [25]. In the current study, pre-incubation of HUVECs with FVa and FXa in
Fig. 4. Inhibition of thrombin and FXa production by PLT in HUVECs. (A) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10 min with the indicated concentrations of PLT or OA (40 μM). Prothrombin was added to a final concentration of 1 μM and prothrombin activation was determined 30 min later, as described in the “Materials and methods” section. (B) HUVECs were pre-incubated with indicated concentrations of PLT or OA (40 μM) for 10 min. TNF-α- (10 ng/ml for 6 h) stimulated HUVECs were incubated with FVIIa (10 nM) and FX (175 nM) in the absence or presence of anti-TF IgG (25 μg/ml) and FXa production was determined as described in the “Materials and methods” section. *p b 0.05 or **p b 0.01 vs. 0 (A) or TNF-α alone (B).
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Fig. 5. Effects of PLT on secretion of PAI-1 by HUVECs stimulated with TNF-α. HUVECs were cultured with PLT or OA (40 μM) in the absence or presence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentrations in culture media were determined as described in the “Materials and methods” section. *p b 0.05 or **p b 0.01 vs. TNF-α alone; n.s., not significant.
the presence of CaCl2 prior to addition of prothrombin resulted in production of thrombin (Fig. 4A). In addition, treatment with PLT resulted in dose-dependent inhibition of production of thrombin from prothrombin (Fig. 4A). According to findings reported by Rao et al., the endothelium provides the functional equivalent of procoagulant phospholipids and supports activation of FX [26], and, in TNF-α stimulated HUVECs, activation of FX by FVIIa occurred in a TF expression-dependent manner [27]. Thus, we investigated the effects of PLT on activation of FX by FVIIa. HUVECs were stimulated with TNF-α for induction of TF expression, and, as shown in Fig. 4B, the rate of FX activation by FVIIa was 18-fold higher in stimulated HUVECs (95.2 ± 8.2 nM) than in non-stimulated HUVECs (5.3 ± 1.2 nM), and this increase in activation was abrogated by anti-TF IgG (13.8 ± 2.8 nM). In addition, pre-incubation with PLT resulted in dosedependent inhibition of FX activation by FVIIa (Fig. 4B). Therefore, these results suggest that PLT can inhibit production of thrombin and FXa. 3.5. Effects of PLT on secretion of PAI-1 or t-PA protein It is well known that TNF-α appears to inhibit the fibrinolytic system in HUVECs by inducing production of PAI-1 and altering the balance between t-PA and PAI-1 is known to modulate coagulation and fibrinolysis [28,29]. To determine the direct
effects of PLT on TNF-α-stimulated secretion of PAI-1, HUVECs were cultured in media with or without PLT in the absence or presence of TNF-α for 18 h. As shown in Fig. 5, treatment with PLT resulted in dose-dependent inhibition of TNF-α-induced secretion of PAI-1 from HUVECs, and these decreases became significant at a PLT dose of 20 μM. TNF-α does not have a significant effect on t-PA production [30] and the balance between plasminogen activators and their inhibitors reflects net plasminogen-activating capacity [4,5,7]; therefore, we investigated the effect of TNF-α with PLT on secretion of t-PA from HUVECs. The results obtained were consistent with those of a prior study reporting a modest decrease in production of t-PA by TNF-α in HUVECs [31]. This decrease was not significantly altered by treatment with PLT (Fig. 6A). Therefore, collectively, these results indicate that the PAI-1/t-PA ratio was increased by TNF-α and that PLT prevented this increase (Fig. 6B). 3.6. Effects of signal pathway inhibitors on basal and TNF-α induced PAI-1 secretion in the presence or absence of PLT It is well known that both NF-κB and ERK dependent pathways are involved in TNF-α induced PAI-1 production [30]. To define the targets of PLT in the signal transduction pathways leading to TNF-α induced PAI-1 expression, we investigated the effects of three signal transduction inhibitors, Emodin (NF-κB inhibitor), PD98059 (ERK inhibitor), and SP600125 (JNK inhibitor) on TNF-α induced PAI-1 expression in the presence or absence of PLT. We first demonstrated the effect of increasing doses of each inhibitor on HUVECs viability, and selected appropriate non-cytotoxic doses. To confirm the specificity of the inhibitors, we checked the cell viability for all agents by MTT assay. We found that the inhibitors combined with TNF-α and/or PLT as used in this study has no effect on HUVECs viability (data not shown). Experiments performed showed that PD98059 and Emodin inhibited TNF-α induced PAI-1 accumulation, which is consistent with the previous study (Fig. 7A) [30]. In addition, we observed that SP600125 did inhibit TNF-α induced PAI-1 secretion (Fig. 7A). Furthermore, neither PD98059 nor Emodin showed any additional inhibitory effects in the presence of PLT (Fig. 7B). However, the inhibitory effects of
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.
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Fig. 7. Effect of signal transduction inhibitors on TNF-α induced PAI-1 secretion. (A) HUVECs were cultured with SP600125 (2 μM), Emodin (2 μg/ml), and PD98059 (10 μM) in the absence or presence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentration in the culture mediums was examined as described in the “Materials and methods” section. (B) the same as (A) except that cells were preincubated with PLT 20 μM. *p b 0.05 as compared to TNF-α alone (A) or TNF-α + PLT (B).
SP600125 were essentially additive with those of PLT (Fig. 7B). These results suggest that the ERK and NF-κB pathways are involved in PLT-mediated inhibition of TNF-α induced PAI-1 expression in HUVECs. No significant effects of the three inhibitors on basal levels of PAI-1 production could be explained by the fact that the activities of ERK, JNK, and NF-κB are relatively low in the unstimulated cells [32,33]. Thus, these results seem to indicate that PLT decreases PAI-1 levels via inhibition of the ERK and NF-κB pathways. However, additional work will be required to elucidate whether PLT has beneficial effects on fibrinolytic systems in vivo. The pre-clinical evaluation of the antithrombotic potential of novel molecules requires the use of reliable and reproducible experimental models. PT, aPTT, fibrin polymerization, and platelet aggregation are the most established and commonly used preparations to determine the efficacy of novel antithrombotic drugs [34,35]. In our experiment, PT and aPTT using human plasma were used to evaluate the antithrombotic effect of PLT. Fibrin polymerization and platelet aggregation are also used to determine the anti-platelet functions of PLT. Data showed that PLT prolonged PT and aPTT values and PLT resulted in a significant decrease in the maximal rate of fibrin polymerization and inhibited platelet aggregation without having cytotoxicity on human endothelial cells. There is ample evidence that inflammation and coagulation are intricately related processes that may considerably affect each other [36,37]. This cross-talk occurs at the levels of platelet activation, fibrin formation, and resolution as well as physiological anticoagulant pathways [36,37]. On the basis of our current experimental studies, it can be hypothesized that inhibitory modulation of coagulation by PLT could give promising anti-inflammatory mediators. Well-designed prospective studies are needed to prove this hypothesis. In conclusion, results of this study demonstrate that PLT inhibited the extrinsic and intrinsic blood coagulation pathways through inhibition of FXa and thrombin production in HUVECs, and that PLT inhibits TNF-α-induced secretion of PAI-1. These results add to previous work on the topic, and should be of interest to those designing pharmacological strategies for the treatment or prevention of vascular diseases.
Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MSIP] (Grant No. 2012-000940). References [1] Coller BS. Antiplatelet agents in the prevention and therapy of thrombosis. Annu Rev Med 1992;43:171–80. [2] Weitz JI. New anticoagulants for treatment of venous thromboembolism. Circulation 2004;110:I19–26. [3] Hoffman MM, Monroe DM. Rethinking the coagulation cascade. Curr Hematol Rep 2005;4:391–6. [4] Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991;30:10363–70. [5] Davie EW. Biochemical and molecular aspects of the coagulation cascade. Thromb Haemost 1995;74:1–6. [6] Esmon CT. Role of coagulation inhibitors in inflammation. Thromb Haemost 2001;86:51–6. [7] Quinn C, Hill J, Hassouna H. A guide for diagnosis of patients with arterial and venous thrombosis. Clin Lab Sci 2000;13:229–38. [8] Levi M, Schultz M, van der Poll T. Coagulation biomarkers in critically ill patients. Crit Care Clin 2011;27:281–97. [9] Han AR, Kim HJ, Shin M, Hong M, Kim YS, Bae H. Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells. Chem Biodivers 2008;5:346–51. [10] Kim SJ, Gao Zhang C, Taek Lim J. Mechanism of anti-nociceptive effects of Asarum sieboldii Miq. radix: potential role of bradykinin, histamine and opioid receptor-mediated pathways. J Ethnopharmacol 2003;88:5–9. [11] Oh J, Hwang IH, Kim DC, Kang SC, Jang TS, Lee SH, et al. Anti-listerial compounds from Asari Radix. Arch Pharm Res 2010;33:1339–45. [12] Quang TH, Ngan NT, Minh CV, Kiem PV, Tai BH, Thao NP, et al. Anti-inflammatory and PPAR transactivational effects of secondary metabolites from the roots of Asarum sieboldii. Bioorg Med Chem Lett 2012;22:2527–33. [13] Stohr JR, Xiao PG, Bauer R. Constituents of Chinese Piper species and their inhibitory activity on prostaglandin and leukotriene biosynthesis in vitro. J Ethnopharmacol 2001;75:133–9. [14] Park IK. Insecticidal activity of isobutylamides derived from Piper nigrum against adult of two mosquito species, Culex pipiens pallens and Aedes aegypti. Nat Prod Res 2012;26:2129–31. [15] Reddy SV, Srinivas PV, Praveen B, Kishore KH, Raju BC, Murthy US, et al. Antibacterial constituents from the berries of Piper nigrum. Phytomedicine 2004;11:697–700.
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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 b c
Department of Anatomy and Histology, College of Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea Department of Cosmetic Science and Technology, Seowon University, Cheongju 361-742, Republic of Korea 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
Article history: Received 10 July 2013 Accepted in revised form 9 August 2013 Available online 22 August 2013 Keywords: Pellitorine Coagulation cascade Fibrinolysis Endothelium
a b s t r a c t Pellitorine (PLT), an active amide compound, is well known to possess insecticidal, antibacterial and anticancer properties. However, the anti-coagulant functions of PLT are not studied yet. Here, the anticoagulant activities of PLT were examined by monitoring activated 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 plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor (TNF)-α activated human umbilical vein endothelial cells (HUVECs). Treatment with PLT resulted in prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, and PLT inhibited production of thrombin and FXa in HUVECs. 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 antithrombotic activities and offers bases for development of a novel anticoagulant. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The leading causes of death (30% of total) in the world are diseases involving the heart and blood vessels and, consequently, thrombosis [1]. Most thromboembolic processes require anticoagulant therapy and this explains the current efforts to develop specific and potent anticoagulant and antithrombotic agents [2]. Primary haemostatic events are triggered in response to damage of the vascular wall by the exposure of blood to the subendothelial extracellular matrix [3,4]. Thrombin is the key effector enzyme of the coagulation system, having many biologically important functions such as the activation of platelets, conversion of fibrinogen to a fibrin network, and feedback amplification of coagulation [4,5]. The ⁎ Corresponding author at: College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82 53 950 8557. E-mail address: [email protected] (J.-S. Bae). 1 First two authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.08.004
precise and balanced generation of thrombin at sites of vascular injury is the result of an ordered series of reactions collectively referred to as blood coagulation [4,5]. Clots are eventually broken down by plasmin, which is activated by tissue-type plasminogen activator (t-PA) from plasminogen. Thrombin is also an activator of inflammation and an inhibitor of fibrinolysis [6]. The hemostatic plug that forms within blood vessels, often within the veins or arteries of the heart, in pathological conditions associated with arterial disease, referred to as a thrombus [6], is a major cause of morbidity and death. Clotting time assays measure the time required to generate thrombin [7] and activated partial thromboplastin time (aPTT) measures the efficacy of the contact activation and common coagulation pathways [7]. Furthermore, the aPTT or prothrombin time (PT) mainly serves to aid the diagnosis of deficiencies in certain factors [8]. Asarum sieboldii Miq. (Aristolochiaceae) is a wide-ranging species found through North America, Europe, and Asia [9]. The radix of A. sieboldii is a traditional herb medicine used as
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remedies for aphthous stomatitis, toothache, and gingivitis and as a local anesthetic agent in Korea and China [10]. Previous studies on the constituents of A. sieboldii have revealed isolation of various types of essential oils, amide, alkaloids, lignans, and flavonoids [9,11,12]. Pellitorine (PLT) was isolated from the methylene chloride soluble fraction of the roots of A. sieboldii using a combination of silica gel column chromatography and recrystallization. A conjugated alkaldienamide, pellitorine was isolated mainly from the Piper species [13]. Pellitorine showed various biological properties including insecticidal [14], lavicidal [14], antibacterial [15], and anti-cancer [16] activities. However, no studies on the anticoagulant activities of PLT have been reported. Therefore, in the current study, we examined the anticoagulant activities of PLT in the production of FXa and thrombin, and their effects on PT and aPTT and on fibrinolytic activity. 2. Materials and methods 2.1. Reagents TNF-α was purchased from Abnova (Taiwan). Anti-tissue factor antibody was purchased from Santa Cruz Biologics (Santa Cruz, CA). c-Jun N-terminal kinase (JNK) inhibitor (SP600125), Nuclear factor (NF)-κB inhibitor (Emodin), and extracellular signal regulated kinase (ERK) inhibitor (PD98059) were purchased from R&D Systems (Minneapolis, MN). Factors V, Vll, Vlla, FX, and FXa, antithrombin III (AT III), prothrombin, and thrombin were obtained from Haematologic Technologies (Essex Junction, VT, USA). aPTT assay reagent and PT reagents were purchased from Fisher Diagnostics (Middletown, Virginia, USA), and the chromogenic substrates, S-2222 and S-2238, were purchased from Chromogenix AB (Sweden). The PAI-1 and t-PA ELISA kits were purchased from American Diagnostica Inc. (Stamford, CT, USA). Other reagents were of the highest commercially available grades. Oleanolic acid (OA) was prepared as described previously [17].
(251.0 g) extractions, respectively. The MC extraction was fractioned by silica gel column chromatography eluting with EtOAc in n-hexane (0–100%, step-wise), to yield twenty fractions (MC1–MC20). MC15 (8.2 g) was chromatographed on silica gel column using a solvent system of EtOAc in n-hexane (15%) to give seven fractions (MC15-1–MC15-7). MC15-4 (16.6 g) was purified by recrystallization from chloroform to afford a needle-type solid, compound 1 [675.0 mg, 0.12% (w/w) of MeOH extract]. The structure of compound 1 (Fig. 1) was identified by a combination of spectroscopic methods and comparisons with the literature data [18]. 2.3. Pellitorine (1) Needles, mp 69 °C; 1H NMR (250 MHz, CDCl3): δ 0.91 (3H, s, H-10), 0.93 (3H, s, H-3′), 0.96 (3H, s, H-4′), 1.32 (4H, m, H-8, H-9), 1.44 (2H, m, H-7), 1.82 (1H, m, H-2′), 2.18 (2H, dd, J = 12.5, 6.2 Hz, H-6), 3.18 (2H, t, J = 12.9, 6.6 Hz, H-1′), 5.18 (1H, d, J = 15.0 Hz, H-2), 5.58 (NH, br s), 6.07 (1H, m, H-5), 6.17 (1H, m, H-4), 7.20 (1H, m, H-3); 13C NMR (63 MHz, CDCl3): δ 14.4 (C-10), 20.5 (C-3′, C-4′), 22.9 (C-9), 28.9 (C-7), 29.0 (C-2′), 31.8 (C-8), 33.3 (C-6), 47.3 (C-1′), 122.0 (C-2), 128.6 (C-4), 141.8 (C-3), 143.8 (C-5), 166.9 (C-1). 2.4. Isolation of plasma Blood samples were taken in the morning from 10 healthy volunteers in fasting status (aged between 24 and 28 years, 4 males and 6 females) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. All subjects gave written informed consent before participation. Healthy subjects did not use addictive substances and antioxidant supplementation, and their diet was balanced (meat and vegetables). Human blood was collected into sodium citrate (0.32% final concentration) and immediately centrifuged (2000 ×g 15 min) to obtain plasma. 2.5. Anticoagulation assay
2.2. Plant material, extraction, and purification Melting points were obtained with an Electrothermal 9100 melting point apparatus (Electrothermal Ltd.). 1H and 13C NMR spectra were obtained on a Bruker ARX spectrometer (250 MHz) and chemical shifts given in δ (ppm) from tetramethylsilane (TMS) as an internal standard. Column chromatography was carried out on silica gel (70–230 mesh, Merck). Thin layer chromatography (TLC) analysis was performed on Kieselgel 60 F254 (Merck 1.05715) aluminum plate; spots were visualized by spraying with 10% aqueous H2SO4 followed by heating. All other chemicals and solvents were of analytical grade and used without further purification. The roots of A. sieboldii were purchased from herbal market at Daegu, Korea, in February 2006. The plant material was identified by Dr. Seung Ho Lee at the College of Pharmacy, Yeungnam University. A voucher specimen (SH0602) was deposited at the herbarium, College of Pharmacy, Yeungnam University. The dried roots of A. sieboldii (6.0 kg) were extracted with MeOH at room temperature for 5 days. After being concentrated, the MeOH extract (564.0 g) was suspended in water and then partitioned successively with methylene chloride (MC) and ethyl acetate (EtOAc) to give MC (298.0 g), EtOAc (15.0 g), and water
aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Germany), according to the manufacturer's instructions as described previously [19]. In brief, citrated normal human plasma (90 μl) was mixed with 10 μl of PLT or OA and incubated for 1 min at 37 °C. aPTT assay reagent (100 μl) was added and incubated for 1 min at 37 °C, and then 20 mM CaCl2 (100 μl) was added. Clotting times were recorded. For PT assays, citrated normal human plasma (90 μl) was mixed with 10 μl of PLT or OA stock and incubated for 1 min at 37 °C. PT assay reagent (200 μl), which has been preincubated for 10 min at 37 °C, was then added and clotting time was recorded. PT results are expressed in seconds and as International Normalized Ratios (INR), and aPTT results are expressed in seconds. INR = (PT sample / PT control)ISI. ISI = international sensitivity index.
Fig. 1. The chemical structure of PLT.
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2.6. Platelet aggregation assay Mouse platelets from platelet-rich plasma (PRP) were washed once with HEPES buffer (5 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH2PO4, 2 mM MgCl2, 5.6 mM glucose, 0.1% BSA (w/v), pH to 7.45). The platelet aggregation study was carried out according to a method previously reported [20]. Washed platelets were incubated with indicated PLT or OA for 3 min, and then stimulated by thrombin (3 U/ml, Sigma) in 0.9% saline solution at 37 °C for 5 min. Platelet aggregation was recorded using an aggregometer (CHRONO-LOG, Havertown, PA, USA). 2.7. Thrombin-catalyzed fibrin polymerization Thrombin-catalyzed polymerization was determined every 6 s for 20 min by monitoring turbidity at 360 nm using a spectrophotometer (TECAN, Switzerland) at ambient temperature. Control plasma and plasma incubated with PLT or OA were trebly diluted TBS (50 mM Tris-buffered physiological saline solution pH 7.4) and clotted with thrombin (final concentration — 0.5 U/ml). The maximum polymerization rate (Vmax, ΔmOD/min) of each absorbance curve was recorded [21]. All experiments were performed three times. 2.8. Cell culture Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA) and were maintained using a previously described method [22,23]. Briefly, the cells were cultured until confluent at 37 °C at 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science).
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rates of color development were converted into FXa concentrations using a standard curve prepared with known dilutions of purified human FXa. 2.11. Thrombin production on the surfaces of HUVECs Measurement of thrombin production by HUVECs was quantitated as previously described [19,23]. Briefly, HUVECs were pre-incubated in 300 μl containing PLT or OA in 50 mM Tris–HCl buffer, 100 pM FVa, and 1 nM FXa for 10 min, followed by addition of prothrombin to a final concentration of 1 μM. After 10 min, duplicate samples (10 μl each) were transferred to a 96-well plate containing 40 μl of 0.5 M EDTA in Tris-buffered saline per well to terminate prothrombin activation. Activated prothrombin was determined by measuring the rate of hydrolysis of S2238 at 405 nm. Standard curves were prepared using the amounts of purified thrombin. 2.12. Thrombin or factor Xa (FXa) activity assay PLT or OA in 50 mM Tris–HCl buffer (pH 7.4) containing 7.5 mM EDTA and 150 mM NaCl was mixed in the presence of 150 μl of AT III (200 nM). The heparins with AT III (200 nM) were dissolved in physiological saline and placed in the sample wells. After incubation at 37 °C for 2 min, thrombin solution (150 μl; 10 U/ml) was added, followed by incubation at 37 °C for 1 min. S-2238 (a thrombin substrate; 150 μl; 1.5 mM) solution was then added and absorbance at 405 nm was monitored for 120 s using a spectrophotometer (TECAN, Switzerland). And FXa activity assay was performed in the same manner as the thrombin activity assay, but using factor Xa (1 U ml/1) and S-2222 as substrates.
2.9. 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, the cells were washed with fresh medium, followed by treatment with PLT. After a 48-h incubation period, the cells were washed, and 100 μl of 1 mg/ml MTT was added, followed by incubation for 4 h. Finally, 150-μl DMSO was added to solubilize the formazan salt formed, the amount of which was determined by measuring the absorbance at 540 nm using a microplate reader (Tecan Austria GmbH, Austria). 2.10. Factor Xa production on the surfaces of HUVECs TNF-α (10 ng/ml for 6 h in serum-free medium) stimulated confluent monolayers of HUVECs (preincubated with the indicated concentrations of PLT or OA for 10 min) in a 96-well culture plate were incubated with FVIIa (10 nM) in buffer B (buffer A supplemented with 5 mg/ml bovine serum albumin [BSA] and 5 mM CaCl2) for 5 min at 37 °C in the presence or absence of anti-TF IgG (25 μg/ml). FX (175 nM) was then added to the cells (final reaction mixture volume, 100 μl) and incubated for 15 min. The reaction was stopped by adding buffer A (10 mM HEPES, pH 7.45, 150 mM NaCl, 4 mM KCl, and 11 mM glucose) containing 10 mM EDTA and the amounts of FXa generated were measured by using a chromogenic substrate. Changes in absorbance at 405 nm over 2 min were monitored using a microplate reader. Initial
Tail bleeding times were measured using the method described by Dejana et al. [19,24]. Briefly, ICR mice were fasted overnight before experiments. One hour after intravenous administration of PLT or OA, tails of mice were transected at 2 mm from their tips. Bleeding time was defined as the time elapsed until bleeding stopped. When the bleeding time exceeded 15 min, bleeding time was recorded as 15 min for the analysis. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University. 2.14. ELISA for PAI-1 and t-PA The concentrations of PAI-1 and t-PA in HUVEC cultured supernatants were determined using ELISA kits (American Diagnostica Inc. CT, USA). 2.15. Statistical analysis Data are expressed as mean ± SEM (standard error of the mean) of at least three independent experiments. Statistical significance between two groups was determined using the Student's t-test. Statistical significance was accepted for p b 0.05.
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recently we published that OA has anticoagulant activities in vitro and in vivo [17].
Table 1 Anticoagulant activity of PLTa. In vitro coagulant assay Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control PLT
Saline 1 μM 2 μM 3 μM 5 μM 10 μM 20 μM 40 μM
30.6 ± 0.6 31.2 ± 0.4 30.8 ± 0.6 31.4 ± 0.8 32.4 ± 1.2 40.6 ± 0.4⁎⁎ 48.2 ± 0.6⁎⁎ 63.4 ± 1.0⁎⁎ 0.5 μg/ml 114.8 ± 1.2⁎⁎
14.0 ± 0.2 14.2 ± 0.4 14.6 ± 0.6 15.0 ± 0.4 15.2 ± 0.2 23.2 ± 0.2 27.4 ± 0.4 28.2 ± 0.6 10 μg/ml 34.6 ± 0.8⁎⁎
1.00 1.03 1.10 1.16 1.20 3.04⁎⁎ 4.38⁎⁎ 4.67⁎⁎ 7.32⁎⁎
OA Heparin
In vivo bleeding time Sample
Dose
Tail bleeding time (s)
n
Control PLT
Saline
41.6 57.6 71.8 74.6 121.5
10 10 10 10 10
OA Heparin
4.5 μg/mouse 9.0 μg/mouse 36.5 μg/mouse 1 mg/mouse
± ± ± ± ±
1.2 0.8⁎⁎ 1.2⁎⁎ 1.4⁎⁎ 1.2⁎⁎
a Each value represents the means ± SEM (n = 10). ⁎⁎ p b 0.01 as compared to control.
3.1. Effects of PLT on aPTT and PT Incubation with PLT resulted in changes in the coagulation properties of human plasma. The anticoagulant properties of PLT in human plasma were tested using aPTT and PT assays; a summary of the results is shown in Table 1. Although the anticoagulant activities of PLT were weaker than those of heparin, aPTT and PT were significantly prolonged by treatment with PLT at concentrations greater than 10 μM. The result showing prolongation of aPTT suggests inhibition of the intrinsic and/or the common pathway, whereas prolongation of PT indicates that PLT could also inhibit the extrinsic coagulation pathway. To confirm these data in vivo, PLT was administered into mouse via intravenous injection. As shown in Table 1, tail bleeding times were significantly prolonged by treatment with PLT. Assuming that the average weight of a mouse was 20 g, and the average blood volume was 2 ml, the amount of PLT injected (4.5 or 9.0 μg per mouse) or OA injected (36.5 μg per mouse) was equivalent to PLT 10, 20 μM or OA 40 μM in peripheral blood.
3. Results and discussion
3.2. Effects of PLT on thrombin-catalyzed platelet aggregation and fibrin polymerization and cellular viability
In this study, we examined the anticoagulant effects of pellitorine (PLT, Fig. 1) for the first time and sought to identify the mechanisms responsible for these effects. Oleanolic acid (OA) was used as a positive control because
The effects of PLT 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. 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.
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Fig. 3. Effects of PLT on inactivation of thrombin and factor Xa. (A) Inhibition of thrombin (Th) by PLT (○) or OA (40 μM, ■) was measured using a chromogenic assay, as described in the “Materials and methods” section. (B) Inhibition of factor Xa (FXa) by PLT (○) or OA (40 μM, ■) was monitored using a chromogenic assay, as described in the “Materials and methods” section. Heparin (●) was used as a positive control. **p b 0.01 vs. 0.
that incubation of human plasma with PLT resulted in a significant decrease in the maximal rate of fibrin 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. To confirm the anticoagulant activities of PLT, thrombincatalyzed platelet aggregation assay was conducted. As shown in Fig. 2B, PLT significantly inhibited mouse platelet aggregation induced by thrombin (final concentration: 3 U/ ml) in a concentration dependent manner. To exclude the possibility that the decrease of the polymerization could be due to direct effect on thrombin leading to decrease in fibrin production rather than polymerization of fibrin formed, reptilase-catalyzed polymerization assay was introduced. Results showed that PLT significantly decreased reptilasecatalyzed polymerization (data not shown). To determine the cellular viability of PLT, cellular viability assay (MTT assay) was performed in HUVECs treated with PLT for 24 h. At concentrations up to 30 μM, PLT did not affect cell viability (Fig. 2C).
3.3. Effects of PLT on the activities of thrombin and FXa To elucidate the mechanism responsible for inhibition of coagulation by PLT, the inhibitory effects of PLT on the activities of thrombin and FXa were measured using chromogenic substrates. In the results presented in Fig. 3A, treatment with PLT resulted in dose-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by the anticoagulant. In addition, we also investigated the effects of PLT on FXa activity. PLT inhibited the effects on FXa activities (Fig. 3B). These results are consistent with the results of our antithrombin assay, and therefore suggest that the antithrombotic mechanisms of PLT appear to be due to inhibition of fibrin polymerization and/or the intrinsic/extrinsic pathway. 3.4. Effects of PLT on production of thrombin and FXa Previously, Sugo et al. reported that endothelial cells are able to support prothrombin activation by FXa [25]. In the current study, pre-incubation of HUVECs with FVa and FXa in
Fig. 4. Inhibition of thrombin and FXa production by PLT in HUVECs. (A) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10 min with the indicated concentrations of PLT or OA (40 μM). Prothrombin was added to a final concentration of 1 μM and prothrombin activation was determined 30 min later, as described in the “Materials and methods” section. (B) HUVECs were pre-incubated with indicated concentrations of PLT or OA (40 μM) for 10 min. TNF-α- (10 ng/ml for 6 h) stimulated HUVECs were incubated with FVIIa (10 nM) and FX (175 nM) in the absence or presence of anti-TF IgG (25 μg/ml) and FXa production was determined as described in the “Materials and methods” section. *p b 0.05 or **p b 0.01 vs. 0 (A) or TNF-α alone (B).
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Fig. 5. Effects of PLT on secretion of PAI-1 by HUVECs stimulated with TNF-α. HUVECs were cultured with PLT or OA (40 μM) in the absence or presence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentrations in culture media were determined as described in the “Materials and methods” section. *p b 0.05 or **p b 0.01 vs. TNF-α alone; n.s., not significant.
the presence of CaCl2 prior to addition of prothrombin resulted in production of thrombin (Fig. 4A). In addition, treatment with PLT resulted in dose-dependent inhibition of production of thrombin from prothrombin (Fig. 4A). According to findings reported by Rao et al., the endothelium provides the functional equivalent of procoagulant phospholipids and supports activation of FX [26], and, in TNF-α stimulated HUVECs, activation of FX by FVIIa occurred in a TF expression-dependent manner [27]. Thus, we investigated the effects of PLT on activation of FX by FVIIa. HUVECs were stimulated with TNF-α for induction of TF expression, and, as shown in Fig. 4B, the rate of FX activation by FVIIa was 18-fold higher in stimulated HUVECs (95.2 ± 8.2 nM) than in non-stimulated HUVECs (5.3 ± 1.2 nM), and this increase in activation was abrogated by anti-TF IgG (13.8 ± 2.8 nM). In addition, pre-incubation with PLT resulted in dosedependent inhibition of FX activation by FVIIa (Fig. 4B). Therefore, these results suggest that PLT can inhibit production of thrombin and FXa. 3.5. Effects of PLT on secretion of PAI-1 or t-PA protein It is well known that TNF-α appears to inhibit the fibrinolytic system in HUVECs by inducing production of PAI-1 and altering the balance between t-PA and PAI-1 is known to modulate coagulation and fibrinolysis [28,29]. To determine the direct
effects of PLT on TNF-α-stimulated secretion of PAI-1, HUVECs were cultured in media with or without PLT in the absence or presence of TNF-α for 18 h. As shown in Fig. 5, treatment with PLT resulted in dose-dependent inhibition of TNF-α-induced secretion of PAI-1 from HUVECs, and these decreases became significant at a PLT dose of 20 μM. TNF-α does not have a significant effect on t-PA production [30] and the balance between plasminogen activators and their inhibitors reflects net plasminogen-activating capacity [4,5,7]; therefore, we investigated the effect of TNF-α with PLT on secretion of t-PA from HUVECs. The results obtained were consistent with those of a prior study reporting a modest decrease in production of t-PA by TNF-α in HUVECs [31]. This decrease was not significantly altered by treatment with PLT (Fig. 6A). Therefore, collectively, these results indicate that the PAI-1/t-PA ratio was increased by TNF-α and that PLT prevented this increase (Fig. 6B). 3.6. Effects of signal pathway inhibitors on basal and TNF-α induced PAI-1 secretion in the presence or absence of PLT It is well known that both NF-κB and ERK dependent pathways are involved in TNF-α induced PAI-1 production [30]. To define the targets of PLT in the signal transduction pathways leading to TNF-α induced PAI-1 expression, we investigated the effects of three signal transduction inhibitors, Emodin (NF-κB inhibitor), PD98059 (ERK inhibitor), and SP600125 (JNK inhibitor) on TNF-α induced PAI-1 expression in the presence or absence of PLT. We first demonstrated the effect of increasing doses of each inhibitor on HUVECs viability, and selected appropriate non-cytotoxic doses. To confirm the specificity of the inhibitors, we checked the cell viability for all agents by MTT assay. We found that the inhibitors combined with TNF-α and/or PLT as used in this study has no effect on HUVECs viability (data not shown). Experiments performed showed that PD98059 and Emodin inhibited TNF-α induced PAI-1 accumulation, which is consistent with the previous study (Fig. 7A) [30]. In addition, we observed that SP600125 did inhibit TNF-α induced PAI-1 secretion (Fig. 7A). Furthermore, neither PD98059 nor Emodin showed any additional inhibitory effects in the presence of PLT (Fig. 7B). However, the inhibitory effects of
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.
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Fig. 7. Effect of signal transduction inhibitors on TNF-α induced PAI-1 secretion. (A) HUVECs were cultured with SP600125 (2 μM), Emodin (2 μg/ml), and PD98059 (10 μM) in the absence or presence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentration in the culture mediums was examined as described in the “Materials and methods” section. (B) the same as (A) except that cells were preincubated with PLT 20 μM. *p b 0.05 as compared to TNF-α alone (A) or TNF-α + PLT (B).
SP600125 were essentially additive with those of PLT (Fig. 7B). These results suggest that the ERK and NF-κB pathways are involved in PLT-mediated inhibition of TNF-α induced PAI-1 expression in HUVECs. No significant effects of the three inhibitors on basal levels of PAI-1 production could be explained by the fact that the activities of ERK, JNK, and NF-κB are relatively low in the unstimulated cells [32,33]. Thus, these results seem to indicate that PLT decreases PAI-1 levels via inhibition of the ERK and NF-κB pathways. However, additional work will be required to elucidate whether PLT has beneficial effects on fibrinolytic systems in vivo. The pre-clinical evaluation of the antithrombotic potential of novel molecules requires the use of reliable and reproducible experimental models. PT, aPTT, fibrin polymerization, and platelet aggregation are the most established and commonly used preparations to determine the efficacy of novel antithrombotic drugs [34,35]. In our experiment, PT and aPTT using human plasma were used to evaluate the antithrombotic effect of PLT. Fibrin polymerization and platelet aggregation are also used to determine the anti-platelet functions of PLT. Data showed that PLT prolonged PT and aPTT values and PLT resulted in a significant decrease in the maximal rate of fibrin polymerization and inhibited platelet aggregation without having cytotoxicity on human endothelial cells. There is ample evidence that inflammation and coagulation are intricately related processes that may considerably affect each other [36,37]. This cross-talk occurs at the levels of platelet activation, fibrin formation, and resolution as well as physiological anticoagulant pathways [36,37]. On the basis of our current experimental studies, it can be hypothesized that inhibitory modulation of coagulation by PLT could give promising anti-inflammatory mediators. Well-designed prospective studies are needed to prove this hypothesis. In conclusion, results of this study demonstrate that PLT inhibited the extrinsic and intrinsic blood coagulation pathways through inhibition of FXa and thrombin production in HUVECs, and that PLT inhibits TNF-α-induced secretion of PAI-1. These results add to previous work on the topic, and should be of interest to those designing pharmacological strategies for the treatment or prevention of vascular diseases.
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