Antithrombotic activities of wogonin and wogonoside via inhibiting platelet aggregation

Fitoterapia 98 (2014) 27–35

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Antithrombotic activities of wogonin and wogonoside via inhibiting platelet aggregation Sae-Kwang Ku a, Jong-Sup Bae b,⁎ a b

Department of Anatomy and Histology, College of Korean 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

a r t i c l e

i n f o

Article history: Received 8 June 2014 Accepted in revised form 5 July 2014 Available online 11 July 2014 Keywords: Wogonin Wogonoside Coagulation cascade Fibrinolysis Endothelium

a b s t r a c t Wogonin (WGN), a flavonoid extracted from Scutellaria baicalensis Georgi, has several biological effects including antioxidant, anti-inflammatory, antiviral, neuroprotective, anxiolytic, and anticancer activities, and the flavonoid wogonoside (WGNS) can be derived from S. baicalensis, as it is a metabolite of wogonin. Here, the anticoagulant activities of WGN(S) were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin (factor IIa, FIIa) and activated factor X (FXa), and the effects of WGN(S) on expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated in tumor necrosis factor (TNF)-α activated human umbilical vein endothelial cells (HUVECs). Treatment with WGN(S) resulted in prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, as well as inhibited production of thrombin and FXa in HUVECs. In addition, WGN(S) inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. WGN(S) also elicited anticoagulant effects in mice. In addition, treatment with WGN(S) resulted in significant reduction of the PAI-1 to t-PA ratio. Collectively, WGN(S) possesses antithrombotic activities and offers a basis for development of a novel anticoagulant. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Anticoagulation is a very common form of medical intervention which is increasingly used for primary or secondary prevention of thromboembolic complications of vascular disease [1]. Oral anticoagulation with vitamin K antagonists (VKAs), mainly warfarin, is almost the unique tool for chronic anticoagulant treatment in clinical practice, although newer anticoagulants have already been approved or evaluated in clinical studies [2]. Anticoagulation with VKAs has been shown to be effective in the prevention and treatment of thrombotic complications in various clinical settings, including atrial fibrillation, venous

⁎ 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).

http://dx.doi.org/10.1016/j.fitote.2014.07.006 0367-326X/© 2014 Elsevier B.V. All rights reserved.

thromboembolism (VTE), acute coronary syndromes and after invasive cardiac procedures [2]. Adequate treatment with VKAs is rather demanding, for both the patients and the health care systems, because periodic laboratory monitoring is necessary to maintain the effect of the drug in a therapeutic range and to minimize adverse events [3]. Bleeding is the most important complication of VKAs and a major concern for both physicians and patients [2]. The coagulation system can be divided into the extrinsic and intrinsic pathway [4]. Activation of the extrinsic pathway is generally considered to initiate both hemostasis and thrombosis. Hemostasis is initiated when blood is exposed to tissue factor located in the adventitia of blood vessels, and thrombosis is initiated when blood is exposed to tissue factor in the necrotic core of the ruptured atherosclerotic plaques, in the subendothelium of injured vessels and on the surface of activated leucocytes attracted to the damaged vessel [5]. The final common mediator of both the intrinsic and extrinsic

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coagulation pathways is thrombin. Thrombin triggers platelet activation, as well as the production of factor V, VIII, and IX, and mediates the proteolytic cleavage of fibrinogen to fibrin and then it binds to fibrin where it remains active [6]. Given the central role of thrombin in the development of a thrombus, many strategies for preventing and treating thromboembolic events have focused on inhibiting thrombin generation or blocking its activity [5,6]. Scutellaria baicalensis Georgi is one of the most popular and multipurpose traditional Chinese medicinal herbs, and it has a high flavonoid content [7]. Wogonin (WGN), a flavonoid extracted from S. baicalensis, has several biological effects including antioxidant, anti-inflammatory, antiviral, neuroprotective, anxiolytic, and anticancer activities [8]. It has been shown to possess antitumor effects in various cancer cells [9], including antiproliferation, cell cycle arrest, induction of apoptosis and differentiation, inhibition of angiogenesis, anti-invasion, and increased sensitivity to apoptosis. Moreover, wogonin has shown therapeutic potential for the treatment of hematologic malignancies [10]. Flavonoid aglycones undergo rapid and extensive metabolism after either oral or intravenous administration. Flavonoid aglycones enter the bloodstream in the form of glucuronide or sulfate conjugates, and very little unchanged aglycone can be found in the plasma, although levels of glucuronic acid conjugates are high. Glucuronidation is thought to affect the biological activity of aglycones by altering the physicochemical properties of flavonoids, which is seen as a detoxification process. The biological activities of these conjugates are thus important [11,12]. The flavonoid wogonoside (WGNS, wogonin 7-O-glucuronide) can also be derived from S. baicalensis, as it is a metabolite of wogonin [13]. It is known to possess antiinflammatory and anti-inflammation-induced angiogenic activities [14,15]; however, its antileukemic properties have not been explored. Inflammation and coagulation play pivotal roles in the pathogenesis of vascular disease [16]. Increasing evidence points to extensive cross-talk between these two systems, whereby inflammation leads not only to activation of coagulation, but coagulation also has a considerable effect on inflammatory activity [16]. Activation of coagulation and fibrin deposition as a consequence of inflammation is known to occur and can be viewed as an essential part of the body’s host defense against infectious agents [16]. Together with the previous report, which demonstrated the potential anti-inflammatory effects of wogonin and wogonoside [8,14,15], we hypothesized that WGN(S) may have anti-coagulant activities. Furthermore, no studies on the anticoagulant activities of WGN(S) have been reported. Therefore, in the current study, we examined the anticoagulant activities of WGN(S) in production of FXa and thrombin, and their effects on PT and aPTT, antiplatelet and fibrinolytic activity. 2. Materials and methods 2.1. Reagents Wogonin (WGN) and wogonoside (wogonin 7-Oglucuronide, WGNS) was purchased from Sigma (St. Louis, MO, USA). TNF-α was purchased from Abnova (Taiwan). Anti-tissue factor antibody was purchased from Santa Cruz Biologics (Santa Cruz, CA). Factor V, Vll, Vlla, FX, FXa,

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). Rivaroxaban (direct FXa inhibitor) and argatroban (direct Flla inhibitor) were purchased from Santa Cruz Inc. (Dallas, Texas, USA). PAI-1 and t-PA ELISA kits were purchased from American Diagnostica Inc. (Stamford, CT, USA). Other reagents were of the highest commercially available grade. 2.2. Isolation of human plasma Human 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 before participation. Healthy subjects did not use addictive substances or 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. 2.3. Anticoagulation assay aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Germany), according to the manufacturer's instructions, as described previously [17]. In brief, citrated normal human plasma (90 μl) was mixed with 10 μl of WGN(S) and incubated for 1 min at 37 °C. aPTT assay reagent (100 μl) was added and incubated for 1 min at 37 °C, followed by addition of 20 mM CaCl2 (100 μl). Clotting times were recorded. For PT assays, citrated normal human plasma (90 μl) was mixed with 10 μl of WGN(S) stock and incubated for 1 min at 37 °C. PT assay reagent (200 μl), which had been pre-incubated 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). 2.4. Platelet aggregation assay Platelet-rich plasma (PRP) was obtained from syngeneic donor mice by double centrifugation (200 g to create platelet/ plasma phase, 500 g to pellet platelets). The platelet-rich plasma was adjusted to a concentration of 1 × 109 platelets/ml with use of a hemocytometer for cell counts. 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) in the presence of 1 mM CaCl2. The platelet aggregation study was carried out according to a method previously reported [18]. Washed platelets were incubated with indicated WGN(S) in TBS for 3 min, and then stimulated by thrombin (3 U/ml, Sigma) in 0.9% saline solution at 37 °C for 5 min or collagen (1 μg/ml) at 37 °C for 15 min. Platelet aggregation was recorded using an aggregometer (Chronolog, Havertown, PA, USA).

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2.5. 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 WGN(S) 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, ΔmOD/min) of each absorbance curve was recorded [19]. All experiments were performed three times. 2.6. Cell culture Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA) and were maintained using a previously described method [20–22]. Briefly, cells were cultured until confluent at 37 °C at 5% CO2 in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science). 2.7. Cell viability assay MTT was used as an indicator of cell viability. Cells were grown in 96-well plates at a density of 5 × 103 /well. After 24 h, cells were washed with fresh medium, followed by treatment with WGN(S). After a 48-h incubation period, 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 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). 2.8. 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 WGN(S) 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 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 into FXa concentrations using a standard curve prepared with known dilutions of purified human FXa. 2.9. Thrombin production on the surfaces of HUVECs Measurement of thrombin production by HUVECs was quantitated as previously described [17,21]. Briefly, HUVECs were pre-incubated in 300 μl containing WGN(S) 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 in order to terminate prothrombin activation.

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Activated prothrombin was determined by measuring the rate of hydrolysis of S2238 at 405 nm. Standard curves were prepared using amounts of purified thrombin. 2.10. Thrombin activity assay WGN(S) 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 μl of AT III (200 nM). Heparins with AT III (200 nM) were dissolved in physiological saline and placed in the sample wells. Following 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). 2.11. 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) and S-2222 as substrates. 2.12. In vivo bleeding time Tail bleeding times were measured using the method described by Dejana et al. [17,23]. Briefly, male ICR mice were fasted overnight before experiments. One hour after intravenous administration of WGN(S), 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 (IRB number; KNU 2012-13). 2.13. Ex vivo clotting time Male ICR mice were fasted overnight and WGN(S) in 0.5% DMSO was administered by intravenous injection. One hour after administration, arterial blood samples (0.1 ml) were withdrawn into 3.8% Na–citrate (1/10; v/v) for ex vivo aPTT and PT determination. Clotting times were performed as described in Section 2.2. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by the 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 Results are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments with duplicate determination. Statistical significance was defined to be based on a p value smaller than 0.05 (SPSS, version 14.0, SPSS Science, Chicago, IL, USA).

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3. Results

with WGN(S) 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 (pH7.4). 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 WGN(S), thrombin-catalyzed platelet aggregation assay was performed. As shown in Fig. 1B, treatment with WGN(S) resulted in significantly inhibited mouse platelet aggregation induced by thrombin (final concentration: 3 U/ml) in a concentration dependent manner. 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 polymerization of fibrin formed, reptilase-catalyzed polymerization assay was performed. Results showed that WGN(S) induced a significant decrease in reptilase-catalyzed polymerization (data not shown). To determine the cellular viability of WGN(S), cellular viability assay (MTT assay) was performed in HUVECs treated with WGN(S) for 24 h. WGN(S) did not affect cell viability at concentrations up to 50 μM (Fig. 1C).

3.1. Effects of WGN(S) on clotting time and bleeding times Incubation of human plasma with WGN(S) changed coagulation properties. The anticoagulant activities of WGN and WGNS were tested using aPTT and PT assays and human plasma (Tables 1 and 2). Although the anticoagulant activities of WGN or WGNS were weaker than those of heparin or warfarin, aPTT and PT were significantly prolonged by WGN or WGNS at concentrations 10 μM WGN or 5 μM WGNS and above. A prolongation of aPTT suggests inhibition of the intrinsic and/or the common pathway, and PT prolongation suggests inhibition of the extrinsic and/or the common pathway. The concentrations of WGN at 55.3 μM and 52.9 μM were required to double clotting time for aPTT and PT respectively, and the concentrations of WGNS at 21.1 μM and 25.9 μM were required to double clotting time for aPTT and PT respectively. Therefore, results obtained in this study indicate that WGN(S) could also inhibit the common coagulation pathway. To confirm these in vitro results, in vivo tail bleeding times were determined. Because an average body weight is 20 g and an average blood volume is 2 ml, WGN administered (5.7 or 11.4 μg/mouse) or WGN administered (9.2 or 18.4 μg/mouse) produced a concentration of approximately 20 or 50 μM in peripheral blood respectively. As shown in Tables 1 and 2, tail bleeding times were significantly prolonged by WGN(S) in comparison to the controls. The anticoagulation in response to WGN(S) was also observed ex vivo in mice with dose-dependent prolongation of the aPTT and PT (Tables 3 and 4).

3.3. Effects of WGN(S) on the activities of thrombin and FXa In order to elucidate the mechanism responsible for inhibition of coagulation by WGN(S), the inhibitory effects of WGN(S) on the activities of thrombin and FXa were measured using chromogenic substrates. Results are shown in Fig. 2A; treatment with WGN(S) resulted in dose-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by the anticoagulant. Direct thrombin inhibitor, argartoban, was used as a positive control. In addition, we also investigated the effects of WGN(S) on activity of FXa. WGN(S) inhibited the activity of FXa (Fig. 2B). Direct FXa inhibitor, rivaroxaban, was used as a positive control. These results are consistent with those of our antithrombin assay, and therefore suggest that the antithrombotic mechanisms of WGN(S) are due to inhibition of fibrin polymerization and/or the intrinsic/extrinsic pathway.

3.2. Effects of WGN(S) on thrombin-catalyzed platelet aggregation and fibrin polymerization The effects of WGN(S) on thrombin-catalyzed fibrin polymerization in human plasma were monitored as changes in absorbance at 360 nm, as described in Section 2. The results, shown in Fig. 1A, demonstrate that incubation of human plasma Table 1 Anticoagulant activity of wogonin.a In vitro coagulant assay Sample

Dose

aPTT (s)

PT (s)

Control Wogonin

Saline 0.1 μM 0.5 μM 1 μM 5 μM 10 μM 20 μM 20 μM 20 μM

25.8 25.2 25.6 25.8 25.4 29.6 35.2 58.4 53.8

12.0 12.2 12.6 12.4 12.2 14.2 16.8 26.6 22.2

Heparin Warfarin

± ± ± ± ± ± ± ± ±

0.6 0.4 0.6 0.8 0.4 0.8⁎ 0.4⁎ 1.2⁎ 1.4⁎

± ± ± ± ± ± ± ± ±

PT (INR) 0.4 0.6 0.2 0.8 0.6 0.8⁎ 0.4⁎ 0.8⁎ 1.4⁎

1.00 1.04 1.11 1.07 1.04 1.45⁎ 2.10⁎ 5.76⁎ 3.87⁎

In vivo bleeding time Sample

Dose

Tail bleeding time (s)

n

Control Wogonin

Saline 5.7 μg/mouse 11.4 μg/mouse 144.0 μg/mouse 12.3 μg/mouse

33.6 35.4 45.2 60.6 55.8

10 10 10 10 10

Heparin Warfarin

a Each value represents the means ± SEM (n = 5). ⁎ p b 0.05 as compared to control.

± ± ± ± ±

1.2 1.0⁎ 1.4⁎ 1.2⁎ 1.4⁎

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Table 2 Anticoagulant activity of wogonoside.a In vitro coagulant assay Sample

Dose

aPTT (s)

PT (s)

Control Wogonoside

Saline 0.1 μM 0.5 μM 1 μM 5 μM 10 μM 20 μM 20 μM 20 μM

25.8 25.6 26.2 26.0 29.8 39.4 49.8 58.4 53.8

12.0 12.4 12.8 12.2 14.6 18.4 20.6 26.6 22.2

Heparin Warfarin

± ± ± ± ± ± ± ± ±

0.6 0.2 0.8 0.4 0.6⁎ 0.6⁎ 0.8⁎ 1.2⁎ 1.4⁎

± ± ± ± ± ± ± ± ±

PT (INR) 0.4 0.2 0.6 0.6 0.4⁎ 0.2⁎ 0.6⁎ 0.8⁎ 1.4⁎

1.00 1.07 1.15 1.04 1.54⁎ 2.56⁎ 3.28⁎ 5.76⁎ 3.87⁎

In vivo bleeding time Sample

Dose

Tail bleeding time (s)

n

Control Wogonoside

Saline 9.2 μg/mouse 18.4 μg/mouse 144.0 μg/mouse 12.3 μg/mouse

33.6 45.2 52.8 60.6 55.8

10 10 10 10 10

Heparin Warfarin

± ± ± ± ±

1.2 1.6⁎ 1.0⁎ 1.2⁎ 1.4⁎

a Each value represents the means ± SEM (n = 5). ⁎ p b 0.05 as compared to control.

3.4. Effects of WGN(S) on production of thrombin and FXa and cellular viability

3.5. Effects of WGN(S) on secretion of PAI-1 or t-PA protein

In a previous study, Sugo et al. reported that endothelial cells are able to support prothrombin activation by FXa [24]. In the current study, pre-incubation of HUVECs with FVa and FXa in the presence of CaCl2 prior to addition of prothrombin resulted in production of thrombin (Fig. 2C). In addition, treatment with WGN(S) resulted in dose-dependent inhibition of production of thrombin from prothrombin (Fig. 2C). According to findings reported by Rao et al., the endothelium provides the functional equivalent of pro-coagulant phospholipids and supports activation of FX [25], and, in TNF-α stimulated HUVECs, activation of FX by FVIIa occurred in a TF expression-dependent manner [26]. Thus, we investigated the effects of WGN(S) on activation of FX by FVIIa. HUVECs were stimulated with TNF-α for induction of TF expression, and, as shown in Fig. 2D, the rate of FX activation by FVIIa was 17-fold higher in stimulated HUVECs (94.5 ± 9.2 nM) than in non-stimulated HUVECs (5.5 ± 1.7 nM), and this increase in activation was abrogated by anti-TF IgG (14.2 ± 2.1 nM). In addition, pre-incubation with WGN(S) resulted in dose-dependent inhibition of FX activation by FVIIa (Fig. 2D). Therefore, these results suggest that WGN(S) can inhibit production of thrombin and FXa.

TNF-α is known to inhibit the fibrinolytic system in HUVECs by inducing production of PAI-I, and altering the balance between t-PA and PAI-1 is known to lead to modulation of coagulation and fibrinolysis [27,28]. In order to determine the direct effects of WGN(S) on TNF-α-stimulated secretion of PAI-1, HUVECs were cultured in media with or without WGN(S) in the absence or presence of TNF-α for 18 h. As shown in Fig. 3A, treatment with WGN(S) resulted in dose-dependent inhibition of TNF-α-induced secretion of PAI-1 from HUVECs, and these decreases became significant at a WGN(S) dose of 10 μM. TNF-α does not have a significant effect on t-PA production [29] and the balance between plasminogen activators and their inhibitors reflects net plasminogen-activating capacity [5,6,30]; therefore, we investigated the effect of TNF-α with WGN(S) on secretion of t-PA from HUVECs. The results obtained were consistent with those of a previous 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 WGN(S) (Fig. 3B). Therefore, collectively, these results indicate that the PAI-1/t-PA ratio was increased by TNF-α and that WGN(S) prevented this increase (Fig. 3C).

Table 3 Ex vivo coagulation time of wogonin.a

Table 4 Ex vivo coagulation time of wogonoside.a

Sample

Dose

aPTT (s)

PT (s)

Control Wogonin

Saline 0.06 μg/mouse 0.3 μg/mouse 0.6 μg/mouse 2.8 μg/mouse 5.7 μg/mouse 11.4 μg/mouse

31.2 30.8 31.0 31.8 32.4 38.2 41.2

15.4 14.8 15.6 15.2 15.4 18.2 20.6

± ± ± ± ± ± ±

1.4 0.6 1.2 1.8 1.6 2.2⁎ 1.8⁎

a Each value represents the means ± SEM (n = 5). ⁎ p b 0.05 as compared to control.

± ± ± ± ± ± ±

0.6 1.0 1.2 0.8 1.2 1.0⁎ 1.8⁎

PT (INR)

Sample

Dose

aPTT (s)

PT (s)

1.00 0.92 1.03 1.10 1.11 1.44⁎ 1.90⁎

Control Wogonoside

Saline 0.1 μg/mouse 0.5 μg/mouse 0.9 μg/mouse 4.6 μg/mouse 9.2 μg/mouse 18.4 μg/mouse

31.2 32.0 32.2 31.4 37.8 46.8 58.4

15.4 15.2 15.8 16.4 19.2 22.4 28.4

± ± ± ± ± ± ±

1.4 0.8 1.0 1.4 1.0⁎ 1.4⁎ 1.6⁎

a Each value represents the means ± SEM (n = 5). ⁎ p b 0.05 as compared to control.

± ± ± ± ± ± ±

PT (INR) 0.6 1.2 1.4 1.8 1.0⁎ 1.2⁎ 1.6⁎

1.00 0.97 1.06 1.15 1.62⁎ 2.28⁎ 3.90⁎

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Fig. 1. Effects of WGN(S) on fibrin polymerization in human plasma. (A) Thrombin-catalyzed fibrin polymerization at the indicated concentrations of WGN(S) was monitored using a catalytic assay, as described in Section 2. The results are Vmax values expressed as percentages versus controls. (B) Effect of WGN(S) on mouse platelet aggregation induced by 3 U/ml thrombin. Data represent the mean ± SEM of three independent experiments performed in triplicate. (C) Effect of WGN(S) on cellular viability was measured by MTT assay. *p b 0.05 vs. Th alone.

Fig. 2. Effects of WGN(S) on inactivation and production of thrombin and factor Xa and cytotoxicity. (A) Inhibition of thrombin (Th) by WGN(S) was measured using a chromogenic assay, as described in Section 2. (B) Inhibition of factor Xa (FXa) by WGN(S) was monitored using a chromogenic assay, as described in Section 2. Argatroban (A) or rivatoxaban (B) was used as positive control. (C) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10 min with the indicated concentrations of WGN(S). Prothrombin was added to a final concentration of 1 μM and prothrombin activation was determined 30 min later, as described in Section 2. (D) HUVECs were pre-incubated with the indicated concentrations of WGN(S) 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 Section 2. *p b 0.05 vs. 0 (A, B, C) or TNF-α alone (D).

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4. Discussion The vascular endothelium provides a number of important functions in order to maintain adequate blood supply to vital organs [32]. These functions include prevention of coagulation, regulation of vascular tonus, orchestration of the migration of blood cells by the expression of adhesion molecules and regulation of vasopermeability [32]. Among these, regulation of hemostatic activity was regulated through a balance of pro- and anticoagulant properties [6]. Dysregulation of the thrombotic and fibrinolytic potential of endothelial cells is actively involved in the development and progression of inflammatory diseases such as sepsis and atherosclerosis [33,34]. A major complication and logical inconsistency arises with thrombin function when inflammatory responses promote coagulation, inhibit anticoagulant responses, and down regulate the fibrinolytic cascade [35]. WGN is one of the major bioactive compounds, isolated from a longstanding Chinese herbal named S. baicalensis. There is evidence indicating that WGN has antioxidant, anti-inflammatory, antiviral, neuroprotective, anxiolytic, and anticancer activities [8]. In this study, we presented WGN(S) as a potent anticoagulant by inhibiting thrombin or FXa and antiplatelet agents by inhibiting platelet aggregation. The anticoagulant activity of WGN(S) was evidenced by the prolongation of the clotting time in plasma-based PT and APTT assays. Additionally, the inhibitory effects by WGN(S) on FXa generation and further thrombin generation support the anticoagulant activities of WGN(S), and the anticoagulant

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activities of wogonoside were better than those of wogonin indicating that glycoside group on wogonoside plays important roles in anticoagulant activities and that wogonoside may be a promising agent for bleeding. It is well known that proinflammatory cytokines play a major role in the development of inflammatory diseases [36,37]. PAI-1, which plays a central role in regulating the fibrinolytic potential of endothelial cells, is produced by endothelial cells stimulated with various inflammatory cytokines, including TNF-α [38,39]. Here, we showed that WGN(S) treatment lowered PAI-1 expression in TNF-α stimulated HUVECs. Thus, these results collectively indicate that WGN(S) amplifies TNF-α induced alterations in the fibrinolytic system by shifting the ratio of PAI-1 to t-PA. Thrombin is the final enzyme in the blood clotting cascade responsible for clot formation and platelet activation [5,40]. Based on the results that WGN(S) could inhibit generation of FXa and thrombin, the anticoagulant activity of WGN(S) was initiated from the inhibition of the penultimate and final enzyme in the blood clotting cascade. The significant progress made in understanding the role of FXa and thrombin in various thrombotic disease states has clearly demonstrated potential therapeutic benefits of blocking these key enzymes in the blood coagulation cascade [41]. A potent and selective small molecule FXa or thrombin inhibitor has the potential to offer substantial therapeutic benefits [41]. WGN(S) exhibits the potency and selectivity required for such a candidate and is currently undergoing additional evaluations.

Fig. 3. Effects of WGN(S) on secretion of PAI-1 and tPA. (A) HUVECs were cultured with WGN(S) 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 Section 2. (B) HUVECs were cultured with WGN(S) 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 Section 2. (C) PAI-1/t-PA ratio in TNF-α activated HUVECs from (A) and (B). *p b 0.05 or **p b 0.01 vs. TNF-α alone or 0; n.s., not significant.

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In a recent study, Lim previously reported that the WGN and WGNS, isolated from S. baicalensis, exhibited anti-inflammatory responses in concanavalin A (Con A) treated cells [14]. He demonstrated that WGN(S) inhibited Con A-stimulated IgE production and lipid peroxidation and WGN has antioxidant and anti-inflammatory activities [8]. There is ample evidence indicating that inflammation and coagulation are intricately related processes that may have a considerable effect on each other [16,33]. This cross-talk occurs at the levels of platelet activation, fibrin formation, and resolution, as well as physiological anticoagulant pathways [16,33]. Based on the previous and current experimental studies, it can be hypothesized that inhibitory modulation of both coagulation and inflammation by WGN(S) could give promising anti-coagulant and antiinflammatory mediators. Flavonoids connected to one or more sugar molecules are known as flavonoid glycosides, while those that are not connected to a sugar molecule are called aglycones. Flavonoid aglycones enter the bloodstream in the form of glucuronide or sulfate conjugates, and very little unchanged aglycone can be found in the plasma, although levels of glucuronic acid conjugates are high [42]. WGN undergoes rapid metabolism and enters the bloodstream mainly in the form of the glucuronide WGNS. Glucuronidation is thought to affect the biological activity of aglycones by altering the physicochemical properties of flavonoids, which is seen as a detoxification process [12], suggesting that WGN will have limited clinical use. Based on the above concept and the current findings that the antithrombotic effects of WGNS were better than those of WGN in vivo, we conclude that the glucuronide metabolite WGNS, like its aglycone wogonin, has better biological activities and the metabolic stabilities of WGNS might be affect its activities in vivo. There are a few advantages associated with using WGN(S) as opposed to pharmaceutical products if WGN(S) is developed as an anticoagulant drug. First of all, using WGN(S) could reduce risk of side effects or toxicity. Most herbal medicines are well tolerated by the patient, with fewer unintended consequences than pharmaceutical drugs. Herbs typically have fewer side effects than traditional medicine, and may be safer to use over time [43]. Second advantage of WGN(S) is lower cost. Herbs cost much less than prescription medications. Research, testing, and marketing add considerably to the cost of prescription medicines. Herbs tend to be inexpensive compared to drugs. Finally, 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 [44,45]. In our experiment, PT and aPTT using human plasma were used to evaluate the antithrombotic effect of WGN(S). Fibrin polymerization and platelet aggregation are also used to determine the anti-platelet functions of WGN(S). Data showed that WGN(S) prolonged PT and aPTT values and WGN(S) 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 [16,33]. This cross-talk occurs at the levels of platelet activation, fibrin formation, and resolution as well as physiological

anticoagulant pathways [16,33]. On the basis of our current experimental studies, it can be hypothesized that inhibitory modulation of coagulation by WGN(S) could give promising anti-inflammatory mediators. Well-designed prospective studies are needed to prove this hypothesis. However, for more than 60 years, well-known anticoagulants such as heparin and coumarin have been mainstays of anticoagulation therapy [46,47]. They are widely available, inexpensive, effective, and have specific antidotes but are regarded as problematic because of their need for careful monitoring. Similar to these anticoagulants, if WGN(S) developed to new anticoagulant, WGN(S) might require monitoring of its therapeutic effect and can also cause thrombosis. Many flavonoids have anti-clotting activities. For example, several flavonoids from Eupatorium odoratum have been isolated and structurally characterized by Triratana et al. [48]. This plant has long been used as a hemostatic in traditional Thai medicine. One compound, 4′,5,6,7-tetramethoxyflavanone, was found to significantly reduce the activated partial thromboplastin time, while having no effect on prothrombin time or thrombin time. This result suggested that this compound could act to enhance-blood coagulation by possibly affecting factors XII, XI, IX, and VIII. Several flavonoids (e.g., baicalein and oroxylin A) were found to be potent inhibitors of NAD(P)H: quinone acceptor oxidoreductase [49]. Most oral anticoagulants are inhibitors of this enzyme and antagonize vitamin K. Consequently, selected flavonoids may be potentially useful anticoagulant drugs. Hispidulin (4′,5,7-trihydroxy-6methoxyflavone), a naturally occurring flavonoid derived from the flowering parts of Arnica montana, inhibited human platelet aggregation stimulated by adenosine monophosphate, arachidonic acid, PAF, and collagen [50]. In conclusion, results of this study demonstrate that WGN(S) inhibited the extrinsic and intrinsic blood coagulation pathways through inhibition of FXa and thrombin production in HUVECs, and that WGN(S) inhibited 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 treatment or prevention of vascular diseases. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government [MSIP] (Grant No. 2013-067053). References [1] Fares A. Winter cardiovascular diseases phenomenon. N Am J Med Sci 2013;5:266–79. [2] Lip GY, Andreotti F, Fauchier L, Huber K, Hylek E, Knight E, et al. Bleeding risk assessment and management in atrial fibrillation patients. Executive Summary of a Position Document from the European Heart Rhythm Association [EHRA], endorsed by the European Society of Cardiology [ESC] Working Group on Thrombosis. Thromb Haemost 2011;106:997–1011. [3] Singer DE, Albers GW, Dalen JE, Fang MC, Go AS, Halperin JL, et al. Antithrombotic therapy in atrial fibrillation: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008;133:546S–92S.

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