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Anti-platelet Effect of Black Tea Extract via Inhibition of TXA2 in Rat
Biomed Sci Letters 2019;25:302-312
Published online December 31, 2019;  https://doi.org/10.15616/BSL.2019.25.4.302
© 2019 The Korean Society For Biomedical Laboratory Sciences.

Ju-Ye Ro* and Hyun-Jeong Cho,**

Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon 35365, Korea
Correspondence to: Hyun-Jeong Cho. Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, 685, Gasuwon-dong, Seo-gu, Daejeon 35365, Korea.
Tel: +82-42-600-8433, Fax: +82-42-600-8408, e-mail: hjcho@konyang.ac.kr
*Graduate student, **Professor.
Received November 4, 2019; Revised November 25, 2019; Accepted November 26, 2019.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract

The aim of this work was to investigate the effect of black tea extract (BTE) on collagen-induced platelet aggregation. In this study, BTE (10~500 μg/mL) was shown to inhibit platelet aggregation via thromboxane A2 (TXA2) downregulation by blocking cyclooxygenase-1 (COX-1) activity. Also, BTE decreased intracellular Ca2+ mobilization ([Ca2+]i). Additionally, BTE enhanced the levels of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are aggregation-inhibiting molecules. BTE inhibited the phosphorylation of phospholipase C (PLC) γ2 and syk activated by collagen. BTE regulated platelet aggregation via cAMP-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) Ser157. The anti-platelet effects of BTE in high fat diet (HFD)-induced obese rats were evaluated. After eight weeks of BTE treatment (300 and 600 mg/kg), the platelet aggregation rate in the treated groups was significantly less than that in the HFD-fed control group. Also, BTE exhibited a hepatoprotective effect and did not exert hepatotoxicity. Therefore, these data suggest that BTE has anti-platelet effects on collagen-stimulated platelet aggregation and may have therapeutic potential for the prevention of platelet-mediated thrombotic diseases.

Keywords : Black tea extract; Cyclic nucleotides; Intracellular Ca2+; Platelet aggregation; Thromboxane A2
INTRODUCTION

Platelets are essential for primary hemostasis and preservation of vascular integrity. However, aberrant platelet activation can lead to the development of cardiovascular disorders such as ischemic stroke, thrombosis, and atherosclerosis (Wald and Law, 2003; Nesbitt et al., 2009).

When platelets are activated by agonists such as thrombin, epinephrine, collagen, or TXA2, platelet surface receptors trigger the activation of phospholipase C to catalyze the release of membrane phosphoinositides (such as phosphatidylinositol 4,5-bisphosphate, PIP2), releasing the second messengers inositol triphosphate (IP3) and diacylglycerol (DG) (Dery et al., 1998). DG is hydrolyzed by DG lipase to produce arachidonic acid (Mahadevappa and Holub, 1986). TXA2 activates other platelets in a positive-feedback loop during platelet aggregation, and is synthesized by activated platelets from arachidonic acid (AA) through the cyclooxygenase (COX) and TXA2 synthase (TXAS) pathways (Zucker and Nachmias, 1985). On the other hand, IP3 increases cytosolic Ca2+ levels by stimulating Ca2+ release from the endoplasmic reticulum (Chueh and Gill, 1986). Plateletsexpress the P2Y12 receptor, which is activated by prothrom-botic factor-like adenosine diphosphate (ADP). Activated P2Y12 inhibits adenylate cyclase, causing a decrease in the levels of cyclic nucleotides and an increase in the intracellularCa2+ level (Nieswandt et al., 2001; Smolenski, 2012). Therefore, cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) are anti-platelet second messengers, and a substance which elevates the levels of cAMP or cGMP may control platelet aggregation. The anti-platelet effects of cAMP and cGMP are mediated by cAMP/A-kinase and cGMP/G-kinase-dependent protein kinase, where phosphorylate substrate proteins, vasodilator-stimulatedphosphoprotein (VASP). VASP is phosphorylated at various sites, and these are phosphorylated with differing kinetics. In A-Kinase, Ser157 is preferred site of phosphorylation. In G-kinase, Ser239 is the preferred phosphorylation site, in vitro (Butt et al., 1994).

Obesity, a major metabolic syndrome, increases the risk of cardiovascular diseases. High fat diet (HFD)-induced obeserat models exhibit vascular dysfunction. Therefore, the obeserat model has been used to study the mechanisms responsible for obesity-related abnormalities (i. e. disorders of cardiovascular function) (Carroll et al., 2006). A previous study suggested that platelet aggregation is greatly increased in HFD-fed obese rats (Monteiro et al., 2012). In this the currentstudy, the antiplatelet effect of BTE on HFD-induced obese rats was investigated.

Tea is the most frequently consumed drink worldwide (Zhang et al., 2007). Four varieties (green, black, white, andoolong) are made from leaves ofCamellia sinensis (Ferrara et al., 2001). Tea leaves contain abundant polyphenols (a type of antioxidant), known as catechin and epigallocatechingallate (Manach et al., 2004). Black tea is more highly oxidized than are other teas during the oxidation process, so most of the catechins are oxidized to thearubigins and theaflavins (Subramanian et al., 1999; Leung et al., 2001). Previous studies demonstrated the beneficial effects of black tea on prevention of cardiovascular disease risk and its ability to lower blood pressure and blood cholesterol level (Vinson, 2000; Davies et al., 2003). In addition, the consumption of abundant dietary flavonoids like theaflavin may be associated with reductions in the risk of stroke and coronary artery disease (Stangl et al., 2006; Mink et al., 2007). However, little is known regarding the role of black tea extract (BTE) on platelet aggregation, and the mechanism of action of BTE on collagen-induced platelet aggregation has not been elucidated. Therefore, the present study was undertaken to determine the effect of BTE on platelet aggre-gation and characterize the underlying mechanisms of action.

MATERIALS AND METHODS

Materials

Camellia sinensis (Assam black tea) was obtained from Ahmad Tea Ltd. (London, UK). Collagen was purchased from Chrono-Log Co. (Havertown, USA). The TXB2 enzyme immunoassay (EIA), lactate dehydrogenase (LDH) cytotoxicity assay, and COX-1 fluorescent kits were bought from Cayman Chemical (Ann Arbor, USA). cAMP- and cGMP-EIA kits were bought from BioVision, Inc. (Milpitas, USA). Antibodies for western blot analysis were purchased from Cell Signaling Technology (Beverly, MA, USA). H&E (hematoxylin and eosin) stain was obtained from YD Diagnostics Corp. (Yongin, South Korea). AST (aspartate aminotransferase) and ALT (alanine aminotransaminase) assay kitswere purchased from Asan Pharm., Co. (Seoul, South Korea). All other chemicals and reagents used in this study were obtained from Sigma Chemical Corp. (St. Louis, USA).

Preparation of Black tea extract (BTE)

Black tea leaves (20 g) were heated in boiling water with 70% ethanol for 5 hours, and blended with a vortex mixer for 10 min. The mixture was centrifuged at 2,000×g for 10 min and the clear supernatant was collected. The sediment was blended with the vortex mixer for 10 min with distilled water. The mixture was centrifuged at 2,000×g for 10 min and the clear supernatant was collected. The above procedures were repeated 5 times. Collected BTE was freeze-dried with a freeze dryer (Ilshin BioBase Co., Ltd., Gyeonggi-do, South Korea) and re-suspended with distilled water to the desired concentrations.

Analysis of the contents of BTE with high-performance liquid chromatography

BTE was dissolved in 80% methanol, and then analyzed by high performance liquid chromatography (HPLC). An Alliance HPCL e2695 Separations Module (Waters Cor-poration, UK) was equipped with a 2998 photodiode array detector. A phenomenex Luna C18 columm (2) (250×4.6 mm, 5 μm) was used at a column temperature of40℃. The mobile phase consisted of water with 0.1% trifluoroacetic acid (TFA) (A) and acetonitrile (ACN) (B) using the following program: A:B = 90:10 (0 min) → 90:10 (10 min) → 40:60 (60 min) → 0:100 (75 min) → 90:10 (90 min). The flow rate was 1.0 mL/min and the sample injection volume was 10 μL. The UV detection was done at 210 nm.

Measurement of platelet aggregation and TXB2

8-week-old male Sprague-Dawley rats were purchased from Taconic Farm., Inc. (Hudson, NY, USA). They were sacrificed with ethyl ether and blood was collected from theabdominal vein with 3.2% sodium citrate (1:9, v/v). Washed platelets (108 cells/mL) were prepared according to previously published methods (Ro et al., 2015). Washed platelets (108 cells/mL) were pre-incubated for 3 min at 37℃ in the presence of2 mM exogenous CaCl2 with or without BTE, then stimulated with collagen (10 μg/mL) for 5 min. Aggregation was monitored using a Chrono-Log aggregometer at a constant stirring speed of1,200 rpm. Aggregation rate was evaluated as an increase in light transmission. To measure TXB2, the aggregation reactions were terminated by the add-ition of ice-cold EDTA (5 mM) and indomethacin (0.2 mM). The amount of TXB2 (a stable metabolite of TXA2) was determined using a TXB2 EIA kit according to the manufacturer's instructions.

TXAS and COX-1 activity assays

Platelets in a suspension buffer containing 1% protease inhibitor were sonicated. Platelet lysates (10 μg protein) werepre-incubated with or without BTE at 37℃ for 5 min. TXAS activity was measured according to previously published methods (Ro et al., 2015). The amount of TXB2, a stable metabolite of TXA2, was determined using a TXB2 EIA kit according to the manufacturer's instructions. Ozagrel (11nM) was used as positive control. COX-1 activity was measured according to previously published methods (Ro et al., 2015).SC-560 (330 nM) and ASA (500 μM) were used as positive control COX-1 inhibitors. COX-1 activity was measured with a Synergy HT multi-model microplate reader (BioTek Instruments, Winooski, USA).

Measurement of cyclic nucleotides (cAMP and cGMP) and [Ca2+]i

After platelet aggregation reactions were terminated by the addition of 80% ice-cold ethanol, cAMP and cGMP were measured using cAMP and cGMP EIA kits (Biovision,Milpitas, CA, USA). To measure [Ca2+]i, fura 2-AM (Sigma Corp.) -loaded washeplatelets were stimulated with collagen (10 μg/mL) for 5 min. Fura 2 fluorescence was measured with an RF-5301 spectrof luorometer (Shimadzu Corp., Kyoto, Japan) with an excitation wavelength that changed every 0.5 sec from 340 to 380 nm; the emission wavelength was set at 510 nm. [Ca2+]i values were calculated using the method published by (Schaeffer and Blaustein, 1989).

Western blot analysis

After platelet aggregation reactions were terminated, platelet lysates (10 μg-proteins) were used for analysis. The effectsof substances on Syk, PLCγ2 and VASP-phosphorylations were analyzed by western blotting. Blots were analyzed by using Fusion SL imaging systems (Vilber Lourmat, France).

Cell toxicity assay

To assess whether BTE is cytotoxic, we examined the effect of BTE on LDH release. Washed platelets were incubated for 5 min at 37℃ with BTE. Platelets were centrifuged at 2,000 ×g at 25℃, and the supernatant was measured with an LDH assay kit according to the manufacturer's instructions.

Animals and diets

Six-week-old male Sprague-Dawley rats were purchased from Taconic Farm., Inc. (Hudson, NY, USA). They were fed a diet consisting of45% fat; the high fat diet (HFD) (TD.06415, Harlan Laboratories Ltd., Indianapolis, USA) was used to rapidly induce obesity. The rats were divided into 4 groups of8 animals each (ND group, Group I, HFD group, Group II; 2 groups administered BTE (300 mg/kg, Group III and 600 mg/kg, Group IV)). Groups II, III, and IVwere grown for 8 weeks on the HFD to induce obesity. BTE was dissolved in mineral water and given orally to HFD-fedobese rats at doses of300 and 600 mg/kg of body weight/day for 6 weeks. The rats had free access to food and water. After treatment with BTE for 6 weeks, the rats were sacrificed after a 12 h starvation period. Platelet aggregation was determined by the above-mentioned methods. Serum AST and ALT levels were measured to assess the effects of BTE on hepatotoxicity using Asan AST and ALT enzyme assay kits. After the blood was drained, the liver was fixed in 3.7% formaldehyde and embedded in paraffin for histological study. Sections were stained with H&E. Images were viewed with a Zeiss 20× lens, and digital images were captured with a Zeiss Axiocam MRC digital camera and Axiovision sof tware. All animal experiments were carried out according to the guidelines of the Institutional AnimalCare and Use Committee of Konyang University (P-15-08-A-01) (Daejeon, South Korea).

Statistical analysis

The results are expressed as means ± S.D. Statistical analysis was performed with a two tailed-unpaired Student'st-test or ANOVA, as appropriate. Ifthere were significant differences between the group means according to ANOVA, each group was compared by Scheffe's method for post hoc tests (Brown, 2005).

RESULTS

contains various polyphenols

Tea leaves have been reported to contain diverse polyphenols, including flavonoids, epigallocatechin gallate (EGCG), and other catechins (Williamson et al., 2011). As shown in Fig. 1 and Table 1, HPLC analysis revealed that BTE (20 mg/mL) contains various compounds, including epigallocatechin (EGC), catechin, caffeine, epigallocatechin gallate (EGCG), coumaric acid, vitexin-2"-O-rhamnoside, hyperoside, astragalin, and theaflavin.

Internal standards and content (mg/g-BTE) analysis of BTE

Standards (0.1 mg/mL)RT (min)Contents (mg/g-BTE)
AECG8.945.45
BCatechin11.651.23
CCaffeine12.0319.07
DEGCG18.079.90
ECoumaric acid19.442.57
FVitexin-2"-O-rhamnoside23.730.74
GHyperoside25.040.71
HAstragalin27.450.28
ITheaflavin35.023.25

BTE inhibits platelet aggregation by reducing TXA2 production

The platelet aggregation rate induced by collagen was 82.0±2.0%, but BTE (10, 100, and 500 μg/mL) significantly reduced platelet aggregation by 67.8±5.2, 30.3±6.3 and 4.3±1.2% (inhibition rate: 17.3, 63.0, and 94.8%), respectively, in a dose-dependent manner (Fig. 2A). TXA2 is one of the most powerful stimulators of platelet aggregation. It plays an important role in the mechanism of platelet activation because it is a potent autacoidal agonist of platelet activation (Bunting et al., 1983). Therefore, we determined whether BTE reduces the production of TXA2 under collagen exposure. The TXB2 level in intact platelets was 1.2 ±0.4 ng/108 platelets, which was markedly increased to 76.8±6.1 ng/108 platelets following collagen induction (Fig. 2B). However, BTE (10, 100, and 500 μg/mL) significantly reduced the levels of TXB2 in a dose-dependent manner (57.4±7.3, 47.8±4.9, and 23.7±2.6 ng/108 platelets, respectively). These results suggest that BTE inhibits collagen-induced platelet aggregation by decreasing TXA2 production.

BTE reduces TXA2 production via decreasing the activity of COX-1 but not TXAS

As shown in Fig. 2C, the effects of BTE were unchangedby TXA2 synthase. Ozagrel (11 nM) decreased TXA2 levels from 112.7±16.8 ng/min/mg-protein to 95.5±5.9 ng/min/mg-protein. As Fig. 2D shows, BTE inhibited COX-1 activity from 2.6±0.1 (control) to 1.7±0.1 (10 μg/mL BTE) and 1.1±0.1 (100 μg/mL BTE) nmol/min/mg-protein. 500 μM of aspirin and 330 nM of SC-560 selectively inhibited COX-1 activity (2.1±0.1 and 1.4±0.3 nmol/min/mg-protein, respectively). These results show that the inhibitory activity of BTE on TXA2 production was mediated by a reduction in COX-1 activity.

BTE inhibits [Ca2+]i by elevation of cyclic nucleotides

cAMP and cGMP are anti-platelet-aggregation second messengers (Cho et al., 2012). In addition, elevated cAMP or cGMP levels decrease the intracellular Ca2+ level, which is an essential factor for platelet aggregation (Ok et al., 2012). Therefore, we investigated whether BTE up-regulates the cellular levels of cAMP or cGMP. As Fig. 3A shows, BTE(10, 100, and 500 μg/mL) significantly increased cAMP levels in a dose-dependent manner (34.6±3.5, 37.4±4.2 and 56.5±6.5 pmol/108 platelets, respectively). Also, BTE significantly increased cGMP levels dose dependently (32.4±2.6, 37.5±3.9, and 38.4±2.2 pmol/108 platelets, respectively) (Fig. 3B). These results indicate that BTE has an anti-platelet effect that involves up-regulating the productionof cAMP and cGMP in collagen-stimulated platelets. As Fig. 3C shows, when Fura-2-loaded platelets were stimulated by collagen (10 μg/mL), [Ca2+]i increased from 69.9±9.8 to 808.6±34.8 nM. However, [Ca2+]i was significantly decreased by BTE in a dose-dependent manner (545.2±91.1, 215.3±42.9, and 110.3±11.0 nM, respectively). Additionally, no signs of toxicity were observed when platelets wereincubated with the concentrations of BTE used in this study (Fig. 3D).

Effects of BTE on phosphorylation of PLC, Syk and VASP Ser157

The effect of BTE on PLCγ2 and/or Syk phosphorylation induced by collagen was investigated. Treatment with BTE eliminated phosphorylation of PLCγ2 and Syk stimulated by collagen (Fig. 4A and B). PGE1 markedly diminished phosphorylation of PLCγ2 as compared to collagen control. These results indicate that BTE inhibits platelet activation via decrease of phosphorylation of PLCγ2 and Syk. The Ser157 phosphorylation of VASP is mediated by cAMP/A-kinase pathway. As shown in Fig. 3A and B, BTE increasedthe level of cyclic nucleotides. Therefore, it was investigated whether BTE can induce VASP Ser157 phosphorylation on platelets. As illustrated in Fig. 4C, BTE activated the phosphorylation of VASP (Ser157), a cAMP/A-kinase substrate. A selective activator of cAMP-dependent protein kinase (PGE1) induced VASP Ser157 phosphorylation.

Fig. 3. Chromatograms for standardization of BTE (20 mg/mL).

A; epicatechin gallate (ECG), B; Catechin, C; Caffeine, D; epigallocatechin gallate (EGCG), E; Coumaric acid, F; Vitexin-2"-O-rhamnoside, G; Hyperoside, H; Astragalin, I; Theaflavin.


Fig. 4. Effects of BTE on collagen-induced platelet aggregation and TXA2 generation.

(A) Effects of BTE on collagen-induced plateletaggregation. Data are expressed as mean ± SD (n = 5). *P<0.05 compared to the collagen control. (B) Effects of BTE on TXA2 generation stimulated by collagen. #P<0.05 compared with intact cells, *P<0.05 compared to collagen-treated control cells. (C) Effect of BTE on TXA2 synthase activity. Data represent means ± SD (n = 3). *P<0.05 compared to control. (D) Effect of BTE on COX-1 activity. Data represent means ± SD (n = 3). *P<0.05 compared to control.


Consumption of BTE has anti-platelet effects in HFD-fed obese rats

After eight weeks of being fed a HFD, the body weight in the HFD-fed groups (groups II, III, and IV) was significantly higher than in the normal-diet group (378.8±23.9 vs. 424.4±12.4* g, 412.5±21.8*, and 418.8±33.1*, respectively,*P<0.05, data not shown). In the normal diet group (group I), the platelet aggregation rate induced by collagen treatment was 69.4±6.2%, and in the HFD-fed group (group II), the rate was significantly increased to 74.2±5.0%. However, when HFD-fed rats were treated with 300 or 600 mg/kg BTE, the platelet aggregation rate was decreased (aggregation rate, 63.2±7.6 and 61.4±5.4%; inhibition rate, 14.8 and 17.3%, respectively) (Fig. 5A). Consistent with the results shown in Fig. 5A, consumption of BTE decreased collagen-stimulated TXA2 generation in HFD-induced obeserats. In group II, the TXB2 level significantly increased from 18.8±5.4 (group I) to 116.9±2.8 pg/106 platelets. However, in groups III and IV, the levels of TXB2 were significantly reduced in a dose-dependent manner (81.8±16.0 and 58.5 ±9.9 pg/106 platelets, respectively) (Fig. 5B). These results demonstrate that consumption of BTE has antiplatelet effectsthat are mediated by regulation of TXA2 production in vivo.

BTE does not exert hepatotoxicity

To test for potential BTE-induced hepatotoxicity, AST and ALT levels were examined after 6 weeks of BTE consumption. As Table 2 shows, the serum levels of AST and ALT in group II and the BTE-treated groups (groups III and IV) were similar to those of group I, even though BTE was used as an anti-platelet agent. As shown in Fig. 5C and D, a mild degree of hepatic steatosis was found in group II when compared with group I. However, administration of BTE resulted in a reduction in hepatic fatty deposition in hepatocytes (Fig. 5E and F) and, correspondingly, in liver weight (Table 2). These results suggest that the highest concentration of BTE used in this study (~600 mg/kg) does notexert hepatotoxicity, and also has a hepato-protective effect.

Effects of BTE on HFD-induced fatty liver and serum AST/ALT levels

GroupsLiver weight (g)Serum (IU/L)

ASTALT
I10.8±1.1 63.5±11.112.7±4.9
II11.7±2.7*66.4±6.313.3±1.5
III10.4±1.6#60.8±8.513.3±5.8
IV9.5±1.8# 60.2±13.214.0±4.3

Data are expressed as mean ± SD (n = 8). Group I, ND-treated rats; group II, HFD-treated obese rats; group III, HFD + BTE 300 mg/kg-treated rats; group IV, HFD + 600 mg/kg-treated rats. *P<0.05 as compared with group I and #P<0.05 as compared with group II


DISCUSSION

In the present study, we provided evidence that BTE has antiplatelet effects via inhibitions of COX-1 and TXA2, and elevation of cellular cAMP level by decreasing [Ca2+]i. In addition, BTE (20 mg/mL) contains various compounds, such as EGC, catechin, EGCG. Previous studies have shown that these compounds act to inhibit platelet aggregation or activation (Landolfi et al., 1984; Stangl et al., 2006; Luceri et al., 2007; Jin et al., 2008). Ali and Afzal (1987) suggested that caffeine inhibits thromboxane formation in whole blood. Also, Ok et al. (Ok et al., 2012) investigated the effect of EGCG on cyclic nucleotide production and vasodilator-stimulated phosphoprotein phosphorylation in collagen-stimulated platelet aggregation. Especially, it is reported that EGCG has anti-platelet effect by inhibiting collagen-mediated PLC γ2 and protein tyrosine phosphorylation, and it involved in reduction of cytosolic calcium mobilization (Jin et al., 2008). EGCG also diminished activities of COX-1 and TXAS in platelet microsomal fraction (Lee et al., 2013). Therefore, we suggest that the anti-platelet effect of BTE is partly attributable to these compounds, although their other peaks were not confirmed (Fig. 1).

Fig. 1. Effects of BTE on cyclic nucleotides, [Ca2+]i mobilization and cytotoxicity.

(A) Effect of BTE on cAMP production in resting and collagen-stimulated platelets. (B) Effect of BTE on cGMP production in resting and collagen-stimulated platelets. (C) Effect of BTE on [Ca2+]i mobilization. Data are expressed as means ± SD (n = 3). #P<0.05 as compared with resting platelets, and *P<0.05 as compared with collagen-stimulated platelets. (D) Effect of BTE on LDH release in platelets. Data are expressed as means ± SD (n = 3).


When platelets were stimulated by collagen, arachidonic acid (AA) converts to TXA2via COX-1 and/or TXAS activity. Thus, the inhibitor of TXA2 production inducers such as COX-1 can be a promising agent to protection of thrombotic disorder cause (Zucker and Nachmias, 1985). Currently, aspirin is the most powerful antiplatelet agent clinically available. It prevents athero-thrombotic vascular diseases by inhibition of platelet aggregation through COX-1 down-regulation and TXA2 generation (Michelson, 2004; Guthikonda et al., 2007). Therefore, inhibition of TXA2 synthesis in activated platelets could have therapeutic efficacy as an anti-platelet treatment. In this study, BTE decreased the production of TXB2 level via inhibition of COX-1 activity (Fig. 2B and D). These results suggest that BTE may have beneficial effects on prevention of thrombotic disorders.

Fig. 2. Effects of BTE on the phosphorylation of PLCγ2, Syk and VASP.

(A) Effect of BTE on PLCγ2 phosphorylation.(B) Effect of BTE on Syk phosphorylation. (C) Effect of BTEon VASP phosphorylation. After platelet aggregation reactions were terminated, platelet lysates (10 μg-proteins) were used for analysis. The effects of substances on Syk, PLCγ2 and VASP-phosphorylations were analyzed by western blotting. Blots were analyzed by using Fusion SL imaging systems (Vilber Lourmat, France). Data are expressed as mean ± S.D. (n=4). #P<0.05 as compared with resting platelets, *P<0.05 as compared with collagen-stimulated platelets, and **P<0.05as compared with collagen + BTE 100 μg/mL-treated platelets.


The Ser157 phosphorylation of VASP is mediated by cAMP/A-kinase pathway. Phosphorylation results in inhibition of Ca2+ release from intracellular stores (Kuo et al., 1980; Butt et al., 1994). Also, activation of Syk-PLCγ2 is required for the production of IP3 and Ca2+ release from endoplasmic reticulum stores. [Ca2+]i-lowering effects by substances may be associated with their inhibitory effect on phosphorylation of Syk and/or PLCγ2 (Nieswandt and Watson, 2003). Thus, the inhibitory effect of BTE on collagen-stimulated increase in [Ca2+]i might be related with the regulation of the phosphorylation of PLCγ2 and VASP (Ser157). Since Ser239 phosphorylation of VASP is mediated by cGMP/G-kinase pathway, future research is certainly required to confirm the effect of BTE on cGMP/G-kinase-mediated pathways.

Previous study reported HFD-induced obesity exhibit vascular dysfunction in rat. In Fig. 5A, the platelet aggregation rate induced by collagen treatment was increased in the HFD-fed group. This result is supported by those of a study by Monteiro et al. (2012). The livers of HFD-induced obese rats showed an accumulation of fatty droplets; for this reason, liver weight is higher in HFD-fed rats than in ND-fed rats (Xu et al., 2003). In the present study, consumption of BTE diminished hyper-aggregated platelet activity and has a hepato-protective effect. Therefore, our findings suggest that BTE may have preventive effects against platelet-derived vascular thrombosis and obesity-related abnormalities (Carroll et al., 2006).

Fig. 5. Effects of BTE on HFD-induced obese rats.

(A) Effect of BTE on platelet aggregation in HFD-induced obese rats. (B) Effect of BTE on TXA2 production in HFD-induced obese rats. Data are expressed as means ± SD (n = 6-8). #P<0.05 as compared with group I and *P<0.05 as compared with group II. Hepatoprotective effects of BTE against hepatic steatosis in HFD-induced obese rats. (C) Normal hepatocytes observed in group I. (D) Mild hepatic fatty deposition in group II hepatocytes. (E, F) BTE treatment resulted in the prevention of hepatic fatty deposition. Group I, ND-treated rats; group II, HFD-treated obese rats; group III, HFD + BTE 300 mg/kg-treated rats; group IV, HFD + 600 mg/kg-treated rats. Sections were stained with hematoxylin-eosin (H&E). All magnifications: 400×. Arrows indicate fatty hepatocytes.


In conclusion, we suggest that BTE has anti-platelet effectson collagen-stimulated platelet aggregation and may have therapeutic potential for the prevention of platelet-mediated thrombotic diseases.

ACKNOWLEDGEMENT

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2010-0024028 & 2016R1C1B2007025).

CONFLICT OF INTEREST

The authors declare no conflict of interests.

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