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Inhibitory Effect of Scopoletin on U46619-induced Platelet Aggregation through Regulation of Ca2+ Mobilization
Biomed Sci Letters 2019;25:123-130
Published online June 30, 2019;  https://doi.org/10.15616/BSL.2019.25.2.123
© 2019 The Korean Society For Biomedical Laboratory Sciences.

Dong-Ha Lee,*

Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, Namseoul University, Cheonan 31020, Korea
Correspondence to: Dong-Ha Lee. Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, Namseoul University, 91, Daehak-ro, Seonghwan-eup, Seobuk-gu, Cheonan-si, Chungcheongnam-do 31020, Korea.
Tel: +82-41-580-2148, Fax: +82-41-580-2932, e-mail: dhlee@nsu.ac.kr
Received February 21, 2019; Revised March 30, 2019; Accepted May 22, 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

Platelet aggregation is essential for hemostatic process in case of blood vessels damages. However, excessive platelet aggregation can cause cardiovascular disorders including atherosclerosis, thrombosis and myocardial infarction. Scopoletin is usually found in the roots of genus Scopolia or Artemisia, and is known to have anticoagulant and anti-malarial effects. This study investigated the effect of scopoletin on human platelet aggregation induced by U46619, an analogue of thromboxane A2 (TXA2). Scopoletin had anti-platelet effects by down-regulating TXA2 and intracellular Ca2+ mobilization ([Ca2+]i), the aggregation-inducing molecules generated in activated platelets. On the other hand, scopoletin increased the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are known to be intracellular Ca2+ antagonists. This resulted in inhibition of fibrinogen binding to αIIb/β3 in U46619-induced human platelet aggregation. In addition, scopoletin inhibited the release of adenosine trisphosphate (ATP) in dose-dependent manner. This result means that the aggregation amplification activity through the granule secretion in platelets was suppressed by scopoletin. Therefore, we demonstrated that scopoletin has a potent antiplatelet effect and is highly likely to prevent platelet-derived vascular disease.

Keywords : Scopoletin, Platelet aggregation, Intracellular Ca2+, Fibrinogen binding
꽌濡

삁냼뙋 쓳吏묒 삁愿씠 넀긽릺뿀쓣 븣 吏삁 留덇컻瑜 삎꽦븯뒗뜲 엳뼱꽌 븘닔쟻씤 諛섏쓳씠떎. 듅엳, 삁냼뙋뿉꽌 꽭룷吏 궡 Ca2+ ([Ca2+]i) 냽룄뒗 삁냼뙋 쓳吏묒쓽 솢꽦솕뿉 빑떖쟻씤 뿭븷쓣 븯硫, [Ca2+]i inositol 1,4, s-trisphosphate (IP3)뿉 쓽빐 궡遺 옣냼씤 dense tubular system뿉꽌遺꽣 닔슜泥대 넻빐 꽭룷吏 궡濡 룞썝맂떎怨 븣젮졇 엳떎(Furuichi and Mikoshiba, 1995). 利앷맂[Ca2+]i뒗 삁냼뙋留됱뿉 쐞移섑븳 phosatidylinositiol 4,5-bisphosphate (PIP2)媛 遺꾪빐릺硫댁꽌 깮꽦릺뒗 Diacyglycerol (DG)怨 븿猿 Ca2+/calmodulin 蹂듯빀泥댁뿉 쓽議댁쟻씤 myosin light chain (20 kDa)쓽 씤궛솕 protein kinase C (PKC)뿉 쓽議댁쟻씤 plecktrin (40 삉뒗 47 kDa)쓽 씤궛솕瑜 씪쑝궡쑝濡쒖뜥 꽭룷怨④꺽 떒諛깆쭏쓽 옱諛곗뿴쓣 쑀룄븯 怨, 寃곌낵쟻쑝濡 삁냼뙋쓣 쓳吏묒떆궎뒗 寃껋쑝濡 븣젮졇 엳떎(Furuichi and Mikoshiba, 1995). 삉븳, DG뒗 DG lipase monoacyglycerol (MG) lipase뿉 쓽빐 닚李⑥쟻쑝濡 媛닔 遺꾪빐릺뼱 arachidonic acid瑜 嫄곗퀜 Thromboxane A2 (TXA2)濡 쟾솚맂떎(Ohkubo et al., 1996). TXA2뒗 삁냼뙋쓣 솢꽦솕떆耳 遺꾨퉬 諛 삎깭 蹂솕瑜 쑀룄븯뒗 寃껋쑝濡 븣젮졇 엳떎(Saitoh et al., 1986). 떎젣濡, TXA2쓽 븞젙븳 쑀궗泥댁씤 U46619 (9,11-dideoxy-9a.la-methanoepoxyprostaglandin F2a)뒗 [Ca2+]i瑜 긽듅떆궎硫댁꽌 myosin light chain pleckstrin쓽 씤궛솕瑜 利앷떆궎뒗 슦닔븳 삁냼뙋 쓳吏 쑀룄젣濡 씠슜맂떎(Cattanco et al., 1991; Su et al., 1999). 씠윭븳 쁽긽 삁븸 궡 吏삁 諛섏쓳쓽 젙긽쟻씤 怨쇱젙씠吏留 삁냼뙋 쓳吏묒씠 怨쇰룄븯寃 씪뼱궇 寃쎌슦뿉 룞留 寃쏀솕 媛숈 떎뼇븳 삁愿 吏덊솚쓽 썝씤씠 릺湲곕룄 븳떎. 뵲씪꽌, 쟻젅븯寃 삁냼뙋 쓳吏묒쓣 뼲젣븯뒗 寃껋씠 떖삁愿怨 吏덊솚쓣 삁諛⑺븯뒗 쑀슜븳 젒洹쇰갑踰뺤씠 맆 닔 엳떎(Cattanco et al., 1991; Su et al., 1999).

Verapamil怨 theophylline 삁냼뙋 쓳吏묒쓽 븘닔 슂냼씤 [Ca2+]i瑜 媛먯냼떆궎뒗 cyclic adenosine monophosphate (cAMP)쓽 닔以쓣 넂엫쑝濡쒖뜥 빆삁냼뙋 湲곕뒫쓣 븳떎. 삉븳, molsidomine怨 nitroprusside 媛숈 삁愿솗옣젣 zaprinast erythro-9-(2-hydroxy-3-nonyl) adenine怨 媛숈 cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) 뼲젣젣뒗 삁냼뙋뿉꽌 cGMP 닔以쓣 넂씤떎(Menshikov et al., 1993). cAMP cGMP쓽 빆삁냼뙋 슚怨쇰뒗 湲곗쭏 떒諛깆쭏 vasodilator-stimulated phosphoprotein (VASP)쓣 씤궛솕떆궎뒗 cAMP cGMP-쓽議댁꽦 떒諛깆쭏 kinase(媛곴컖 A-kinase G-kinase)뿉 쓽빐 留ㅺ컻맂떎(Kawada et al., 1997; Calderwood et al., 2000). VASP뒗 actin 寃고빀 떒諛깆쭏濡쒖꽌 遺꾨퉬 遺李⑹뿉 湲명빆옉슜쓣 븯硫, VASP쓽 씤궛솕뒗 αIIb/β3쓽 솢꽦뿉 湲곗뿬븯뒗 VASP 移쒗솕룄瑜 뼲젣븯뒗 寃껋뿉 愿젴맂떎怨 븣젮졇 엳떎(Smolenski et al., 1998).

Scopolia 삉뒗 Artemisia 냽 떇臾쇱쓽 肉뚮━뿉꽌 쓷엳 諛쒓껄릺뒗 Scopoletin 留먮씪由ъ븘 諛⑹ 諛 쓳怨 諛⑹ 슚怨쇨 엳뒗 寃껋쑝濡 븣젮졇 엳떎(Obasi et al., 1994; Towler and Weathers, 2015). 洹몃윭굹, 삁냼뙋 쓳吏묒뿉꽌쓽 scopoletin쓽 뿭븷 諛 U46619뿉 쓽빐 쑀諛쒕릺뒗 궗엺 삁냼뙋 쓳吏묒뿉 븳 scopoletin쓽 湲곗쟾뿉 빐꽌뒗 븣젮吏 諛붽 뾾떎. 蹂 뿰援щ뒗 scopoletin쓽 빆삁냼뙋 옉슜쓣 紐낇솗엳 븯湲 쐞빐 sco- poletin씠 U46619媛 쑀룄븳 삁냼뙋 쓳吏묎낵 愿젴맂 떎뼇븳 씤옄뿉 誘몄튂뒗 쁺뼢쓣 솗씤븯怨좎옄 븯떎.

옱猷 諛 諛⑸쾿

떎뿕옱猷

Scopoletin (PIN:7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one, Chemical formula: C10H8O4, Molar mass: 192.16 g/moL)뒗 Avention Corporation (Seoul, Korea)뿉꽌 援ъ엯븯떎(Fig. 1). U46619뒗 Chrono-Log Corporation (Havertown, PA, USA)뿉꽌 엯닔븯떎. TXB2, ATP, cAMP 諛 cGMP enzymeimmunoassay (EIA) kit뒗 Cayman Chemical (AnnArbor, MI, USA)뿉꽌 援ъ엯뻽떎. Fura 2-AM 諛 Fibrinogen Alexa Fluor 488 젒빀泥대뒗 Invitrogen (Eugene, OR, USA)쑝濡쒕꽣 援ъ엯븯떎.

Fig. 1.

The structure of scopoletin. PIN:7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one, Chemical formula: C10H8O4, Molar mass: 192.16 g/moL.


궗엺 꽭泥 삁냼뙋 젣議

궗엺 삁냼뙋 뭾遺 삁옣(platelet rich plasma, PRP) 븳援 쟻떗옄 삁븸썝(Suwon, Korea)뿉꽌 엯닔븯떎. 씠쟾뿉 諛쒗몴맂 諛⑸쾿(Kwon and Lee, 2017)뿉 뵲씪꽌 꽭泥 삁냼뙋(108 cells/mL)쓣 以鍮꾪뻽뒿땲떎. PRP瑜 1,300 G뿉꽌 10遺 룞븞 썝떖 遺꾨━븯뿬 삁냼뙋쓣 쉷뱷븯怨, 씠寃껋쓣 꽭泥 셿異⑹븸 (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM glucose 諛 1 mM EDTA, pH 6.9)쑝濡 2쉶 꽭泥숉븯떎. 꽭泥숇맂 삁냼뙋쓣 108 cells/mL쓽 理쒖쥌 냽룄濡 쁽긽 셿異⑹븸(138 mM NaCl, 5.5 mM 湲猷⑥퐫뒪, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2 諛 0.25% gelatin, pH 7.4)뿉 쁽긽떆耳곕떎. 삩뿉꽌 삁냼뙋 쓳吏묒씠 씪뼱굹뒗 寃껋쓣 뵾븯湲 쐞빐 紐⑤뱺 젅李⑤ 25°C꽌 닔뻾븯떎. 씠 떎뿕 궓꽌슱븰援먯쓽 Institutional Review Board (1041479-HR-201803-003)쓽 듅씤쓣 諛쏆븘 닔뻾븯떎.

삁냼뙋 쓳吏묐뒫 諛 TXA2 깮꽦웾 痢≪젙

꽭泥 삁냼뙋(108 cells/mL)쓣 37°C뿉꽌 3遺꾧컙 諛곗뼇븯怨, 2 mM쓽 CaCl2瑜 泥④븳 썑, U46619 (0.5 μM)濡 쓳吏묒쓣 쑀룄븯뿬 5遺 룞븞 솗씤븯떎. 쓳吏묒 1,000 rpm뿉꽌 aggregometer (Chrono-Log, Corp., Havertown, PA, USA)瑜 궗슜븯뿬 痢≪젙븯떎. 媛곴컖쓽 쓳吏묐뒫 鍮 닾怨쇱쑉씠 利앷릺뒗 젙룄濡 룊媛릺뿀떎. TXA2 깮꽦웾 TXA2쓽 븞젙븳 궗 궛臾쇱씤 TXB2쓽 뼇쑝濡 솗씤븯怨, ice-cold EDTA (5 mM) 諛 indomethacin (0.2 mM)쓣 泥④븯뿬 諛섏쓳쓣 以묐떒떆궓 썑, TXB2 EIA kit (Cayman Chemical)瑜 궗슜븯뿬 痢≪젙븯떎.

꽭룷吏 궡 Ca2+ ([Ca2+])쓽 痢≪젙

PRP瑜 5 μM쓽 Fura 2-AM怨 븿猿 37°C뿉꽌 60遺 룞븞 諛곗뼇븯떎. Fura 2-AM 鍮쏆뿉 誘쇨컧븯湲 븣臾몄뿉 PRP媛 뱾뼱엳뒗 뒠釉뚮 븣猷⑤몃뒆 샇씪濡 뜮 긽깭瑜 쑀吏븯떎. 쐞뿉 紐낆떆븳 젅李⑤ 궗슜븯뿬 Fura 2濡 異⑹쟾맂 꽭泥 삁냼뙋(108 cells/mL)瑜 젣議고븯怨, 2 mM CaCl2 議댁옱 븯뿉 37°C 뿉꽌 3遺꾧컙 빆삩 泥섎━븳 썑, U46619 (0.5 μM)瑜 泥섎━븯뿬 5遺 룞븞 솗씤븯떎. Fura 2쓽 삎愿묒 遺꾧킅 삎愿 痢≪젙湲 (SFM 25, BioTeck Instrument, Italy)瑜 궗슜븯뿬 痢≪젙븯怨, [Ca2+]i 寃곌낵뒗 湲곗〈 끉臾몄쓽 諛⑸쾿쓣 궗슜븯뿬 怨꾩궛븯떎 (Schaeffer and Blaustein, 1989).

Cyclic nulceotides (cAMP 諛 cGMP) 깮꽦웾 痢≪젙

꽭泥 삁냼뙋(108 cells/mL)쓣 37°C뿉꽌 3遺꾧컙 諛곗뼇븯怨, 2 mM쓽 CaCl2瑜 泥④븳 썑, U46619 (0.5 μM)濡 옄洹뱁븯뿬 5遺 룞븞 쓳吏묒쓣 쑀룄븯떎. 1 M HCl瑜 泥④븯뿬 諛섏쓳쓣 醫낃껐븯怨, cAMP 諛 cGMP瑜 EIA kit瑜 궗슜븯뿬 Synergy HT Multi-Model Microplate Reader (BioTek Instruments, Winooski, VT, USA)瑜 넻빐 痢≪젙븯떎.

αIIb/β3뿉 븳 fibrinogen 寃고빀뒫 痢≪젙

Alexa Flour 488-human fibrinogen (30 μg/mL)씠 寃고빀맂 꽭泥 삁냼뙋(108 cells/mL)쓣 2 mM쓽 CaCl2瑜 泥④븳 썑, U46619 (0.5 μM)濡 옄洹뱁븯떎. 諛섏쓳쓣 醫낃껐떆궎湲 쐞빐 0.5% paraformaldehyde媛 븿쑀맂 phosphate-buffered saline (PBS, pH 7.4)瑜 泥④븯떎. 씠 怨쇱젙 鍮쏆쓣 李⑤떒븯硫댁꽌 닔뻾릺뿀怨, fibrinogen 寃고빀뒫 쑀꽭룷遺꾩꽍湲(BD Biosciences, San Jose, CA, USA)瑜 궗슜븯뿬 닔뻾븯쑝硫, CellQuest software (BD Biosciences)濡 遺꾩꽍븯떎.

ATP 諛⑹텧웾 痢≪젙

꽭泥 삁냼뙋(108 cells/mL)뿉 2 mM쓽 CaCl2瑜 泥④븳 썑, U46619 (0.5 μM)濡 옄洹뱁븯뿬 5遺 룞븞 쓳吏묒쓣 쑀룄븯떎. 諛섏쓳씠 醫낃껐릺怨 4°C뿉꽌 10遺꾧컙 1,000 G濡 썝떖 遺꾨━븳 썑, 긽벑븸쓣 遺꾨━븯뿬 ATP 諛⑹텧웾 痢≪젙뿉 궗슜븯떎. ATP 諛⑹텧웾 luminometer (BioTek Instruments濡 ATP assay kit瑜 궗슜븯뿬 痢≪젙븯떎.

넻怨 遺꾩꽍

寃곌낵뒗 룊洹± 몴以 렪李⑤줈 굹깉怨, 넻怨 遺꾩꽍 뼇痢≪쓽 unpaired Student’s t-test 삉뒗 ANOVA瑜 궗슜븯뿬 쟻젅븯寃 닔뻾릺뿀떎. ANOVA뿉 뵲瑜 吏묐떒 룊洹 媛꾩뿉 쑀쓽븳 李⑥씠媛 엳떎硫 媛 吏묐떒쓣 Scheffe쓽 諛⑸쾿쑝濡 鍮꾧탳븯떎.

寃 怨

삁냼뙋 쓳吏묐뒫뿉 븳 scopoletin쓽 슚怨

U46619뿉 쓽빐 쑀룄릺뒗 궗엺 삁냼뙋쓽 쓳吏묐룄瑜 떎뼇븳 냽룄뿉꽌 솗씤븯怨, 0.5 μM 씠긽뿉꽌 理쒕濡 쓳吏묒쓣 쑀룄븯뒗 寃껋쓣 솗씤븯떎(Fig. 2A). 뵲씪꽌, 0.5 μM U46619쓣 쓳吏묒쓣 쑀룄븯뒗 臾쇱쭏쓽 냽룄濡 궗슜븯떎. Fig. 2B뿉 굹궦 諛붿 媛숈씠, U46619뿉 쓽빐 쑀룄 맂 삁냼뙋 쓳吏묒 80.5±2.1%씠뿀吏留, scopoletin (50, 100, 300 諛 500 μM) 삁냼뙋 쓳吏묒쓣 74.0±1.7, 71.3±2.9, 44.0±3.6 諛 1.8±1.5%쑝濡 쑀쓽븯寃 뼲젣븯떎. 洹몃━怨, scopoletin쓽 half-maximal inhibitory concentration (IC50) 빟 326.3 μM씠뿀쑝硫(Fig. 2C), 삁냼뙋뿉 誘몄튂뒗 scopoletin쓽 룆꽦 쑀쓽븳 슚怨쇨 뾾쓬쓣 솗씤븯떎(Data not shown). 씠 寃곌낵뒗 scopoletin씠 꽭룷룆꽦씠 뾾씠 삁냼뙋 쓳吏 뼲젣젣濡쒖꽌 湲곕뒫븷 닔 엳쓬쓣 쓽誘명븳떎.

Fig. 2.

Effects of scopoletin on platelet aggregation. (A) Concentration threshold of U46619 on human platelet aggregation. (B) Effects of scopoletin on platelet aggregation stimulated by U46619. (C) Half-maximal inhibitory concentration (IC50) value of scopoletin in U46619-stimulated human platelet aggregation. Data are expressed as means ± SD (n=4). *P<0.05, **P<0.001 compared with the U46619-stimulated platelets.


Scopoletin씠 TXB2 [Ca2+] 뿉 誘몄튂뒗 쁺뼢

넀긽씠 뾾뒗 긽깭쓽 삁냼뙋뿉꽌 TXB2쓽 뼇 3.0±0.8 ng/108 삁냼뙋씠뿀怨 삁냼뙋쓣 U46619 (0.5 μM)濡 옄洹뱁뻽쓣 븣 48.9±5.4 ng/108 cells쑝濡 겕寃 利앷뻽떎. 洹몃윭굹, scopoletin (50, 100, 300 諛 500 μM) 利앷븳 TXB2쓽 뼇쓣 냽룄 쓽議댁쟻쑝濡 쑀쓽븯寃 媛먯냼떆耳곕떎(Fig. 3A). 삉븳, Fig. 3B뿉꽌 蹂 닔 엳벏씠, scopoletin (50, 100, 300 諛 500 μM) U46619뿉 쓽빐 利앷맂[Ca2+]i瑜 媛뺣젰븯寃 뼲젣븯떎.

Fig. 3.

Effects of scopoletin on TXB2 production and [Ca2+]i (A) Effects of scopoletin on TXB2 production stimulated by U46619. (B) Effects of scopoletin on [Ca2+]i stimulated by U46619. Data are expressed as means ± SD (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the U46619-stimulated platelets.


Scopoletin씠 cAMP 諛 cGMP 깮꽦뿉 誘몄튂뒗 쁺뼢

cAMP cGMP 紐⑤몢 [Ca2+]i쓣 媛먯냼떆궡쑝濡쒖뜥 삁냼뙋 쓳吏묒쓽 쓬꽦 議곗젅옄濡 湲곕뒫븳떎怨 븣젮졇 엳떎(Kawada et al., 1997). 뵲씪꽌, scopoletin씠 cAMP 삉뒗 cGMP쓽 깮꽦뿉 쁺뼢쓣 誘몄튂뒗吏 솗씤븯떎. Fig. 4A뿉 굹궦 諛붿 媛숈씠, scopoletin cAMP 닔以쓣 4.2±0.2 pmoL/108 cells뿉꽌 7.3±0.7 pmoL/108 cells쑝濡 媛뺥븯寃 利앷떆耳곕떎. 洹몃━怨, cGMP룄 scopoletin뿉 쓽빐 5.9±0.4 pmoL/108 cells뿉꽌 16.3±2.1 0.4 pmoL/108 cells쑝濡 겕寃 利앷릺뿀떎(Fig. 4B). 씠윭븳 寃곌낵뒗 scopoletin씠 U46619濡 쑀룄맂 삁냼뙋뿉꽌 cAMP 諛 cGMP쓽 깮꽦쓣 利앷떆궡쑝濡쒖뜥 쓳吏묒뼲젣뿉 湲곕뒫븯떎뒗 寃껋쓣 쓽誘명븳떎.

Fig. 4.

Effects of scopoletin on cyclic nucleotides production. (A) Effects of scopoletin on cAMP production stimulated by U46619. (B) Effects of scopoletin on cGMP production stimulated by U46619. Data are expressed as means ± SD (n=4). *P<0.05, **P<0.001 compared with the U46619-stimulated platelets.


Scopoletin씠 αIIb/β3뿉 븳 fibrinogen 寃고빀뒫뿉 誘몄튂뒗 쁺뼢

꽭룷吏 궡 cyclic nulceotides쓽 깮꽦利앷뒗 αIIb/β3뿉 븳 fibrinogen 寃고빀쓣 뼲젣븿쑝濡쒖뜥 삁냼뙋 솢꽦솕瑜 뼲젣븳떎怨 븣젮졇 엳떎(Horstrup et al., 1994; Barragan et al., 2003). 뵲씪꽌, scopoletin씠 αIIb/β3뿉 寃고빀븯뒗 fibrinogen쓣 뼲젣븯뒗吏 솗씤븯떎. Fig. 5A뿉꽌 蹂 닔 엳벏씠, U46619뒗 넀긽씠 뾾뒗 삁냼뙋怨 鍮꾧탳븯뿬 αIIb/β3뿉 寃고빀븯뒗 fibrinogen쓣 0.7±0.1%뿉꽌 76.9±0.9%濡 媛뺣젰븯寃 利앷떆耳곕떎. 洹몃윭굹, scopoletin U46619뿉 쓽빐 利앷맂 αIIb/β3뿉 븳 fibrinogen쓽 寃고빀뒫쓣 理쒕 18.8±0.6%源뚯 냽룄 쓽議댁쟻쑝 濡 媛뺥븯寃 뼲젣븯떎(Fig. 5B).

Fig. 5.

Effects of scopoletin on fibrinogen binding. (A) Effects of scopoletin on fibrinogen binding. Stimulated by U46619. a, Intact platelets; b, U46619 (0.5 μM); c, U46619 (0.5 μM) + scopoletin (50 μM); d, U46619 (0.5 μM) + scopoletin (100 μM); e, U46619 (0.5 μM) + scopoletin (300 μM); f, U46619 (0.5 μM) + scopoletin (500 μM). (B) Effects of scopoletin on U46619-stimulated fibrinogen binding (%). These data were expressed as the means ± SD (n=4), aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the U46619-stimulated platelets.


Scopoletin씠 ATP 諛⑹텧웾뿉 誘몄튂뒗 쁺뼢

Fig. 6뿉 굹궦 諛붿 媛숈씠, U46619媛 쑀룄븳 삁냼뙋뿉꽌쓽 ATP 닔以 넀긽씠 뾾뒗 삁냼뙋뿉꽌쓽 寃곌낵 鍮꾧탳븯쓣 븣 빟 78諛곌 넂 7.8±0.2 μM씠뿀떎. 洹몃윭굹, scopoletin (50, 100, 300 諛 500 μM) U46619뿉 쓽빐 利앷맂 ATP 諛⑹텧쓣 7.4±0.2, 5.4±0.2, 3.9±0.1 諛 1.3±0.1 μM源뚯 냽룄 쓽議댁쟻쑝濡 뼲젣븯떎. Scopoletin (500 μM) U46619뿉 쓽븳 ATP 諛⑹텧웾쓣 83.3%쓽 뼲젣쑉濡 媛뺣젰븯寃 뼲젣븳 寃껋씠떎.

Fig. 6.

Effects of scopoletin on ATP release. Data are expressed as means ± SD (n=4). (n=4), aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the U46619-stimulated platelets.


怨 李

삁냼뙋쓽 솢꽦솕媛 씪뼱궇 븣, phospholipase C-γ2 (PLC-γ2)뒗 삁냼뙋留됱뿉꽌 phosphatidylinositol 4,5-bisphosphate (PIP2)瑜 IP3 diacylglycerol (DG)濡 媛닔遺꾪빐븳떎. 깮꽦맂 IP3뒗 dense tubular system쑝濡쒕꽣 Ca2+ 룞썝쓣 쑀룄븯怨, DG뒗 DG-쓽議댁꽦 protein kinase C瑜 솢꽦솕떆궓떎(Berridge and Irvine, 1989). 利앷맂 [Ca2+]i뒗 Ca2+/calmodulin 쓽議댁꽦 떒諛깆쭏씤 myosin light chain (20 kDa) 諛 pleckstrin(40 삉뒗 47kDa)쓽 씤궛솕瑜 珥덈옒븯뿬 寃곌낵쟻쑝濡 삁냼뙋 쓳吏묒쓣 쑀룄븳떎(Nishikawa et al., 1980). 삉븳, thromboxane A2 (TXA2)뒗 삁냼뙋 쓳吏 諛 썑냽 삁냼뙋 솢꽦솕瑜 옄洹뱁븯뒗 媛뺣젰븳 삁愿 닔異뺤젣濡쒖꽌, cyclooxygenase (COX) thromboxane A synthase (TXAS) 媛숈 슚냼쓽 옉슜뿉 쓽빐 arachidonic acid濡쒕꽣 PGH2瑜 嫄곗퀜 깮꽦맂떎(Okwu et al., 1992). 蹂 뿰援ъ뿉꽌, scopoletin 궗엺 삁냼뙋뿉꽌 TXA2 깮꽦쓣 냽룄 쓽議댁쟻쑝濡 媛먯냼떆耳곌퀬, 洹 寃곌낵[Ca2+]i瑜 냽룄 쓽議댁쟻쑝濡 뼲젣븯뿬 理쒖쥌쟻쑝濡 삁냼뙋 쓳吏묒쓣 뼲젣븯떎. 씠뒗 TXA2쓽 븞젙븳 쑀궗泥댁씤 U46619 (9,11-dideoxy-9a.la-methanoepoxyprostaglandin F2a)媛 [Ca2+]i쓽 긽듅옉슜쓣 넻빐 myosines light chain pleckstrin쓽 씤궛솕瑜 利앷떆궎뒗 슦닔븳 삁냼뙋 쓳吏 쑀룄젣濡 궗슜맂떎뒗 꽑뻾 뿰援ъ 씪移섑븳떎(Cattanco et al., 1991; Su et al., 1999). 肉먮쭔 븘땲씪, cyclic nulceotides뒗 Ca2+ 쑀엯쓣 媛먯냼떆궎怨 cAMP- 諛 cGMP- 쓽議댁꽦 떒諛깆쭏 kinase瑜 솢꽦솕븿쑝濡쒖뜥 삁냼뙋 쓳吏묒쓣 뼲젣븳떎怨 븣젮졇 엳떎(Kuo et al., 1980). 蹂 뿰援ъ뿉꽌, scopoletin 삁냼뙋뿉꽌 cAMP cGMP 깮꽦쓣 媛뺥븯寃 利앷떆耳곌퀬, 씠 寃곌낵뒗 scopoletin뿉 쓽븳 cyclic nucleotides쓽 利앷媛 [Ca2+]i瑜 議곗젅븿쑝濡쒖뜥 삁냼뙋 쓳吏묒뿉 以묒떖쟻씤 뿭븷쓣 븷 닔 엳쓬쓣 굹궦떎. cAMP cGMP뒗 adenylyl cyclase/guanyl cyclase 삉뒗 cyclic nucleotide phosphodiesterases (PDEs)쓽 솢꽦솕뿉 쓽議댄븳떎(Gao et al., 2015). PDE 솢꽦쓽 뼲젣뒗 삁냼뙋 쓳吏묒뿉꽌 cyclic nucleotides쓽 닔以쓣 利앷떆궎湲곗뿉, PDE 뼲젣젣뒗 삁쟾利앹뿉 븳 移섎즺쟻 슚怨쇰 媛吏 닔 엳떎(Haslam et al., 1999). 떎젣濡, triflusal, cilostazol, dipyridamole怨 媛숈 PDE 뼲젣젣뒗 엫긽쟻쑝濡 cyclic nucleotides쓽 깮궛쓣 利앷떆궎뒗 빆삁냼뙋젣濡 궗슜맂떎(Menshikov et al., 1993). 蹂 뿰援ъ뿉꽌, scopoletin cAMP cGMP쓽 깮궛쓣 利앷떆궎뒗 빆삁냼뙋 臾쇱쭏濡쒖꽌 媛뒫꽦씠 엳쑝硫, 洹 湲곗쟾쓣 솗떎엳 諛앺엳湲 쐞빐꽌뒗 異붽쟻씤 뿰援ш 븘슂븯떎.

삁냼뙋뿉꽌 利앷맂 cAMP 諛 cGMP뒗 VASP ser157 諛 ser239쓽 씤궛솕瑜 넻빐 삁냼뙋 솢꽦솕瑜 뼲젣븳떎怨 蹂닿퀬릺뿀怨, VASP뒗 cAMP/cGMP 쓽議댁꽦 PKA/PKG쓽 二쇰맂 湲곗쭏濡 옉슜븯뿬 삁냼뙋쓽 遺꾨퉬 젒李 듅꽦쓣 議곗젅븯뿬 삁냼뙋 솢꽦솕瑜 뼲젣븯뒗 寃껋쑝濡 븣젮졇 엳떎(Wangorsch et al., 2011; Napeñas et al., 2013). 삉븳, VASP 씤궛솕뒗 integrin αIIb/β3쓽 솢꽦솕瑜 뼲젣븯뿬 寃곌낵쟻쑝濡 삁냼뙋 쓳吏묒쓣 뼲젣븳떎. 蹂 뿰援ъ뿉꽌, scopoletin U46619뿉 쓽빐 옄洹밸맂 삁냼뙋뿉꽌 αIIb/β3뿉 븳 fibrinogen 寃고빀뒫쓣 쑀쓽븯寃 뼲젣븯쑝硫, 씠뒗 cAMP 諛 cGMP 깮꽦쓽 利앷 愿젴씠 엳뒗 寃껋쑝濡 蹂댁씤떎. 삉븳, 삁냼뙋 궡 dense body濡쒕꽣쓽 ATP 諛⑹텧웾 利앷뒗 삁냼뙋 쓳吏묒쓽 利앺룺뿉 愿뿬븯뒗 寃껋쑝濡 븣젮졇 엳떎(Holmsen and Day, 1970; Wentworth et al., 2006). 蹂 뿰援ъ뿉꽌, scopoletin ATP 諛⑹텧웾쓣 냽룄 쓽議댁쟻쑝濡 뼲젣븯怨, 씠뒗 삁냼뙋쓽 쓳吏묒씠 利앺룺릺뒗 寃껋쓣 뼲젣떆궎뒗 뜲 湲곗뿬븯떎怨 깮媛곷맂떎. 씠 寃곌낵뱾쓣 넻빐, 슦由щ뒗 scopoletin媛 U46619 쑀룄쓽 궗엺 삁냼뙋 쓳吏묒뿉 엳뼱꽌 뼲젣 슚怨쇰 媛吏꾨떎뒗 寃껋쓣 遺꾨챸엳 븯떎. 蹂 뿰援щ뒗, scopoletin쓽 빆삁냼뙋 슚怨쇱뿉 븳 湲곗〈쓽 뿰援ъ 떎瑜닿쾶, 넗겮굹 伊먯쓽 삁냼뙋씠 븘땶 궗엺 삁냼뙋뿉 븳 scopoletin쓽 쓳吏묒뼲젣 슚怨쇰 洹쒕챸븳 寃껋씠硫, 삁냼뙋 쓳吏묐뒫 肉 븘땲씪 愿젴맂 뿬윭 씤옄뱾쓣 遺꾩꽍븿쑝濡쒖뜥 湲곗쟾쓣 솗씤븯湲곗뿉 李⑤퀎쟻 쓽誘멸 엳떎怨 궗猷뚮맂떎(Okada et al., 1995; Wu et al., 2001).

寃곕줎쟻쑝濡, scopoletin 궗엺 삁냼뙋 쓳吏묒쓣 뼲젣븯뒗 쑀슚븳 臾쇱쭏씠硫, 씠 옉슜뿉 엳뼱꽌 꽭룷吏 궡濡쒖쓽 Ca2+ 룞썝쓣 議곗젅븯뒗 寃껋씠 以묒슂븯寃 옉슜맂떎怨 궗猷뚮맂떎. 뵲씪꽌, 蹂 뿰援щ뒗 scopoletin씠 Ca2+ 룞썝쓽 議곗젅쓣 넻빐 삁냼뙋 쓳吏묒쓣 빐븯뒗 슚怨쇱쟻씤 삁쟾 삁諛 諛 移섎즺젣濡쒖꽌쓽 媛쒕컻 媛뒫꽦씠 엳쓬쓣 젣떆븳떎.

ACKNOWLEDGEMENT

씠 끉臾몄 2018뀈룄 궓꽌슱븰援 븰닠뿰援щ퉬 吏썝뿉 쓽빐 뿰援щ릺뿀쓬.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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