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Thrombus Formation Inhibition of Esculetin through Regulation of Cyclic Nucleotides on Collagen-Induced Platelets
Biomed Sci Letters 2021;27:270-276
Published online December 31, 2021;  https://doi.org/10.15616/BSL.2021.27.4.270
© 2021 The Korean Society For Biomedical Laboratory Sciences.

Dong-Ha Lee†,*

Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, Namseoul University, Chungcheongnam-do 31020, Korea
Correspondence to: *Professor.
Corresponding author: 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: 041-580-2932, e-mail: dhlee@nsu.ac.kr
Received October 5, 2021; Accepted November 22, 2021.
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
Physiological agents trigger a signaling process called "inside-out signaling" and activated platelets promote adhesion, granule release, and conformational changes of glycoprotein IIb/IIIa (αIIb/β3). Activated αIIb/β3 interacts with fibrinogen and initiates a second signaling step called "external signaling". These two signaling pathways can cause hemostasis or thrombosis, and thrombosis is a possible medical problem in arterial and venous vessels, and platelet-mediated thrombosis is a major cause of cardiovascular disease (CVD). Therefore, modulating platelet activity is important for platelet-mediated thrombosis and cardiovascular disease. Esculetin is a coumarin-based physiologically active 6,7-dihydroxy derivative known to have pharmacological activity against obesity, diabetes, renal failure and CVD. Although some studies have confirmed the effects of esculetin in human platelet activation and experimental mouse models, it is not clear how esculetin has antiplatelet and antithrombotic effects. We confirmed the effect and mechanism of action of escultein on human platelets induced by collagen. As a result, esculetin decreased Ca2+ recruitment through upregulation of inositol 1, 4, 5-triphosphate receptor. In addition, esculetin upregulates cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-dependent pathways and inhibits fibrinogen binding and thrombus contraction. Our results demonstrate the antiplatelet effect and antithrombotic effect of esculetin in human platelets. Therefore, we suggest that esculetin could be a potential phytochemical for the prevention of thrombus-mediated CVD.
Keywords : Esculetin, cAMP/cGMP, Intracellular Ca2+, Fibrinogen binding, Fbrin clot
꽌 濡

젙긽쟻씤 닚솚뿉꽌 collagen 꽟쑀뒗 삁냼뙋뿉 寃고빀븷 닔 뾾吏留 궡뵾 踰쎌씠 넀긽릺뼱 collagen 꽟쑀媛 끂異쒕릺寃 릺硫, collagen 꽟쑀媛 삁냼뙋 留 닔슜泥댁쓽 integrin α2β1 諛 glycoprotien VI뿉 寃고빀븿쑝濡쒖뜥 inside-out signaling 寃쎈줈瑜 넻빐 삁냼뙋쓽 솢꽦솕瑜 쑀룄븳떎(Chen and Kahn, 2003). 삁냼뙋 솢꽦솕媛 씪뼱굹硫, 꽭룷吏 궡 phospholipase Cγ2뒗 phosphatidylinositol 4,5 bisphosphate瑜 inositol diacylglycerol怨 1,4,5-trisphosphate (IP3)濡 媛닔遺꾪빐 릺뒗뜲, 궡뵾踰쎌씠 넀긽릺硫 collagen씠 끂異쒕릺뼱 삁냼뙋쓽 integrin α2β1 諛 glycoprotien VI뿉 寃고빀븿쑝濡쒖뜥 삁냼뙋 솢꽦솕媛 씪뼱굹뒗 룞븞 phospholipase Cγ2뒗 phosphatidylinositol 4,5 bisphosphate瑜 inositol diacylglycerol怨 1,4,5-trisphosphate (IP3)濡 遺꾪빐븯怨, IP3뒗 냼룷泥대줈遺꽣 移쇱뒛쓣 꽭룷吏 궡濡 씠룞떆궓떎(Farndale, 2006). 솢꽦솕맂 삁냼뙋뿉꽌 씠윭븳 떊샇 쟾떖 씠踰ㅽ듃뒗 glycoprotein IIb/IIIa (integrin αIIb/β3)쓽 援ъ“쟻 蹂솕瑜 씪쑝耳 삁쟾 삎꽦 諛 삁냼뙋 留ㅺ컻 삁쟾 닔異뺤쓣 쑀諛쒗빀땲떎(Phillips et al., 2001). 뵲씪꽌 삁냼뙋 吏삁뿉 븘닔쟻씤 슂냼씤 룞떆뿉 삁쟾利앹쓽 쐞뿕쓣 궡룷븯怨 엳뒿땲떎. CVD 솚옄쓽 삁쟾利앹쓣 슚怨쇱쟻쑝濡 媛먯냼떆궎湲 쐞빐 빆삁냼뙋젣媛 씠슜릺湲 엳吏留 씪遺 빟臾쇰뱾쓽 遺옉슜뱾룄 蹂닿퀬릺怨 엳떎(Jackson, 2011). 뵲씪꽌 깉濡쒖슫 빆삁냼뙋젣 媛쒕컻쓣 쐞빐 떎뼇븳 빟슜떇臾쇱쓽 빆삁냼뙋 솢꽦뿉 二쇰ぉ븯怨 엳떎. 삉븳, 理쒓렐 肄붾줈굹19 諛깆떊쑝濡 뇤젙留λ룞 삁쟾利(CVST), 異쒗삁꽦 뇤議몄쨷 벑쓽 遺옉슜 궗濡媛 蹂닿퀬릺怨 엳뼱, 뼢썑 肄붾줈굹19 諛깆떊쓽 궗슜뿉 빆삁쟾젣쓽 蹂묓뻾 궗슜쓣 쐞빐꽌씪룄 븞젙쟻씤 빆삁쟾 꽦遺꾩쓽 洹쒕챸씠 떆湲됲븯떎(Kantarcioglu et al., 2021).

젙긽쟻씤 삁븸 닚솚뿉꽌 궡뵾 꽭룷뒗 鍮꾪솢꽦 삁냼뙋 긽깭瑜 쑀吏븯뒗 nitric acid prostaglandin I2瑜 諛⑹텧븯뒗뜲, 씠 臾쇱쭏뱾 삁냼뙋뿉 옉슜븯뿬 cAMP 諛 cGMP 닔以쓣 利앷떆궓떎. 씠 以 cAMP protein kinase A (PKA)쓽 솢꽦솕瑜, cGMP뒗 protein kinase G (PKG)쓽 솢꽦솕瑜 쑀룄븯뿬 vasodilator-stimulated phosphoprotein (VASP) 諛 inositol 1,4,5-triphosphate receptor (IP3R)쓽 씤궛솕瑜 씪쑝궓떎(Schwarz et al., 2011). IP3R쓽 씤궛솕瑜 넻빐 IP3R쓽 鍮꾪솢꽦솕媛 씠猷⑥뼱吏硫, 씠뒗 dense tubular system쑝濡쒕꽣 꽭룷吏 궡濡쒖쓽 Ca2+ 룞썝쓣 뼲젣븯뒗 寃곌낵瑜 珥덈옒븳떎(Cavallini et al., 1996; Quinton and Dean, 1992). 삁냼뙋뿉꽌, VASP뒗 cAMP-쓽議댁꽦씤 VASP Ser157 諛 cGMP-쓽議댁꽦씤 VASP Ser239媛 엳쑝硫 씠뱾쓽 씤궛솕媛 씪뼱궇 븣, αIIb/β3 솢꽦솕 諛 actin filament 솢꽦씠 뼲젣릺뒗 寃껋쑝濡 蹂닿퀬맂 諛 엳떎(Laurent et al., 1999; Sudo et al., 2003). 뵲씪꽌, cyclic nucleotides瑜 議곗젅븯뒗 빟슜 떇臾쇱 삁냼뙋 留ㅺ컻 삁쟾利앹뿉 궗슜맆 媛뒫꽦씠 엳떎.

ScopoliaArtemisia 냽쓽 떇臾 肉뚮━뿉 二쇰줈 븿쑀릺뼱 엳떎怨 蹂닿퀬맂 esculetin 빆떦눊, 빆뿼利, 빆븫 벑쓽 뿬윭 빟由ы븰쟻 슚怨쇰 媛吏怨 엳떎(Karthika et al., 2012). 紐뉖챺 뿰援ъ뿉 뵲瑜대㈃, esculetin 떖삁愿怨꾩뿉 蹂댄샇 슚怨쇨 엳떎怨 蹂닿퀬릺뿀怨, esculetin쓣 寃쎄뎄濡 쟾泥섎━ 븯쓣 븣 isoproterenol濡 쑀룄븳 떖洹쇨꼍깋利앹쓽 伊먯뿉꽌 anti-lipoperoxidative 슚怨쇰 굹궡뿀쑝硫, 씠뒗 free radical 냼嫄 듅꽦 븣臾몄씪 寃껋쑝濡 蹂댁븯떎(Liang et al., 2017). 떎瑜 뿰援ъ뿉꽌 esculetin쓽 슚怨쇰뒗 궗엺 삁냼뙋 솢꽦솕 諛 떎뿕쟻 留덉슦뒪 紐⑤뜽뿉꽌 議곗궗릺뿀쑝硫, esculetin collagen 諛 arachidonic acid濡 쑀룄븳 궗엺 삁냼뙋쓽 쓳吏묒쓣 뼲젣븯湲곕룄 븯떎(Hsia et al., 2019). 洹몃윭굹, thrombin 諛 U46619 媛숈 떎瑜 옉슜젣뿉뒗 슚怨쇨 굹굹吏 븡븯怨, esculetin媛 삁냼뙋 솢꽦쓣 뼲젣븯뒗 湲곗쟾룄 遺덈텇紐낇븳 긽깭씠떎. 蹂 뿰援щ뒗 collagen 쑀룄쓽 삁냼뙋뿉꽌 esculetin媛 cyclic nucleotides 諛 愿젴 떊샇 遺꾩옄瑜 議곗젅븿쑝濡쒖뜥 삁냼뙋 솢꽦솕 諛 삁쟾 삎꽦쓣 뼲젣븯뒗吏瑜 솗씤븯떎.

옱猷 諛 諛⑸쾿

떆猷

Avention Corporation (Incheon, Korea)뿉꽌 Esculetin쓣 젣怨듯븯떎(Fig. 1). Collagen Chrono-Log Corporation (Havertown, PA, USA)濡쒕꽣 젣怨듬컺븯怨, cAMP怨 cGMP Enzyme Immunoassay (EIA) kits, serotonin EIA kit 諛 ATP assay kit뒗 Cayman Chemical (Ann Arbor, Miami, USA)濡쒕꽣 援ъ엯븯뿬 궗슜븯떎. Invitrogen (Eugene, OR, USA)쑝濡쒕꽣 Alexa Fluor 488-conjugate fibrinogen怨 Fura 2-AM쓣 젣怨듬컺븯떎. Westen blotting뿉 씠슜릺뒗 antibody뱾 Cell Signaling (Beverly, MA, USA)뿉꽌 援ъ엯븯뿬 궗슜븯떎. Enhanced chemiluminescence solution (ECL) 諛 polyvinylidene difluoride (PVDF) membrane쓽 떆빟 General Electric Healthcare (Chalfont St. Giles, Buckinghamshire, UK)濡쒕꽣 솗蹂댄븯떎.

Fig. 1. The structure of esculetin. PIN: 6,7-Dihydroxy-2H-1-benzopyran-2-one, Chemical formula: C9H6O4, Molar mass: 178.14 g/moL.

궗엺 삁냼뙋 遺쑀븸 以鍮

븳 쟻떗옄궗 삁븸썝(Suwon, Korea)뿉꽌 젣怨듬컺 궗엺쓽 삁냼뙋 뭾遺 삁옣(PRP)쑝濡 떎뿕쓣 닔뻾븯떎. 꽭泥 삁냼뙋 湲곗〈쓽 以鍮 諛⑸쾿뿉 뵲씪꽌 以鍮꾨릺뿀떎(Shin et al., 2019). PRP瑜 1,300 ×g濡 10遺 룞븞 썝떖遺꾨━븯怨, 븯痢듭쓽 삁냼뙋쓣 쉷뱷븯뿬 꽭泥숈셿異⑹븸(138 mM NaCl, 12 mM NaHCO3, 2.7 mM KCl, 5.5 mM glucose, 0.36 mM NaH2PO4, 諛 1 mM Na2EDTA, pH 6.9)濡 2踰 꽭泥숉븯떎. 씠썑 쁽긽셿異⑹븸(138 mM NaCl, 12 mM NaHCO3, 2.7 mM KCl, 5.5 mM glucose, 0.36 mM NaH2PO4, 0.25% gelatin, pH 7.4)쓣 궗슜븯뿬 理쒖쥌쟻쑝濡 108 cells/mL媛 릺寃 옱援ъ꽦븯떎. 삩뿉꽌 삁냼뙋 쓳吏묒씠 쑀諛쒕맆 닔 엳湲곗뿉 긽湲곗쓽 紐⑤뱺 怨쇱젙쓣 떎삩뿉꽌 吏꾪뻾븯쑝硫, 궓꽌슱븰援 Institutional Review Board (IRB)쓽 듅씤(1041479-HR-201803-003)쓣 嫄곗퀜 떎뿕씠 씠猷⑥뼱議뚮떎.

Cyclic nulceotides(cAMP 諛 cGMP) 깮꽦웾쓽 痢≪젙

꽭泥숉븳 궗엺 삁냼뙋뿉 떎뼇븳 냽룄쓽 esculetin瑜 쟾泥섎━븯떎. 37℃뿉꽌 3遺꾧컙 諛곗뼇븯怨, 2 mM쓽 CaCl2瑜 泥④븳 떎쓬, 2.5 μg/mL쓽 collagen瑜 泥④빐二쇱뼱 5遺 룞븞 諛섏쓳븯떎. 1 M쓽 HCl瑜 泥④븯뿬 諛섏쓳쓣 젙吏븯怨, cAMP 諛 cGMP ELISA kits瑜 궗슜븯뿬 ELISA-reader (Molecular devices, San Jose, CA, USA)뿉꽌 cAMP 諛 cGMP 닔以쓣 痢≪젙븯떎.

꽭룷 궡 Ca2+ 룞썝웾쓽 痢≪젙

5 μM쓽 Fura 2-AM瑜 PRP뿉 꽔怨 37oC뿉꽌 60遺 룞븞 諛곗뼇븳 떎쓬, 꽭泥 셿異⑹븸쑝濡 2踰 꽭泥숉븯떎. 씠 썑, 쁽긽셿異⑹븸쑝濡 理쒖쥌 냽룄媛 108 cells/mL媛 릺룄濡 옱援ъ꽦븯怨, 떎뼇븳 냽룄쓽 esculetin쓣 쟾泥섎━븯떎. 37℃뿉꽌 3遺 룞븞 諛곗뼇븯怨, 2 mM CaCl2瑜 泥④븳 떎쓬, 2.5 μg/mL쓽 collagen濡 泥④빐 二쇱뼱 5遺 룞븞 諛섏쓳븯떎. BioTeck Instrument궗(Winooski, Vermont, USA)쓽 삎愿묐텇愿묎킅룄怨(SFM 25, USA)濡 珥덇린 媛 340~380 nm쓽 뿬湲고뙆옣怨 510 nm쓽 諛⑹텧 뙆옣뿉꽌 Fura 2쓽 삎愿묒쓣 痢≪젙븯떎. Grynkiewicz쓽 諛⑸쾿쓣 씠슜븯뿬 꽭룷吏덈줈 룞썝릺뒗 Ca2+쓣 궛異쒗븯떎(Grynkiewicz et al., 1985).

삁냼뙋 留 αIIb/β3뿉 븳 fibrinogen binding 젙룄쓽 痢≪젙

꽭泥숇맂 궗엺 삁냼뙋뿉 떎뼇븳 냽룄쓽 esculetin쓣 쟾泥섎━븯떎. 37℃뿉꽌 3遺꾧컙 諛곗뼇븯怨, 2 mM쓽 CaCl2瑜 泥④븳 떎쓬, 2.5 μg/mL쓽 collagen 15 μg/mL쓽 Alexa Flour 488-human fibrinogen 泥④빐二쇱뼱 5遺 룞븞 諛섏쓳븯떎. 0.5% paraformaldehyde媛 븿쑀맂 phosphate-buffered saline (pH 7.4)瑜 泥④븿쑝濡쒖뜥 諛섏쓳쓣 젙吏븯怨, BD Bioscience 궗(San Jose, CA, USA)쓽 쑀꽭룷 遺꾩꽍湲(FACS)濡 fibrinogen binding 젙룄瑜 痢≪젙븯떎. 빆泥닿 媛吏 삎愿묒쓣 쑀吏떆궎湲 쐞븯뿬 긽湲곗쓽 怨쇱젙쓣 뼱몢슫 솚寃쎌뿉꽌 吏꾪뻾븯怨, Cell-Quest software (BD Biosciences)瑜 遺꾩꽍봽濡쒓렇옩쑝濡 궗슜븯떎.

삁냼뙋씠 留ㅺ컻븳 fibrin clot 삎꽦뒫 痢≪젙

400 μL쓽 PRP瑜 polyethylene tube濡 삷湲 떎쓬, 뿬윭 냽룄쓽 esculetin쓣 쟾泥섎━븯떎. 37℃뿉꽌 3遺꾧컙 諛곗뼇븯怨 2 mM쓽 CaCl2瑜 泥④븳 떎쓬, fibrin clot 삎꽦쓣 쑀諛쒗븯湲 쐞빐 0.05 U/mL쓽 thrombin瑜 꽔뼱 37℃뿉꽌 15遺 룞븞븯떎. Fibrin clot쓽 씠誘몄瑜 뵒吏꽭 移대찓씪濡 珥ъ쁺븯怨, Image J Software (v1.46, National Institutes of Health, USA)쓽 봽濡쒓렇옩쑝濡 fibrin clot씠 삎꽦맂 硫댁쟻쓣 怨꾩궛븯떎.

Immunoblotting

꽭泥숉븳 궗엺 삁냼뙋뿉 떎뼇븳 냽룄쓽 esculetin瑜 쟾泥섎━븯떎. 37℃뿉꽌 3遺꾧컙 諛곗뼇븯怨, 2 mM쓽 CaCl2瑜 泥④븳 떎쓬, 2.5 μg/mL쓽 collagen瑜 泥④빐二쇱뼱 5遺 룞븞 諛섏쓳븯떎. 媛숈 뼇쓽 lysis buffer瑜 泥④븯뿬 諛섏쓳쓣 젙吏븯怨, BCA assay kit (Pierce Biotechnology, Illinois, USA)濡 떒諛깆쭏 냽룄瑜 젙웾븯떎. 20 μg쓽 떒諛깆쭏쓣 븿쑀븳 삁냼뙋 슜빐臾쇱쓣 SDS-PAGE (8%)뿉꽌 쟾湲곗쁺룞븯怨, PVDF membrane쑝濡 삷寃⑥꽌 빆泥대컲쓳쓣 吏꾪뻾븯떎. 1李 빆泥대뒗 1:1,000쓽 씗꽍諛곗닔쓽 1李 빆泥댁 1:2,000쓽 씗꽍諛곗닔쓽 2李 빆泥대줈 諛섏쓳떆궓 떎쓬. ECL 떆빟(General Electric Healthcare, Buckinghamshire, UK)쑝濡 떆媛곹솕瑜 쑀諛쒗븳 썑, Image J Software (v1.46, National Institutes of Health, USA)쓽 봽濡쒓렇옩쑝濡 諛대뱶쓽 硫댁쟻쓣 怨꾩궛븯떎.

넻怨 遺꾩꽍

4쉶 씠긽쓽 諛섎났 떎뿕쓣 븯怨 룊洹 ± 몴以 렪李⑤줈 떎뿕 寃곌낵瑜 몴떆빐 二쇱뿀떎. 媛 洹몃9 媛꾩쓽 李⑥씠뒗 Student's t-test 삉뒗 ANOVA瑜 씠슜븯뿬 遺꾩꽍븯怨, P<0.05씤 寃쎌슦뒗 넻怨꾩쟻쑝濡 쑀쓽븳 寃껋쑝濡 蹂댁븯떎. 遺꾩궛 遺꾩꽍뿉 뵲씪 洹몃9 룊洹 媛꾩뿉 쑀쓽븳 李⑥씠媛 엳쓣 寃쎌슦뿉뒗 Scheffe쓽 諛⑸쾿쓣 씠슜븯뿬 媛 洹몃9쓣 鍮꾧탳遺꾩꽍븯떎.

寃곌낵 諛 怨좎같

Cyclic nucleotides 깮꽦뿉 誘몄튂뒗 esculetin쓽 슚怨

씠쟾쓽 뿰援щ 넻빐, cyclic nucleotides(cAMP 諛 cGMP)媛 cAMP- 諛 cGMP-쓽議댁꽦쓽 protein kinase(PKA 諛 PKG)瑜 솢꽦솕떆궡쑝濡쒖뜥 꽭룷吏 궡濡 룞썝릺뒗 Ca2+쓣 媛먯냼븯怨, 삁냼뙋쓽 쓳吏묒쓣 뼲젣븳떎怨 븣젮吏 諛 엳떎(Kuo et al., 1980). 蹂 뿰援ъ뿉꽌뒗 esculetin씠 cAMP 諛 cGMP쓽 cyclic nucleotides 깮꽦뿉 愿뿬븯뒗吏 솗씤븯떎. Fig. 2A뿉 굹궦 寃곌낵濡 蹂대㈃, esculetin뿉 쓽빐 cAMP 깮꽦씠 3.92±0.41 pmoL/108 cells뿉꽌 9.20±0.88 pmoL/108 cells源뚯 媛뺥븯寃 利앷맂 寃껋쓣 븣 닔 엳떎. 삉븳, esculetin뒗 cGMP 깮꽦웾룄 6.53±0.46 pmoL/108 cells뿉꽌 8.54±0.39 pmoL/108 cells源뚯 냽룄쓽議댁쟻쑝濡 利앷떆궓 寃껋쓣 솗씤븯떎(Fig. 2B). 씠 寃곌낵뱾쓣 넻빐 esculetin씠 collagen 쑀룄쓽 삁냼뙋뿉꽌 cAMP 諛 cGMP쓽 깮꽦웾쓣 냽룄쓽議댁쟻쑝濡 媛뺥븯寃 利앷떆궎硫댁꽌 삁냼뙋쓽 湲곕뒫뿉 愿뿬븳떎뒗 寃껋쓣 솗씤븯떎.

Fig. 2. Effects of esculetin on cyclic nucleotides production. (A) Effects of esculetin on cAMP production stimulated by collagen. (B) Effects of esculetin on cGMP production stimulated by collagen. Data are expressed as means ± SD (n=4). *P<0.05, **P< 0.001 compared with the collagen-stimulated platelets.

꽭룷 궡 Ca2+ 룞썝 諛 IP3R 씤궛솕뿉 誘몄튂뒗 esculetin쓽 슚怨

cAMP 諛 cGMP쓽 cyclic nucleotide씠 利앷븷 븣, 쓽議댁꽦 떒諛깆쭏 kinase씤 PKA 諛 PKG瑜 솢꽦솕떆궎怨, 씠뒗 愿젴맂 湲곗쭏뱾쓣 씤궛솕븯뒗 寃껋쑝濡 븣젮졇 엳쑝硫, 洹 以 inositol 1,4,5-triphosphate receptor (IP3R)媛 엳떎(Schwarz et al., 2001). IP3R뒗 삁냼뙋 궡 dense tubular system뿉 쐞移섑븯뒗 Ca2+ 넻濡쒕줈꽌 inositol 1,4,5-trisphosphate (IP3) 寃고빀맆 븣, 꽭룷吏 궡濡쒖쓽 Ca2+ 룞썝([Ca2+]i)쓣 쑀룄븳떎. [Ca2+]i씠 利앷븯硫 꽭룷怨④꺽 떒諛깆쭏濡 븣젮吏 myosin light chain怨 pleckstrin쓽 씤궛솕媛 쑀諛쒕릺寃 릺硫, 씠 븣 삁냼뙋 궡 怨쇰┰ 遺꾨퉬 諛 P-selectin 諛쒗쁽씠 씪뼱굹 삁냼뙋 솢꽦솕 諛 쓳吏묒씠 릺뒗뜲 以묒슂븯寃 뿭븷쓣 븳떎(VargaSzabo et al., 2009).

蹂 뿰援ъ뿉꽌뒗 esculetin媛 [Ca2+]i뿉 뼱뼡 쁺뼢쓣 誘몄튂뒗 吏 솗씤븯怨, 洹 寃곌낵, Fig. 3A뿉 蹂댁뿬以 諛붿 媛숈씠, collagen뿉 쓽빐 100.7±0.3 nM뿉꽌 590.6±18.3 nM源뚯 利앷맂 [Ca2+]i씠 뿬윭 슜룄쓽 esculetin(50, 100, 200 諛 300 μM)瑜 泥④븯쓣 븣 媛 媛 432.5±10.0 nM, 275.6±12.3 nM, 152.1±3.2 nM 諛 120.4±3.6 nM濡 냽룄 쓽議댁쟻쑝濡 媛먯냼맂 寃껋쓣 븣 닔 엳떎(Fig. 3A). 洹몃━怨, esculetin媛 [Ca2+]i쓣 議곗젅븯뒗 떒諛깆쭏씤 IP3R쓽 씤궛솕뿉 쁺뼢쓣 二쇱뿀뒗吏 솗씤븯怨, Fig. 3B濡 蹂 닔 엳뒗 諛붿 媛숈씠, esculetin씠 collagen濡 쑀룄븳 삁냼뙋뿉꽌 IP3R瑜 냽룄 쓽議댁쟻쑝濡 씤궛솕떆耳곕떎뒗 寃껋쓣 븣 닔 엳떎. 씠 寃곌낵뱾 esculetin씠 cAMP/cGMP 깮꽦 諛 PKA/PKG쓽 솢꽦쓣 利앷떆耳 IP3R쓽 씤궛솕瑜 쑀룄븿쑝濡쒖뜥 dense tubular system뿉꽌 꽭룷吏덈줈 룞썝릺뒗 Ca2+瑜 뼲젣븳떎뒗 寃껋쓣 蹂댁뿬以떎.

Fig. 3. Effects of esculetin on [Ca2+]i mobilization and IP3R phosphorylation. (A) Inhibitory effects of esculetin on collagen-induced [Ca2+]i mobilization. (B) Effects of esculetin on collagen-induced IP3R phosphorylation. Measurement of [Ca2+]i mobilization and Western blotting was described in "Materials and Methods" section. The data are expressed as the mean ± standard deviation (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, **P< 0.001 compared with the collagen-stimulated platelets.

VASP 씤궛솕뿉 誘몄튂뒗 esculetin쓽 슚怨

씠쟾쓽 뿰援щ뱾쓣 넻빐, VASP媛 삁냼뙋 遺꾨퉬 諛 젏李⑹쓣 議곗젅븿쑝濡쒖뜥 삁냼뙋 솢꽦솕뿉 愿뿬븯뒗 cAMP/cGMP-쓽議댁꽦 PKA/PKG쓽 二쇱슂 湲곗쭏 以 븯굹씪뒗 寃껉낵, VASP쓽 씤궛솕뿉 쓽빐 integrin αIIb/β3쓽 솢꽦솕媛 뼲젣릺뼱 삁냼뙋 쓳吏묒쓣 빐븳떎怨 븣젮졇 엳떎(Napeñas et al., 2013; Wangorsch et al., 2011). 蹂 뿰援ъ뿉꽌뒗, collagen 쑀룄쓽 삁냼뙋뿉꽌 esculetin뿉 쓽빐 cAMP 諛 cGMP 깮꽦씠 냽룄 쓽議댁쟻쑝濡 利앷맂 寃껋쓣 븣 닔 엳뿀怨(Fig. 2), 異붽쟻쑝濡 esculetin뿉 쓽빐 cAMP-쓽議댁꽦쓽 VASP Ser157 諛 cGMP-쓽議댁꽦쓽 VASP Ser239쓽 씤궛솕媛 뼱뼡 쁺뼢쓣 諛쏅뒗吏 솗씤빐 蹂댁븯떎. 洹 寃곌낵, Fig. 4뿉꽌 굹궦 諛붿 媛숈씠, escultein VASP Ser157 VASP Ser239쓽 씤궛솕瑜 냽룄 쓽議댁쟻쑝濡 利앷떆耳곗쑝硫, 씠 寃곌낵뒗 escultein媛 cAMP 諛 cGMP쓽 깮꽦쓣 利앷떆궓 寃껋씠 VASP쓽 씤궛솕濡 씠뼱議뚮떎뒗 寃껋쓣 蹂댁뿬以떎.

Fig. 4. Effects of esculetin on VASP phosphorylation. Western blotting was determined as described in "Materials and Methods" section. The data are expressed as the mean ± standard deviation (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the collagen-stimulated platelets.

αIIb/β3뿉 븳 fibrinogen binding뿉 誘몄튂뒗 esculetin쓽 슚怨

Integrin αIIb/β3뿉 쓽븳 삁냼뙋 궡 떊샇 쟾떖 꽭룷 怨④꺽쓽 蹂삎쓣 쑀諛쒗븿쑝濡쒖뜥 삁냼뙋 利앹떇 諛 삁쟾 깮꽦쓣 씪쑝궎뒗 寃껋쑝濡 蹂닿퀬맂 諛 엳떎(Topol et al., 1999). 젙긽쟻씤 쑕吏湲 삁냼뙋뿉꽌뒗 αIIb/β3媛 궙 移쒗솕룄쓽 긽깭濡 議댁옱븯떎媛 쓳吏묒쑀룄젣뿉 쓽빐 삁냼뙋씠 솢꽦솕릺硫 inside-out 떊샇쟾떖 寃쎈줈瑜 嫄곗퀜 αIIb/β3쓽 移쒗솕룄瑜 利앷릺뒗 履쎌쑝濡 援ъ“쟻씤 蹂솕媛 씪뼱궃떎(Phillips et al., 2001). αIIb/β3쓽 移쒗솕룄媛 利앷릺硫 fibrinogen쓽 寃고빀씠 옒 씪뼱궓쑝濡쒖뜥 異붽쟻씤 삁냼뙋쓽 삎깭 蹂솕 젏李⑹쓣 넻빐 쓳吏묒씠 珥됰컻맂떎.

蹂 뿰援ъ뿉꽌, esculetin씠 fibrinogen씠 αIIb/β3뿉 寃고빀븯뒗 젙룄뿉 뼱뼡 쁺뼢쓣 誘몄튂뒗吏 솗씤븯怨, collagen뿉 쓽빐 αIIb/β3뿉 븳 fibrinogen 寃고빀룄媛 1.0±0.2%뿉꽌 90.8±0.5%源뚯 利앷릺뿀떎(Fig. 5A-b, b, 5B). 洹몃윭굹, esculetin (50, 100, 200 諛 300 μM)쓣 泥섎━븯쓣 븣, fibrinogen쓽 寃고빀 젙룄媛 媛 媛 70.2±1.0%, 59.1±1.4%, 24.1±0.4% 諛 17.4±0.6%源뚯 냽룄 쓽議댁쟻쑝濡 뼲젣릺뒗 寃껋쓣 솗씤븯떎(Fig. 5A-c~f, 5B). 씠뒗 esculetin뿉 쓽빐 깮꽦씠 利앷맂 cyclic nucleotide뱾씠 VASP 씤궛솕瑜 쑀諛쒗븯怨, integrin αIIb/β3쓽 移쒗솕룄瑜 뼥뼱쑉由쇱쑝濡쒖뜥 fibrinogen 寃고빀쓣 뼲젣븳 寃껋쑝濡 蹂댁뿬 吏꾨떎.

Fig. 5. Effects of esculetin on collagen-induced fibrinogen binding. (A) The flow cytometry histograms on fibrinogen binding. a, Intact platelets (base); b, collagen (2.5 μg/mL); c, collagen(2.5 μg/mL) + esculetin (50 µM); d, collagen (2.5 μg/mL) + esculetin (100 µM); e, collagen (2.5 μg/mL) + esculetin (200 µM); f, collagen (2.5 μg/mL) + esculetin (300 µM) (B) Effects of esculetin on collagen-induced fibrinogen binding (%). Measurement of fibrinogen binding was described in "Materials and Methods" section. The data are expressed as the mean ± standard deviation (n=4). aP<0.05 compared with no-stimulated platelets, **P<0.001 compared with the collagen-stimulated platelets.

삁냼뙋 留ㅺ컻 fibrin clot 삎꽦뿉 誘몄튂뒗 esculetin쓽 슚怨

Fibrin clot쓽 삎꽦 삁愿쓽 넀긽맂 遺遺꾩씠 쉶蹂듬릺뒗 怨쇱젙쓽 留덉留 떒怨꾨줈, 넀긽遺쐞뿉 솢꽦솕맂 삁냼뙋씠 紐⑥뿬뱾뼱 30~60遺꾧컙 닔異뺤쓣 씪쑝궎怨 留뚮뱾뼱吏 plug瑜 떦湲곕㈃꽌 留뚮뱾뼱吏꾨떎. 씠 븣, αIIb/β3뿉 븳 fibrinogen 寃고빀 젙룄媛 삁냼뙋씠 留ㅺ컻븳 fibrin clot 삎꽦뿉꽌 以묒슂븯硫, αIIb/β3 移쒗솕룄瑜 媛먯냼떆궎뒗 臾쇱쭏뱾씠 삁쟾 삎꽦룄 留됰뒗떎怨 븣젮졇 엳떎(Topol et al., 1999). 삉븳, 삁냼뙋 솢꽦珥됱쭊젣瑜 궗슜븯뿬 αIIb/β3쓽 솢꽦쓣 넂씠寃 맆 븣, fibrinogen쓽 寃고빀룄媛 利앷릺뼱 fibrin clot씠 옒 삎꽦맂떎.

蹂 뿰援ъ뿉꽌뒗 thrombin쑝濡 쑀룄븳 fibrin clot 삎꽦뿉꽌 esculetin씠 誘몄튂뒗 슚怨쇰 솗씤븯怨, Fig. 6A뿉 젣떆븳 寃곌낵 媛숈씠, thrombin쓣 泥섎━븯쓣 븣 fibrin clot씠 媛뺥븯寃 삎꽦릺뿀吏留 esculetin뿉 쓽빐 fibrin clot쓽 삎꽦씠 냽룄 쓽議댁쟻쑝濡 뼲젣맂 寃껋쓣 蹂 닔 엳떎, Esculetin(100, 200 諛 300 μM)쓽 뼲젣쑉쓣 솗씤빐 蹂대㈃ 媛곴컖 10.1%, 47.8% 諛 77.0%濡 솗씤릺뿀떎(Fig. 6B). 씠 寃곌낵뱾 esculetin媛 cAMP/cGMP 깮꽦 利앷瑜 넻빐 VASP Ser157/VASP Ser239 씤궛솕瑜 쑀諛쒗븯怨, αIIb/β3瑜 移쒗솕룄瑜 媛먯냼떆궡쑝濡쒖뜥 fibrin clot 삎꽦 媛먯냼쓽 寃곌낵濡 굹궃 寃껋쑝濡 蹂댁씤떎.

Fig. 6. Effects of esculetin on fibrin clot retraction. (A) Effects of esculetin on thrombin-retracted fibrin clot photographs (B) Effects of esculetin on thrombin-retracted fibrin clot area. Quantification of fibrin clot retraction was described in "Materials and Methods" section. The data are expressed as the mean ± standard deviation (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, compared with the thrombin-stimulated platelets, **P<0.001 compared with the thrombin-stimulated platelets.
ACKNOWLEDGEMENT

Funding for this paper was provided by Namseoul University year 2021.

CONFLICT OF INTEREST

The author declares no conflict of interest.

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