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Anti-platelet effects of Artesunate through Regulation of Cyclic Nucleotide on Collagen-induced Human Platelets
Biomed Sci Letters 2023;29:41-47
Published online March 31, 2023;  https://doi.org/10.15616/BSL.2023.29.1.41
© 2023 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: 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
*Professor.
Received December 23, 2022; Accepted March 28, 2023.
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
Discovery of new substance that can regulate platelet aggregation or suppress aggregation will aid in the prevention and treatment of cardiovascular diseases. Artesunate is a compound from plant roots of Artemisia or Scopolia, and its effects have shown to be promising in areas of anticancer and Alzheimer's disease. However, the role and mechanisms by which artesunate affects the aggregation of platelets, and the formation of a thrombus are currently not understood. This study examined the ways artesunate affects platelets activation and thrombus formation induced by collagen. As a result, cAMP and cGMP production were increased significantly by artesunate relative to the doses, as well as phosphorylated VASP and IP3R, substrates to cAMP-dependent kinase and cGMP-dependent kinase, in a significant manner. The Ca2+ normally mobilized from the dense tubular system was inhibited due to IP3R, phosphorylation from artesunate, and phosphorylated VASP aided in inhibiting platelet activity via αIIb/β3 platelet membrane inactivation and inhibiting fibrinogen binding. Finally, artesunate inhibited thrombin-induced thrombus formation. Therefore, we suggest that artesunate has importance with cardiovascular diseases stemming from the abnormal platelets activation and thrombus formation by acting as an effective prophylactic and therapeutic agent.
Keywords : Artesunate, Platelet, Cyclic nucleotide, Intracellular Ca2+, Fibrinogen binding
꽌 濡

삁븸 媛 옣湲곗 議곗쭅쑝濡 궛냼 쁺뼇遺꾩쓣 怨듦툒븯硫 끂룓臾 젣嫄곗쓽 湲곕뒫쓣 넻빐 깮議댁뿉 븘닔쟻씤 臾쇱쭏씠硫, 씠 뿭븷쓣 젙긽쟻쑝濡 닔뻾븯湲 쐞빐 썝솢븳 삁븸 닚솚씠 씠猷⑥뼱졇빞 븳떎. 뵲씪꽌, 삁愿씠 넀긽릺뒗 寃쎌슦 삁븸 넀떎쓣 理쒖냼솕븷 肉 븘땲씪 젙긽쟻씤 닚솚쓣 쑀吏븯湲 쐞빐 鍮좊Ⅴ寃 吏삁 諛섏쓳씠 씪뼱굹硫, 삁냼뙋 솢꽦솕 怨쇱젙씠 洹 떆옉젏쑝濡 옉슜븳떎(Jackson, 2011). 洹몃윭굹, 삁냼뙋쓽 鍮꾩젙긽쟻 삉뒗 怨쇰떎븳 솢꽦솕뒗 삁쟾利, 뇤議몄쨷 諛 二쎌긽 룞留 寃쏀솕利 벑쓽 떖삁愿 吏덊솚쓣 씪쑝궎뒗 썝씤씠 맂떎. 洹몃윴 씠쑀濡, 삁냼뙋쓽 솢꽦솕瑜 議곗젅븯硫 삁쟾 깮꽦쓣 빐븯뒗 臾쇱쭏쓣 諛쒓뎬븯뒗 寃껋씠 떖삁愿 吏덊솚쓣 삁諛⑺븯怨 移섎즺븯뒗뜲 엳뼱꽌 以묒슂븯떎(Schwartz et al., 1990).

삁愿씠 넀긽씠 씪뼱궇 븣, 닚솚븯뜕 삁냼뙋씠 넀긽맂 遺쐞濡 紐⑥뿬뱾뼱 삁愿 솢꽦쑀룄젣(collagen, ADP 諛 arachidonic acid 벑)瑜 留뚮궓쑝濡쒖뜥 솢꽦솕媛 씪뼱궃떎. 씠븣, 삁냼뙋 留됱쓽 phospholipase C媛 솢꽦솕릺뼱 phosphatidylinositol 4,5-bisphosphate 媛숈 씤吏吏덉쓣 inositol 1,4,5-triphosphate (IP3)怨 diacylglycerol (DG)濡 媛닔 遺꾪빐릺怨 븯怨, 깮꽦씠 利앷맂 IP3뒗 삁냼뙋 궡쓽 dense tubular system뿉 쐞移섑븯뒗 Ca2+ 梨꾨꼸쓣 뿴뼱꽌 꽭룷吏 궡 Ca2+ 냽룄媛 媛뺥븯寃 利앷븯寃 븳떎(Payrastre et al., 2000). 삉븳, 삁냼뙋씠 솢꽦솕릺뒗 怨쇱젙뿉꽌, 꽭룷吏 留됱쓽 씤吏吏덈줈遺꽣 arachidonic acid媛 媛닔 遺꾪빐릺뼱 굹삤硫, 씠 怨쇱젙뿉꽌 TXA synthase cyclooxygenase-1 슚냼뱾뿉 쓽빐 TXA2濡 쟾솚릺뼱 삁냼뙋쑝濡쒕꽣 遺꾨퉬릺뼱 굹삩떎(Morello et al., 2009; Jennings, 2009). 꽭룷 諛뽰쑝濡 遺꾨퉬맂 TXA2뒗 떎瑜 삁냼뙋 留됱쓽 닔슜泥댁뿉 寃고빀릺뼱 異붽쟻씤 삁냼뙋 솢꽦솕瑜 珥됱쭊븯뒗 옉슜젣濡 湲곕뒫븳떎(Sabatine and Jang, 2000).

젙긽쟻쑝濡 삁븸 닚솚씠 씪뼱궇 븣, 삁愿 궡뵾꽭룷뿉꽌 遺꾨퉬릺뒗 prostanglandin I2 諛 nitric oxide뒗 삁냼뙋 궡 cAMP 삉뒗 cGMP 깮꽦쓣 湲곗씤븳떎. cAMP 깮꽦 利앷뿉 쓽빐 솢꽦솕릺뒗 Protein kinase A (PKA) cGMP 깮꽦 利앷뿉 쓽빐 솢꽦솕릺뒗 protein kinase G (PKG)뒗 vasodilator-stimulated phosphoprotein (VASP) IP3 receptor (IP3R) 媛숈 湲곗쭏 떒諛깆쭏쓣 씤궛솕븳떎怨 븣젮졇 엳떎(Schwarz et al., 2001). IP3R媛 씤궛솕맆 븣 IP3R쓣 鍮꾪솢꽦솕媛 씪뼱굹 dense tubular system濡쒕꽣 꽭룷吏덈줈 룞썝릺뒗 Ca2+ 냽룄媛 뼲젣븳떎(Quinton and Dean, 1992; Cavallini et al., 1996). PKA 諛 PKG쓽 二쇰맂 湲곗쭏濡쒖꽌 븣젮吏 VASP뒗 씤궛솕맆 븣 慣IIb/棺3쓽 솢꽦솕瑜 빐븯硫댁꽌 actin filament쓽 떊옣쓣 뼲젣븳떎(Laurent et al., 1999; Sudo et al., 2003). 뵲씪꽌, IP3R 씤궛솕瑜 넻빐 씪뼱굹뒗 Ca2+ 룞썝쓽 뼲젣 VASP쓽 씤궛솕瑜 넻빐 慣IIb/棺3 솢꽦 뼲젣뒗 삁냼뙋 솢꽦 議곗젅쓣 룊媛븯뒗뜲 以묒슂븯떎.

Artesunate뒗 Artemisia 냽 떇臾쇱쓽 二쇱슂 솢꽦 꽦遺 以 빆留먮씪由ъ븘젣濡 븣젮吏 artemisinn쓽 쑀룄泥대줈꽌 궙 룆꽦怨 궡꽦쓣 듅吏뺤쑝濡 븯뒗 깉濡쒖슫 빆留먮씪由ъ븘젣濡 븣젮졇 엳떎(Thanaketpaisarn et al., 2011). 삉븳, artesunate뒗 빆醫낆뼇 솢꽦쓣 넻빐 엫긽뿉꽌 쟾씠꽦 쓳깋醫 솚옄뿉寃 꽦怨듭쟻씤 移섎즺젣濡 궗슜릺뿀떎(Thanaketpaisarn et al., 2011). 븵 꽑 뿰援ъ뿉꽌 artesunate뒗 뇤議몄쨷쓽 뼇쓣 겕寃 以꾩씠怨 떊寃쏀븰쟻 쉶蹂듭쓣 媛뒫븯寃 븯뿬 솚옄쓽 깮議댁쓣 뼢긽떆궎뒗 寃껋쑝濡 蹂닿퀬맂 諛 엳떎(Lu et al., 2018). 洹몃윭굹, 삁냼뙋 솢꽦솕뿉 븳 artesunate쓽 뿭븷怨 湲곗쟾 븘吏 諛쒓껄릺吏 븡븯떎. 蹂 뿰援ъ뿉꽌뒗 nucelotide쓽 議곗젅쓣 넻븳 移쇱뒛 룞썝 諛 怨쇰┰ 遺꾨퉬瑜 넻븳 artesunate쓽 빆삁냼뙋 슚怨쇰 솗씤븯怨좎옄 븯떎.

옱猷 諛 諛⑸쾿

떆빟

Artesunate뒗 Avention Corporation (Incheon, Korea)濡쒕꽣 솗蹂댄븯떎(Fig. 1). Collagen쓣 Chrono-Log Corporation (Havertown, PA, USA)쑝濡쒕꽣 援ъ엯븯떎. Cayman Chemical (Ann Arbor, MI, USA)濡쒕꽣 cAMP cGMP assay kit瑜 솗蹂댄븯떎. Fura 2-AM Invitrogen (Eugene, OR, USA)濡쒕꽣 援ъ엯븯떎. Anti-棺-actin, anti-phospho-IP3R type I, anti-total VASP, anti-phospho-VASP Ser157, anti-phospho-VASP Ser239, anti-rabbit IgG-HRP-conjugate 벑쓽 빆泥댁 lysis buffer 벑쓣 Cell Signaling (Beverly, MA, USA)濡쒕꽣 援ъ엯븯떎. Thermos fisher Scientific Corp. (Middlesex County, MA, USA)濡쒕꽣 polyvinylidene difluoride (PVDF) membrane瑜 젣怨듬컺븯怨, Invitrogen Molecular Probes濡쒕꽣 Fibrinogen Alexa Fluor 488 젒빀泥 諛 ECL (Enhanced chemiluminescence solution)瑜 젣怨듬컺븯떎.

Fig. 1. The structure of artesunate. PIN: 6-hydroxy-7-methoxy-2H-chromen-2-one, Chemical formula: C10H8O4, Molar mass: 192.17 g/moL.

꽭泥 삁냼뙋쓽 젣議

븳 쟻떗옄궗 寃쎄린삁븸썝(Suwon, Korea)쑝濡쒕꽣 삁냼뙋 뭾遺 삁옣(Platelet Rich Plasma, PRP)쓣 젣怨듬컺븘 궗슜븯떎. 꽭泥 삁냼뙋 븵꽑 뿰援ъ뿉꽌 닔뻾븯뜕 諛⑸쾿쓣 李멸퀬븯뿬 以鍮꾪븯떎(Shin et al., 2019). 以鍮꾨맂 PRP뒗 10遺 媛 1,300 횞g뿉꽌 썝떖 遺꾨━븿쑝濡쒖뜥 삁냼뙋 遺꾪쉷쑝濡 紐⑥쑝怨, 꽭泥 셿異⑹븸(138 mM NaCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 2.7 mM KCl, 5.5 mM glucose 諛 1 mM Na2EDTA, pH 6.9)뿉 쓽빐 2踰덉쓽 꽭泥숈쓣 븯떎. 씠젃寃 以鍮꾨맂 꽭泥 삁냼뙋쓣 쁽긽 셿異⑹븸(138 mM NaCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 2.7 mM KCl, 0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH 7.4)뿉꽌 108 cells/mL쓽 냽룄濡 씗꽍븯뿬 遺쑀븯떎. 삩뿉꽌 쑀諛쒕릺뒗 삁냼뙋 쓳吏묒쓣 留됯린 쐞븳 議곗튂濡쒖꽌 紐⑤뱺 怨쇱젙쓣 25꼦뿉꽌 닔뻾븯쑝硫, 궓꽌슱븰援 깮紐낆쑄由 湲곌 떖쓽쐞썝쉶(IRB)쓽 듅씤쓣 諛쏆븘꽌 씠 떎뿕쓣 닔뻾븯떎(1041479-HR-201803-003).

깮꽦맂 cyclic nucleotides (cAMP 諛 cGMP) 痢≪젙

궗엺쓽 꽭泥 삁냼뙋(108 cells/mL)쓣 37꼦뿉꽌 3遺 媛 諛곗뼇븯怨, 2 mM CaCl2瑜 泥④븳 떎쓬 collagen (2.5 關g/mL)쓣 泥섎━븯뿬 5遺 媛 옄洹뱁븯떎. 1 M HCl瑜 泥섎━븿쑝濡쒖뜥 諛섏쓳쓣 젙吏븯怨, cAMP 삉뒗 cGMP assay kit瑜 궗슜븯뿬 ELISA-reader (Molecular Devices, San Jose, CA, USA)瑜 넻빐 깮꽦맂 cAMP 삉뒗 cGMP쓣 痢≪젙븯떎.

꽭룷吏 궡쓽 Ca2+ 룞썝웾 痢≪젙

PRP뿉 5 關M쓽 Fura 2-AM瑜 泥④븯뿬 37꼦뿉꽌 60遺 媛 諛곗뼇븳 썑, 쐞뿉꽌 뼵湲 븳 諛⑸쾿쓣 李멸퀬빐꽌 꽭泥 삁냼뙋(108 cells/mL)쓣 以鍮꾪븯뿬 37꼦뿉꽌 3遺 媛 諛곗뼇븯떎. 씠 썑, 2 mM CaCl2瑜 泥④븳 떎쓬 collagen (2.5 關g/mL)쓣 泥섎━븯뿬 5遺 媛 옄洹뱁븯떎. Fura 2씠 굹궡뒗 삎愿묒쓣 Hitachi궗쓽 遺꾧킅 삎愿 愿묐룄怨(F7500, Seoul, Korea)瑜 궗슜븯뿬 痢≪젙븯떎. 340 nm쓽 excitation 뙆옣怨 510 nm쓽 emission 뙆옣쓣 꽕젙븯뿬 삎愿묐룄瑜 痢≪젙븯怨, Grynkiewicz쓽 諛⑸쾿쓣 궗슜븯뿬 Ca2+ 룞썝웾쓣 遺꾩꽍븯떎(Grynkiewicz et al., 1985).

Western Immunoblot 떎뿕

꽭泥숉븯뿬 以鍮꾪븳 궗엺 삁냼뙋(108 cells/mL)쓣 37꼦뿉꽌 3遺 媛 諛곗뼇븯怨, 2 mM CaCl2瑜 泥④븳 썑 collagen (2.5 關g /mL)쓣 泥섎━븯뿬 옄洹뱁븯떎. 씠 썑, 1x lysis buffer瑜 泥④븯뿬 諛섏쓳쓣 젙吏븯떎. BCA protein assay kit (Thermo-scientific, IL, USA)瑜 넻빐 슜빐맂 삁냼뙋쓽 떒諛깆쭏 냽룄瑜 痢≪젙븯떎. 떒諛깆쭏(20 關g)쑝濡 8% SDS-PAGE뿉 쟾湲곗쁺룞 븯怨, PVDF 留됱뿉 씠룞떆耳곕떎. 1李 빆泥대줈 1:1,000쓽 씗꽍諛곗닔瑜, 2李 빆泥대줈 1:2,000쓽 씗꽍諛곗닔瑜 궗슜븯뿬 깋옣怨좎뿉꽌 븯猷 룞븞 泥섎━븯떎. ECL 떆빟(Invitrogen Molecular Probes)뿉꽌 諛섏쓳떆궡쑝濡쒖뜥 떒諛깆쭏쓣 떆媛곹솕븯떎.

Fibrinogen뿉 븳 寃고빀 젙룄 痢≪젙

꽭泥숉븯뿬 以鍮꾪븳 삁냼뙋(108 cells/mL)쓣 37꼦뿉꽌 3遺 媛 諛곗뼇븯怨, 2 mM CaCl2 30 關g/mL 냽룄쓽 fibrinogen Alexa Fluor 488 젒빀泥댁 諛섏쓳떆궓 썑, collagen (2.5 關g/mL)쓣 泥섎━븯뿬 5遺 媛 옄洹뱁븯떎. 씠 썑, 0.5% paraformaldehyde媛 븿쑀릺뼱 엳뒗 phosphate-buffered saline (PBS, pH 7.4)瑜 泥④븯뿬 諛섏쓳쓣 젙吏븯떎. 鍮쏆쓣 李⑤떒븳 긽깭뿉꽌 쐞 怨쇱젙쓣 닔뻾븯怨, 쑀꽭룷遺꾩꽍湲(FACS, BD Bioscience, San Jose, CA, USA)瑜 넻빐 fibrinogen쓽 寃고빀 젙룄瑜 痢≪젙븳 썑, Cell-Quest 냼봽듃썾뼱(BD Bioscience, San Jose, CA, USA)瑜 넻빐 遺꾩꽍븯떎.

삁냼뙋 留ㅺ컻 fibrin clot 깮꽦웾 痢≪젙

삁븸뿉꽌 遺꾨━븳 PRP (500 關L)뿉 2 mM CaCl2怨 thrombin (0.05 U/mL)쓣 泥④븳 썑 37꼦뿉꽌 15遺 룞븞 諛곗뼇븯떎. 삎꽦맂 Fibrin clot쓣 digital camera瑜 궗슜븯뿬 珥ъ쁺븯怨, ImageJ 냼봽듃썾뼱(v1.46, National Institutes of Health, Bethesda, MD, USA)瑜 넻빐 쓳怨좊맂 쁺뿭쓣 궛異쒗븯떎.

넻怨 遺꾩꽍

蹂 떎뿕쓽 寃곌낵뱾 룊洹 짹 몴以렪李⑤ 궗슜븯뿬 넻怨꾩쿂由 븯떎. 넻怨 遺꾩꽍 ANOVA 삉뒗 Student's t-test瑜 씠슜븯뿬 遺꾩꽍븯뿬 P<0.05씤 寃쎌슦 쑀쓽꽦씠 엳떎怨 뙋떒븯怨, 遺꾩궛 遺꾩꽍뿉 뵲씪 遺꾩꽍븯뿬꽌 洹몃9 룊洹좉컙쓽 쑀쓽꽦씠 굹굹뒗 寃쎌슦, Scheffe쓽 諛⑸쾿쓣 궗슜븯뿬 洹몃9 媛 쑀쓽꽦쓣 鍮꾧탳븯떎.

寃곌낵 諛 怨좎같

Artesunate씠 cyclic nucleotides 깮꽦뿉 誘몄튂뒗 쁺뼢

씠쟾 뿰援щ 넻빐, cyclic nucleotides(cAMP 諛 cGMP)뒗 꽭룷吏 궡 Ca2+ 룞썝쓣 뼲젣븯硫, cAMP 諛 cGMP 쓽議댁꽦 떒諛깆쭏 kinase(PKA 諛 PKG)瑜 솢꽦솕릺寃 븿쑝濡쒖뜥 삁냼뙋 쓳吏묒쓣 뼲젣븳떎怨 蹂닿퀬릺뿀떎(Kuo et al., 1980). 蹂 뿰援ъ뿉꽌뒗 artesunate媛 cAMP굹 cGMP 깮꽦뿉 엳뼱 뼱뼡 쁺뼢쓣 겮移섎뒗吏 솗씤븯떎. Fig. 2뿉 굹궦 寃곌낵뿉꽌 蹂대벏씠, collagen쓣 떒룆 泥섎━븯硫 intact cell怨 鍮꾧탳븯뿬 cAMP cGMP 깮꽦웾쓽 쑀쓽쟻씤 蹂솕瑜 솗씤븷 닔 뾾떎. 洹몃윭굹, artesunate瑜 泥④븯怨 collagen쓣 泥섎━븯硫 3.92짹0.41 pmoL/108 cells씠뿀뜕 cAMP 깮꽦웾씠 5.79짹0.42 pmoL/108 cells濡 쑀쓽븯寃 利앷 떆 릺뒗 寃껋쓣 솗씤븷 닔 엳떎(Fig. 2A). 삉븳, cGMP 깮꽦웾룄 artesunate뿉 쓽븯뿬 6.91짹0.63 pmoL/108 cells뿉꽌 9.19짹1.33 pmoL/108 cells濡 媛뺥븯寃 利앷릺뿀떎(Fig. 2B). 씠윭븳 寃곌낵뱾쓣 넻빐 collagen쑝濡 쑀룄븳 삁냼뙋뿉꽌 artesunate媛 cAMP 諛 cGMP쓽 깮꽦웾쓣 쑀쓽븯寃 利앷떆궎硫 삁냼뙋쓽 湲곕뒫뿉 愿뿬븿쓣 븣 닔 엳떎.

Fig. 2. The effects of artesunate on cyclic nucleotides production. (A) Effects of artesunate on cAMP production. (B) Effects of artesunate on cGMP production. Measurement of cAMP and cGMP production were described in "Materials and Methods" section. The results are expressed as mean 짹 SD (n=4). *P<0.05, **P<0.001 compared with the collagen-induced platelets.

Artesunate씠 꽭룷 궡쓽 Ca2+ 룞썝怨 IP3R쓽 씤궛솕뿉 誘몄튂뒗 쁺뼢

깮꽦씠 利앷맂 cAMP 諛 cGMP뒗 쓽議댁꽦 kinase씤 PKA 諛 PKG瑜 솢꽦솕븯뿬 떎瑜 湲곗쭏뱾쓣 씤궛솕븳떎怨 븣젮졇 엳怨, 洹 以 inositol 1, 4, 5-triphosphate receptor (IP3R)쓽 씤궛솕瑜 쑀諛쒗븳떎怨 蹂닿퀬릺뿀떎(Schwarz et al., 2001). 삁냼뙋 궡 dense tubular system쓽 留됱뿉 議댁옱븯뒗 IP3R뒗 inositol 1,4,5-trisphosphate (IP3)씠 寃고빀뿉 쓽빐 뿴由щʼn, 씠 怨쇱젙뿉꽌 꽭룷吏 궡濡쒖쓽 Ca2+ 룞썝([Ca2+]i)쓣 쑀룄븿쑝濡쒖뜥 꽭룷怨④꺽 떒諛깆쭏쓽 醫낅쪟씤 myosin light chain 諛 pleckstrin쓽씤궛솕瑜 넻빐 삁냼뙋 궡 怨쇰┰ 遺꾨퉬瑜 珥됱쭊븯뿬 삁냼뙋 솢꽦솕 諛 쓳吏묒쓣 쑀룄븳떎怨 蹂닿퀬릺뿀떎(VargaSzabo et al., 2009).

蹂 뿰援ъ뿉꽌뒗 artesunate媛 [Ca2+]i뿉 誘몄튂뒗 쁺뼢쓣 궡렣蹂댁븯떎. 洹 寃곌낵, Fig. 3A뿉꽌 굹궦 諛붿 媛숈씠, collagen뿉 쓽빐 [Ca2+]i씠 100.1짹0.3 nM뿉꽌 537.9짹12.7 nM濡 利앷븯怨, artesunate뒗 利앷맂 [Ca2+]i瑜 냽룄 쓽議댁쟻쑝濡 媛먯냼븯떎(Fig. 3A). 洹몃━怨, 슦由щ뒗 artesunate媛 [Ca2+]i 議곗젅뿉 愿뿬븯뒗 IP3R뿉 쁺뼢쓣 誘몄튂뒗吏 痢≪젙븯떎. 洹 寃곌낵, Fig. 3B뿉꽌 굹궦 諛붿 媛숈씠, collagen쑝濡 쑀룄븳 삁냼뙋뿉꽌 artesunate媛 IP3R쓽 씤궛솕瑜 냽룄 쓽議댁쟻쑝濡 쑀룄떆궎뒗 寃껋쓣 솗씤븯떎. 씠뒗 artesunate濡 씤빐 利앷맂 cAMP/cGMP 깮꽦 諛 PKA/PKG쓽 솢꽦솕媛 IP3R쓽 씤궛솕瑜 쑀諛쒗븿쑝濡쒖뜥 dense tubular system [Ca2+]i瑜 媛먯냼떆궓떎뒗 寃껋쓣 蹂댁뿬以떎.

Fig. 3. The effects of artesunate on intracellular Ca2+ mobilization and IP3R phosphorylation. (A) Effects of artesunate on intracellular Ca2+ mobilization. (B) Effects of artesunate on IP3R phosphorylation. Measurement of intracellular Ca2+ mobilization and IP3R phosphorylation were described in "Materials and Methods". The results are expressed as mean 짹 SD (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the collagen-induced platelets.

Artesunate媛 VASP 씤궛솕뿉 誘몄튂뒗 쁺뼢

씠쟾 뿰援щ뱾뿉꽌 VASP媛 cAMP/cGMP-쓽議댁꽦 PKA/PKG쓽 二쇱슂 湲곗쭏 以 븯굹濡쒖꽌 삁냼뙋 遺꾨퉬 諛 젏李⑹쓣 議곗젅븯硫, VASP媛 씤궛솕릺硫 integrin 慣IIb/棺3쓽 솢꽦솕媛 뼲젣맖쑝濡쒖뜥 삁냼뙋 쓳吏묒씠 빐맂떎怨 蹂닿퀬릺뿀떎(Wangorsch et al., 2011; Nape챰as et al., 2013). 蹂 뿰援ъ뿉꽌뒗 artesunate媛 collagen 쑀룄쓽 삁냼뙋뿉꽌 cAMP 諛 cGMP 깮꽦쓣 냽룄 쓽議댁쟻쑝濡 利앷븳떎뒗 寃껋쓣 솗씤븯怨(Fig. 2), 異붽쟻쑝濡 artesunate媛 cAMP-쓽議댁꽦씤 VASP Ser157 諛 cGMP-쓽議댁꽦씤 VASP Ser239쓣 씤궛솕뿉 愿뿬븯뒗吏 궡렣蹂댁븯떎. 洹 寃곌낵, Fig. 4뿉꽌 蹂 닔 엳벏씠, artesunate뒗 VASP Ser157 諛 VASP Ser239 씤궛솕瑜 냽룄 쓽議댁쟻쑝濡 利앷떆耳곕떎. 듅엳, VASP Ser239뿉 鍮꾪빐 VASP Ser157쓽 씤궛솕媛 뜑 媛뺥븯寃 쑀諛쒕릺뿀뒗뜲, 씠뒗 artesunate뿉 쓽븳 cAMP 諛 cGMP쓽 깮꽦 利앷媛 VASP쓽 씤궛솕濡 씠뼱吏꾨떎뒗 寃껋쓣 蹂댁뿬以떎.

Fig. 4. The effects of artesunate on VASP phosphorylation. Termination of the reaction was carried out using a 1x lysis buffer. A BCA protein kit was used to measure the concentration of proteins from platelet lysates. Measurement of VASP phosphorylation was described in "Materials and Methods". The results are expressed as mean 짹 SD (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the collagen-stimulated platelets.

Artesunate媛 慣IIb/棺3뿉 븳 fibrinogen 寃고빀뿉 誘몄튂뒗 쁺뼢

Integrin 慣IIb/棺3쓣 留ㅺ컻濡 씪뼱굹뒗 떊샇 쟾떖 삁냼뙋 꽭룷 怨④꺽뿉 蹂삎쓣 쑀룄븯뿬 삁냼뙋 솢꽦솕 삁쟾 깮꽦쓣 쑀諛쒗븳떎怨 蹂닿퀬맂 諛 엳떎(Topol et al., 1999). 쑕吏湲 삁냼뙋뿉꽌 慣IIb/棺3뒗 궙 移쒗솕룄 긽깭濡 議댁옱븯떎媛, 쓳吏묒쑀룄젣 쓽빐 inside-out 떊샇쟾떖 寃쎈줈媛 솢꽦솕릺硫, 援ъ“쟻씤 蹂솕媛 씪뼱굹 慣IIb/棺3쓽 移쒗솕룄媛 利앷맂떎怨 븣젮졇 엳떎(Phillips et al., 2001). 慣IIb/棺3쓽 移쒗솕룄媛 利앷맆 븣, fibrinogen媛 닔슜泥댁씤 慣IIb/棺3뿉 寃고빀븿쑝濡쒖뜥 異붽쟻씤 삁냼뙋 怨④꺽쓽 삎깭 蹂솕 젏李⑹쓣 넻빐 삁냼뙋 쓳吏묒쓽 利앺룺떆궓떎.

蹂 뿰援ъ뿉꽌뒗 慣IIb/棺3뿉 븳 fibrinogen쓽 寃고빀뿉 븯뒗뜲 artesunate媛 誘몄튂뒗 쁺뼢쓣 솗씤븯떎. Fig. 5A뿉꽌 蹂댁뿬吏뒗 諛붿 媛숈씠, collagen쓣 泥④瑜 넻빐 慣IIb/棺3뿉 븳 fibrinogen쓽 寃고빀 젙룄媛 1.3짹0.1%뿉꽌 76.8짹2.5%濡 媛뺥븯寃 利앷릺뿀떎(Fig. 5A-b, 5B). 洹몃윭굹, artesunate (50~ 300 關M)瑜 쟾泥섎━ 븯쓣 븣, fibrinogen 寃고빀 젙룄媛 냽룄 쓽議댁쟻쑝濡 뼲젣릺뿀怨, 듅엳 300 關M쓽 artesunate fibrinogen 寃고빀쓣 18.2짹0.9%源뚯 媛뺥븯寃 뼲젣븯떎(Fig. 5A-c~f, 5B). 씠윭븳 寃곌낵뒗, artesunate뿉 쓽빐 씤궛솕맂 VASP媛 integrin 慣IIb/棺3쓽 移쒗솕룄瑜 뼲젣븳 寃껋쑝濡 蹂댁뿬吏꾨떎. 삉븳, artesunate씠 삁냼뙋 꽭룷뿉 룆꽦쓣 媛吏뒗 吏 LDH (lactate dehydrogenase) 遺꾨퉬瑜 넻빐 痢≪젙븯怨, artesunate (50, 100, 200, 300 關M)씠 궗엺 삁냼뙋뿉 誘몄튂뒗 쑀쓽븳 룆꽦 솗씤븷 닔 뾾뿀떎(data not shown). 씠윭븳 寃곌낵뒗 artesunate씠 꽭룷룆꽦쓣 媛吏吏 븡怨 삁냼뙋쓽 솢꽦솕瑜 議곗젅븳떎뒗 寃껋쓣 쓽誘명븳떎.

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

Artesunate媛 삁냼뙋 留ㅺ컻쓽 fibrin clot 삎꽦뿉 誘몄튂뒗 쁺愿

슦由 紐몄쓽 삁쟾 넀긽맂 삁愿씠 쉶蹂듬맆 븣 븘닔쟻쑝濡 留뚮뱾뼱吏뒗뜲, 솢꽦솕맂 삁냼뙋씠 넀긽맂 삁愿뿉 紐⑥씠 硫 30~60遺꾩씠 吏굹 닔異뺤씠 씪뼱굹怨 깮꽦맂 plug瑜 떦寃 fibrin clot쓣 삎꽦븳떎. 씠 怨쇱젙뿉꽌 慣IIb/棺3怨 fibrinogen쓽 긽샇 옉슜씠 삁냼뙋씠 留ㅺ컻븯뒗 fibrin clot쓣 삎꽦뿉 엳뼱 以묒슂븳 뿭븷쓣 븯怨, 慣IIb/棺3 솢꽦쓣 빐븯뒗 臾쇱쭏씠 삁쟾 삎꽦쓣 媛뺥븯寃 뼲젣븳떎怨 蹂닿퀬릺뿀떎(Topol et al., 1999). 삁냼뙋 솢꽦쑀룄젣濡 慣IIb/棺3瑜 솢꽦솕븯硫 씠뿉 븳 fibrinogen쓽 寃고빀씠 利앺룺릺怨 fibrin clot 삎꽦쑝濡 씠뼱吏꾨떎.

蹂 뿰援ъ뿉꽌뒗 artesunate씠 thrombin濡 쑀룄븳 fibrin clot 삎꽦뿉 誘몄튂뒗 쁺뼢쓣 궡렣蹂댁븯떎. Fig. 6A뿉꽌 蹂 닔 엳벏씠, thrombin 옄洹뱀뿉 쓽빐 媛뺥븯寃 삎꽦맂 fibrin clot쓣 100 關M 씠긽쓽 artesunate씠 냽룄 쓽議댁쟻쑝濡 뼲젣븯떎. Artesunate (100, 200 諛 300 關M)뿉꽌 27.0%, 69.9% 諛 82.6%濡 fibrin clot씠 媛곴컖 뼲젣릺뿀쓬쓣 솗씤븷 닔 엳떎(Fig. 6B). 씠윭븳 寃곌낵뒗 artesunate媛 cAMP/cGMP 깮꽦 諛 VASP Ser157/VASP Ser239 씤궛솕 慣IIb/棺3瑜 移쒗솕룄 뼲젣瑜 넻빐 fibrin clot 삎꽦쓣 빐븳 寃껋쑝濡 蹂댁뿬吏꾨떎.

Fig. 6. Effects of artesunate on platelet-mediated fibrin clot formation. (A) Effects of artesunate on thrombin-retracted fibrin clot photographs (B) Effects of artesunate on thrombin-retracted fibrin clot area. Measurement of platelet-mediated fibrin clot formation was described in "Materials and Methods". The results are expressed as mean 짹 SD (n=4). aP<0.05 compared with no-stimulated platelets, *P<0.05, **P<0.001 compared with the thrombin-induced platelets.

ACKNOWLEDGEMENT

Funding for this paper was provided by Namseoul University year 2022.

CONFLICT OF INTEREST

No conflict of interest.

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