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ORIGINAL ARTICLE Korean J Clin Lab Sci. 2017;49(2):99-107 https://doi.org/10.15324/kjcls.2017.49.2.99 pissn 1738-3544 eissn 2288-1662 Korean J Clin Lab Sci. Vol. 49, No. 2, June 2017 99 The Inhibitory Effects of Cordycepin on Phosphoproteins including PI3K, Akt, and p38 Hyuk-Woo Kwon 1, Dong-Ha Lee 2 1 College of Veterinary Medicine, Kyungpook National University, Daegu, Korea 2 Department of Biomedical Laboratory Science, Korea Nazarene University, Cheonan, Korea PI3K, Akt, p38 을포함한인산화단백질에대한 Cordycepin 의억제효과 권혁우 1, 이동하 2 1 경북대학교수의학대학, 2 나사렛대학교임상병리학과 A species of Cordyceps, an ingredient in Chinese traditional medicine well-known for its major component, cordycepin (3'-deoxyadenosine), has been known to have antiplatelet effects; however, its effects on regulation of phosphoprotein have not been fully elucidated. In this study, we investigated how cordycepin regulates the phosphoprotein, including phosphatidylinositol 3-kinase (PI3K)/Akt and p38, to inhibit platelet aggregation, which are concerned with fibrinogen binding to glycoprotein IIb/IIIa ( IIb/ 3) and granule secretion in platelets. Our finding suggests that cordycepin inhibits collagen-induced platelet aggregation with 261.1 M of IC 50 and also inhibits fibrinogen binding to IIb/ 3 by a suppression of PI3K/Akt phosphorylation in a dose dependent manner. In addition, cordycepin further showed to inhibit collagen-induced p38 phosphorylation, reducing granule secretion (i.e. ATP- and serotonin-release) and thromboxane A 2 (TXA 2) production without regulating cyclooxygenase-1 (COX-1) and thromboxane A synthase (TXAS) activities, as well as phospholipase C- 2 (PLC- 2) phosphorylation. In conclusion, these results demonstrate that cordycepin-mediated antiplatelet effects were due to the inhibition of fibrinogen binding to IIb/ 3 via the suppression of PI3K/Akt phosphorylation and inhibition of granule secretion & TXA 2 production by suppressing p38 phosphorylation. These results strongly indicate that cordycepin might have therapeutic or preventive potential for platelet aggregation-mediated disorders, regulating the phosphoprotein, including PI3K/Akt and p38. Key words: Cordycepin, Platelets, PI3K, Akt, p38 Corresponding author: Dong-Ha Lee Department of Biomedical laboratory Science, Korea Nazarene University, 48 Wolbong-ro, Seobuk-gu, Cheonan 31172, Korea Tel: 82-41-570-1680 Fax: 82-41-570-4258 E-mail: sec38@hanmail.net This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2017 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: April 7, 2017 Revised 1 st : April 21, 2017 Revised 2 nd : April 29, 2017 Accepted: May 1, 2017 서론혈관벽의손상시야기되는혈소판응집은지혈을위한필수적인과정이지만, 과도한경우에고지혈증과함께혈전증, 동맥경화증, 뇌줄중, 심근경색과같은심혈관계질환을유발하는요인이되기도한다 [1]. 그러므로, 혈소판응집을저해하는물질에 대한탐색및기전연구는심혈관계질환에대한예방및치료로이어질수있다 [2]. 다양한 agonists (collagen, thrombin, ADP) 은혈소판막의 integrin인 glycoprotein IIb/IIIa ( IIb/ 3) 를활성화시킴으로써 fibrinogen과의결합을유도하고, 이러한 inside-out signaling 은결과적으로 outside-in signaling 을야기하여혈소판의응집과혈소판내과립방출

100 Hyuk-Woo Kwon and Dong-Ha Lee. Inhibitory Effects of Cordycepin on Phosphoproteins 을촉진한다 [3,4]. IIb/ 3 활성화는 phosphatidylinositol 3-kinase (PI3K) 와 Akt같은 phosphoprotein 의인산화와관련이있으며 [5], 이들 phosphoprotein 의인산화에의해혈소판내세포골격의재조직화와과립방출이일어난다고알려져있다 [6]. 그러므로, PI3K/Akt 인산화의억제는항혈소판효과를보이는물질또는성분을평가하는데유용하다. 그예로, Abciximab, etifibatide, lamifiban, 그리고 tirofiban과같은 IIb/ 3 antagonist 들이항혈소판물질로이용되고있다 [7]. 또한, Collagen 은혈관손상시에혈관벽에서노출되는물질로서, 혈소판막의 collagen 수용체인 glycoprotein (GP)VI를통해 phospholipase C- 2 (PLC- 2 ) 의인산화반응을촉진함으로써혈소판을활성화시킨다 [8]. 활성화된 PLC- 2 가 phosphatidylinositol 4, 5-bisphosphate (PIP 2) 를 inositol 1, 4, 5-trisphosphate (IP 3 ) 와 diacylglycerol (DG) 로가수분해하고, 생성된 IP 3 는혈소판내 DTS (Dense Tubular System) 의 IP 3 수용체와결합하여 Ca 2+ channel을열어 free Ca 2+ ([Ca 2+ ] i) 의동원을야기한다 [9]. 혈소판내증가된 [Ca 2+ ] i 은 calmodulin 과결합하여 myosin light chain kinase를활성화시켜세포골격을이루는단백질인 myosin light chain을인산화함으로써혈소판의형태변화를일으켜응집을유도한다 [10,11]. 한편, DG 는 DG-와 monoacylglycerol (MG)-lipase에의해분해되며 arachidonic acid (AA) 를생성시키는데, AA가 cyclooxygenase-1 (COX-1) 과 thromboxane A synthase (TXAS) 의효소작용에의해 TXA 2 로바뀐다. 이때생성된 TXA 2 는주변의다른혈소판막수용체에결합하여혈소판응집을촉진하는강력한 agonist 로작용한다고알려져있다 [12]. 뿐만아니라, 과립방출을통해방출되는 ATP 및 serotonin 등의물질이혈소판응집에있어서중요하며, 이들은 MAPK (p38, ERK, 그리고 JNK) phosphoprotein 의인산화와관련되어있다고알려져있다. 특히, MAPK 중에 p38는혈소판내과립방출및 myosin light chain의활성화를자극함으로써 clot retraction 을유도하는데중요하게작용한다 [13]. 그러므로, 이들을어떻게조절하는지확인하는것이항혈소판효과를가지는물질과성분을평가하는데있어서매우유용하다. Cordyceps는진균속에속하는종으로서중국의전통약제로알려져있으며, 염증과암에있어서유용하게처방되어오고있다 [14]. 혈소판의활성에관해서는, Cordyceps militaris의유효성분으로알려진 cordycepin (3'-deoxyadenosine, Figure 1) 이 camp와 cgmp에의존적으로 [Ca 2+ ] i 을억제함으로써혈소판활성을저해한다고보고된바있다 [15]. 또한, 우리의앞선연구에서는, Cordyceps militaris 를 n-butanol 로추출한 extracts (WIB801C) 가혈소판막의 IIb/ 3 에 fibrinogen 이결합하는것을억제한다고보고된바있다 [16]. 본연구에서는, cordycepin 이 fibrinogen binding에관여한다고알려진 PI3K/Akt와 TXA 2 생성및과립방출에관여한다고알려진 p38 와같은 phosphoprotein 의인산화를어떻게조절하며혈소판응집을억제에관여하는지규명하고자하였다. 재료및방법 1. 실험재료 Collagen은 Chrono-Log 사 (Havertown, PA, USA) 에서, TXB 2 enzyme immunoassay (EIA) kits와 COX fluorescent activity assay kit는 Cayman Chemical 사 (Ann Arbor, MI, USA) 로부터구입하였다. Cordycepin 및그밖의시약들은 Sigma Aldrich 사 (Saint Louis, MO, USA) 에서구입하였고, Serotonin EIA kit는 Labor Diagnostika Nord 사 (Nordhorn, Germany) 에서 ATP assay kit는 Biomedical Research Service Center (Buffalo, NY, USA) 에서구입하였다. Western blotting 용 antibody들과 lysis buffer는 Cell Signaling (Beverly, MA, USA) 에서구입하였고, Polyvinylidene difluoride (PVDF) membrane와 Enhanced chemiluminesence solution (ECL) 는 GE Healthcare (Buckinghamshire, UK) 에서, 그리고 Fibrinogen Alexa Fluor 488 conjugate는 Invitrogen Molecular Probes (Eugene, OR, USA) 에서구입하였다. 2. 실험방법 1) 세척혈소판준비 Acid-citrate-dextrose solution (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose) 로항응고처리된 human platelet-rich plasma (PRP) 를한국적십자혈액원 (Changwon, Figure 1. Chemical structure of cordycepin (3'-deoxyadenosine).

Korean J Clin Lab Sci. Vol. 49, No. 2, June 2017 101 Korea) 으로부터제공받았다. 미량의적혈구를제거하기위해 PRP를 125 g에서 10분간원심분리한후, 1,300 g에서 10 분간원심분리하여 platelet pellets을얻었다. 이것을 washing buffer (138 mm NaCl, 2.7 mm KCl, 12 mm NaHCO 3, 0.36 mm NaH 2 PO 4, 5.5 mm glucose, and 1 mm EDTA, ph 6.5) 로두번세척하고. 세척된혈소판을 suspension buffer (138 mm NaCl, 2.7 mm KCl, 12 mm NaHCO 3, 0.36 mm NaH 2PO 4, 0.49 mm MgCl 2, 5.5 mm glucose, 0.25% gelatin, ph 6.9) 로재구성하여최종 10 8 /ml 농도가되게하였다. 위에있는모든과정은낮은온도에서일어날수있는혈소판응집을피하기위하여 25 C에서수행하였다. 이실험은 The Korea National Institute for Bioethics Policy Public Institutional Review Board (Seoul, Korea) 의승인을받아수행되었다 (PIRB12-072). 2) 혈소판응집반응의측정세척혈소판 (10 8 /ml) 에여러농도의 cordycepin 을첨가하여 37 C에서 3분간전처리한후, 10 g/ml collagen으로응집을유도하고 5분간측정하였다. 응집은 1,000 rpm stirring speed에서 560CA aggregometer (Chrono-Log, Havertown, PA, USA) 로측정하였고, 응집능은빛투과도의증가된정도로산출하였다. Suspension buffer를투과도 0% 의기준값으로사용하였고, cordycepin는 suspension buffer (ph 6.9) 에녹여서사용하였다. 여 37 C에서 3분간전처리한후, 30 g/ml Alexa Flour 488-human fibrinogen을첨가하여 10 g/ml collagen으로응집을유도하고 5분간반응시켰다. 0.5% paraformaldehyde in phosphate-buffered saline으로반응을정지시키고, 혈소판에결합한 Alexa Fluor 488-fibrinogen 의형광을 FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) 로측정하였고결과값을 cellquest software로분석하였다. 5) ATP 방출의측정세척혈소판 (10 8 /ml) 에여러농도의 cordycepin 을첨가하여 37 C에서 3분간전처리한후, 10 g/ml collagen 으로응집을유도하고 5분간반응시켰다. Ice-cold 1 mm EDTA 으로반응을정지한후, 원심분리하여상층으로방출된 ATP 를 ATP assay kit를사용하여 Synergy HT multi-model microplate reader (BioTek Instruments, USA) 로측정하였다. 6) Serotonin 방출의측정세척혈소판 (10 8 /ml) 에여러농도의 cordycepin 을첨가하여 37 C에서 3분간전처리한후, 10 g/ml collagen 으로응집을유도하고 5분간반응시켰다. Ice-cold 1 mm EDTA 으로반응을정지한후, 원심분리하여상층으로방출된 serotonin 을 serotonin EIA kit를사용하여 synergy HT multi-model microplate reader (BioTek Instruments, USA) 로측정하였다. 3) Western blot법을이용한 phosphoprotein 의인산화측정세척혈소판 (10 8 /ml) 에여러농도의 cordycepin 을첨가하여 37 C에서 3분간전처리한후, 10 g/ml collagen으로응집을유도하고 5분간반응시켰다. 동량의 lysis buffer를첨가함으로써반응을정지시켰다. 혈소판용해물을 bicinchoninic acid protein assay kit (Pierce Biotechnology, IL, USA) 를사용하여단백질정량하였고동량의단백질 (15 g) 을분석에사용하였다. Cordycepin이 phosphoprotein (PI3K, Akt, p38, PLC- 2 ) 의인산화에미치는효과를 western blot법으로측정하였다. 전기영동에 6-8% SDS-PAGE 를사용하였고 PVDF membrane 에단백질을 transfer 하였으며, transfer 한 membrane 은 ECL 시약을반응시켜발색하였다. Quantity One, Version 4.5 (Bio-Rad, Hercules, CA, USA) 를사용하여 band의밀도를분석하였다. 4) 혈소판에대한 fibrinogen 결합능측정세척혈소판 (10 8 /ml) 에여러농도의 cordycepin 을첨가하 7) TXB 2 생성량의측정세척혈소판 (10 8 /ml) 에여러농도의 cordycepin 을첨가하여 37 C에서 3분간전처리한후, 10 g/ml collagen 으로응집을유도하고 5분간반응시켰다. Ice-cold 1 mm EDTA 와 0.2 mm indomethacin 을첨가하여반응을정지시켰다. TXA 2 의안정한대사체인 TXB 2 의양을 TXB 2 EIA kit 를사용하여 synergy HT multi-model microplate reader (BioTek Instruments, USA) 로측정하였다. 8) Cyclooxygenase-1 (COX-1) 의활성측정세척혈소판 (10 8 /ml) 에 1% protease inhibitor cocktail (Sigma Aldrich, USA) 를첨가하여 sonicator (Bandelin Electronic, Bandelin, Germany) 로 100% 감도에서 20초동안 10번 sonication 하여혈소판용해물을만들었다. 이를 4 C에서 12,000 g로 15분간원심분리하여세포 debris를제거한상층에 cordycepin 을첨가하여 37 C에서 30분간전처리한후, COX-1 활성을 COX activity assay kit를사용하여 synergy

102 Hyuk-Woo Kwon and Dong-Ha Lee. Inhibitory Effects of Cordycepin on Phosphoproteins HT multi-model microplate reader (BioTek Instruments, USA) 로측정하였다. 9) Thromboxane A Synthase (TXAS) 의활성측정세척혈소판 (10 8 /ml) 에 1% protease inhibitor cocktail (Sigma Aldrich, USA) 를첨가하여 sonicator (Bandelin Electronic, Bandelin, Germany) 로 100% 감도에서 20초동안 10번 sonication 하여혈소판용해물을만들었다. 이를 4 C에서 12,000 g로 15분간원심분리하여세포 debris를제거한상층에 cordycepin 을첨가하여 37 C에서 30분간전처리한후, prostaglandin H 2 (PGH 2 ) 를첨가하여 37 C에서 1분간효소반응을일으키고 1 M citric acid로정지시켰다. 1 N NaOH 를첨가하여중화시킨후, TXA 2 의안정한대사체인 TXB 2 의양을 TXB 2 EIA kit를사용하여 synergy HT multi-model microplate reader (BioTek Instruments, USA) 로측정하였다. 10) 통계분석측정된모든실험결과들은 mean±sem로처리하여 analysis of variance (ANOVA) 로분석하였다. 그룹간의평균에유의적인차이가있을경우, Newman-Keuls method로비교하여각그룹간에표기하였다. p<0.05 일때유의적인의미가있는것으로판단하였다. 결과 1. Cordycepin 이 collagen 유도혈소판응집에미치는효과혈소판을최대치로응집시키는 collagen의농도가약 10 g/ml이었기에, 본실험에서는 10 g/ml collagen으로응집을유도하였다 [17]. 혈소판에 collagen 을첨가하여응집을유도하였을때, 응집율이 72.4±2.2% 로나타났고, cordycepin 을여러농도 (50 500 M) 로첨가하였을때에농도의존적으로강하게응집이억제되는결과를확인하였다 (Figure 2A). 이때, cordycepin의 the half maximal inhibitory concentration (IC 50) 값은 261.1 M (Figure 2B) 로산출되었다. 이는 cordycepin 이강력한항혈소판효과를가지고있는물질임을보여주는결과이다. 2. Cordycepin 이 PI3K- 및 Akt-인산화와 fibrinogen 결합에미치는효과 Figure 2. Effect of cordycepin on collagen-induced platelet aggregation, and its IC 50 value (A) Effect of cordycepin on collagen-induced platelet aggregation. (B) IC 50 value of cordycepin on collagen-induced platelet aggregation. The data are expressed as the mean±sem (N=4). *p<0.05, **p<0.001 versus the collagen-stimulated platelets. 리고, collagen 에의해증가된 PI3K의인산화가 cordycepin 에의해농도의존적으로감소되었다 (Figure 3A lane 3,4). 또한, collagen은 PI3K 표적분자인 Akt의인산화를증가시켰지만 (Figure 3B lane 1,2), 이또한, cordycepin 을첨가하였을경우에농도의존적으로감소되었다 (Figure 3B lane 3,4). 이는 collagen에의해촉진되는 phosphoprotein (PI3K, Akt) 의인산화를 cordycepin 이억제한다는것을보여주는결과이다. 이에더하여, cordycepin이 IIb/ 3 에대한 fibrinogen의결합에미치는효과를확인하였을때, collagen 이 intact cell에비해 77.2±5.5% 로증가시킨것을 (Figure 4A-a,b and 4B), cordycepin이 15.1±0.6% 까지억제하는결과도확인하였다 (Figure 4A-c and 4B). Figure 3A의 lane 1, 2에서보여지는바와같이, collagen 은 intact cell에비하여 PI3K의인산화를강하게증가시켰다. 그

Korean J Clin Lab Sci. Vol. 49, No. 2, June 2017 103 Figure 3. Effects of cordycepin on PI3K- and Akt-phosphorylation. (A) Effects of cordycepin on PI3K phosphorylation. (B) Effects of cordycepin on Akt phosphorylation. The phosphorylation was determined as described in Methods. These data were expressed as the mean±sem (N=4). a p<0.05 versus the non-stimulated platelets, *p<0.05 versus the collagen-stimulated platelets,**p<0.001 versus the collagen-stimulated platelets. Figure 4. Effects of cordycepin on collagen-induced fibrinogen binding. (A) The flow cytometry histograms on collagen-induced fibrinogen binding. (a) Intact platelets (base); (b) Collagen (10 g/ml); (c) Collagen (10 g/ml) + cordycepin (500 M). (B) Effects of cordycepin on collagen-induced fibrinogen binding (%). The reactions were determined as described in Methods. The data are expressed as the mean±sem (N=4). a p<0.05 versus non-stimulated platelets, **p<0.001 versus the collagen-stimulated platelets.

104 Hyuk-Woo Kwon and Dong-Ha Lee. Inhibitory Effects of Cordycepin on Phosphoproteins 3. Cordycepin 이 p38의인산화와 ATP, serotonin 의방출에미치는효과혈소판내과립방출의유도체로알려진 MAPK의하나인 p38 phosphoprotein 의인산화가 cordycpein 에의해어떻게조절되는지확인한결과, collagen 에의해촉진된 p38의인산화가 cordycepin 에의해농도의존적으로강하게억제되는것을확인할수있었다 (Figure 5A). 또한, 혈소판내과립방출정도를확인하는데이용되는지표로서 ATP 와 serotonin 방출결과를확인한결과, collagen은 3.91±0.21 M 로 intact cell에비해 43.4배정도 ATP 방출을촉진하였고, cordycepin 이이를 58.1% (1.64±0.06 M) 로강하게감소시켰다 (Figure 5B). Serotonin 방출에있어서는, intact cell에서 11.53±1.58 ng/10 8 platelets 정도였던 serotonin 양을 collagen이 84.67±6.51 ng/10 8 platelets까지강하게방출하게하였고, cordycepin 에의해 62.9% (31.38±1.97 ng/10 8 platelets) 로억제되었다 (Figure 5C). 4. Cordycepin 이 TXA 2 생성과 COX-1 와 TXAS 활성및 PLC- 2 의인산화에미치는효과 ATP, serotonin 와함께 p38의인산화에영향을받는다고알려진 TXA 2 생성에있어 cordycepin 이어떤영향을미치는지도확인하였다. 그결과, Figure 6A에서보여지는바와같이, intact cell에서 0.6±0.1 ng/10 8 platelets였던 TXA 2 양이 collagen에의해 60.1±1.0 ng/10 8 platelets로강하게증가하였고, cordycepin은 collagen에의해증가한 TXA 2 를 17.7±0.7 ng/10 8 platelets 로 70.5% 억제하였다. TXA 2 생성은혈소판막 에있는 PIP 2 가 PLC- 2 에의해분해되어나오는 AA로부터 COX-1 과 TXAS의효소작용에의해만들어진다 [17,18]. 따라서, 이들에미치는 cordycepin 의효과를함께살펴보았다. 그결과, Figure 6B 및 6C에서보여주는바와같이, cordycepin 이 COX-1 과 TXAS 활성에유의적인영향을미치지못한다는것을알수있었다. 또한, PLC- 2 인산화에있어서도, collagen이강하게증가시킨 PLC- 2 인산화에 cordycepin 이유의적인영향을미치지못하였음을확인하였다 (Figure 6D lane 2,3,4). 고찰혈소판의활성에있어서, cordycepin (3'-deoxyadenosine, Figure 1) 에관련된작용들이보고된바가있지만 [15,16], phosphoprotein 의인산화에미치는효과에대한연구가미흡하였기에, 본연구에서는이를확실히규명하고자하였다. 그결과, collagen이유도한혈소판응집에있어서 cordycepin 이 261.1 M의 IC 50 으로농도의존적으로강력하게응집을억제하고있음을보여주었고 (Figure 2), 이는몇가지중요한 phosphoprotein (PI3K, Akt, p38) 의인산화에의한것임을확인할수있었다. 우선, cordycepin 은 collagen 에의해증가된 PI3K 와 Akt의인산화를농도의존적으로감소시켰고 (Figure 3), 또한 IIb/ 3 에대한 fibrinogen 결합을농도의존적으로억제하였다. PI3K와 Akt의인산화는 IIb/ 3 를활성화하여 fibrinogen 결합을증가시킴으로써혈소판응집을유도한다는연구들이보고된바있다 [5,20]. 따라서, cordycepin 에의한혈소판응집의억제는 cordycepin 에의해 PI3K/Akt phophoprotein 의인산화가억제됨으로써 IIb/ 3 의비활성화에의한 fibrinogen 결 Figure 5. Effects of cordycepin on p38 phosphorylation, and ATP-and serotonin-release. (A) Effects of cordycepin on p38 phosphorylation in collagen-induced platelets. The phosphorylation was determined as described in Methods. (B) Effects of cordycepin on ATP release in collagen-induced platelets. (C) Effects of cordycepin on serotonin release in collagen-induced platelets. ATP and serotonin release were measured using EIA kits. These data were expressed as the mean±sem (N=4). a p<0.001 versus the non-stimulated platelets, **p<0.001 versus the collagen-stimulated platelets.

Korean J Clin Lab Sci. Vol. 49, No. 2, June 2017 105 Figure 6. Effects of cordycepin on collagen-induced TXA 2 production, COX-1 and TXAS activities, and PLC- 2 phosphorylation (A) Effects of cordycepin on collagen-induced TXA 2 production. TXA 2 was determined by TXB 2 EIA kit as described in Methods. (B) Effects of cordycepin on COX-1 activity in platelet lysates. (C) Effects of cordycepin on TXAS activity in platelet lysates. COX-1 and TXAS activity was determined by COX activity assay and TXB 2 EIA kit as described in Methods. (D) Effect of PLC- 2 phosphorylation. The phosphorylation was determined as described in Methods. These data were expressed as the mean±sem (N=4). a p<0.05 versus the non-stimulated platelets, **p<0.001 versus the collagen-stimulated platelets. 합이감소한결과로보여진다. 또한, 혈소판억제작용에대한물질이나성분을평가할때, 중요한지표로확인하는것이강력한 autacoid로알려진 TXA 2 의생성량이다. Collagen이유도하는혈소판의활성화과정에서, collagen이혈소판막에있는 collagen 수용체인 glycoprotein (GP) VI에결합하면 tyrosine kinase가활성화됨으로써 phospholipase C- 2 (PLC- 2 ) 이인산화를일으킨다 [8]. 이는막에위치한 phosphatidylinositol 4, 5-bisphosphate (PIP 2 ) 를분해하여 diacylglycerol (DG) 를방출시키고, DG-와 monoacylglycerol (MG)-lipase 의효소작용으로 arachidonic acid (AA) 가되는데, 이AA가 cyclooxygenase-1 (COX-1) 과 thromboxane A synthase (TXAS) 의효소작용에의해 TXA 2 로전환되어다른혈소판을응집하는강력한 agonist로작용한다 [9,12]. 따라서, TXA 2 의생성을저해하는물질들이항혈소판물질로서유용하게이용되고있다. 그예로, COX-1 의활성을저해하는 aspirin, TXAS의활성을억제하는 ozagrel과같은물질들이있다 [18,19]. 또한, 우리의앞선연구에서, 항혈소판효과를나타내는물질들 (caffeic acid, EGCG, chlorogenic acid, total sapinin form Korea Red Ginseng) 에서도 TXA 2 의생성이억제되는것을확인할수있다 [17,21-23]. 그러나, cordycepin 이어떤과정을통해 TXA 2 의생성을억제하였는지에대한연구결과는불명확하다. Mei-Chi등의연구에의하면 [24], MAPK

106 Hyuk-Woo Kwon and Dong-Ha Lee. Inhibitory Effects of Cordycepin on Phosphoproteins 중하나인 p38의인산화가 AA방출과 TXA 2 의생성에있어서결정적으로작용하여혈소판응집을일으키는것으로알려져있다. 뿐만아니라, p38의인산화가 ATP 및 serotonin 방출과같은혈소판과립방출을유도하고, myosin light chain의활성화를자극하여 clot retraction 을유도하는데중요하게작용한다고보고되어있다 [13]. 본연구에서는, cordycepin 이 p38의인산화에어떻게관여하고있는지규명하고, 이를통한과립방출 (ATP, secretion) 의정도와 TXA 2 의생성에미치는영향을살펴보았다. 아울러, TXA 2 의생성에관여하는 COX-1 및 TXAS의활성과 PLC- 2 의인산화에 cordycepin 이관여하는지확인함으로써그상관관계를분명히하고자하였다. 그결과, collagen 이촉진시킨 p38의인산화가 cordycepin 에의하여농도의존적으로강하게감소되었고 (Figure 4A), 이와함께과립방출의지표인 ATP 및 serotonin의방출이강하게억제되었고 (Figure 4B, 4C), TXA 2 의생성량도 cordycepin 에의해감소되는것을확인할수있었다 (Figure 5A). 그러나, TXA 2 생성에관여하는효소인 COX-1 과 TXAS의활성은 cordycepin 에의해영향을받지않았고, PLC- 2 의인산화도 cordycepin 에의한변화를확인하지못하였다. 이는, cordycepin 에의한 TXA 2 생성량감소가 COX-1 및 TXAS의활성과 PLC- 2 의인산화의조절에의한것이아님을의미하며, cordycepin 가 p38의인산화를억제하여과립방출 (ATP 및 serotonin 방출 ) 과함께 TXA 2 생성량을감소시킴으로써혈소판응집을저해한것을의미한다. 결론적으로, cordycepin은 PI3K/Akt, p38과같은 phophoprotein 의인산화를억제시킴으로써, IIb/ 3 에대한 fibrinogen 결합및과립방출과 TXA 2 생성량을감소시켜혈소판응집을저해하였음이규명되었다. 따라서, cordycepin 은혈소판응집으로인한심혈관계질환에있어서치료및예방약물로유용한가치가있다고여겨진다. 요약진균속에속하는종인 Cordyceps는중국의전통약제로서, 그유효성분인 cordycepin 이혈소판응집에관여한다는보고가있지만 phosphoprotein 조절에관련된연구는미흡하다. 본연구에서는, cordycepin이 fibrinogen binding에관여한다고알려진 PI3k/Akt 와 TXA 2 분비및과립방출에관여한다고알려진 p38와같은 phosphoprotein 의인산화를어떻게조절하며혈소판응집을억제시키는지규명하고자하였다. 그결과, cordycepin가 261.1 M 의 IC 50 으로 collagen 이유도한혈소판응집을강력하게억제하였고, PI3K와 Akt의인산화를감소 시키며 IIb/ 3 에대한 fibrinogen 결합을농도의존적으로억제하였다. 또한, cordycepin 은 collagen 이촉진시킨 p38의인산화를억제함으로써, 과립방출의지표인 ATP 과 serotonin 의방출을억제하였고 COX-1 과 TXAS의활성및 PLC- 2 인산화에대한영향없이 TXA 2 생성량을감소시켰다. 따라서, cordycepin은 PI3K/Akt, p38와같은 phosphoprotein의인산화를억제함으로써혈소판응집억제를나타내는항혈전치료및예방약물로서유용한가치가있다고여겨진다. Acknowledgements: This Research was supported by the Korea Nazarene University Research Grants 2017 and a grant (NRF-2011-0012143 to Hwa-Jin Park) from Basic Science Research Program via the National Research Foundation of Korea funded by the Ministry of Education (Science and Technology, Korea). Funding: Korea Nazarene University Research Grants 2017. Conflict of interest: None REFERENCES 1. Lee MR, Choi JH, Yang Y, Oh KS, Jeong TS, Lee CH, et al. Attenuation of Atherosclerosis by 3,4-Dihydroxy-Hydrocinnamic Acid in Rabbits by Partial Inhibition of ACAT. Korean J Clin Lab Sci. 2016;48(4):280-286. 2. Schwartz SM, Heinmark RL, Majesky MW. Developmental mechanisms underlying pathology of arteries. Physiol Rev. 1990;70(4):1177-1209. 3. Payrastre B, Missy K, Trumel C, Bodin S, Plantavid M, Chap H. The integrin alpha IIb/beta 3 in human platelet signal transduction. Biochem Pharmacol. 2000;60(8):1069-1074. 4. Phillips DR, Nannizzi-Alaimo L, Prasad KS. Beta3 tyrosine phosphorylation in alphaiibbeta3 (platelet membrane GP IIb-IIIa) outside-in integrin signaling. Thromb Haemost. 2001; 86(1):246-258. 5. Morello F, Perino A, Hirsch E. Phosphoinositide 3-kinase signalling in the vascular system. Cardiovasc Res. 2009;82(2): 261-271. 6. Jennings LK. Role of platelets in atherothrombosis. Am J Cardiol. 2009;103(3 Suppl):4-10. 7. Sabatine MS, Jang IK. The use of glycoprotein IIb/IIIa inhibitors in patients with coronary artery disease. Am J Med. 2000; 109(3):224-237. 8. Wonerow P, Obergfell A, Wilde JI, Bobe R, Asazuma N, Brdicka T, et al. Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets. Biochem J. 2002;364(3): 755-765. 9. Quinton TM, Dean WL. Cyclic AMP-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor inhibits

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