Biomol Ther (Seoul) 2015; 23(1): 60-70  https://doi.org/10.4062/biomolther.2014.086
Effect of Cordycepin-Enriched WIB801C from Cordyceps militaris Suppressing Fibrinogen Binding to Glycoprotein IIb/IIIa
Dong-Ha Lee1,†,ψ, Hyun-Hong Kim1,†, Deok Hwi Lim1, Jong-Lae Kim2, and Hwa-Jin Park1,*

1Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering, Inje University, Gimhae 621-749

2Natural Product Research Team, Central Research Center, Whanin Pharm. Co., Ltd., Suwon 443-766, Republic of Korea

*Corresponding Author: E-mail: mlsjpark@inje.ac.kr, Tel: +82-55-320-3538, Fax: +82-55-334-3426
Received: July 20, 2014; Revised: November 1, 2014; Accepted: November 4, 2014; Published online: January 1, 2015.
© The Korean Society of Applied Pharmacology. All rights reserved.

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

In this study, we investigated the effects of cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha on collagen-stimulated platelet aggregation. CE-WIB801C dose dependently inhibited collagen-induced platelet aggregation, and had a synergistic effect together with cordycepin (W-cordycepin) from CE-WIB801C on the inhibition of collagen-induced platelet aggregation. CE-WIB801C and cordycepin stimulated the phosphorylation of VASP (Ser157) and the dephosphorylation of PI3K and Akt, and inhibited the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/β3) and the release of ATP and serotonin in collagen-induced platelet aggregation. A-kinase inhibitor Rp-8-Br-cAMPS reduced CE-WIB801C-, and cordycepin-increased VASP (Ser157) phosphorylation, and increased CE-WIB801C-, and cordycepin-inhibited the fibrinogen binding to αIIb/β3. Therefore, we demonstrate that CE-WIB801C-, and cordycepin-inhibited fibrinogen binding to αIIb/β3 are due to stimulation of cAMP-dependent phosphorylation of VASP (Ser157), and inhibition of PI3K/Akt phosphorylation. These results strongly indicate that CE-WIB801C and cordycepin may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Keywords: CE-WIB801C, Cordycepin, VASP (Ser157), PI3K/Akt, Fibrinogen binding
INTRODUCTION

Platelet aggregation is caused by “inside-out signaling” and “outside-in signaling”, which is absolutely essential for the formation of a hemostatic plug when normal blood vessels are injured. However, platelet aggregation can also cause circulatory disorders, such as thrombosis, atherosclerosis, and myocardial infarction (Schwartz et al., 1990).

Various agonists (i.e. collagen, thrombin, ADP) induce “inside-out signaling” to bind fibrinogen to glycoprotein IIb/IIIa (αIIb/β3), platelet membrane integrin, then subsequently cause “outside-in signaling” by binding fibrinogen to αIIb/β3 (van Willigen and Akkerman, 1991; Payrastre et al., 2000; Phillips et al., 2001). Vasodilator-stimulated phosphoprotein (VASP) phosphorylation inhibits VASP affinity for contractile protein filamentous actin, and fibrinogen binding to αIIb/β3 to inhibit the final common pathway for platelet aggregation (Laurent et al., 1999; Sudo et al., 2003). αIIb/β3 activation is also stimulated by phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation (Morello et al., 2009). In special, the interaction between αIIb/β3 and plasma fibrinogen is known to involve in [Ca2+]i mobilization, tyrosine phosphorylation of Src and Syk, activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C-γ2 phosphorylation, cytoskeleton reorganization and granule secretion (Clutton et al., 2001; Jennings, 2009). Therefore, the stimulation of VASP phosphorylation, and the inhibition of PI3K/Akt phosphorylation are very useful for evaluating the antiplatelet effect of substances or compounds. For instance, a major catechin analogue, (-)-epigallocatechin- 3-gallate from green tea, is known to produce cAMP via adenylate cyclase activation and subsequently phosphorylates VASP (Ser157) through cAMP- dependent protein kinase (A-kinase) activation to inhibit [Ca2+]i mobilization (Ok et al., 2012), and caffeic acid, a phenolic acid, is reported to phosphorylate VASP (Ser157) through cAMP/A-kinase pathway to inhibit [Ca2+]i mobilization (Lee et al., 2014a). In addition, Abciximab, etifibatide, tirofiban, and lamifiban are known as αIIb/β3 antagonists (Lincoff et al., 2000; Sabatine and Jang, 2000).

In previous reports (Cho et al., 2006, 2007a, 2007b), we suggested that cordycepin (3′-deoxyadenosine, Fig. 1) inhibits [Ca2+]i mobilization in collagen-, TXA2 analogue U46619-, and tumor promoter thapsigargin-activated human platelets. In addition, cordycepin strongly inhibited the phosphorylation of Ca2+-dependent proteins (myosin light chain and pleckstrin) by suppressing collagen-, and U46619-elevated [Ca2+]i mobilization (Cho et al., 2006; 2007a). In recent, we prepared cordycepin-enriched WIB801C (Compound from 2008 First Project of Bioteam, Whanin Pharm. Co., Ltd., Suwon, Korea), a n-butanol extract from Cordyceps militaris-hypha (Lee et al., 2014b), and reported that cordycepin-enriched (CE) WIB801C has an antiplatelet effect by inhibiting collagen-induced [Ca2+]i mobilization via cAMP-dependent phosphorylation of inositol 1, 4, 5-trisphosphate receptor (IP3R) in human platelet (Lee et al., 2014b). In this study, we investigated the novel effects of CE-WIB801C on the phosphorylation of VASP and dephosphorylation of PI3K and Akt affecting on fibrinogen binding to αIIb/β3.

MATERIALS AND METHODS
Materials

Collagen was purchased from Chrono-Log Co. (Havertown, PA., USA). ATP assay kit was purchased from Biomedical Research Service Center (Buffalo, NY., USA). Cordycepin, A-kinase inhibitor Rp-8-Br-cAMPS, cGMP-dependent protein kinase (G-kinase) inhibitor Rp-8-Br-cGMPS, A-kinase activator pCPT-cAMP, and G-kinase activator 8-Br-cGMP were obtained from Sigma Chemical Corporation (St. Louis, MO., USA). Serotonin ELISA kit was purchased from Labor Diagnostika Nord GmbH & CO. (Nordhorn, Germany). Anti-VASP, anti-phosphor- VASP (Ser157), anti-phosphor-VASP (Ser239), anti-PI3K, anti-phosphor- PI3K, anti-Akt, anti-phosphor-Akt, and anti-rabbit IgG-horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling (Beverly, MA., USA). Poly-vinylidene difluoride (PVDF) membrane was from GE Healthcare (Piseataway, NJ., USA). Enhanced chemiluminesence solution (ECL) was from GE Healthcare (Chalfont St., Giles, Buckinghamshire, UK). Fibrinogen Alexa Fluor 488 conjugate was obtained from Invitrogen Molecular Probes (Eugene, OR., USA).

Preparation of CE-WIB801C and quantity of cordycepin

The preparation of CE-WIB801C was performed according to the method of our previous report (Lee et al., 2014b). Culture- solution of Cordyceps militaris-hypha was concentrated with a rotary vacuum evaporator (Eyela N3000, Rikakikai Co. Ltd., Tokyo, Japan) at 60°C. The concentrate was extracted by extraction-shaker (Cosmos 660, Kyungseo Co. Ltd., Seoul, Korea) at 60°C two times with n-butanol, which was filtered two times using a filter paper (Advantec No.2). The filtrate was completely concentrated by an evaporator (Eyela N3000, Rikakikai Co. Ltd., Tokyo, Japan) under reduced pressure (40°C), and was lyophilized and stored at −20°C until used. The quantity of cordycepin in CE-WIB801C was determined with calibration curve of (authentic cordycepin : 50, 100, 200, and 400 μg/mL) by HPLC as described in our previous report (Lee et al., 2014b).

Purification of cordycepin in CE-WIB801C with prep-HPLC

The purification of cordycepin from CE-WIB801C was performed according to the Lee method (Lee et al., 2014c). The methanol (50%) extract from CE-WIB801C was dissolved with 50% methanol and then purified by prep-HPLC. An Agilent 1100 liquid chromatography system (Palo Alto, CA, USA), equipped with vacuum degasser, quaternary gradient pump, autosampler and DAD, connected to an Agilent ChemStation software. A Jupiter C18 column (250 mm×21.2 mm id, 5 μm) were used at a column temperature of 25°C. The mobile phase consisted of water (A) and methanol with 0.01M KH2PO4 (B) using the following program: 0-30 min, 15% B. The flow rate was at 25 mL/min and sample injection volume was 1.5 mL. The UV detection was operated at 254 nm. The purified cordycepin were freeze-dried using a freeze dryer (Clean-vac 24T, Biotron, Korea) to obtain powder, which were analyzed and calculated by analytic HPLC above condition. The cordycepin was dissolved in distilled water, and used to investigate the effects on platelet aggregation. In this study, the cordycepin from CE-WIB801C was called as W-cordycepin to differentiate from authentic cordycepin.

Preparation of washed human platelets

Human platelet-rich plasma (PRP) anti-coagulated with acid-citrate-dextrose solution (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose) were obtained from Korean Red Cross Blood Center (Changwon, Korea). PRP was centrifuged for 10 min at 125×g to remove a little red blood cells, and was centrifuged for 10 min at 1,300×g to obtain the platelet pellets. The platelets were washed twice with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM glucose, and 1 mM EDTA, pH 6.5). The washed platelets were then resuspended in suspension buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH 6.9) to a final concentration of 5×108/mL. All of the above procedures were carried out at 25°C to avoid platelet aggregation from any effect of low temperature. The Korea National Institute for Bioethics Policy Public Institutional Review Board (Seoul, Korea) approved these experiments (PIRB12-072).

Measurement of platelet aggregation

Washed platelets (108/mL) were preincubated for 3 min at 37°C in the presence of 2 mM CaCl2 with or without substances (CE-WIB801C, authentic cordycepin, W-cordycepin, and so on), then stimulated with collagen (10 μg/mL) for 5 min. Aggregation was monitored using an aggregometer (Chrono- Log Corporation, Havertown, PA., USA) at a constant stirring speed of 1,000 rpm. Each aggregation rate was calculated as an increase in light transmission. The suspension buffer was used as the reference (transmission 0).

Western blot for analysis of VASP-, PI3K-, and Akt-phosphorylations

Washed platelets (108/mL) were preincubated with or without substances in the presence of 2 mM CaCl2 for 3 min and then stimulated with collagen (10 μg/mL) for 5 min at 37°C in an aggregometer (Chrono-Log, Corp., Havertown, PA., USA) at a constant stirring speed of 1,000 rpm. The reactions were terminated by adding an equal volume (250 μL) of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM serine/threonine phosphatase inhibitor β-glycerophosphate, 1 mM ATPase, alkaline and acid phosphatase, and protein phosphotyrosine phosphatase inhibitor Na3VO4, 1 μg/mL serine and cysteine protease inhibitor leupeptin, and 1 mM serine protease and acetylcholinesterase inhibitor phenylmethanesulfonyl fluoride, pH 7.5). Platelet lysates containing the same protein (20 μg) were used for the analysis. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce Biotechnology, IL., USA). The effects of substances on VASP-, PI3K-, and Akt-phosphorylation were analyzed using Western blotting. A 6–8% SDS-PAGE was used for electrophoresis and a PVDF membrane was used for protein transfer from the gel. The dilutions for anti-VASP, anti-phosphor-VASP (Ser157), antiphosphor- VASP (Ser239), anti-PI3K, anti-phosphor-PI3K, anti- Akt, anti-phosphor-Akt, and anti-rabbit IgG-HRP were 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, and 1:10000, respectively. The membranes were visualized using ECL. The blots were analyzed using the Quantity One, Ver. 4.5 (Bio- Rad, Hercules, CA., USA).

Determination of fibrinogen binding to αIIb/β<sub>3</sub>

Washed platelets (108/mL) were preincubated for 3 min at 37°C with or without substances in the presence of 2 mM CaCl2 and then stimulated with collagen (10 μg/mL) in the presence of Alexa Flour 488-human fibrinogen (30 μg/mL) for 5 min at 37°C. The reaction was stopped by the addition of 0.5% paraformaldehyde in phosphate-buffered saline, and the samples were placed in the dark. Alexa Fluor 488-fibrinogen binding to platelets was determined using flow cytometry (BD Biosciences, San Jose, CA., USA) and fibrinogen binding to αIIb/β3 was analyzed using cellQuest software.

Determination of ATP secretion

Washed platelets (108/mL) were preincubated for 3 min at 37°C with or without substances in the presence of 2 mM CaCl2 and then stimulated with collagen (10 μg/mL) for 5 min at 37°C in an aggregometer (Chrono-Log, Corp., Havertown, PA., USA) at a constant stirring speed of 1,000 rpm. The reaction was terminated by the addition of ice-cold 2 mM EDTA, the samples were centrifuged, and supernatants were used for the assay of ATP secretion from dense body. ATP secretion was measured with a luminometer (BioTek Instruments, Winooski, VT., USA) using ATP assay kit.

Determination of serotonin secretion

Washed platelets (108/mL) were preincubated for 3 min at 37°C with or without substances in the presence of 2 mM CaCl2 and then stimulated with collagen (10 μg/ml) for 5 min at 37°C in an aggregometer (Chrono-Log, Corp., Havertown, PA., USA) at a constant stirring speed of 1,000 rpm. The reaction was terminated by the addition of ice-cold 2 mM EDTA, the samples were centrifuged and supernatants were used for the assay of serotonin secretion. Serotonin secretion was measured with a Synergy HT Multi-Model Microplate Reader (BioTek Instruments, Winoosku, VT., USA) using serotonin ELISA kit (Labor Diagnostika Nord GmbH & CO., Nordhorn, Germany).

Statistical analysis

The experimental results are expressed as the mean ± S.E.M. accompanied by the number of observations. The data were assessed using an analysis of variance (ANOVA). If the analysis indicated significant differences between the group means, then each group was compared according to the Newman-Keuls method. p<0.05 was considered to be statistically significant.

RESULTSComposition of cordycepin in CE-WIB801C

The quantity of cordycepin in WIB801C was about 8.2% (81.98 ± 1.37 mg/g-WIB801C (Lee et al., 2014b). It is known that whole fruiting body myelia of Cordyceps militaris contains 0.16% of cordycepin, and whole fruiting body, stroma, and larva of Cordyceps sinensis does not contain cordycepin (Yue et al., 2008). Accordingly, the cordycepin level in WIB801C that we used in this study is very higher than those in whole fruiting body myelia of Cordyceps militaris, or in whole fruiting body, stroma, and larva of Cordyceps sinensis. Thus, WIB801C is named as cordycepin-enriched WIB801C (CE-WIB801C) in this report. The yield of cordycepin from CE-WIB801C with prep-HPLC was 80.7% (Lee et al., 2014c).

Effects of CE-WIB801C on collagen-induced platelet aggregation

The concentration of collagen-induced maximal platelet aggregation was approximately 10 μg/mL (Lee et al., 2014a). Therefore, collagen (10 μg/mL) was used as the platelet agonist in this study. The light transmission in response to various concentrations of CE-WIB801C (100, 200, 400 μg/mL) in intact platelet was 4.3 ± 0.6% (at 100 μg/mL of CE-WIB801C), 4.0 ± 0.0% (at 200 μg/mL of CE-WIB801C), and 4.3 ± 0.6 (at 400 μg/mL of CE-WIB801C), respectively, which were not significantly different from that (4.0 ± 1.0%) in resting platelets. These mean that CE-WIB801C alone did not affect on platelet aggregation as compared with that in intact platelets without CE-WIB801C. When washed platelet platelets (108/mL) were activated with collagen (10 μg/mL) in the presence of 2mM CaCl2, the aggregation was increased to 78.1 ± 1.7% (Fig. 2). However, various concentrations (100, 200, 400 μg/mL) of CE-WIB801C dose dependently inhibited collagen-induced platelet aggregation (Fig. 2).

Effects of CE-WIB801C and cordycepin on VASP phosphorylation

Because 200 and 400 μg/mL of CE-WIB801C (Fig. 2), and 500 μM cordycepin (Cho et al., 2007a) significantly inhibited collagen-induced platelet aggregation, we used 200 and 400 μg/mL of CE-WIB801C, and 500 μM cordycepin. CE- WIB801C alone (200, 400 μg/mL), and cordycepin alone (500 μM) (Fig. 3A lane 2, 3, and 4) potently increased p-VASP (Ser157) as compared with that (Fig. 3A lane 1) of intact platelets, control. Collagen increased weakly the phosphorylation of Ser157 [p- VASP (Ser157)] at 50 kDa of VASP (Fig. 3C lane 2) as compared with that of intact platelets (Fig. 3C lane 1), which involves in feedback inhibition by collagen (Gambaryan et al., 2010). Akinase activator pCPT-cAMP stimulated VASP phosphorylation, indicating VASP phosphorylation (Ser157) is dependent on cAMP in collagen-induced platelet aggregation (Fig. 3C lane 6). CE-WIB801C (400 μg/mL), and cordycepin (500 μM) also increased p-VASP (Ser157) (Fig. 3C lane 4, 7), and CEWIB801C (400 μg/mL) increased to 78.1% the ratio of p-VASP (Ser157) to VASP in collagen-induced platelet aggregation (Table 1). A-kinase inhibitor Rp-8-Br-cAMPS decreased to 23.0% CE-WIB801C-elevated p-VASP (Ser157) (Fig. 3C lane 5 and Table 1). With regard to the p-VASP (Ser239), G-kinase activator increased p-VASP (Ser239) (Fig. 3D lane 6). CE-WIB801C alone (200, 400 μg/mL), and cordycepin alone (500 μM) (Fig. 3B lane 2, 3, and 4) also potently increased p-VASP (Ser239) as compared with that (Fig. 3B lane 1) of intact platelets, control. However, CE-WIB801C (200, 400 μg/mL), and cordycepin (500 μM) did not almost increase the p-VASP (Ser239) (Fig. 3D lane 3, 4, and 7) as compared with that (Fig. 3D lane 2) by collagen. G-kinase inhibitor Rp-8-Br-cGMPS did not change p-VASP (Ser239) in the presence of CE-WIB801C (400 μg/mL) or cordycepin (500 μM) in collagen-induced platelet aggregation (Fig. 3D lane 5, 8).

Effects of CE-WIB801C and cordycepin on fibrinogen binding to αIIb/β<sub>3</sub>

Next, we investigated whether the VASP phosphorylation by CE-WIB801C involved in inhibition of fibrinogen binding to αIIb/β3. As shown in Fig. 4, collagen activated fibrinogen binding to αIIb/β3 (Fig. 4A–b), and increased the degree of fibrinogen binding to αIIb/β3 up to 77.2 ± 5.5% as compared with that (5.4 ± 0.2%) of intact platelets, basal (Table 1). However, CE-WIB801C inhibited collagen-activated fibrinogen binding to αIIb/β3 (Fig. 4A–c, 4B), and its inhibitory degree was 89.2% as compared with that (77.2 ± 5.5%) by collagen (Table 1). Cordycepin also potently inhibited collagen-activated fibrinogen binding to αIIb/β3 (Fig. 4A–d and 4B). Because the inhibition of αIIb/β3 is resulted from cAMP/A-kinase- and cGMP/G-kinase- mediated VASP phosphorylation, and it is known that cAMP- and cGMP-increasing compounds involve in inhibition of αIIb/β3 (Horstrup et al., 1994; Barragan et al., 2003), we investigated whether the inhibition of fibrinogen binding to αIIb/β3 by CE-WIB801C was contributed to which kinase of Akinase and G-kinase. A-kinase activator pCPT-cAMP and G-kinase activator 8-Br-cGMP inhibited collagen-induced fibrinogen binding to αIIb/β3, respectively (Fig. 5A–e, f and Fig. 5B). It was confirmed that cAMP/A-kinase and cGMP/G-kinase pathway involve in inhibition of fibrinogen binding to αIIb/β3 in collagen-induced platelet aggregation. CE-WIB801C-inhibited fibrinogen binding to αIIb/β3 (Fig. 4A–c and Fig. 4B) was elevated by A-kinase inhibitor Rp-8-Br-cAMPS (Fig. 5A–a and Fig. 5B), and its stimulatory degree was 386.7% as compared with that (8.3%) by both CE-WIB801C and collagen (Table 1). However, CE-WIB801C-inhibited fibrinogen binding to αIIb/β3 (Fig. 4A–c and Fig. 4B) was not almost elevated by G-kinase inhibitor Rp-8-Br-cGMPS (Fig. 5A–b and Fig. 5B), and its stimulatory degree was 6.0% as compared with that (8.3%) by both CE-WIB801C and collagen (Table 1).

Effects of CE-WIB801C on PI3K/Akt phosphorylation

As apposed to the phosphorylated VASP, PI3K/Akt phosphorylation stimulates αIIb/β3 activation and fibrinogen binding (Zhang et al., 1996; Chen et al., 2004). Thus, we investigated the effect of CE-WIB801C, and cordycepin on phosphorylation of PI3K and its downstream molecule Akt. CE-WIB801C alone (200, 400 μg/mL), and cordycepin alone (500 μM) almost did not change p-PI3K (Fig. 6A lane 2, 3, and 4) as compared with that (Fig. 6A lane 1) of intact platelets, control. PI3K inhibitor wortmannin suppressed collagen-induced PI3K phosphorylation (Fig. 6B lane 5). Collagen potently phosphorylated PI3K (Fig. 6B lane 2) as compared with that (Fig. 6B lane 1) of intact platelets, however, CE-WIB801C (Fig. 6B lane 3, 4) and cordycepin (Fig. 6B lane 6) inhibited collagen-induced PI3K phosphorylation. CE-WIB801C alone (200, 400 μg/mL), and cordycepin alone (500 μM) almost did not change p-Akt (Fig. 6C lane 2, 3, and 4) as compared with that (Fig. 6C lane 1) of intact platelets, control. Collagen elevated the phosphorylation of PI3K target molecule Akt (Fig. 6D lane 2), however, CE-WIB801C (Fig. 6D lane 3, 4) and cordycepin (Fig. 6D lane 5) inhibited collagen-induced Akt phosphorylation.

Effects of CE-WIB801C on ATP and serotonin release

Collagen, ADP, and thrombin that activate αIIb/β3 release ATP and serotonin out of dense bodies to aggregate platelets, which due to the [Ca2+]i mobilization by fibrinogen binding to αIIb/β3 (Weiss et al., 1974; Michal and Motamed, 1976; Kamruzzaman et al., 2013). Because CE-WIB801C also inhibited [Ca2+]i mobilization (Lee et al., 2014b), and fibrinogen binding to αIIb/β3 (Fig. 4A–c), we investigated whether CE-WIB801C, and cordycepin have inhibitory effect on collagen-elevated ATP and serotonin release. As shown in Fig. 7A, collagen released to 3.91 ± 0.24 μM ATP from platelets, it’s the stimulatory degree was 4,244% as compared with that (0.09 ± 0.02 μM) in intact platelets, control (Fig. 7A). However, CE-WIB801C, and cordycepin potently inhibited ATP release by collagen (Fig. 7A).

With regard to serotonin release, as shown in Fig. 7B, collagen potently released serotonin to 84.7 ± 6.5 ng/108 platelets, and its stimulatory degree was 636.5% as compared with that (11.5 ± 1.6 ng/108 platelets) in intact platelets, control (Fig. 7B). However, CE-WIB801C, and cordycepin inhibited collagen-elevated serotonin release (Fig. 7B).

Effects of cordycepin, W-cordycepin on collagen-induced platelet aggregation

CE-WIB801C (100, 200, 400 μg/mL) dose dependently inhibited collagen-induced platelet aggregation (Fig. 2). To investigate whether cordycepin (W-cordycepin) of CE-WIB801C involved in inhibition of collagen-induced platelet aggregation by CE-WIB801C, we purified cordycepin from CE-WIB801C with prep-HPLC (Lee et al., 2014c). The concentration of cordycepin (Mw. 251.14) was 81.98 ± 1.37 mg/g-CE-WIB801C (8.2%) (Lee et al., 2014b). The concentration of CE-WIB801C that inhibited collagen-induced platelet aggregation was at least 100, 200, 400 μg/mL (Fig. 2, Lee et al., 2014b). In this study, therefore, to investigate the synergistic effect of cordycepin, or W-cordycepin with CE-WIB801C on collagen-induced platelet aggregation, we used 33, 66, 132 μM of cordycepin, or W-cordycepin corresponding to cordycepin concentration that contains in CE-WIB801C (100, 200, 400 μg/mL). As the results, as shown in Fig. 8A and 8C, the light transmissions (4.3 ± 0.6∼5.0 ± 1.0%) in response to various concentration (33, 66, 132 μM) of cordycepin, and W-cordycepin alone was not significantly different from that (4.0 ± 1.0%) in resting platelets. However, cordycepin and W-cordycepin inhibited collagen-induced platelet aggregation in a dose (33, 66, 132 μM) dependent manner. With regard to the synergistic effect of cordycepin or W-cordycepin with CE-WIB801C on collagen-induced platelet aggregation. As shown in Fig. 8B and 8D, when platelets were activated by collagen in the presence of both CE-WIB801C (200 μg/mL) and cordycepin (33, 66, 132 μM) (Fig. 8B) or W-cordycepin (33, 66, 132 μM) (Fig. 8D), the aggregation was dose dependently decreased as compared with that (42.1 ± 2.4%) by CE-WIB801C (200 μg/mL) alone. The aggregation (21.5 ± 1.8%) by both cordycepin (132 μM) and CE-WIB801C (200 μg/mL) was lower as compared with that (42.1 ± 2.4%) by CE-WIB801C (200 μg/mL) alone in collagen-induced platelet aggregation. In addition, the aggregation (14.4 ± 2.7%) by both W-cordycepin (132 μM) and CE-WIB801C (200 μg/mL) was lower as compared with that (42.1 ± 2.4%) by CE-WIB801C (200 μg/mL) alone in collagen-induced platelet aggregation. Furthermore, the inhibitory degree of collagen-induced platelet aggregation by CE-WIB801C together with cordycepin or W-cordycepin was also increased in a dose dependent manner. Therefore, it is thought that cordycepin or W-cordycepin with CE-WIB801C have a synergistic inhibitory effect on collagen- induced platelet aggregation, and the inhibition (Fig. 2) is resulted from antiplatelet function of W-cordycepin in CE-WIB801C.

DISCUSSION

A downstream pathway of both cAMP/A-kinase and cGMP/G-kinase involves in VASP phosphorylation to inhibit fibrinogen binding to αIIb/β3. Ser157 at 50 kDa of VASP is phosphorylated by the cAMP/A-kinase pathway, whereas Ser239 at 50 kDa of VASP is phosphorylated by the cGMP/G-kinase pathway (Horstrup et al., 1994; Smolenski et al., 1998). CE-WIB801C and cordycepin phosphorylated A-kinase substrate VASP (Ser157) in intact platelets, and collagen-activated platelets. It is unknown whether CE-WIB801C increases cAMP level in intact platelets. Cordycepin (500 μM) increased cAMP level about 100 fmol/109 platelets in intact platelets (Cho et al., 2007a). We could not explain how the cAMP level (100 fmol/109 platelets) that was increased by cordycepin (500 μM) involved in VASP (Ser157) phosphorylation in intact platelets. However, this phenomenon could be explained from the report (Eigenthaler et al., 1992) that a very small elevation in cAMP is enough to activate most A-kinase. CE-WIB801C elevated cAMP level to 18.1 ± 1.0 pmol/109 platelets (Lee et al., 2014b), and cordycepin (500 μM) increased cAMP level to 7.5 pmol/109 platelets in collagen-induced platelet aggregation (Cho et al., 2007a). Therefore, it is thought that CE-WIB801C and cordycepin increase the level of cAMP, and subsequently involve in VASP (Ser157) phosphorylation via A-kinase activation in collagen-induced platelet aggregation. If not so, CE-WIB801C-, and cordycepin-induced VASP (Ser157) phosphorylation would not be inhibited by A-kinase inhibitor Rp- 8-Br-cAMPS in collagen-induced platelet aggregation. With regard to VASP (Ser239) phosphorylation, CE-WIB801C and cordycepin phosphorylated G-kinase substrate VASP (Ser239) in intact platelets, but did not phosphorylate that in collagen-induced platelet aggregation. Unfortunately, we could not explain the different phenomena of VASP (Ser239) phosphorylation caused by CE-WIB801C and cordycepin in intact platelets and collagen-activated platelets, which are necessary to study in the future. It is known that A-kinase plays predominately in the cGMP-phosphorylated VASP to inhibit platelet aggregation (Li et al., 2003), which reflects that CE-WIB801C-, and cordycepin-indcued VASP (Ser157) phosphorylation was inhibited by A-kinase inhibitor Rp-8-Br-cAMPS, and was dependent on cAMP/A-kinase pathway.

A-kinase inhibitor Rp-8-Br-cAMPS increased CE-WIB801C-, and cordycepin-inhibited fibrinogen binding to αIIb/β3. This means that CE-WIB801C and cordycepin inhibit fibrinogen binding to αIIb/β3via cAMP/A-kinase pathway. In this report, we have established that the inhibitory effect by CE-WIB801C and cordycepin on collagen-induced αIIb/β3 activation is due to cAMP/A-kinase-dependent VASP (Ser157) phosphorylation. It is known that the phosphorylation of PI3K and Akt involves in αIIb/β3 activation, an index of fibrinogen binding to αIIb/β3, in collagen-, ADP-, thrombin-induced platelet activation (Morello et al., 2009). cAMP-elevating agents (i.e. cilostamide, cilostazole, and forskolin) are known to inhibit PI3K- and Akt-phosphorylation in collagen-induced platelet aggregation (Hayashi and Sudo, 2009) and cAMP/A-kinase pathway is known to involve in suppression of αIIb/β3 activation (van Willigen and Akkerman, 1991; Payrastre et al., 2000; Shattil and Newman, 2004). Our findings show that CE-WIB801C (Lee et al., 2014b), and cordycepin (Cho et al., 2007a) increases cAMP and subsequently stimulates the phosphorylation of VASP (Ser157), on the contrary, inhibits the phosphorylation of both PI3K and Akt to inhibit fibrinogen binding to αIIb/β3 on collagen-induced platelet aggregation.

A lot of agonists such as collagen, thrombin and ADP mobilize [Ca2+]i to phosphorylate Ca2+/calmodulin-dependent myosin light chain (20 kDa), which involves in granule secretion such as ATP and serotonin (Nishikawa et al., 1980; Kaibuchi et al., 1982).

The inhibition of ATP and serotonin secretion by CE-WIB801C and cordycepin is associated with the elevation of cAMP level and the inhibition of [Ca2+]i mobilization, which also supports the facts that CE-WIB801C induced the cAMP-dependent phosphorylation of IP3R to inhibit [Ca2+]i mobilization (Lee et al., 2014b). In our another study, we observed that cordycepin inhibits [Ca2+]i mobilization by phosphorylating IP3R in collagen-induced platelet aggretion (Data not shown). In addition, inhibition of 20kDa phosphorylation by cordycepin (Cho et al., 2006; 2007a) might be resulted in inhibition of ATP and serotonin release by cordycepin.

The purified W-cordycepin from CE-WIB801C, and cordycepin inhibited collagen-induced platelet aggregation. In addition, because W-cordycepin, and cordycepin had a synergistic inhibitory effect with CE-WIB801C on collagen-induced platelet aggregation, it is thought that the inhibition of collagen-induced platelet aggregation by CE-WIB801C might be resulted from the inhibitory effect by at least W-cordycepin. If so, this reflects the possibility that W-cordycepin in CE-WIB801C would directly or indirectly involve in phosphorylation of VASP (Ser157), and dephosphorylation of PI3K and Akt to inhibit fibrinogen binding to αIIb/β3.

Platelet aggregation is generated at site of vascular wall injury, and is involved in the formation of thrombus. During the formation of thrombus, platelets release cell growth proteins such as platelet-derived growthfactor (PDGF), and vascular endothelial growth factor (VEGF) (Holash et al., 1999; Castro-Malaspina et al., 1981). It is well established that PDGF and VEGF induce the proliferation of fibroblast, vascular smooth cells, and epithelial cells, and subsequently enhance the rate of atherosclerosis lesion progression (Seppä et al., 1982; Schwartz and Ross, 1984; Packham and Mustad, 1986; Schwartz and Reidy, 1987; Nagai et al., 2005). The progression of atherosclerosis is strongly induced by inflammatory cell such as monocyte/macrophage, and neutrophil (Philips et al., 2005). Although CE-WIB801C and cordycepin have anti-platelet effects, if CE-WIB801C and cordycepin do not inhibit inflammation by leukocyte, the progression of atherosclerosis lesion would be generated at site of vascular wall injury, and a question for antiplatelet effects of CE-WIB801C or cordycepin might be raised. Kim et al. (2006) reported that cordycepin inhibits lipopolysaccharide-induced inflammation by inhibiting Akt phosphorylation and NF-κB activity. In addition, cordycepin is known to inhibit protein synthesis by attenuating Akt/mTOR signaling pathway in NIH3T3 fibroblast, which means that cordycepin has an inhibitory effect on proliferation of NIH3T3 fibroblast (Wong et al., 2010). Considering our results and these two previous reports that cordycepin inhibits platelet aggregation, inflammation, and fibroblast proliferation, it is thought that CE-WIB801C, and cordycepin may have antithrombotic-, and antiatherosclerotic-effects without inflammation and progression of atherosclerotic lesion at site of vascular wall injury. Therefore, CE-WIB801C and cordycepin is highlighted as an non-toxic antiplatelet compound.

Antiplatelet drugs such as thienopyridine derivatives (i.e. ticlopidine, clopidogrel) have characteristics that phosphorylate VASP, inhibit [Ca2+]i mobilization, and inhibit αIIb/β3 activation, which is mediated by cAMP or cGMP (Barragan et al., 2003). Therefore, it is thought that CE-WIB801C and cordycepin as well as thienopyridine derivatives may also represent a useful tool in the therapy and prevention of vascular diseases associated with platelet aggregation.

Figures
Fig. 1. Chemical structure of cordycepin (3′-deoxyadenosine).
Fig. 2. Effects of CE-WIB801C on collagen-induced platelet aggregation. Measurement of platelet aggregation was carried out as described in “Materials and Methods.” The data are expressed as the mean ± S.E.M. (n=4). **p<0.001 versus the collagen-stimulated platelets.
Fig. 3. Effects of CE-WIB801C and cordycepin on VASP phosphorylation. (A) Effect of CE-WIB801C and cordycepin on p-VASP (Ser157). (B) Effect of CE-WIB801C and cordycepin on p-VASP (Ser239). Lane 1, Intact platelets (base); Lane 2, CE-WIB801C (200 μg/mL); Lane 3, CE-WIB801C (400 μg/mL); Lane 4, Cordycepin (500 μM). (C) Effect of CE-WIB801C and cordycepin on p-VASP (Ser157). Lane 1, Intact platelets (base); Lane 2, Collagen (10 μg/mL); Lane 3, Collagen (10 μg/mL)+CE-WIB801C (200 μg/mL); Lane 4, Collagen (10 μg/mL)+CEWIB801C (400 μg/mL); Lane 5, Collagen (10 μg/mL)+CE-WIB801C (400 μg/mL)+Rp-8-Br-cAMPS (250 μM); Lane 6, Collagen (10 μg/mL)+pCPT-cAMP (1 mM); Lane 7, Collagen (10 μg/mL)+cordycepin (500 μM); Lane 8, Collagen (10 μg/mL)+cordycepin (500 μM)+Rp-8-BrcAMPS (250 μM). (D) Effect of CE-WIB801C and cordycepin on p-VASP (Ser239). Lane 1, Intact platelets (base); Lane 2, Collagen (10 μg/mL); Lane 3, Collagen (10 μg/mL)+CE-WIB801C (200 μg/mL); Lane 4, Collagen (10 μg/mL)+CE-WIB801C (400 μg/mL); Lane 5, Collagen (10 μg/mL)+CE-WIB801C (400 μg/mL)+Rp-8-Br-cGMPS (250 μM); Lane 6, Collagen (10 μg/mL)+8-Br-cGMP (1 mM); Lane 7, Collagen (10 μg/mL)+cordycepin (500 μM); Lane 8, Collagen (10 μg/mL)+cordycepin (500 μM)+Rp-8-Br-cGMPS (250 μM). Western blotting was performed as described in “Materials and Methods.” The data are expressed as the mean ± S.E.M. (n=3). **p<0.001 versus the collagen-stimulated platelets, ††p<0.001 versus the collagen-stimulated platelets in the presence of CE-WIB801C (400 μg/mL), ##p<0.001 versus the collagen-stimulated platelets in the presence of cordycepin (500 μM).
Fig. 4. Effects of CE-WIB801C and cordycepin on collagen-induced fibrinogen binding. (A) The flow cytometry histograms on fibrinogen binding. a, Intact platelets (base); b, Collagen (10 μg/mL); c, Collagen (10 μg/mL)+CE-WIB801C (400 μg/mL); d, Collagen (10 μg/mL)+cordycepin (500 μM). (B) Effects of CE-WIB801C and cordycepin on collagen-induced fibrinogen binding (%). Determination of fibrinogen binding to αIIb/β3 was carried out as described in “Materials and Methods.” The data are expressed as the mean ± S.E.M. (n=4). ap<0.05 versus non-stimulated platelets, **p<0.001 versus the collagen-stimulated platelets.
Fig. 5. Effects of CE-WIB801C and cordycepin on collagen-induced fibrinogen binding in the presence of A-kinase inhibitor (Rp-8-BrcAMPS), or G-kinase inhibitor (Rp-8-Br-cGMPS). (A) The flow cytometry histograms on fibrinogen binding. a, Collagen (10 μg/mL)+CEWIB801C (400 μg/mL)+Rp-8-Br-cAMPS (250 μM); b, Collagen (10 μg/mL)+CE-WIB801C (400 μg/mL)+Rp-8-Br-cGMPS (250 μM); c, Collagen (10 μg/mL)+cordycepin (500 μM)+Rp-8-Br-cAMPS (250 μM); d, Collagen (10 μg/mL)+cordycepin (500 μM)+ Rp-8-Br-cGMPS (250 μM); e, Collagen (10 μg/mL)+pCPT-cAMP (1 mM); f, Collagen (10 μg/mL)+8-Br-cGMP (1 mM). (B) Effects of CE-WIB801C and cordycepin on collagen-induced fibrinogen binding (%) in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) or G-kinase inhibitor (Rp-8-Br-cGMPS). Determination of fibrinogen binding to αIIb/β3 was carried out as described in “Materials and Methods.” The data are expressed as the mean ± S.E.M. (n=4). **p<0.001 versus the collagen-stimulated platelets, ††p<0.001 versus the collagen-stimulated platelets in the presence of CEWIB801C (400 μg/mL). #p<0.05 versus the collagen-stimulated platelets in the presence of cordycepin (500 μM). ##p<0.001 versus the collagen- stimulated platelets in the presence of cordycepin (500 μM).
Fig. 6. Effects of CE-WIB801C and cordycepin on PI3K phosphorylation and Akt. (A) Effects of CE-WIB801C and cordycepin on PI3K phosphorylation in intact platelets. Lane 1, Intact platelets (base); Lane 2, CE-WIB801C (200 μg/mL); Lane 3, CE-WIB801C (400 μg/mL); Lane 4, Cordycepin (500 μM). (B) Effects of CE-WIB801C and cordycepin on PI3K phosphorylation. Lane 1, Intact platelets (base); Lane 2, Collagen (10 μg/mL); Lane 3, Collagen (10 μg/mL)+CE-WIB801C (200 μg/mL); Lane 4, Collagen (10 μg/mL)+CE-WIB801C (400 μg/mL); Lane 5, Collagen (10 μg/mL)+Wortmannin (10 μM); Lane 6, Collagen (10 μg/mL)+Cordycepin (500 μM). (C) Effects of CE-WIB801C and cordycepin on Akt phosphorylation in intact platelets. Lane 1, Intact platelets (base); Lane 2, CE-WIB801C (200 μg/mL); Lane 3, CEWIB801C (400 μg/mL); Lane 4, Cordycepin (500 μM). (D) Effects of CE-WIB801C and cordycepin on Akt phosphorylation. Lane 1, intact platelets (base); Lane 2, collagen (10 μg/mL); Lane 3, collagen (10 μg/mL)+CE-WIB801C (200 μg/mL); Lane 4, collagen (10 μg/mL)+CE-WIB801C (400 μg/mL); Lane 5, Collagen (10 μg/mL)+cordycepin (500 μM). The data are expressed as the mean ± S.E.M. (n=3). ap<0.05 versus non-stimulated platelets, *p<0.05, **p<0.001 versus the collagen-stimulated platelets.
Fig. 7. Effects of CE-WIB801C and cordycepin on ATP and serotonin release. (A) Effect of CE-WIB801C and cordycepin on ATP release in collagen-induced platelets. (B) Effect of CE-WIB801C and cordycepin on serotonin release in collagen-induced platelets. ATP and serotonin secretion were measured as described in “Materials and Methods.” The data are expressed as the mean ± S.E.M. (n=4). ap<0.05 versus non-stimulated platelets, **p<0.001 versus the collagen-stimulated platelets.
Fig. 8. Effects of cordycepin or W-cordycepin on collagen-induced platelet aggregation. (A) Effects of cordycepin on collagen-induced platelet aggregation. (B) The synergistic inhibitory effects of cordycepin with CE-WIB801C on collagen-induced platelet aggregation. (C) Effects of W-cordycepin on collagen-induced platelet aggregation. (B) The synergistic inhibitory effects of W-cordycepin with CE-WIB801C on collagen-induced platelet aggregation. Measurement of platelet aggregation was carried out as described in “Materials and Methods.” The data are expressed as the mean ± S.E.M. (n=4). **p<0.05 versus the collagen-stimulated platelets. The light transmission and inhibitory degree by cordycepin or W-cordycepin, and cordycepin or W-cordycepin plus CE-WIB801C were expressed as a percentage of that by collagen (100% : aggregation, 0% : inhibition).
Tables

Changes of p-VASP (Ser157)/VASP ratio and fibrinogen binding

p-VASP (Ser157)/VASPΔ (%)Fibrinogen binding (%)Δ (%)
Collagen (10 μg/mL)2.10 ± 0.20077.2 ± 5.50
CE-WIB801C (400 μg/mL)+Collagen (10 μg/mL)3.74 ± 0.81+78.11)08.3 ± 0.8−89.22)0
CE-WIB801C (400 μg/mL) +Rp-8-Br-cAMPS (250 μM)+Collagen (10 μg/mL)2.88 ± 0.57-−23.03)40.4 ± 1.1-+386.74)
CE-WIB801C (400 μg/mL) +Rp-8-Br-cGMPS (250 μM)+Collagen (10 μg/mL)3.84 ± 0.79-+2.75)8.8 ± 0.5-+6.06)

Data were from Fig. 3C, 3D, 5B.

to

Δ (%)=[(CE-WIB801C+Collagen)-Collagen]/Collagen×100,

to

Δ (%)=[(CE-WIB801C+Rp-8-Br-cAMPS+Collagen)-(CE-WIB801C+Collagen)]/(CE-WIB801C+Collagen)×100,

to

Δ (%)=[(CE-WIB801C+Rp-8-Br-cGMPS+Collagen)-(CE-WIB801C+Collagen)]/(CE-WIB801C+Collagen)×100.

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