1Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering, Inje University, Gimhae 621-749
2Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon 302-718
3Department of Medical Laboratory Science, Dong-Eui Institute of Technology, Busan 614-715
4Bioscience & Biotechnology Team, Central Research Center, Whanin Pharm. Co., Ltd., Suwon 443-766, Republic of Korea
In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of
Platelet aggregation is absolutely essential for the formation of a hemostatic plug when normal blood vessels are injured. However, the interactions between platelets and collagen can also cause circulatory disorders, such as thrombosis, atherosclerosis, and myocardial infarction (Schwartz
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In addition, we investigated the effects of cordycepin-enriched (CE)-WIB801C on upregulation of aggregation-inhibiting molecules (i.e. cAMP, cGMP), and downregulation of aggregation-inducing molecules (Ca2+, TXA2). In special, we set out to investigate in this study whether CE-WIB801C has inhibitory effect on collagen-induced [Ca2+]i mobilization, and which, if any, cAMP and cGMP is responsible for the IP3R phosphorylation to exert Ca2+-antagonistic effect.
Collagen was purchased from Chrono-Log Co. (Havertown, PA., USA). Fura 2-AM, and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO., USA). TXB2, cAMP and cGMP enzyme immunoassay (EIA) kit, and cyclooxygenase (COX) fluorescent activity assay kit were purchased from Cayman Chemical Co. (Ann Arbor, MI., USA). Anti-phosphor-IP3-receptor, anti-rabbit IgG-horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling (Beverly, MA., USA). Polyvinylidene difluoride (PVDF) membrane was from GE Healthcare (Piseataway, NJ., USA). Enhanced chemiluminesence solution (ECL) was from GE Healthcare (Chalfont St, Giles, Buckinghamshire, UK).
WIB801C was dissolved in 50% methanol, for the first time, and then it was analyzed by high performance liquid chromatography (HPLC). An Agilent 1100 liquid chromatography system (Palo Alto, CA., USA), equipped with vacuum degasser, quaternary gradient pump, autosampler and diode array detector, connected to an Agilent ChemStation software. A Zorbax octadecylsilane (ODS) C18 column (250 mm×4.6 mm id, 5 μm) and a Zorbax ODS C18 guard column (12.5 mm×4.6 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 1.0 ml/min and sample injection volume was 10 μL. The UV detection was operated at 254 nm. To detect and analyze the nucleoside analogue, we used various concentrations (cordycepin: 50, 100, 200, and 400 μg/ml; adenosine: 4, 20, 100, and 200 μg/ml; adenine: 10, 20, 50, 100, 200 μg/ml) of each authentic compounds (cordycepin, adenosine, and adenine) in duplicate with HPLC, then the calibration curves were constructed by plotting the peak area against the concentration of each analyte with regression analysis, and we calculated linear equation from the calibration curve (Table 1).
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.
Washed platelets (108/ml) were preincubated for 3 min at 37°C in the presence of 2 mM CaCl2 with or without substances, 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).
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 for platelet aggregation. The aggregation was terminated by the addition of 80% ice-cold ethanol. cAMP and cGMP were measured with synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT., USA) using cAMP and cGMP EIA kits.
PRP was incubated with 5 μM Fura 2-AM at 37°C for 60 min. Because Fura 2-AM is light sensitive, the tube containing the PRP was covered with aluminum foil during loading. The Fura 2-loaded washed platelets were prepared using the procedure described above and 108 platelets/ml were preincubated for 3 min at 37°C with or without substances in the presence of 2 mM CaCl2, then stimulated with collagen (10 μg/ml) for 5 min for evaluation of [Ca2+]i. Fura 2 fluorescence was measured with a spectrofluorometer (SFM 25; Bio-Teck Instrument, Italy) with an excitation wavelength that was changed every 0.5 sec from 340 to 380 nm; the emission wavelength was set at 510 nm. The [Ca2+]i values were calculated using the method of Schaeffer (Schaeffer and Blaustein, 1989).
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. 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-gly cerophosphate, 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 (15 μg) were us ed for analysis. Protein concentrations were measured by using bicinchoninic acid protein assay kit (Pierce Biotechnology, USA). The effects of substances on IP3R phosphorylation were analyzed by 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-phosphor-IP3R and anti-rabbit IgG-HRP were 1:1000 and 1:10000, respectively. The membranes were visualized using ECL. Blots were analyzed by using the Quantity One, Ver. 4.5 (Bio-Rad, Hercules, CA., USA).
Washed platelets (108/ml) were preincubated with or without substances for 3 min in the presence of 2 mM CaCl2, and activated for 5 min with collagen (10 μg/ml). The reactions were terminated by the addition of ice-cold EDTA (5 mM) and indomethacin (0.2 mM). The amount of TXB2, a stable metabolite of TXA2, was determined with synergy HT multi-model microplate reader (BioTek Instruments, Winoosku, VT., USA) using a TXB2 EIA kit.
Washed platelets (108/ml) with 1% protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO., USA) were sonicated 10 times at sensitivity 100% for 20 seconds on ice with a model HD2070 sonicator (Bandelin Electronic, Bandelin, Germany) to obtain platelet lysates. The homogenates were centrifuged at 12,000×g for 15 min at 4°C to remove cell debris. The supernatant was used to measure COX-1 activity. The platelet lysates were pre-incubated with or without substances at 37°C for 30 min. COX-1 activity was measured with synergy HT multi-model microplate reader (BioTek Instruments, Winooski, VT., USA) using COX fluorescent activity assay kit.
Washed platelets (108/ml) with 1% protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO., USA) were sonicated 10 times at sensitivity 100% for 20 seconds on ice with a model HD2070 sonicator (Bandelin Electronic, Bandelin, Germany) to obtain platelet lysates. Next, the homogenates were centrifuged at 12,000×g for 15 min at 4°C to remove cell debris. The platelet lysates were pre-incubated with or without substances at 37°C for 30 min. The reaction was initiated by the addition of prostaglandin H2 (PGH2) and allowed to proceed for 1 min at 37°C. The reaction was then terminated by the addition of 1M citric acid. After neutralization with 1N NaOH, the concentration of thromboxane B2 (TXB2), a stable metabolite of TXA2, was determined with synergy HT multi-model microplate reader (BioTek Instruments, Winoosku, VT., USA) using TXB2 EIA kit.
The experimental results are expressed as the mean ± S.E.M. accompanied by the number of observations. Data were assessed by analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared by the Newman-Keuls method.
We analyzed the composition of cordycepin in WIB801C with HPLC, as shown in Fig. 2A, two peaks (peak 1, 2) mainly were observed. The retention time of peak 1 was 6.6 min, and peak 2 was 14.8 min (Fig. 2A, Table 1). We detected cordycepin and analyzed its composition. As shown in Fig. 2B, the retention times of authentic compounds, cordycepin, adenosine, and adenine, were 14.8, 11.7, and 6.6 min in order. The retention times of peak 1, and peak 2 were in accord with those of authentic adenine and cordycepin, but a certain peak corresponding authentic adenosine was not almost observed in WIB801C. Calibration curve were linear over the range of 50 to 400 μg/ml for cordycepin, and 10 to 200 μg/ml for adenine with r2>0.9996 (Table 1). With regard to contents of cordycepin and adenine calculated from calibration curve, as shown in Table 1, the content of peak 2 corresponding to cordycepin was 81.98 ± 1.37 mg/g-WIB801C (about 8.2%), and the content of peak 1 corresponding to adenine was 16.21 ± 0.25 mg/g-WIB801C (about 1.62%).
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The concentration of collagen-induced maximal platelet aggregation was approximately 10 μg/ml (Lee
As shown in Table 2, collagen decreased intracellular cAMP level from 5.2 ± 0.4 pmoL/109 platelets (basal level) to 2.8 ± 0.5 pmoL/109 platelets, which was reduced to 46.2% as compared with that of basal level (Table 2). When platelets, however, were incubated in the presence of both CE-WIB801C and collagen, 400 μg/ml of CE-WIB801C increased cAMP level from 2.8 ± 0.5 pmoL/109 platelets to 18.1 ± 1.0 pmoL/109 platelets (Table 2). This result suggests that CE-WIB801C (400 μg/ml) increased collagen-decreased cAMP level to 546.4% (Table 2). On the other hand, collagen decreased intracellular cGMP level from 4.0 ± 0.3 pmoL/109 platelets (basal level) to 3.0 ± 0.4 pmoL/109 platelets (Table 2). Collagen reduced basal cGMP level to 25.0% to aggregate platelets (Table 2). When platelets, however, were incubated in the presence of both CE-WIB801C (400 μg/ml) and collagen (10 μg/ml), the cGMP level was increased to 53.3% as compared with that (3.0 ± 0.4 pmoL/109 platelets) achieved by collagen (10 μg/ml) alone (Table 2).
As shown in Fig. 4A, collagen increased [Ca2+]i level from 106.6 ± 2.1 nM (basal level) to 536.6 ± 45.0 nM. However, CEWIB801C (400 μg/ml) decreased collagen-elevated [Ca2+]i (536.6 ± 45.0 nM) to 124.9 ± 2.6 nM (Fig. 4A). This suggests that CEWIB801C decreased collagen-elevated [Ca2+]i level to 76.7% (Fig. 4A). The level of [Ca2+]i in the presence of both collagen and CE-WIB801C was 124.9 ± 2.6 nM, however, which level was dose dependently increased by A-kinase inhibitor Rp-8-Br-cAMPS (50 to 250 μM) and was increased to 254.9 ± 9.4 nM (104.1%) (Fig. 4B). On the other hand, the level of [Ca2+]i in the presence of both collagen and CE-WIB801C was not increased by G-kinase inhibitor Rp-8-Br-cGMPS (50 to 250 μM) (Fig. 4C). Because [Ca2+]i reduction is resulted from cAMP/A-kinase-phosphorylated IP3R, we next investigated whether CE-WIB801C involves in phosphorylation of IP3R.
The phosphorylation (p-IP3R) of IP3R and the ratio of p-IP3R to β-actin were increased in the presence of A-kinase activator pCPT-cAMP (1 mM) (Fig. 5 lane 7), and G-kinase activator 8-Br-cGMP (1 mM) (Fig. 5 lane 8) as compared with collagen alone. These mean that cAMP/A-kinase and cGMP/G-kinase involve in IP3R phosphorylation. As shown in Fig. 5 lane 3 and 4, p-IP3R and the ratio of p-IP3R to β-actin were dose dependently increased in the presence of both collagen and CE-WIB801C (200 and 400 μg/ml). However, the ratio (3.15) of p-IP3R to β-actin by both collagen and CE-WIB801C (400 μg/ml) was decreased to 1.71 (45.7%) in the presence of A-kinase inhibitor Rp-8-Br-cAMPS (Fig. 5 lane 5, Table 3). On the other hand, the ratio (3.15, Fig. 5 lane 4) of p-IP3R to β-actin by both collagen and CE-WIB801C (400 μg/ml) was not almost decreased in the presence of G-kinase inhibitor Rp-8-Br-cGMPS (Fig. 5 lane 6, Table 3). Accordingly, cAMP/A-kinase-dependent IP3R phosphorylation exclusively contributed to the inhibitory effect of [Ca2+]i mobilization achieved by CE-WIB801C on collagen-activated platelets.
The TXA2 (determined as TXB2) level in intact platelets was 0.6 ± 0.1 ng/108 platelets, and collagen (10 μg/ml) ma rkedly increased TXA2 level to 60.1 ± 1.0 ng/108 platelets (Fig. 6A). This suggests that collagen increased TXA2 production to 9,917% (Fig. 6A). However, CE-WIB801C potently reduced TXA2 production to 5.5 ± 0.5 ng/108 platelets (90.8% inhibition at 400 μg/ml) (Fig. 6A). TXA2 production is concerned with COX-1 and TXAS, which convert 20:4 to TXA2 (Patrono, 1994; Cipollone
CE-WIB801C contained mainly adenine (Fig. 1A) and cordycepin (Fig. 1C), and inhibited collagen-induced platelet ag g regation, which is thought by cordycepin in WIB801C because authentic cordycepin inhibited collagen-induced platelet aggregation in a dose dependent manner (Fig. 7A), but authentic adenine did not inhibited collagen-induced platelet aggregation (Fig. 7B). It is established that cordycepin inhibits collagen-induced platelet aggregation in our previous report (Cho
Therefore, even though it is thought that CE-WIB801C might involve in inhibition of COX-1 or TXAS to suppress collagen-produced TXA2 level, because CE-WIB801C did not inhibit COX-1 and TXAS activities in a cell free system, it is inferred that CE-WIB801C would not directly involve in inhibition of COX-1 and TXAS activities to reduce TXA2 production in collagen-induced platelet aggregation.
In real, TXA2 precursor 20:4 is generated from DG/monoacylglycerol (MG)
CE-WIB801C more increased exclusively cAMP than cGMP on collagen-induced platelet aggregation, which trend is as well as those by phenolic compounds such as epigallocatechin-3-gallate (Ok
The Ca2+-antagonistic reaction by cAMP or cGMP is mediated by A-kinase/IP3R phosphorylation or G-kinase/IP3R phosphorylation. CE-WIB801C elevated IP3R phosphorylation, and this was inhibited by A-kinase inhibitor Rp-8-Br-cAMPS only, not G-kinase inhibitor Rp-8-Br-cGMPS. In addition, CE-WIB801C-decreased [Ca2+]i was increased by A-kinase inhibitor Rp-8-Br-cAMPS in collagen-activated platelets. Accordingly, it is an evidence that the inhibition of [Ca2+]i mobilization by CEWIB801C is obviously due to cAMP/A-kinase-dependent IP3R phosphorylation. Otherwise, CE-WIB801C-reduced [Ca2+]i would not be increased by A-kinase inhibitor Rp-8-Br-cAMPS, and CE-WIB801C-elevated IP3R phosphorylation would not be decreased by A-kinase inhibitor Rp-8-Br-cAMPS.
Antiplatelet drugs such as thienopyridine derivatives (i.e. ticlopidine, clopidogrel) have characteristics that inhibit [Ca2+]i mobilization, which is mediated by cAMP or cGMP (Barragan