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
In this study, we investigated the effects of cordycepin-enriched (CE)-WIB801C, a n-butanol extract of
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
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
In previous reports (Cho
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).
The preparation of CE-WIB801C was performed according to the method of our previous report (Lee
The purification of cordycepin from CE-WIB801C was performed according to the Lee method (Lee
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×
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).
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).
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.
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.
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).
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.
The quantity of cordycepin in WIB801C was about 8.2% (81.98 ± 1.37 mg/g-WIB801C (Lee
The concentration of collagen-induced maximal platelet aggregation was approximately 10 μg/mL (Lee
Because 200 and 400 μg/mL of CE-WIB801C (Fig. 2), and 500 μM cordycepin (Cho
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
As apposed to the phosphorylated VASP, PI3K/Akt phosphorylation stimulates αIIb/β3 activation and fibrinogen binding (Zhang
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
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).
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
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
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/β3
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
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
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
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