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Inhibitory effects of black soybean on platelet activation mediated through its active component of adenosine
Thrombosis Research 131 (2013) 254–261
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Regular Article
Inhibitory effects of black soybean on platelet activation mediated through its active component of adenosine Keunyoung Kim a, Kyung-Min Lim b, Hyun-Jung Shin b, Dae-Bang Seo b, Ji-Yoon Noh a, Seojin Kang a, Han Young Chung c, Sue Shin d, Jin-Ho Chung a, Ok-Nam Bae e,⁎ a
College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea AMOREPACIFIC CO/R&D Center, Gyeonggi-do 446-729, Republic of Korea College of Science and Technology, Korea University, Sejong City 339-700, Republic of Korea d Department of Laboratory Medicine, Boramae Hospital, 156-707, Republic of Korea e College of Pharmacy, Hanyang University, Gyeonggi-do 426-791, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 20 July 2012 Received in revised form 26 November 2012 Accepted 2 January 2013 Available online 17 January 2013 Keywords: Adenosine Black soybean Anti-platelet activity Isoflavones
a b s t r a c t Owing to the beneficial health effects on human cardiovascular system, soybeans and soy-related products have been a focus of intensive research. Soy isoflavones are known to be primarily responsible for the soy-related biological effects including anti-platelet activity but its in vivo relevancy has not been fully verified. Here we compared the role of adenosine, an active ingredient abundant in black soybean (BB) extract, in the anti-platelet effects of BB, to that of soy isoflavones. At the concentrations existing in BB, isoflavones such as genistein and daidzein could not attenuate collagen-induced platelet aggregation, however, adenosine significantly inhibited platelet aggregation with an equivalent potency to BB, suggesting that adenosine may be the major bioactive component. Consistently, the anti-aggregatory effects of BB disappeared after treatment of adenosine receptor antagonists. The effects of BB are mediated by adenosine through intracellular cAMP and subsequent attenuation of calcium mobilization. Of note, adenosine and BB significantly reduced platelet fibrinogen binding and platelet adhesion, other critical events for platelet activation, which were not affected by isoflavones. Taken together, we demonstrated that adenosine might be the major active ingredient for BB-induced anti-platelet activity, which will shed new light on the roles of adenosine as a bioactive compound in soybeans and soy-related food. © 2013 Elsevier Ltd. All rights reserved.
Introduction A link between the consumption of soy-related products and low incidence of chronic diseases has long been recognized and supported by several clinical and epidemiological studies [1–4]. To identify the active ingredients for the health benefits of soy products, many efforts have been made both in pharmacological and nutritional field. Various bioactive ingredients, such as isoflavones, soy proteins and soy saponins have been identified in soybeans and suggested to be responsible for the biological activities of soybeans [2,3,5]. Especially, soy isoflavones including genistein and daidzein have been considered as the primary active components for anti-osteoporosis [6,7], Abbreviations: AC, adenylyl cyclase; ACD, acid-citrate-dextrose; anti-GP Ib PE Ab, phycoerythrin-labeled monoclonal antibody against human glycoprotein Ib; BB, black soybean; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; PAC-1 FITC, fluorescein isocyanate labeled PAC-1; PBS, phosphate-buffered saline; PGE1, prostaglandin E1; PRP, platelet rich plasma; WP, washed platelets; YB, yellow soybean. ⁎ Corresponding author at: College of Pharmacy, Hanyang University, 55 Hanyangdaehakro, Sangnokgu, Ansan, Gyeonggi-do 426-791, Republic of Korea. Tel.: +82 31 400 5805; fax: +82 31 400 5958. E-mail address: [email protected] (O.-N. Bae). 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.01.002
anti-diabetes [8,9], anti-cancer effects [10,11] and cardiovascular benefits of soybean [4,12–14]. However, the authenticity of soy isoflavones as a true major active ingredient in soy or soy-related food is being questioned, especially for anti-platelet effects of soybeans. Increased systemic bioavailability of genistein and daidzein did not result in equally enhanced antiplatelet effects [15]. Anti-platelet activities of genistein and daidzein were observed at the concentration range of 10 to 30 μM [16–19], which has a large margin from their actual contents in soybeans. In addition, other active components in soybeans are newly being discovered to be effective in attenuating platelet activation, including anthocyanin or soy saponin [20–23]. Considering the diversity and heterogeneity of soybeans, there is a huge probability that components other than isoflavones may contribute to the anti-platelet effects of soy foods [3,5]. Recently, we demonstrated that black soybean (Glycine max; BB), which has shown superior biological activities to yellow or green soybeans such as anti-oxidant, anti-cancer, and anti-inflammatory effects [24–26], has potent inhibitory effect on platelet aggregation both in vitro human platelets and in vivo rat thrombosis models [27]. The active principle of anti-aggregating effect of BB was suggested to be adenosine through activity-guided fractionation and NMR/ESI-MS
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analyses. However, the contribution of adenosine to the inhibitory effect of BB on platelet activation and the exact mechanism underlying has not been elucidated in details. Adenosine is an endogenous molecule accumulating in the extracellular space in response to cell damage or metabolic stress [28]. Generally, adenosine plays protective roles including anti-inflammatory [29], anti-ischemic [30], and neuroprotective effects [31]. In cardiovascular system, adenosine increases vascular reactivity [32], promotes angiogenesis [33], decreases vascular adhesion [34], suppresses endothelial tissue factor expression [35] and inhibits platelet aggregation [36,37]. Despite the diverse biological activities of adenosine, the studies regarding the roles of food-based adenosine as an active ingredient in foods or dietary supplements are limited. To our best knowledge, the role of adenosine has not been addressed in the health effects from soybean or soy-related food. In this study, we compared the role of soy isoflavones and adenosine in the anti-platelet effects of BB. In addition, the effects of BB and adenosine on intra-platelet signaling pathways and other aspects of platelet activation were examined to provide a detailed mechanistic explanation of anti-platelet effect of BB.
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centrifugation at 500 g for 10 min. Platelets were washed with Tyrode buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl2, 10.0 mM HEPES, 5.0 mM glucose, 12.0 mM NaHCO3, 0.34 mM Na2HPO4, 2 mM CaCl2 and 0.3% BSA, pH 7.4) containing 1 μM PGE1 and 10% ACD. After centrifugation at 500 g 10 min, platelets were resuspended with Tyrode buffer, and the cell number was adjusted to 3 × 10 8 cells/ml. The final CaCl2 concentration was adjusted to 2 mM prior to use. Platelet aggregation measurement Platelet aggregation was determined by turbidometric method using an aggregometer (Chrono-log, Havertown, PA). After incubation with soybean extracts, adenosine, genistein or daidzein for 10 min at 37 °C, WP was loaded on the aggregometer and stimulated with collagen (1–4 μg/ml) for 6 min. The concentration of collagen selected for each individual platelet sample was the lowest showing maximum (>80%) aggregation. Platelet aggregation was measured by light transmission, with 100% calibrated as the absorbance of Tyrode buffer and 0% calibrated as the absorbance of WP. Measurement of intracellular calcium levels
Materials and methods Materials Trisodium citrate, citric acid, dimethyl sulfoxide (DMSO), ethanol, adenosine, genistein, daidzein, 2′,5′-dideoxyadenosine (DDA), SQ22538, urethane, ferric chloride, HEPES, glucose, NaCl, KCl, MgCl2, NaHCO3, Na2HPO4, CaCl2, KH2PO4, Tris–HCl, EDTA, triton X-100, prostaglandin E1 (PGE1) and bovine serum albumin (BSA) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO). SCH 58261 and 8-(3-Chlorostyryl) caffeine (CSC) were purchased from Tocris Biosciences (Bristol, UK). Collagen was from Chrono-log Co. (Harvertown, PA). Phycoerythrinlabeled monoclonal antibody against human CD42b (anti-CD42b-PE) and fluorescein isocyanate labeled PAC-1 (PAC-1 FITC) was from BD bioscience (San Diego, CA). Fluo-4 AM, calcein-AM and Alexa Fluor 488 conjugated fibrinogen was from Invitrogen (Eugene, OR) and cAMP ELISA kit was from Cayman Chemical Co. (Ann Arbor, MI). Protease inhibitor cocktail was from Calbiochem (San Diego, CA). Soybean extraction Black soybean (black seed coat and a green cotyledon) and yellow soybean (yellow seed coat and a yellow cotyledon) were from Boeun and Goesan, Korea. Dry matured soybeans were extracted for 3–5 hr at 50-60 °C with 20% ethanol. The extraction was repeated three times, and left for 12 hr at room temperature. After filtration and concentration under reduced pressure, the extract was lyophilized. The final yield of black soybean and yellow soybean extracts were 7.6% and 8.7%, respectively and the resultant powder was stored at −20 °C. The black soybean extract (BB) consisted of protein (1.0%), lipid (1.2%), carbohydrate (86.8%), ash (10.2%) and water (0.8%), and the content of total phenolic compounds was 1.29%. For in vitro experiments soybean extracts were dissolved in water.
Intracellular calcium change was determined using fluo-4 AM with flow cytometry. Fluo-4 AM (5 μM) was loaded to platelets in the presence of PGE1 (1 μM) for 45 min at 37 °C in dark. Then platelets were spun-down by centrifugation at 300 g for 10 min, and resuspended with Tyrode buffer. Dye loaded platelets were incubated with soybean extracts or adenosine for 10 min and stimulated with collagen (1–4 μg/ml) for 6 min. The reaction was terminated by dilution with Tyrode buffer. After incubated with anti-CD42b-PE for 20 min as platelet identifiers, platelets were analyzed on the FACSCalibur (BD Biosciences, San Jose, CA) equipped with argon lasor (λex 488 nm). Data from 5,000 events were collected and analyzed using CellQuest Pro software (BD Biosciences). Measurement of cAMP Platelets were incubated with soybean extracts or adenosine for 10 min and stimulated with collagen (1–4 μg/ml) for 6 min. The reaction was terminated by adding HCl (final 0.1 M) and platelets were lysed by sonication for 60 s in ultrasonic processor (Sonics and Materials Inc., Newtown, CT). The cAMP level in each samples were measured using a commercial cAMP ELISA kit, following the procedure provided by the manufacturer. Measurement of GP IIb/IIIa activation or fibrinogen binding GP IIb/IIIa activation was determined with flow cytometry using PAC-1, a GP IIb/IIIa activation specific antibody. Platelets were incubated with soybean extracts, adenosine, genistein or daidzein for 10 min and stimulated with collagen (1–4 μg/ml) for 6 min. The reaction was terminated by dilution with Tyrode buffer. Platelets were incubated with anti-CD42b-PE for 20 min and analyzed on FACSCalibur as described above. To determine fibrinogen binding, Alexa Fluor 488 conjugated fibrinogen was used instead of PAC-1.
Preparation of human platelets Measurement of platelet-collagen adhesion Human blood was collected from healthy male volunteers (18–25 years old) who had not taken any drugs for at least 14 days with an approval from the Ethics Committee of Health Service Center at Seoul National University. Blood was anti-coagulated with acidcitrate-dextrose (ACD; 85 mM trisodium citrate, 71 mM citric acid, 111 mM glucose) for preparation of washed platelets (WP). All procedures were conducted at room temperature and the use of glass containers and pipettes was avoided. Platelet rich plasma was prepared by centrifugation for 15 min at 150 g, and platelets were pelleted by
For measurement of platelet adhesion, 96 well plates were coated with collagen (40 μg/ml) for 1 hr, blocked with phosphate-buffered saline (PBS; 1 mM KH2PO4, 154 mM NaCl, 3 mM Na2HPO4, pH 7.4) containing 5% BSA for 1 hr, and washed 3 times with PBS. Platelets were stained with calcein-AM (2.5 μM) in for 15 min at 37 °C in dark. Calcein-AM loaded platelets were incubated with soybean extracts, adenosine, genistein or daidzein for 10 min and 50 μl of platelets were placed onto collagen-coated well for 30 min at room temperature.
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Non-adherent platelets were removed by washing with PBS, and adherent platelets were lysed with lysis buffer (1% triton X-100, 150 mM NaCl, 10 mM Tris–HCl, 5 mM EDTA, and protease inhibitor cocktail) for 10 min at 4 °C. Fluorescence was measured with a microplate reader (λex = 492 nm, λem = 535 nm, TECAN Spectrafluor, Grödig, Austria). Statistical analysis All the data are shown as mean±SEM and the data were subjected to one-way analysis of variance followed by Duncan's multiple ranged tests to determine which means were significantly different from the control. Statistical analysis was performed using SPSS software (Chicago, IL). In all cases, p value of b0.05 was used to determine significance. Results Inhibitory effect of BB against collagen-induced platelet adhesion and aggregation To address the anti-platelet effects of BB in platelets, we employed a freshly isolated washed platelet (WP) system, while we have used platelet-rich plasma (PRP) system in our recent report [27]. Platelets were pretreated with BB extract (0 to 100 μg/mL), and stimulated by collagen, a representative aggregatory agonist. BB significantly inhibited collagen-induced platelet adhesion (Fig. 1A), a key phenomenon during platelet activation in patho-physiology [38,39]. BB extract also significantly attenuated aggregation in a concentration-dependent manner (Fig. 1B), and the inhibitory pattern was well correlated to BB effect on PRP system. In addition, yellow soybean (YB) did not induce any protection against collagen-stimulated platelet adhesion and aggregation. Intracellular pathways for anti-platelet effects of BB Next, we investigated the mechanism for the inhibitory effect of BB on platelet activation. Collagen activates GPVI receptor which initiates IP3 increase and subsequent calcium mobilization from intraplatelet storage, which is a key molecular mediator for platelet activation [40]. Meanwhile, intracellular cAMP can decrease collagen-induced calcium increases through the blockade of IP3 receptor [41]. BB prevented the decrease of cAMP level of collagen-stimulated human platelets in a concentration dependent manner (Fig. 2A). In line with increased cAMP level, BB significantly decreased collagen-induced intracellular calcium increase (Fig. 2B). While BB showed significant effects on cAMP and calcium level, YB did not affect these intraplatelet signaling, confirming the superior anti-platelet effect of BB. Based on the recent reports that platelet adhesion receptor expression play pivotal roles in platelet aggregation and activation [39,40,42], we have further examined the
protective role of BB in platelet adhesion receptor. BB significantly reduced collagen-induced GP IIb/IIIa expression (Fig. 2C), and subsequent fibrinogen binding (Fig. 2D). We recently suggested that adenosine may be important in anti-aggregatory effect of BB through activity-guided fractionation [27]. In order to examine the role of adenosine in BB-induced platelet protection, we examined BB effect in the presence of adenosine receptor antagonists, SCH 58261 and 8-(3-Chlorostyryl) caffeine (CSC). Confirming our previous result in PRP system, SCH 58261 and CSC significantly reduced BB-induced platelet inhibition (Fig. 2E), showing that adenosine plays a key role in anti-platelet activity of BB. Anti-platelet activities of adenosine To investigate the mechanism of adenosine action in BB activity, we examined anti-platelet activities of adenosine. When platelets were pretreated with adenosine (0 to 1 μM), collagen-stimulated aggregation was significantly reduced (Fig. 3A). Notably, the concentrationresponse pattern of adenosine is consistent with that of BB extract (0 to 100 μg/mL), based on the equivalent adenosine concentration in BB extract (0.36%). Collagen stimulation of platelets increased intracellular calcium as observed in flow cytometric analysis, and the increased calcium level was significantly reversed by adenosine pretreatment (Fig. 3B). Adenosine inhibited collagen-induced GP IIb/IIIa expression (Fig. 3C), reflecting that adenosine can block the downstream activation pathway following calcium increase. Collagen-induced cAMP decrease was reversed by adenosine, suggesting that increased cAMP plays a key role in the anti-platelet activity of adenosine (Fig. 3D). cAMP restoring activity of adenosine is mediated via cAMP synthesis by adenylyl cyclase (AC), as evidenced by abolition of cAMP increase by AC inhibitors (DDA and SQ22536) (Fig. 3E). To address if cAMP signaling is responsible for anti-platelet activity of adenosine, the effects of AC inhibitors on intracellular calcium and aggregation were examined. As expected, AC inhibitors reversed the inhibitory effects of adenosine in collagen-induced calcium mobilization (Fig. 3F) and aggregation (Fig. 3G), suggesting that cAMP is the key pathway in adenosinemediated anti-platelet effects. Role of adenosine-cAMP in anti-platelet effects of BB Next, we investigated if the inhibitory effect of BB in platelet activation is reversed by inhibition of cAMP synthesis. AC inhibitors reversed the inhibitory effects of adenosine in collagen-induced platelet adhesion (Fig. 4A) and aggregation (Fig. 4B). In addition, the inhibitory effects of BB on intracellular calcium were inhibited by DDA or SQ22536 (Fig. 4C). Consistently, AC inhibitor inhibited cAMP increase by BB (Fig. 4D), confirming that BB effect on platelet activation is mediated by adenosine-cAMP signaling.
Fig. 1. Effects of BB on adhesion and aggregation in human washed platelets. (A) Various concentrations of BB or YB (100 μg/ml) were treated for 10 min, and human platelets were stimulated with collagen (1–4 μg/ml) for 6 min. Platelet aggregation was measured using lumi-aggregometer. (B) Calcein-loaded platelets were exposed to BB or YB (100 μg/ml) for 10 min and placed onto collagen-coated microwells for 30 min. Adhered platelets were analyzed by calcein fluorescence. Values are means±SEM, n=3–4. The asterisk represents significant differences from the corresponding control, Pb 0.05.
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Fig. 2. Inhibitory effects of BB on platelet activation pathways. (A) Various concentrations of BB or YB (100 μg/ml) were added to platelets for 10 min and intracellular cAMP level was determined after collagen stimulation. Intracellular cAMP level in collagen-stimulated platelets was determined using commercial cAMP ELISA kit. (B) Effect of BB or YB on collagen-induced intracellular calcium increase was examined in flow cytometry. (C) Various concentrations of BB were added to platelets for 10 min and GP IIb/IIIa expression was determined after collagen stimulation. (D) Fibrinogen binding was examined by flow cytometry after pretreatment of BB and collagen stimulation. (E) BB (100 μg/ml) was treated for 10 min in the presence of adenosine receptor antagonist SCH 58261 (300 nM) or 8-(3-Chlorostyryl) caffeine (CSC; 1 μM), and platelet aggregation was initiated with collagen. Values are means ± SEM, n= 3–4. The asterisk represents significant differences from the corresponding control, P b 0.05. The pound sign represents significant differences from BB treated group, P b 0.05.
Here we demonstrated that adenosine plays important roles in anti-platelet effects of BB, however, previous studies showed that isoflavones in soybean can inhibit platelet aggregation [16–19]. In order to identify which ingredient is more important in anti-platelet effect of BB, isoflavones (genistein or daidzein) or adenosine at the equivalent concentration included in BB extract was treated to platelets. Surprisingly, while adenosine (1 μM) treatment significantly reduced collagen-induced platelet aggregation, treatment of genistein (0.3 μM) or daidzein (0.3 μM), the major bioactive isoflavones known to be responsible for soybean-related health effects, failed to attenuate platelet aggregation (Fig. 5A). Even the combined treatment of isoflavones (0.3 μM genistein with 0.3 μM daidzein) did not inhibit collagen-stimulated platelet aggregation, suggesting that adenosine rather than isoflavones accounted for the protective role of BB in platelet activation. Of note, isoflavones of genistein or daidzein could not inhibit collagen-induced GP IIb/IIIa expression and platelet adhesion (Fig. 5B) or platelet adhesion (Fig. 5C), suggesting that adenosine may act as the major component in inhibitory effect of BB on platelet adhesion as well.
Discussion In this investigation, we have demonstrated that adenosine plays a pivotal role in the inhibitory effect of black soybean (BB) on collageninduced platelet aggregation and adhesion (Fig. 6). In human washed platelets, we showed that adenosine decreased intracellular calcium mobilization through cAMP elevation. Notably, in addition to platelet aggregation, BB displayed a wide-spectrum of anti-platelet effects, modulating the overall aspects of platelet activation including GPIIb/ IIIa expression, fibrinogen binding and platelet adhesion, which may lead to an efficient control of thrombosis. With this study, we believe that important evidence was provided supporting the possible role of adenosine as a key mediator of soy-related biological effects. Previously, most of the biological effects from soybeans have been suggested to be mediated by their unique component of isoflavones, which showed diverse health effects such as anti-diabetes, anticancer and anti-osteoporosis [2,6,10,12]. In platelets, soy isoflavones, such as genistein and daidzein, can modulate thromboxane A2 receptor density, serotonin secretion and aggregation [16–19]. The concentration
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Fig. 3. Effects of adenosine on platelet activation. (A) Platelets were treated with different concentrations of adenosine for 10 min, and collagen-induced platelet aggregation was examined. Representative tracing (left) and dose–response graph (right) from more than 3 independent experiments are shown. (B) Platelets were treated with different concentrations of adenosine and Fluo-4 elicited intracellular calcium increase was determined after collagen stimulation by flow cytometry. Representative tracing (left) and dose– response graph (right) from more than 3 independent experiments are shown. (C and D) Effect of adenosine on adhesion receptor GP IIb/IIIa expression (C) or on intracellular cAMP level (D) was examined following collagen stimulation. (E to G) Treatment of DDA (100 μM) or SQ22536 (20 μM) for 10 min reversed adenosine effect on cAMP level (E), intracellular calcium level (F) and aggregation (G) in collagen-stimulated human platelets. Values are means ± SEM, n = 3–4. The asterisk represents significant differences from the corresponding control, P b 0.05. The pound sign represents significant differences from adenosine-only treatment, P b 0.05.
range of genistein required for the inhibitory effect of platelet aggregation is reported to be higher than 30 μM [18], or around 37 μM (10 μg/mL in their original article) [19]. Guerrero et al., reported that IC50 of genistein was 11.9 ± 1.5 μM against collagen-induced platelet aggregation [16]. Interestingly, here we found that isoflavones are not protective against collagen-induced platelet aggregation at the concentration (0.3 μM) as contained in BB (Fig. 5A). Consistent with our results, Gooderham et al. observed that consumption of soy proteins which was rich in genistein and daidzein had no effects on platelet aggregation in human male volunteers. Plasma concentration of genistein was determined to be 906.8 nmol/L and they concluded that sub-micromolar isoflavones might not be sufficient to exert the biological activities on platelet aggregation [15]. There are several reports showing that the human plasma concentration of isoflavones can be increased following soy-related food, for example, 43–312 ng/mL in females consuming diets of soy-based food [43], 40–240 ng/mL in Japanese adults who
frequently consume soy food [44], and 980 ng/mL in infants fed with soy-based formula [45]. However, these plasma ranges are far below the effective concentration of isoflavones for anti-platelet activities, reflecting that components other than isoflavones might play key roles in anti-thrombotic effect of soy-food. In the present study, adenosine rather than isoflavones was important for BB-induced platelet inhibition. The effective concentration of adenosine (IC50 = 1.85 ± 0.86 μM) well matches its actual content in BB extract (0.36%), explaining the anti-platelet potency of BB. This point can be further supported by the anti-platelet potencies of soybean extracts with different contents of adenosine and isoflavones. The BB extracted with 20% EtOH (BB20) showed higher anti-platelet activity than that with 70% EtOH (BB70). Notably, adenosine contents are 0.5% in BB20 and 0.04% in BB70, while those of isoflavone are similar between BB20 and BB70 (0.09% and 0.11%, respectively), supporting that the anti-platelet effects of soybean extracts were correlated better with the adenosine contents than those of isoflavones.
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Fig. 4. Inhibitory effect of BB on platelet activation: role of adenylcyclase inhibition. (A) Adenylate cyclase inhibition by DDA (100 μM) or SQ22536 (20 μM) for 10 min reversed the effects of BB on platelet adhesion to collagen. (B to D) Adenylate cyclase inhibition by DDA (100 μM) or SQ22536 (20 μM) for 10 min reversed the effects of BB platelet aggregation (B), intracellular calcium level (C), and cAMP level (D) in collagen-stimulated human platelets. Values are means ± SEM, n = 3–4. The asterisk represents significant differences from the corresponding control, P b 0.05. The pound sign represents significant differences from BB-only treatment, P b 0.05.
In our previous report [27], we observed that single or multiple administration of BB to rats (50, 100 mg/kg) significantly inhibited thrombus formation, and these dose range were relevant to daily consumption of soybean foods (20–80 g) [3] or BB (720.7 mg) [46]. This indicates that daily intake of BB might be able to manifest a protective effect against thrombosis-associated cardiovascular diseases. Although human kinetic data of adenosine after consumption of BB are currently unavailable, we believe that adenosine contained in BB may have contributed to anti-thrombotic effects of BB in vivo, based on the followings; 1) adenosine increases in plasma following BB administration to rats, and 2) the oral intake of BB results in higher plasma level of adenosine than that of pure adenosine. The plasma level of adenosine can be modified by internal and external factors like diseases, diets and drugs. For example, diprydamole which inhibits the
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adenosine reuptake by red blood cells can increase the plasma level of adenosine in hypoxia [47]. Exact contribution of adenosine to the anti-thrombotic effects of BB may be clarified when the mechanism underlying the increased bioavailability of adenosine following BB consumption is elucidated in the future. While many researches on soybean and related products have focused on the positive health effects of isoflavones, the concerns about the safety of soy isoflavones were proposed due to their hormonemimic effects [48]. Besides the safety issue, the roles of soy isoflavones have been questioned for some of soy-related health effects [5,49], and the more dominant roles of other active ingredients in soybeans were suggested. Considering the diversity and heterogeneity of soybeans and products [5,50], components other than isoflavones may be bioactive and more important for the beneficial effects of soy foods. It is necessary to examine the other bioactive ingredient(s) and to understand the actual mechanism underlying the health benefits of soy products to identify the true bioactive components. Here we demonstrated that adenosine may be an important mediator for soy-related anti-platelet effects. Adenosine has diverse biological functions and the modulation of adenosine metabolism and/or its receptor function are considered as a possible therapeutic target for infection, autoimmunity, ischemia and degenerative diseases [29–34,36]. Besides the endogenous formation and accumulation, adenosine naturally occurs and can be exogenously taken up from food via nucleoside transporter [51]. Nevertheless, studies addressing adenosine as an active food ingredient are limited [52,53]. Considering the potent biological activities of adenosine, it will be worthy to examine food-based adenosine as a potential candidate of active ingredients in foods or herbal medicine. In this study, BB and adenosine can attenuate GP IIb/ IIIa expression, fibrinogen binding and adhesion as well as platelet aggregation, while genistein or daidzein could not affect these events. Up to now, most of the researches on pathological platelet activation have focused on platelet aggregation rather than platelet adhesion. However, the expression of adhesion molecules such as GP IIb/IIIa and resultant adhesion is an essential process in platelet activation [39,40]. GPIIb/IIIa represents a soluble fibrinogen biding site, contributing to post-ligand occupancy signaling in platelets leading to further signal transduction and cytoskeletal protein alteration, resulting in platelet spreading and shape change [54,55]. In addition to platelet adhesion, the pretreatment of BB significantly inhibited granular secretion and phosphatidylserine (PS) exposure induced by collagen (data not shown). Mediators secreted from platelets and exposed PS promote thrombosis through inducing secondary platelet activation, plateletleukocyte interaction, vasoconstriction and coagulation. In this regard, the holistic modulation of diverse features of pathological platelet activation by BB and adenosine might be effective in preventing thrombotic events and related cardiovascular diseases.
Fig. 5. Comparison of the anti-platelet effects of adenosine and isoflavones. (A to C) Adenosine (1 μM), genistein (0.3 μM), daidzein (0.3 μM) and combination of genistein and daidzein (0.3 μM for both compound) were treated for 10 min, and collagen-induced platelet aggregation (A), GPIIb/IIIa expression (B) or platelet adhesion (D) was examined. Values are means± SEM, n=3–4. The asterisk represents significant differences from the corresponding control, Pb 0.05.
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Fig. 6. Suggested mechanism for the inhibitory effect of Black soybean on collagen-stimulated platelet activation.
In conclusion, we have shown that the anti-platelet effect of BB is mediated by adenosine-cAMP signaling, resulting in decreased intraplatelet calcium mobilization, GPIIb/IIIa expression, fibrinogen binding, and platelet aggregation/adhesion. We believe that this study has provided 1) the mechanistic explanation for the anti-platelet effects of BB and 2) the evidence that adenosine may act as a principle ingredient in health benefits by soybeans and soy related food. Conflict of interest statement No author of this study had financial or personal relationships with other people, manufacturers or organizations that could inappropriately influence this work. Acknowledgements This work was supported by the National Research Foundation of Korea, which is funded by the Korean government Grant (20120000844), and by a Basic Science Research Program (2012R1A1A3013240). References [1] Beavers KM, Jonnalagadda SS, Messina MJ. Soy consumption, adhesion molecules, and pro-inflammatory cytokines: a brief review of the literature. Nutr Rev 2009;67: 213–21. [2] Messina M. A brief historical overview of the past two decades of soy and isoflavone research. J Nutr 2010;140:1350S–4S. [3] Omoni AO, Aluko RE. Soybean foods and their benefits: potential mechanisms of action. Nutr Rev 2005;63:272–83. [4] Rivas M, Garay RP, Escanero JF, Cia Jr P, Cia P, Alda JO. Soy milk lowers blood pressure in men and women with mild to moderate essential hypertension. J Nutr 2002;132: 1900–2. [5] Kang J, Badger TM, Ronis MJ, Wu X. Non-isoflavone phytochemicals in soy and their health effects. J Agric Food Chem 2010;58:8119–33. [6] Ishimi Y. Dietary equol and bone metabolism in postmenopausal Japanese women and osteoporotic mice. J Nutr 2010;140:1373S–6S. [7] Song Y, Paik HY, Joung H. Soybean and soy isoflavone intake indicate a positive change in bone mineral density for 2 years in young Korean women. Nutr Res 2008;28:25–30. [8] Pipe EA, Gobert CP, Capes SE, Darlington GA, Lampe JW, Duncan AM. Soy protein reduces serum LDL cholesterol and the LDL cholesterol:HDL cholesterol and
[9]
[10] [11] [12] [13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24]
[25]
apolipoprotein B:apolipoprotein A-I ratios in adults with type 2 diabetes. J Nutr 2009;139:1700–6. Lu MP, Wang R, Song X, Chibbar R, Wang X, Wu L, et al. Dietary soy isoflavones increase insulin secretion and prevent the development of diabetic cataracts in streptozotocin-induced diabetic rats. Nutr Res 2008;28:464–71. Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 2002;3:364–73. Jian L. Soy, isoflavones, and prostate cancer. Mol Nutr Food Res 2009;53:217–26. Anthony MS, Clarkson TB, Williams JK. Effects of soy isoflavones on atherosclerosis: potential mechanisms. Am J Clin Nutr 1998;68:1390S–3S. Pase MP, Grima NA, Sarris J. The effects of dietary and nutrient interventions on arterial stiffness: a systematic review. Am J Clin Nutr 2011;93:446–54. Marsh TG, Straub RK, Villalobos F, Hong MY. Soy protein supports cardiovascular health by downregulating hydroxymethylglutaryl-coenzyme A reductase and sterol regulatory element-binding protein-2 and increasing antioxidant enzyme activity in rats with dextran sodium sulfate-induced mild systemic inflammation. Nutr Res 2011;31:922–8. Gooderham MH, Adlercreutz H, Ojala ST, Wahala K, Holub BJ. A soy protein isolate rich in genistein and daidzein and its effects on plasma isoflavone concentrations, platelet aggregation, blood lipids and fatty acid composition of plasma phospholipid in normal men. J Nutr 1996;126:2000–6. Guerrero JA, Lozano ML, Castillo J, Benavente-Garcia O, Vicente V, Rivera J. Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor. J Thromb Haemost 2005;3:369–76. Guerrero JA, Navarro-Nunez L, Lozano ML, Martinez C, Vicente V, Gibbins JM, et al. Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells. Br J Clin Pharmacol 2007;64:133–44. Kondo K, Suzuki Y, Ikeda Y, Umemura K. Genistein, an isoflavone included in soy, inhibits thrombotic vessel occlusion in the mouse femoral artery and in vitro platelet aggregation. Eur J Pharmacol 2002;455:53–7. Nakashima S, Koike T, Nozawa Y. Genistein, a protein tyrosine kinase inhibitor, inhibits thromboxane A2-mediated human platelet responses. Mol Pharmacol 1991;39:475–80. Choung MG, Baek IY, Kang ST, Han WY, Shin DC, Moon HP, et al. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J Agric Food Chem 2001;49:5848–51. Garcia-Alonso M, Rimbach G, Rivas-Gonzalo JC, De Pascual-Teresa S. Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A–studies in platelets, monocytes, and human endothelial cells. J Agric Food Chem 2004;52:3378–84. Shi J, Arunasalam K, Yeung D, Kakuda Y, Mittal G, Jiang Y. Saponins from edible legumes: chemistry, processing, and health benefits. J Med Food 2004;7:67–78. Xu B, Chang SK. Total phenolics, phenolic acids, isoflavones, and anthocyanins and antioxidant properties of yellow and black soybeans as affected by thermal processing. J Agric Food Chem 2008;56:7165–75. Kumar V, Rani A, Dixit AK, Pratap D, Bhatnagar D. A comparative assessment of total phenolic content, ferric reducing-anti-oxidative power, free radical-scavenging activity, vitamin C and isoflavones content in soybean with varying seed coat colour. Food Res Int 2010;43:323–8. Takahashi R, Ohmori R, Kiyose C, Momiyama Y, Ohsuzu F, Kondo K. Antioxidant activities of black and yellow soybeans against low density lipoprotein oxidation. J Agric Food Chem 2005;53:4578–82.
K. Kim et al. / Thrombosis Research 131 (2013) 254–261 [26] Xu B, Chang SK. Antioxidant capacity of seed coat, dehulled bean, and whole black soybeans in relation to their distributions of total phenolics, phenolic acids, anthocyanins, and isoflavones. J Agric Food Chem 2008;56:8365–73. [27] Kim K, Lim KM, Kim CW, Shin HJ, Seo DB, Lee SJ, et al. Black soybean extract can attenuate thrombosis through inhibition of collagen-induced platelet activation. J Nutr Biochem 2011;22:964–70. [28] Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov 2008;7:759–70. [29] Ernst PB, Garrison JC, Thompson LF. Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology. J Immunol 2010;185:1993–8. [30] Lappas CM, Day YJ, Marshall MA, Engelhard VH, Linden J. Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1ddependent NKT cell activation. J Exp Med 2006;203:2639–48. [31] Fredholm BB, Chen JF, Masino SA, Vaugeois JM. Actions of adenosine at its receptors in the CNS: insights from knockouts and drugs. Annu Rev Pharmacol Toxicol 2005;45: 385–412. [32] Fredholm BB. Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 2007;14:1315–23. [33] Clark AN, Youkey R, Liu X, Jia L, Blatt R, Day YJ, et al. A1 adenosine receptor activation promotes angiogenesis and release of VEGF from monocytes. Circ Res 2007;101: 1130–8. [34] Yang D, Zhang Y, Nguyen HG, Koupenova M, Chauhan AK, Makitalo M, et al. The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J Clin Invest 2006;116:1913–23. [35] Deguchi H, Takeya H, Urano H, Gabazza EC, Zhou H, Suzuki K. Adenosine regulates tissue factor expression on endothelial cells. Thromb Res 1998;91:57–64. [36] Johnston-Cox HA, Yang D, Ravid K. Physiological implications of adenosine receptor-mediated platelet aggregation. J Cell Physiol 2011;226:46–51. [37] Anfossi G, Russo I, Massucco P, Mattiello L, Cavalot F, Balbo A, et al. Adenosine increases human platelet levels of cGMP through nitric oxide: possible role in its antiaggregating effect. Thromb Res 2002;105:71–8. [38] Andrews RK, Berndt MC. Platelet physiology and thrombosis. Thromb Res 2004;114: 447–53. [39] Calderwood DA. Integrin activation. J Cell Sci 2004;117:657–66. [40] Abrams CS. Intracellular signaling in platelets. Curr Opin Hematol 2005;12:401–5.
261
[41] Tertyshnikova S, Fein A. Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell. Proc Natl Acad Sci U S A 1998;95:1613–7. [42] Li R, Mitra N, Gratkowski H, Vilaire G, Litvinov R, Nagasami C, et al. Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations. Science 2003;300:795–8. [43] Morton MS, Wilcox G, Wahlqvist ML, Griffiths K. Determination of lignans and isoflavonoids in human female plasma following dietary supplementation. J Endocrinol 1994;142:251–9. [44] Adlercreutz H, Markkanen H, Watanabe S. Plasma concentrations of phytooestrogens in Japanese men. Lancet 1993;342:1209–10. [45] Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE. Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 1997;350:23–7. [46] USDA. U.S. dry bean statistics, 1970–2010. U.S. Department of Agriculture; 2011. [47] Saito H, Nishimura M, Shinano H, Makita H, Tsujino I, Shibuya E, et al. Plasma concentration of adenosine during normoxia and moderate hypoxia in humans. Am J Respir Crit Care Med 1999;159:1014–8. [48] Munro IC, Harwood M, Hlywka JJ, Stephen AM, Doull J, Flamm WG, et al. Soy isoflavones: a safety review. Nutr Rev 2003;61:1–33. [49] Greaves KA, Parks JS, Williams JK, Wagner JD. Intact dietary soy protein, but not adding an isoflavone-rich soy extract to casein, improves plasma lipids in ovariectomized cynomolgus monkeys. J Nutr 1999;129:1585–92. [50] Erdman Jr JW, Badger TM, Lampe JW, Setchell KD, Messina M. Not all soy products are created equal: caution needed in interpretation of research results. J Nutr 2004;134:1229S–33S. [51] Casado FJ, Lostao MP, Aymerich I, Larrayoz IM, Duflot S, Rodriguez-Mulero S, et al. Nucleoside transporters in absorptive epithelia. J Physiol Biochem 2002;58:207–16. [52] Agarwal KC. Therapeutic actions of garlic constituents. Med Res Rev 1996;16:111–24. [53] Kuo MC, Chang CY, Cheng TL, Wu MJ. Immunomodulatory effect of Antrodia camphorata mycelia and culture filtrate. J Ethnopharmacol 2008;120:196–203. [54] Calderwood DA, Yan B, de Pereda JM, Alvarez BG, Fujioka Y, Liddington RC, et al. The phosphotyrosine binding-like domain of talin activates integrins. J Biol Chem 2002;277:21749–58. [55] Jennings LK. Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 2009;102:248–57.
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Regular Article
Inhibitory effects of black soybean on platelet activation mediated through its active component of adenosine Keunyoung Kim a, Kyung-Min Lim b, Hyun-Jung Shin b, Dae-Bang Seo b, Ji-Yoon Noh a, Seojin Kang a, Han Young Chung c, Sue Shin d, Jin-Ho Chung a, Ok-Nam Bae e,⁎ a
College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea AMOREPACIFIC CO/R&D Center, Gyeonggi-do 446-729, Republic of Korea College of Science and Technology, Korea University, Sejong City 339-700, Republic of Korea d Department of Laboratory Medicine, Boramae Hospital, 156-707, Republic of Korea e College of Pharmacy, Hanyang University, Gyeonggi-do 426-791, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 20 July 2012 Received in revised form 26 November 2012 Accepted 2 January 2013 Available online 17 January 2013 Keywords: Adenosine Black soybean Anti-platelet activity Isoflavones
a b s t r a c t Owing to the beneficial health effects on human cardiovascular system, soybeans and soy-related products have been a focus of intensive research. Soy isoflavones are known to be primarily responsible for the soy-related biological effects including anti-platelet activity but its in vivo relevancy has not been fully verified. Here we compared the role of adenosine, an active ingredient abundant in black soybean (BB) extract, in the anti-platelet effects of BB, to that of soy isoflavones. At the concentrations existing in BB, isoflavones such as genistein and daidzein could not attenuate collagen-induced platelet aggregation, however, adenosine significantly inhibited platelet aggregation with an equivalent potency to BB, suggesting that adenosine may be the major bioactive component. Consistently, the anti-aggregatory effects of BB disappeared after treatment of adenosine receptor antagonists. The effects of BB are mediated by adenosine through intracellular cAMP and subsequent attenuation of calcium mobilization. Of note, adenosine and BB significantly reduced platelet fibrinogen binding and platelet adhesion, other critical events for platelet activation, which were not affected by isoflavones. Taken together, we demonstrated that adenosine might be the major active ingredient for BB-induced anti-platelet activity, which will shed new light on the roles of adenosine as a bioactive compound in soybeans and soy-related food. © 2013 Elsevier Ltd. All rights reserved.
Introduction A link between the consumption of soy-related products and low incidence of chronic diseases has long been recognized and supported by several clinical and epidemiological studies [1–4]. To identify the active ingredients for the health benefits of soy products, many efforts have been made both in pharmacological and nutritional field. Various bioactive ingredients, such as isoflavones, soy proteins and soy saponins have been identified in soybeans and suggested to be responsible for the biological activities of soybeans [2,3,5]. Especially, soy isoflavones including genistein and daidzein have been considered as the primary active components for anti-osteoporosis [6,7], Abbreviations: AC, adenylyl cyclase; ACD, acid-citrate-dextrose; anti-GP Ib PE Ab, phycoerythrin-labeled monoclonal antibody against human glycoprotein Ib; BB, black soybean; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; PAC-1 FITC, fluorescein isocyanate labeled PAC-1; PBS, phosphate-buffered saline; PGE1, prostaglandin E1; PRP, platelet rich plasma; WP, washed platelets; YB, yellow soybean. ⁎ Corresponding author at: College of Pharmacy, Hanyang University, 55 Hanyangdaehakro, Sangnokgu, Ansan, Gyeonggi-do 426-791, Republic of Korea. Tel.: +82 31 400 5805; fax: +82 31 400 5958. E-mail address: [email protected] (O.-N. Bae). 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.01.002
anti-diabetes [8,9], anti-cancer effects [10,11] and cardiovascular benefits of soybean [4,12–14]. However, the authenticity of soy isoflavones as a true major active ingredient in soy or soy-related food is being questioned, especially for anti-platelet effects of soybeans. Increased systemic bioavailability of genistein and daidzein did not result in equally enhanced antiplatelet effects [15]. Anti-platelet activities of genistein and daidzein were observed at the concentration range of 10 to 30 μM [16–19], which has a large margin from their actual contents in soybeans. In addition, other active components in soybeans are newly being discovered to be effective in attenuating platelet activation, including anthocyanin or soy saponin [20–23]. Considering the diversity and heterogeneity of soybeans, there is a huge probability that components other than isoflavones may contribute to the anti-platelet effects of soy foods [3,5]. Recently, we demonstrated that black soybean (Glycine max; BB), which has shown superior biological activities to yellow or green soybeans such as anti-oxidant, anti-cancer, and anti-inflammatory effects [24–26], has potent inhibitory effect on platelet aggregation both in vitro human platelets and in vivo rat thrombosis models [27]. The active principle of anti-aggregating effect of BB was suggested to be adenosine through activity-guided fractionation and NMR/ESI-MS
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analyses. However, the contribution of adenosine to the inhibitory effect of BB on platelet activation and the exact mechanism underlying has not been elucidated in details. Adenosine is an endogenous molecule accumulating in the extracellular space in response to cell damage or metabolic stress [28]. Generally, adenosine plays protective roles including anti-inflammatory [29], anti-ischemic [30], and neuroprotective effects [31]. In cardiovascular system, adenosine increases vascular reactivity [32], promotes angiogenesis [33], decreases vascular adhesion [34], suppresses endothelial tissue factor expression [35] and inhibits platelet aggregation [36,37]. Despite the diverse biological activities of adenosine, the studies regarding the roles of food-based adenosine as an active ingredient in foods or dietary supplements are limited. To our best knowledge, the role of adenosine has not been addressed in the health effects from soybean or soy-related food. In this study, we compared the role of soy isoflavones and adenosine in the anti-platelet effects of BB. In addition, the effects of BB and adenosine on intra-platelet signaling pathways and other aspects of platelet activation were examined to provide a detailed mechanistic explanation of anti-platelet effect of BB.
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centrifugation at 500 g for 10 min. Platelets were washed with Tyrode buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl2, 10.0 mM HEPES, 5.0 mM glucose, 12.0 mM NaHCO3, 0.34 mM Na2HPO4, 2 mM CaCl2 and 0.3% BSA, pH 7.4) containing 1 μM PGE1 and 10% ACD. After centrifugation at 500 g 10 min, platelets were resuspended with Tyrode buffer, and the cell number was adjusted to 3 × 10 8 cells/ml. The final CaCl2 concentration was adjusted to 2 mM prior to use. Platelet aggregation measurement Platelet aggregation was determined by turbidometric method using an aggregometer (Chrono-log, Havertown, PA). After incubation with soybean extracts, adenosine, genistein or daidzein for 10 min at 37 °C, WP was loaded on the aggregometer and stimulated with collagen (1–4 μg/ml) for 6 min. The concentration of collagen selected for each individual platelet sample was the lowest showing maximum (>80%) aggregation. Platelet aggregation was measured by light transmission, with 100% calibrated as the absorbance of Tyrode buffer and 0% calibrated as the absorbance of WP. Measurement of intracellular calcium levels
Materials and methods Materials Trisodium citrate, citric acid, dimethyl sulfoxide (DMSO), ethanol, adenosine, genistein, daidzein, 2′,5′-dideoxyadenosine (DDA), SQ22538, urethane, ferric chloride, HEPES, glucose, NaCl, KCl, MgCl2, NaHCO3, Na2HPO4, CaCl2, KH2PO4, Tris–HCl, EDTA, triton X-100, prostaglandin E1 (PGE1) and bovine serum albumin (BSA) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO). SCH 58261 and 8-(3-Chlorostyryl) caffeine (CSC) were purchased from Tocris Biosciences (Bristol, UK). Collagen was from Chrono-log Co. (Harvertown, PA). Phycoerythrinlabeled monoclonal antibody against human CD42b (anti-CD42b-PE) and fluorescein isocyanate labeled PAC-1 (PAC-1 FITC) was from BD bioscience (San Diego, CA). Fluo-4 AM, calcein-AM and Alexa Fluor 488 conjugated fibrinogen was from Invitrogen (Eugene, OR) and cAMP ELISA kit was from Cayman Chemical Co. (Ann Arbor, MI). Protease inhibitor cocktail was from Calbiochem (San Diego, CA). Soybean extraction Black soybean (black seed coat and a green cotyledon) and yellow soybean (yellow seed coat and a yellow cotyledon) were from Boeun and Goesan, Korea. Dry matured soybeans were extracted for 3–5 hr at 50-60 °C with 20% ethanol. The extraction was repeated three times, and left for 12 hr at room temperature. After filtration and concentration under reduced pressure, the extract was lyophilized. The final yield of black soybean and yellow soybean extracts were 7.6% and 8.7%, respectively and the resultant powder was stored at −20 °C. The black soybean extract (BB) consisted of protein (1.0%), lipid (1.2%), carbohydrate (86.8%), ash (10.2%) and water (0.8%), and the content of total phenolic compounds was 1.29%. For in vitro experiments soybean extracts were dissolved in water.
Intracellular calcium change was determined using fluo-4 AM with flow cytometry. Fluo-4 AM (5 μM) was loaded to platelets in the presence of PGE1 (1 μM) for 45 min at 37 °C in dark. Then platelets were spun-down by centrifugation at 300 g for 10 min, and resuspended with Tyrode buffer. Dye loaded platelets were incubated with soybean extracts or adenosine for 10 min and stimulated with collagen (1–4 μg/ml) for 6 min. The reaction was terminated by dilution with Tyrode buffer. After incubated with anti-CD42b-PE for 20 min as platelet identifiers, platelets were analyzed on the FACSCalibur (BD Biosciences, San Jose, CA) equipped with argon lasor (λex 488 nm). Data from 5,000 events were collected and analyzed using CellQuest Pro software (BD Biosciences). Measurement of cAMP Platelets were incubated with soybean extracts or adenosine for 10 min and stimulated with collagen (1–4 μg/ml) for 6 min. The reaction was terminated by adding HCl (final 0.1 M) and platelets were lysed by sonication for 60 s in ultrasonic processor (Sonics and Materials Inc., Newtown, CT). The cAMP level in each samples were measured using a commercial cAMP ELISA kit, following the procedure provided by the manufacturer. Measurement of GP IIb/IIIa activation or fibrinogen binding GP IIb/IIIa activation was determined with flow cytometry using PAC-1, a GP IIb/IIIa activation specific antibody. Platelets were incubated with soybean extracts, adenosine, genistein or daidzein for 10 min and stimulated with collagen (1–4 μg/ml) for 6 min. The reaction was terminated by dilution with Tyrode buffer. Platelets were incubated with anti-CD42b-PE for 20 min and analyzed on FACSCalibur as described above. To determine fibrinogen binding, Alexa Fluor 488 conjugated fibrinogen was used instead of PAC-1.
Preparation of human platelets Measurement of platelet-collagen adhesion Human blood was collected from healthy male volunteers (18–25 years old) who had not taken any drugs for at least 14 days with an approval from the Ethics Committee of Health Service Center at Seoul National University. Blood was anti-coagulated with acidcitrate-dextrose (ACD; 85 mM trisodium citrate, 71 mM citric acid, 111 mM glucose) for preparation of washed platelets (WP). All procedures were conducted at room temperature and the use of glass containers and pipettes was avoided. Platelet rich plasma was prepared by centrifugation for 15 min at 150 g, and platelets were pelleted by
For measurement of platelet adhesion, 96 well plates were coated with collagen (40 μg/ml) for 1 hr, blocked with phosphate-buffered saline (PBS; 1 mM KH2PO4, 154 mM NaCl, 3 mM Na2HPO4, pH 7.4) containing 5% BSA for 1 hr, and washed 3 times with PBS. Platelets were stained with calcein-AM (2.5 μM) in for 15 min at 37 °C in dark. Calcein-AM loaded platelets were incubated with soybean extracts, adenosine, genistein or daidzein for 10 min and 50 μl of platelets were placed onto collagen-coated well for 30 min at room temperature.
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Non-adherent platelets were removed by washing with PBS, and adherent platelets were lysed with lysis buffer (1% triton X-100, 150 mM NaCl, 10 mM Tris–HCl, 5 mM EDTA, and protease inhibitor cocktail) for 10 min at 4 °C. Fluorescence was measured with a microplate reader (λex = 492 nm, λem = 535 nm, TECAN Spectrafluor, Grödig, Austria). Statistical analysis All the data are shown as mean±SEM and the data were subjected to one-way analysis of variance followed by Duncan's multiple ranged tests to determine which means were significantly different from the control. Statistical analysis was performed using SPSS software (Chicago, IL). In all cases, p value of b0.05 was used to determine significance. Results Inhibitory effect of BB against collagen-induced platelet adhesion and aggregation To address the anti-platelet effects of BB in platelets, we employed a freshly isolated washed platelet (WP) system, while we have used platelet-rich plasma (PRP) system in our recent report [27]. Platelets were pretreated with BB extract (0 to 100 μg/mL), and stimulated by collagen, a representative aggregatory agonist. BB significantly inhibited collagen-induced platelet adhesion (Fig. 1A), a key phenomenon during platelet activation in patho-physiology [38,39]. BB extract also significantly attenuated aggregation in a concentration-dependent manner (Fig. 1B), and the inhibitory pattern was well correlated to BB effect on PRP system. In addition, yellow soybean (YB) did not induce any protection against collagen-stimulated platelet adhesion and aggregation. Intracellular pathways for anti-platelet effects of BB Next, we investigated the mechanism for the inhibitory effect of BB on platelet activation. Collagen activates GPVI receptor which initiates IP3 increase and subsequent calcium mobilization from intraplatelet storage, which is a key molecular mediator for platelet activation [40]. Meanwhile, intracellular cAMP can decrease collagen-induced calcium increases through the blockade of IP3 receptor [41]. BB prevented the decrease of cAMP level of collagen-stimulated human platelets in a concentration dependent manner (Fig. 2A). In line with increased cAMP level, BB significantly decreased collagen-induced intracellular calcium increase (Fig. 2B). While BB showed significant effects on cAMP and calcium level, YB did not affect these intraplatelet signaling, confirming the superior anti-platelet effect of BB. Based on the recent reports that platelet adhesion receptor expression play pivotal roles in platelet aggregation and activation [39,40,42], we have further examined the
protective role of BB in platelet adhesion receptor. BB significantly reduced collagen-induced GP IIb/IIIa expression (Fig. 2C), and subsequent fibrinogen binding (Fig. 2D). We recently suggested that adenosine may be important in anti-aggregatory effect of BB through activity-guided fractionation [27]. In order to examine the role of adenosine in BB-induced platelet protection, we examined BB effect in the presence of adenosine receptor antagonists, SCH 58261 and 8-(3-Chlorostyryl) caffeine (CSC). Confirming our previous result in PRP system, SCH 58261 and CSC significantly reduced BB-induced platelet inhibition (Fig. 2E), showing that adenosine plays a key role in anti-platelet activity of BB. Anti-platelet activities of adenosine To investigate the mechanism of adenosine action in BB activity, we examined anti-platelet activities of adenosine. When platelets were pretreated with adenosine (0 to 1 μM), collagen-stimulated aggregation was significantly reduced (Fig. 3A). Notably, the concentrationresponse pattern of adenosine is consistent with that of BB extract (0 to 100 μg/mL), based on the equivalent adenosine concentration in BB extract (0.36%). Collagen stimulation of platelets increased intracellular calcium as observed in flow cytometric analysis, and the increased calcium level was significantly reversed by adenosine pretreatment (Fig. 3B). Adenosine inhibited collagen-induced GP IIb/IIIa expression (Fig. 3C), reflecting that adenosine can block the downstream activation pathway following calcium increase. Collagen-induced cAMP decrease was reversed by adenosine, suggesting that increased cAMP plays a key role in the anti-platelet activity of adenosine (Fig. 3D). cAMP restoring activity of adenosine is mediated via cAMP synthesis by adenylyl cyclase (AC), as evidenced by abolition of cAMP increase by AC inhibitors (DDA and SQ22536) (Fig. 3E). To address if cAMP signaling is responsible for anti-platelet activity of adenosine, the effects of AC inhibitors on intracellular calcium and aggregation were examined. As expected, AC inhibitors reversed the inhibitory effects of adenosine in collagen-induced calcium mobilization (Fig. 3F) and aggregation (Fig. 3G), suggesting that cAMP is the key pathway in adenosinemediated anti-platelet effects. Role of adenosine-cAMP in anti-platelet effects of BB Next, we investigated if the inhibitory effect of BB in platelet activation is reversed by inhibition of cAMP synthesis. AC inhibitors reversed the inhibitory effects of adenosine in collagen-induced platelet adhesion (Fig. 4A) and aggregation (Fig. 4B). In addition, the inhibitory effects of BB on intracellular calcium were inhibited by DDA or SQ22536 (Fig. 4C). Consistently, AC inhibitor inhibited cAMP increase by BB (Fig. 4D), confirming that BB effect on platelet activation is mediated by adenosine-cAMP signaling.
Fig. 1. Effects of BB on adhesion and aggregation in human washed platelets. (A) Various concentrations of BB or YB (100 μg/ml) were treated for 10 min, and human platelets were stimulated with collagen (1–4 μg/ml) for 6 min. Platelet aggregation was measured using lumi-aggregometer. (B) Calcein-loaded platelets were exposed to BB or YB (100 μg/ml) for 10 min and placed onto collagen-coated microwells for 30 min. Adhered platelets were analyzed by calcein fluorescence. Values are means±SEM, n=3–4. The asterisk represents significant differences from the corresponding control, Pb 0.05.
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Fig. 2. Inhibitory effects of BB on platelet activation pathways. (A) Various concentrations of BB or YB (100 μg/ml) were added to platelets for 10 min and intracellular cAMP level was determined after collagen stimulation. Intracellular cAMP level in collagen-stimulated platelets was determined using commercial cAMP ELISA kit. (B) Effect of BB or YB on collagen-induced intracellular calcium increase was examined in flow cytometry. (C) Various concentrations of BB were added to platelets for 10 min and GP IIb/IIIa expression was determined after collagen stimulation. (D) Fibrinogen binding was examined by flow cytometry after pretreatment of BB and collagen stimulation. (E) BB (100 μg/ml) was treated for 10 min in the presence of adenosine receptor antagonist SCH 58261 (300 nM) or 8-(3-Chlorostyryl) caffeine (CSC; 1 μM), and platelet aggregation was initiated with collagen. Values are means ± SEM, n= 3–4. The asterisk represents significant differences from the corresponding control, P b 0.05. The pound sign represents significant differences from BB treated group, P b 0.05.
Here we demonstrated that adenosine plays important roles in anti-platelet effects of BB, however, previous studies showed that isoflavones in soybean can inhibit platelet aggregation [16–19]. In order to identify which ingredient is more important in anti-platelet effect of BB, isoflavones (genistein or daidzein) or adenosine at the equivalent concentration included in BB extract was treated to platelets. Surprisingly, while adenosine (1 μM) treatment significantly reduced collagen-induced platelet aggregation, treatment of genistein (0.3 μM) or daidzein (0.3 μM), the major bioactive isoflavones known to be responsible for soybean-related health effects, failed to attenuate platelet aggregation (Fig. 5A). Even the combined treatment of isoflavones (0.3 μM genistein with 0.3 μM daidzein) did not inhibit collagen-stimulated platelet aggregation, suggesting that adenosine rather than isoflavones accounted for the protective role of BB in platelet activation. Of note, isoflavones of genistein or daidzein could not inhibit collagen-induced GP IIb/IIIa expression and platelet adhesion (Fig. 5B) or platelet adhesion (Fig. 5C), suggesting that adenosine may act as the major component in inhibitory effect of BB on platelet adhesion as well.
Discussion In this investigation, we have demonstrated that adenosine plays a pivotal role in the inhibitory effect of black soybean (BB) on collageninduced platelet aggregation and adhesion (Fig. 6). In human washed platelets, we showed that adenosine decreased intracellular calcium mobilization through cAMP elevation. Notably, in addition to platelet aggregation, BB displayed a wide-spectrum of anti-platelet effects, modulating the overall aspects of platelet activation including GPIIb/ IIIa expression, fibrinogen binding and platelet adhesion, which may lead to an efficient control of thrombosis. With this study, we believe that important evidence was provided supporting the possible role of adenosine as a key mediator of soy-related biological effects. Previously, most of the biological effects from soybeans have been suggested to be mediated by their unique component of isoflavones, which showed diverse health effects such as anti-diabetes, anticancer and anti-osteoporosis [2,6,10,12]. In platelets, soy isoflavones, such as genistein and daidzein, can modulate thromboxane A2 receptor density, serotonin secretion and aggregation [16–19]. The concentration
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Fig. 3. Effects of adenosine on platelet activation. (A) Platelets were treated with different concentrations of adenosine for 10 min, and collagen-induced platelet aggregation was examined. Representative tracing (left) and dose–response graph (right) from more than 3 independent experiments are shown. (B) Platelets were treated with different concentrations of adenosine and Fluo-4 elicited intracellular calcium increase was determined after collagen stimulation by flow cytometry. Representative tracing (left) and dose– response graph (right) from more than 3 independent experiments are shown. (C and D) Effect of adenosine on adhesion receptor GP IIb/IIIa expression (C) or on intracellular cAMP level (D) was examined following collagen stimulation. (E to G) Treatment of DDA (100 μM) or SQ22536 (20 μM) for 10 min reversed adenosine effect on cAMP level (E), intracellular calcium level (F) and aggregation (G) in collagen-stimulated human platelets. Values are means ± SEM, n = 3–4. The asterisk represents significant differences from the corresponding control, P b 0.05. The pound sign represents significant differences from adenosine-only treatment, P b 0.05.
range of genistein required for the inhibitory effect of platelet aggregation is reported to be higher than 30 μM [18], or around 37 μM (10 μg/mL in their original article) [19]. Guerrero et al., reported that IC50 of genistein was 11.9 ± 1.5 μM against collagen-induced platelet aggregation [16]. Interestingly, here we found that isoflavones are not protective against collagen-induced platelet aggregation at the concentration (0.3 μM) as contained in BB (Fig. 5A). Consistent with our results, Gooderham et al. observed that consumption of soy proteins which was rich in genistein and daidzein had no effects on platelet aggregation in human male volunteers. Plasma concentration of genistein was determined to be 906.8 nmol/L and they concluded that sub-micromolar isoflavones might not be sufficient to exert the biological activities on platelet aggregation [15]. There are several reports showing that the human plasma concentration of isoflavones can be increased following soy-related food, for example, 43–312 ng/mL in females consuming diets of soy-based food [43], 40–240 ng/mL in Japanese adults who
frequently consume soy food [44], and 980 ng/mL in infants fed with soy-based formula [45]. However, these plasma ranges are far below the effective concentration of isoflavones for anti-platelet activities, reflecting that components other than isoflavones might play key roles in anti-thrombotic effect of soy-food. In the present study, adenosine rather than isoflavones was important for BB-induced platelet inhibition. The effective concentration of adenosine (IC50 = 1.85 ± 0.86 μM) well matches its actual content in BB extract (0.36%), explaining the anti-platelet potency of BB. This point can be further supported by the anti-platelet potencies of soybean extracts with different contents of adenosine and isoflavones. The BB extracted with 20% EtOH (BB20) showed higher anti-platelet activity than that with 70% EtOH (BB70). Notably, adenosine contents are 0.5% in BB20 and 0.04% in BB70, while those of isoflavone are similar between BB20 and BB70 (0.09% and 0.11%, respectively), supporting that the anti-platelet effects of soybean extracts were correlated better with the adenosine contents than those of isoflavones.
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Fig. 4. Inhibitory effect of BB on platelet activation: role of adenylcyclase inhibition. (A) Adenylate cyclase inhibition by DDA (100 μM) or SQ22536 (20 μM) for 10 min reversed the effects of BB on platelet adhesion to collagen. (B to D) Adenylate cyclase inhibition by DDA (100 μM) or SQ22536 (20 μM) for 10 min reversed the effects of BB platelet aggregation (B), intracellular calcium level (C), and cAMP level (D) in collagen-stimulated human platelets. Values are means ± SEM, n = 3–4. The asterisk represents significant differences from the corresponding control, P b 0.05. The pound sign represents significant differences from BB-only treatment, P b 0.05.
In our previous report [27], we observed that single or multiple administration of BB to rats (50, 100 mg/kg) significantly inhibited thrombus formation, and these dose range were relevant to daily consumption of soybean foods (20–80 g) [3] or BB (720.7 mg) [46]. This indicates that daily intake of BB might be able to manifest a protective effect against thrombosis-associated cardiovascular diseases. Although human kinetic data of adenosine after consumption of BB are currently unavailable, we believe that adenosine contained in BB may have contributed to anti-thrombotic effects of BB in vivo, based on the followings; 1) adenosine increases in plasma following BB administration to rats, and 2) the oral intake of BB results in higher plasma level of adenosine than that of pure adenosine. The plasma level of adenosine can be modified by internal and external factors like diseases, diets and drugs. For example, diprydamole which inhibits the
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adenosine reuptake by red blood cells can increase the plasma level of adenosine in hypoxia [47]. Exact contribution of adenosine to the anti-thrombotic effects of BB may be clarified when the mechanism underlying the increased bioavailability of adenosine following BB consumption is elucidated in the future. While many researches on soybean and related products have focused on the positive health effects of isoflavones, the concerns about the safety of soy isoflavones were proposed due to their hormonemimic effects [48]. Besides the safety issue, the roles of soy isoflavones have been questioned for some of soy-related health effects [5,49], and the more dominant roles of other active ingredients in soybeans were suggested. Considering the diversity and heterogeneity of soybeans and products [5,50], components other than isoflavones may be bioactive and more important for the beneficial effects of soy foods. It is necessary to examine the other bioactive ingredient(s) and to understand the actual mechanism underlying the health benefits of soy products to identify the true bioactive components. Here we demonstrated that adenosine may be an important mediator for soy-related anti-platelet effects. Adenosine has diverse biological functions and the modulation of adenosine metabolism and/or its receptor function are considered as a possible therapeutic target for infection, autoimmunity, ischemia and degenerative diseases [29–34,36]. Besides the endogenous formation and accumulation, adenosine naturally occurs and can be exogenously taken up from food via nucleoside transporter [51]. Nevertheless, studies addressing adenosine as an active food ingredient are limited [52,53]. Considering the potent biological activities of adenosine, it will be worthy to examine food-based adenosine as a potential candidate of active ingredients in foods or herbal medicine. In this study, BB and adenosine can attenuate GP IIb/ IIIa expression, fibrinogen binding and adhesion as well as platelet aggregation, while genistein or daidzein could not affect these events. Up to now, most of the researches on pathological platelet activation have focused on platelet aggregation rather than platelet adhesion. However, the expression of adhesion molecules such as GP IIb/IIIa and resultant adhesion is an essential process in platelet activation [39,40]. GPIIb/IIIa represents a soluble fibrinogen biding site, contributing to post-ligand occupancy signaling in platelets leading to further signal transduction and cytoskeletal protein alteration, resulting in platelet spreading and shape change [54,55]. In addition to platelet adhesion, the pretreatment of BB significantly inhibited granular secretion and phosphatidylserine (PS) exposure induced by collagen (data not shown). Mediators secreted from platelets and exposed PS promote thrombosis through inducing secondary platelet activation, plateletleukocyte interaction, vasoconstriction and coagulation. In this regard, the holistic modulation of diverse features of pathological platelet activation by BB and adenosine might be effective in preventing thrombotic events and related cardiovascular diseases.
Fig. 5. Comparison of the anti-platelet effects of adenosine and isoflavones. (A to C) Adenosine (1 μM), genistein (0.3 μM), daidzein (0.3 μM) and combination of genistein and daidzein (0.3 μM for both compound) were treated for 10 min, and collagen-induced platelet aggregation (A), GPIIb/IIIa expression (B) or platelet adhesion (D) was examined. Values are means± SEM, n=3–4. The asterisk represents significant differences from the corresponding control, Pb 0.05.
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Fig. 6. Suggested mechanism for the inhibitory effect of Black soybean on collagen-stimulated platelet activation.
In conclusion, we have shown that the anti-platelet effect of BB is mediated by adenosine-cAMP signaling, resulting in decreased intraplatelet calcium mobilization, GPIIb/IIIa expression, fibrinogen binding, and platelet aggregation/adhesion. We believe that this study has provided 1) the mechanistic explanation for the anti-platelet effects of BB and 2) the evidence that adenosine may act as a principle ingredient in health benefits by soybeans and soy related food. Conflict of interest statement No author of this study had financial or personal relationships with other people, manufacturers or organizations that could inappropriately influence this work. Acknowledgements This work was supported by the National Research Foundation of Korea, which is funded by the Korean government Grant (20120000844), and by a Basic Science Research Program (2012R1A1A3013240). References [1] Beavers KM, Jonnalagadda SS, Messina MJ. Soy consumption, adhesion molecules, and pro-inflammatory cytokines: a brief review of the literature. Nutr Rev 2009;67: 213–21. [2] Messina M. A brief historical overview of the past two decades of soy and isoflavone research. J Nutr 2010;140:1350S–4S. [3] Omoni AO, Aluko RE. Soybean foods and their benefits: potential mechanisms of action. Nutr Rev 2005;63:272–83. [4] Rivas M, Garay RP, Escanero JF, Cia Jr P, Cia P, Alda JO. Soy milk lowers blood pressure in men and women with mild to moderate essential hypertension. J Nutr 2002;132: 1900–2. [5] Kang J, Badger TM, Ronis MJ, Wu X. Non-isoflavone phytochemicals in soy and their health effects. J Agric Food Chem 2010;58:8119–33. [6] Ishimi Y. Dietary equol and bone metabolism in postmenopausal Japanese women and osteoporotic mice. J Nutr 2010;140:1373S–6S. [7] Song Y, Paik HY, Joung H. Soybean and soy isoflavone intake indicate a positive change in bone mineral density for 2 years in young Korean women. Nutr Res 2008;28:25–30. [8] Pipe EA, Gobert CP, Capes SE, Darlington GA, Lampe JW, Duncan AM. Soy protein reduces serum LDL cholesterol and the LDL cholesterol:HDL cholesterol and
[9]
[10] [11] [12] [13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24]
[25]
apolipoprotein B:apolipoprotein A-I ratios in adults with type 2 diabetes. J Nutr 2009;139:1700–6. Lu MP, Wang R, Song X, Chibbar R, Wang X, Wu L, et al. Dietary soy isoflavones increase insulin secretion and prevent the development of diabetic cataracts in streptozotocin-induced diabetic rats. Nutr Res 2008;28:464–71. Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 2002;3:364–73. Jian L. Soy, isoflavones, and prostate cancer. Mol Nutr Food Res 2009;53:217–26. Anthony MS, Clarkson TB, Williams JK. Effects of soy isoflavones on atherosclerosis: potential mechanisms. Am J Clin Nutr 1998;68:1390S–3S. Pase MP, Grima NA, Sarris J. The effects of dietary and nutrient interventions on arterial stiffness: a systematic review. Am J Clin Nutr 2011;93:446–54. Marsh TG, Straub RK, Villalobos F, Hong MY. Soy protein supports cardiovascular health by downregulating hydroxymethylglutaryl-coenzyme A reductase and sterol regulatory element-binding protein-2 and increasing antioxidant enzyme activity in rats with dextran sodium sulfate-induced mild systemic inflammation. Nutr Res 2011;31:922–8. Gooderham MH, Adlercreutz H, Ojala ST, Wahala K, Holub BJ. A soy protein isolate rich in genistein and daidzein and its effects on plasma isoflavone concentrations, platelet aggregation, blood lipids and fatty acid composition of plasma phospholipid in normal men. J Nutr 1996;126:2000–6. Guerrero JA, Lozano ML, Castillo J, Benavente-Garcia O, Vicente V, Rivera J. Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor. J Thromb Haemost 2005;3:369–76. Guerrero JA, Navarro-Nunez L, Lozano ML, Martinez C, Vicente V, Gibbins JM, et al. Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells. Br J Clin Pharmacol 2007;64:133–44. Kondo K, Suzuki Y, Ikeda Y, Umemura K. Genistein, an isoflavone included in soy, inhibits thrombotic vessel occlusion in the mouse femoral artery and in vitro platelet aggregation. Eur J Pharmacol 2002;455:53–7. Nakashima S, Koike T, Nozawa Y. Genistein, a protein tyrosine kinase inhibitor, inhibits thromboxane A2-mediated human platelet responses. Mol Pharmacol 1991;39:475–80. Choung MG, Baek IY, Kang ST, Han WY, Shin DC, Moon HP, et al. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J Agric Food Chem 2001;49:5848–51. Garcia-Alonso M, Rimbach G, Rivas-Gonzalo JC, De Pascual-Teresa S. Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A–studies in platelets, monocytes, and human endothelial cells. J Agric Food Chem 2004;52:3378–84. Shi J, Arunasalam K, Yeung D, Kakuda Y, Mittal G, Jiang Y. Saponins from edible legumes: chemistry, processing, and health benefits. J Med Food 2004;7:67–78. Xu B, Chang SK. Total phenolics, phenolic acids, isoflavones, and anthocyanins and antioxidant properties of yellow and black soybeans as affected by thermal processing. J Agric Food Chem 2008;56:7165–75. Kumar V, Rani A, Dixit AK, Pratap D, Bhatnagar D. A comparative assessment of total phenolic content, ferric reducing-anti-oxidative power, free radical-scavenging activity, vitamin C and isoflavones content in soybean with varying seed coat colour. Food Res Int 2010;43:323–8. Takahashi R, Ohmori R, Kiyose C, Momiyama Y, Ohsuzu F, Kondo K. Antioxidant activities of black and yellow soybeans against low density lipoprotein oxidation. J Agric Food Chem 2005;53:4578–82.
K. Kim et al. / Thrombosis Research 131 (2013) 254–261 [26] Xu B, Chang SK. Antioxidant capacity of seed coat, dehulled bean, and whole black soybeans in relation to their distributions of total phenolics, phenolic acids, anthocyanins, and isoflavones. J Agric Food Chem 2008;56:8365–73. [27] Kim K, Lim KM, Kim CW, Shin HJ, Seo DB, Lee SJ, et al. Black soybean extract can attenuate thrombosis through inhibition of collagen-induced platelet activation. J Nutr Biochem 2011;22:964–70. [28] Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov 2008;7:759–70. [29] Ernst PB, Garrison JC, Thompson LF. Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology. J Immunol 2010;185:1993–8. [30] Lappas CM, Day YJ, Marshall MA, Engelhard VH, Linden J. Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1ddependent NKT cell activation. J Exp Med 2006;203:2639–48. [31] Fredholm BB, Chen JF, Masino SA, Vaugeois JM. Actions of adenosine at its receptors in the CNS: insights from knockouts and drugs. Annu Rev Pharmacol Toxicol 2005;45: 385–412. [32] Fredholm BB. Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 2007;14:1315–23. [33] Clark AN, Youkey R, Liu X, Jia L, Blatt R, Day YJ, et al. A1 adenosine receptor activation promotes angiogenesis and release of VEGF from monocytes. Circ Res 2007;101: 1130–8. [34] Yang D, Zhang Y, Nguyen HG, Koupenova M, Chauhan AK, Makitalo M, et al. The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J Clin Invest 2006;116:1913–23. [35] Deguchi H, Takeya H, Urano H, Gabazza EC, Zhou H, Suzuki K. Adenosine regulates tissue factor expression on endothelial cells. Thromb Res 1998;91:57–64. [36] Johnston-Cox HA, Yang D, Ravid K. Physiological implications of adenosine receptor-mediated platelet aggregation. J Cell Physiol 2011;226:46–51. [37] Anfossi G, Russo I, Massucco P, Mattiello L, Cavalot F, Balbo A, et al. Adenosine increases human platelet levels of cGMP through nitric oxide: possible role in its antiaggregating effect. Thromb Res 2002;105:71–8. [38] Andrews RK, Berndt MC. Platelet physiology and thrombosis. Thromb Res 2004;114: 447–53. [39] Calderwood DA. Integrin activation. J Cell Sci 2004;117:657–66. [40] Abrams CS. Intracellular signaling in platelets. Curr Opin Hematol 2005;12:401–5.
261
[41] Tertyshnikova S, Fein A. Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell. Proc Natl Acad Sci U S A 1998;95:1613–7. [42] Li R, Mitra N, Gratkowski H, Vilaire G, Litvinov R, Nagasami C, et al. Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations. Science 2003;300:795–8. [43] Morton MS, Wilcox G, Wahlqvist ML, Griffiths K. Determination of lignans and isoflavonoids in human female plasma following dietary supplementation. J Endocrinol 1994;142:251–9. [44] Adlercreutz H, Markkanen H, Watanabe S. Plasma concentrations of phytooestrogens in Japanese men. Lancet 1993;342:1209–10. [45] Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE. Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 1997;350:23–7. [46] USDA. U.S. dry bean statistics, 1970–2010. U.S. Department of Agriculture; 2011. [47] Saito H, Nishimura M, Shinano H, Makita H, Tsujino I, Shibuya E, et al. Plasma concentration of adenosine during normoxia and moderate hypoxia in humans. Am J Respir Crit Care Med 1999;159:1014–8. [48] Munro IC, Harwood M, Hlywka JJ, Stephen AM, Doull J, Flamm WG, et al. Soy isoflavones: a safety review. Nutr Rev 2003;61:1–33. [49] Greaves KA, Parks JS, Williams JK, Wagner JD. Intact dietary soy protein, but not adding an isoflavone-rich soy extract to casein, improves plasma lipids in ovariectomized cynomolgus monkeys. J Nutr 1999;129:1585–92. [50] Erdman Jr JW, Badger TM, Lampe JW, Setchell KD, Messina M. Not all soy products are created equal: caution needed in interpretation of research results. J Nutr 2004;134:1229S–33S. [51] Casado FJ, Lostao MP, Aymerich I, Larrayoz IM, Duflot S, Rodriguez-Mulero S, et al. Nucleoside transporters in absorptive epithelia. J Physiol Biochem 2002;58:207–16. [52] Agarwal KC. Therapeutic actions of garlic constituents. Med Res Rev 1996;16:111–24. [53] Kuo MC, Chang CY, Cheng TL, Wu MJ. Immunomodulatory effect of Antrodia camphorata mycelia and culture filtrate. J Ethnopharmacol 2008;120:196–203. [54] Calderwood DA, Yan B, de Pereda JM, Alvarez BG, Fujioka Y, Liddington RC, et al. The phosphotyrosine binding-like domain of talin activates integrins. J Biol Chem 2002;277:21749–58. [55] Jennings LK. Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 2009;102:248–57.