http://informahealthcare.com/pltISSN: 0953-7104 (print), 1369-1635 (electronic)

Platelets, Early Online: 1–6! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2014.898747

ORIGINAL ARTICLE

Strawberry extract presents antiplatelet activity by inhibition ofinflammatory mediator of atherosclerosis (sP-selectin, sCD40L, RANTES,and IL-1b) and thrombus formation

Marcelo Alarcon1,2*, Eduardo Fuentes1,2*, Natalia Olate1, Simon Navarrete1, Gilda Carrasco2,3, & Ivan Palomo1,2

1Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on

Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile, 2Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule,

Talca, Chile, and 3Department of Horticulture, Faculty of Agricultural Sciences, Universidad de Talca. Talca, Chile

Abstract

Cardiovascular disease prevention is of high priority in developed countries. Healthy eatinghabits including the regular intake of an antithrombotic diet (fruit and vegetables) maycontribute to prevention. Platelet function is a critical factor in arterial thrombosis and theeffect strawberries have is still unclear. Therefore, the aim of this study was to systematicallyexamine the action of strawberries in preventing platelet activation and thrombus formation.Strawberry extract concentration-dependently (0.1–1 mg/ml) inhibited platelet aggregationinduced by ADP and arachidonic acid. At the same concentrations as strawberry inhibitsplatelet aggregation, it significantly decreased sP-selectin, sCD40L, RANTES, and IL-1b levels.The strawberry may exert significant protective effects on thromboembolic-related disorders byinhibiting platelet aggregation. Also, this suggests that antithrombotic activity may have novelanti-inflammatory effects.

Keywords

Antiplatelet, antithrombotic, IL-1b, P-selectin,RANTES, sCD40L, strawberry

History

Received 30 July 2013Revised 17 February 2014Accepted 24 February 2014Published online 18 April 2014

Introduction

Platelets are involved in the formation of thrombus that play animportant role in heart attacks, strokes, and peripheral vasculardisease [1]. Given that platelets play a central role in theatherosclerotic-related inflammatory response and the subsequentthrombotic event, a variety of antiplatelet agents have beendeveloped [2, 3]. Antiplatelet drugs are members of a class ofpharmaceuticals that decrease platelet aggregation and inhibitthrombus formation. Antiplatelet drugs can be classified intothree groups: (i) prevent cardiovascular diseases (CVD) (primaryprevention), (ii) treat an acute disease, and (iii) treat a chronicdisease (secondary prevention) [4].

The most commonly used antiplatelet agent is aspirin, otheroral agents may also be prescribed (ticlopidine, clopidogrel,dipyridamole, abciximab, and eptifibatide, among others) [5].Each antiplatelet agent affects platelets in slightly different waysand may have specific side effects, such as internal bleeding andgastrointestinal adverse effects, among others [6, 7].

Moreover, recent analyses suggest that there is a concept of thecardio protective properties of the dietary habits within theMediterranean regions [8]. In this context, together with regularphysical activity and smoking cessation, both coronary heartdisease and strokes could be avoided by healthy food choices that

are consistent with the Mediterranean Diet [9]. Within aMediterranean Diet, a higher intake of fruit and vegetables mayprotect against CVD and support current dietary guidelines toincrease fruit and vegetable intake [10, 11]. Preliminary studieshave demonstrated that platelet anti-aggregant activity of fruit(red grapes, strawberries, kiwis, and pineapples) and vegetables(garlic, onions, green onions, melons, and tomatoes) [12–16].

There is evidence suggesting that dietary strawberry powder(containing several key nutrients) may be associated withreduced risk factors for CVD, stroke, and diabetes in obesevolunteers [17–19]. Yet, the mechanisms by which the strawberrymay interfere with platelet function remain to be fully elucidated.The present study was aimed at investigating the effect of aqueousextract from the strawberry on platelet inflammatory mediators ofatherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b) andin vivo thrombus formation.

Materials and methods

Reagents

Sodium chloride (p.a.) was obtained from Arquimed (Santiago,Chile). Adenosine 50-diphosphate (ADP), thrombin receptoractivator peptide 6 (TRAP-6), arachidonic acid (AA), rosebengal, prostaglandin E1 (PGE1), and acetylsalicylic acid (ASA)were obtained from Sigma-Aldrich (St. Louis, MO). Collagentype I was obtained from Chrono-Log corp. (Havertown, PA).

Processing material

The variety of strawberry called Camarosa was selected for thestudy. Ripe fruits were obtained from the Centro Regional deAbastecimiento in Talca, Chile.

*These authors contributed equally to this work and are the first authors.

Correspondence: G. Ivan Palomo, Ph.D., Department of ClinicalBiochemistry and Immunohematology, Faculty of Health Sciences,Universidad de Talca, Chile., Talca, Chile. E-mail:ipalomo@utalca.cl Eduardo Fuentes, Ph.D. Student, Immunology andHaematology Laboratory, Faculty of Health Sciences, Universidad deTalca, Talca, Chile. E-mail: edfuentes@utalca.cl

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Preparation of extract

Total aqueous extract from the strawberries was obtainedaccording to Fuentes et al. [20]. Briefly, the samples werecomminuted in a blender, sonicated, and centrifuged for10 minutes at 700 g. Then the supernatant was filteredand lyophilized at �45 �C (freeze dried), and stored at �80 �Cuntil use.

Preparation of human washed platelet suspensions

The protocol was authorized by the ethics committee ofUniversidad de Talca in accordance with the Declaration ofHelsinki (approved by the 18th World Medical Assembly inHelsinki, Finland, 1964). Written informed consent was obtainedfrom six young healthy volunteers who agreed to participate in thestudy. Venous blood samples were taken according to the ‘‘BritishCommittee for Standards in Hematology’’ [21]. The samples wereplaced in 3.2% citrate tubes (9:1 v/v) by phlebotomy with avacuum tube system (Becton Dickinson Vacutainer Systems,Franklin Lakes, NJ, USA) and needle of 21 G [21]. Samples fromeach volunteer were processed independently for each assay,and centrifuged (DCS-16 Centrifugal Presvac RV) at 240 g for10 minutes to obtain platelet-rich plasma (PRP). Then two-thirdsof PRP was removed and centrifuged (10 minutes at 650 g).The pellet was washed with HEPES-Tyrode’s buffer containingPGE1 (120 nmol/l). Washed platelets were prepared in HEPES-Tyrode’s buffer at a concentration 200� 106 platelets/ml(Bayer Advia 60 Hematology System, Tarrytown, NY). Plateletswere kept at 4 �C during all the isolation steps after blooddrawing.

Platelet aggregation assay

Platelet aggregation was monitored by light transmission accord-ing to Born and Cross [22], using a lumi-aggregometer. Briefly,480ml of washed platelets (200� 109 platelets/l) were pre-incubated with 20 ml of saline, ASA (0.3 mmol/l), or strawberryextract (0.1–1 mg/ml) for 3 minutes. Following this, 20 ml ofagonist (ADP 8 mmol/l, collagen 15 mg/ml, TRAP-6 30mmol/l, orAA 1 mmol/l) was added and platelet aggregation was registeredfor 6 min. All measurements were performed in triplicate. Plateletaggregation results (maximal amplitude [%], slope, area under thecurve (AUC) and lag time [s]) were determined by the AGGRO/LINK (Chrono-Log, Havertown, PA). Strawberry extract inhib-ition of the maximal platelet aggregation was expressed as apercentage of the control (saline).

To evaluate the stability, the strawberry extract was subjectedto 100 �C for 1 hour, kept at 4 �C for 5 minutes, and then used inplatelet aggregation in vitro studies.

Platelet inflammatory mediators

Soluble CD40 ligand (sCD40L), RANTES, and IL-1b weredetermined using human quantikine ELISA kits (R&D systems,Minneapolis, MN) and soluble P-selectin (sP-selectin) wasdetermined by ELISA according to the manufacturer’s instruc-tions (Invitrogen Corporation, Carlsbad, CA). Briefly, washedplatelets were pretreated with saline, ASA (0.3 mmol/l), orstrawberry extract (0.5–1 mg/ml) for 15 minutes at 37 �C andthen stimulated with thrombin (2 U/ml) for 45 minutes at 37 �C.Supernatants were collected after centrifugation (2000 g,10 minutes, 4 �C) and stored at �70 �C until use.

In vivo murine model of thrombosis

Thrombosis in mice was performed by photochemical injuryusing a modification in the methods described by Przyklenk and

Whittaker [23]. Briefly, C57BL/6 mice (12–16 weeks old) wereanesthetized with a combination of tribromoethanol (270 mg/kg)and xylazine (13 mg/kg). The mesentery was exposed byperforming a central incision in the abdomen, permitting thevisualization of thrombus development in mesenteric vessels.Thrombosis was induced by an injection of 50 mg/kg rose bengalthrough the tail vein followed by illumination of the exposedmesenteric area with a diode laser (1.5-mW over objective lens,532 nm, diameter of 5mm2). Blood flow was monitored for60 minutes and stable occlusion was defined as a blood flow of0 ml/minute for 3 minutes. Saline (control group, n¼ 6), ASA(200 mg/kg, n¼ 6), or strawberry extract (200 mg/kg, n¼ 6) wereadministered intraperitoneally 30 minutes before the experiment.Rectal temperatures were similar and within the physiologicalrange in all experimental animals throughout the experimentalperiod.

Thrombus size analysis

After laser exposure, the injury image generated was recordedwith a charge-coupled device camera (Lumenera Corporation,Ottawa, ON). The image was analyzed with ImageJ software(NIH, Bethesda, MD). The region of interest (ROI) was defined asthe target artery that included a portion of the target artery that islarger than the maximum injured area. Using software tools,thrombus size was measured in the ROI.

Statistical analysis

Data were analyzed using SPSS version 17.0 (SPSS, Inc.,Chicago, IL) and expressed as mean ± standard error ofmean (SEM). Three or more independent experiments wereperformed for the different assays. Results were expressed aspercent inhibition or as a percentage of control (as 100%).Differences between groups were analyzed by the Student pairedor unpaired t-test and a one-way analysis of variance (ANOVA)using Tukey’s post-hoc test. p Values 50.05 were consideredsignificant.

Results and discussion

Effects of strawberry extract on platelet aggregation

The effects of strawberry extract on ADP-, collagen-, TRAP-6,and AA- induced platelet aggregation are presented in the Table I.Strawberry extract inhibited platelet aggregation induced by ADPand AA, respectively, but to a different extent. Considering thedifferent agonists tested in this study, the inhibition of plateletaggregation by the strawberry extract was in the following order:AA (65 ± 5%, p50.05)4ADP (55 ± 4%, p50.05) 4TRAP-6

Table I. Antiplatelet activity of aqueous extract from strawberry.

ADP Collagen TRAP-6 AA

Maximum aggregation (%)Strawberry extract 48 ± 3* 88 ± 5 88 ± 4 36 ± 5*Negative control 86 ± 5 89 ± 4 91 ± 6 85 ± 5

SlopeStrawberry extract 66 ± 4* 106 ± 6 118 ± 7 41 ± 5*Negative control 109 ± 12 111 ± 16 101 ± 9 116 ± 12

AUCStrawberry extract 220 ± 28* 334 ± 19 430 ± 10 134 ± 15*Negative control 383 ± 21 299 ± 25 474 ± 51 398 ± 62

Lag time (s)Strawberry extract 29 ± 1* 43 ± 2 25 ± 2 42 ± 2*Negative control 10 ± 1 46 ± 1 23 ± 1 33 ± 1

Values are presented as mean ± SEM (n¼ 6). ADP 8mmol/l, Collagen1.5 mg/ml, TRAP-6 30mmol/l, and AA 1 mmol/l. Extract at 1 mg/ml.*p50.05 vs. negative control (saline 0.9%). AUC: area under the curve.

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(10 ± 5%, p40.05) 4 collagen (2 ± 2%, p40.05) as comparedwith the control. Therefore, strawberry extract in a concentration-dependently (0.1, 0.5, and 1 mg/ml) inhibited human plateletaggregation stimulated by AA and ADP (Figure 1). Thus, theplatelet aggregation induced by AA in the presence of strawberryextract at 0.5 and 1 mg/ml was inhibited by 23 ± 5 and 65 ± 5%(p50.05), respectively. While platelet aggregation stimulatedby ADP was inhibited by 23 ± 5, 39 ± 6, and 55 ± 4% at 0.1, 0.5,and 1 mg/ml (p50.05). Furthermore strawberry extract, both withAA- and ADP-induced platelet aggregation inhibited slope, AUCand lag time as compared with the control (p50.05).

After being subjected to 100 �C, strawberry extract retainedits ability to inhibit platelet activation induced by shear force,similar results were reported by Naemura et al. [24]. Therefore,the thermal stability of strawberry extract, unlike the thermalstability of Solanum lycopersicum [25], is dependent on theagonist used.

Effects of strawberry extract on platelet sP-selectin,sCD40L, RANTES, and IL-1b

It has been described in the last decade that the secretionof platelet pro-inflammatory molecules (sCD40L, RANTES,sP-selectin, and IL-1b) may play a pathogenic role in both thelong-term atherosclerotic process, and in the triggering andpropagation of acute coronary syndromes [26, 27]. In this study,we have demonstrated, for the first time that strawberry extractinhibited platelet inflammatory mediators of atherosclerosis(sP-selectin, sCD40L, RANTES, and IL-1b).

In non-stimulated platelets, P-selectin, sCD40L, RANTES,and IL-1b release were minimal, whereas it was markedlyenhanced after stimulation with thrombin (2 U/ml). Pretreatmentof washed platelet with the strawberry extract (0.5–1 mg/ml)significantly inhibited thrombin-induced P-selectin expression.Concretely, thrombin-induced sP-selectin release was inhibitedby 23 ± 4 (p50.05) and 37 ± 4% (p50.01), in the presenceof strawberry extract at 0.5 and 1 mg/ml, respectively(Figure 2A).

Moreover, as platelets are considered the major source ofsCD40L in blood and sCD40L play a pivotal role in inflammationand atherosclerosis [28], we examined the effect of strawberryextract on platelet sCD40L release. As observed in Figure 2(B),

Figure 2. Effects of strawberry extract on release of sP-selectin, sCD40L, RANTES, and IL-1b from platelets. Washed platelets were pretreated withsaline, ASA (acetylsalicylic acid) 0.3 mmol/l or strawberry extract (0.5–1 mmol/l) for 15 minutes at 37 �C and then stimulated by thrombin (2 U/ml) fordetermination of sP-selectin (A), sCD40L (B), RANTES (C), and IL-1b (D). The graph depicts the mean ± SEM of n¼ 6 experiments. *p50.05 and**p50.01 as compared with the thrombin activated group, analyzed by Student’s t-test.

Figure 1. Strawberry extract concentration-dependently inhibited plateletaggregation. Effect of strawberry extract on ADP (8mmol/l) and AA(1 mmol/l) induced platelet aggregation. Results were expressed as %inhibition (mean ± SEM, n¼ 6).

DOI: 10.3109/09537104.2014.898747 Strawberry extract presents antiplatelet activity 3

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we found that strawberry extract significantly reduced thrombin-induced sCD40L platelet release by 24 ± 3 (p50.05) and 43 ± 3%(p50.01) at concentrations of 0.5 and 1 mg/ml, respectively.

It has been suggested that alteration of the level of theRANTES affects the progress of atherogenesis through theinflammatory pathway in patients with cardiovascular risk factors[29]. Moreover, IL-1b has been identified as a major mediator ofplatelet-induced activation and endothelial dysfunction [30]. Inthis study, the strawberry extract inhibited two important plateletmediators of inflammation, both RANTES and IL-1b. Thus, thestrawberry extract at 1 mg/ml attenuated the effect of thrombin-induced RANTES and IL-1b release by 41 ± 4 (p50.05) and37 ± 4% (p50.01), respectively (Figure 2C and D). In this way,regular consumption of strawberries for 6 weeks may provideprotection against high carbohydrate/fat meal-induced increase ofinflammatory mediators (e.g. IL-1b) [31].

Effects of strawberry extract on arterial thrombusformation in vivo

All animals in this study showed similar physiological values forrectal temperature before and after of thrombosis model amonggroups (data not shown).

Despite the benefits associated with current antiplatelet therapy,significant clinical limitations are associated with the use of aspirinand P2Y12 ADP receptor antagonist [32]. In this study, thestrawberry extract presented inhibitory activity on human plateletsaggregation and each constituent may possess multiply targets, andthey may exert pleiotropic and synergistic effects. Thus, thisprevents the access of the arachidonic aid to and the interactions ofADP with P2Y12 and with P2Y1, resulting in an inhibition ofplatelet thrombus formation under arterial flow [33–35]. It isknown that strawberry is rich in polyphenols. We hypothesized thatthe mechanisms of antiplatelet action could be via the activation ofPPARs, an increase of cAMP levels and inhibition of phospholip-ases C, NADPH oxidase, platelet isoprostanes, NF-kB andthromboxane A2, among others [36–38].

The dramatic inhibitory effects of the strawberry extract onplatelet aggregation, and P-selectin, sCD40L, RANTES, and IL-1b release supported the assessment of strawberry extracteffectiveness to inhibit in vitro thrombus formation [39]. Takinginto consideration all these findings, we provided further in vivoevidence of such in vitro-related strawberry extract- antithrombo-tic effects.

To examine the in vivo antithrombotic activity of thestrawberry extract, we evaluated the effects of the strawberry

Figure 3. Strawberry extract inhibited arterial thrombosis formation. (A) Representative images of thrombus formation after laser irradiation in thesaline control group (n¼ 6); ASA, acetylsalicylic acid (200 mg/kg, n¼ 6) and strawberry extract (200 mg/kg, n¼ 6) to 60 minutes. (B) Time coursechanges of thrombus growth rate as described in Materials and methods section. I, intima; M, media; A, adventitia.

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extract on laser-injured thrombus formation in mice mesentericartery in vivo. As shown in Figure 3, in untreated mice (control),the mesenteric artery was totally blocked by a stablebulky thrombus at 20 minutes. In contrast, further analysisrevealed that the time taken to form the artery thrombosis(42500 mm2) was drastically prolonged in strawberry extract-treated mice compared with mice receiving the same volume ofvehicle. Thus, one intraperitoneally bolus injection of strawberryextract (200 mg/kg) 30 minutes before laser injury pre-vented thrombus formation over 60 minutes after laser injury,only achieving a maximum of occlusion of 35 ± 2% (p50.001vs. control) (Figure 3). Although, in this study we used adifferent strawberry variety than the one published byother studies, the same antithrombotic effects have beenidentified [24].

Although antiplatelet drugs have proven to be beneficial inpatients with clinical evidence of CVD, the outcome still remainspoor. Currently, ASA is the gold standard for secondaryprevention of stroke of vascular origin; however, it is frequentlyassociated with serious adverse effects (internal bleeding andgastrointestinal adverse effects, among others) [6] and itseffectiveness in primary prevention is still a matter of debate[40]. In this study, using a murine model of real-time throm-bus formation, we demonstrate that the strawberry extractinhibited arterial thrombus growth at the same concentration asthat of aspirin, a widely used antiplatelet agent as shown in theFigure 3.

According to the previous results, aqueous fraction fromSolanum lycopersicum (70 mg/kg) had a 20% reduction frombaseline ex vivo platelet aggregation response in acute humanstudy [41]. Therefore, according to the same antiplatelet effectin vitro and in vivo by strawberry it is possible to estimate that theamount of strawberry extract necessary in humans for provenantiplatelet effects is about 70 mg/kg. However, additional studiesare needed to confirm the strawberry extract dose to observe acuteand chronic antiplatelet effects in humans.

Conclusion

In this study, we have demonstrated that aqueous extract fromstrawberries displayed a range of antiplatelet activities targetingdifferent platelet activation responses in vitro and inhibition ofthrombus formation. Based on these effects, it is possible toconsider the strawberry as a functional food that could providehealth benefits beyond basic nutrition.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of this article. This work wasfunded by the CONICYT REGIONAL/GORE MAULE/CEAP/R09I2001,Interdisciplinary Excellence Research Program on Healthy Aging(PIEI-ES), and supported by grant no. 1130216 (I. P., M. G., R. M., M.A., and J. C.) from Fondecyt, Chile.

References

1. Gregg D, Goldschmidt-Clermont PJ. Cardiology patient page.Platelets and cardiovascular disease. Circulation 2003;108:e88–e90.

2. Collins B, Hollidge C. Antithrombotic drug market. Nat Rev DrugDiscov 2003;2:11–12.

3. Palomo I, Toro C, Alarcon M. The role of platelets in thepathophysiology of atherosclerosis (Review). Mol Med Rep 2008;1:179–184.

4. Harrington RA, Hodgson PK, Larsen RL. Cardiology patient page.Antiplatelet therapy. Circulation 2003;108:e45–e47.

5. Smart S, Aragola S, Hutton P. Antiplatelet agents and anaesthesia.Continuing Education in Anaesthesia, Critical Care & Pain 2007;7:157–161.

6. Barrett NE, Holbrook L, Jones S, Kaiser WJ. Future innovations inanti-platelet therapies. Br J Pharmacol 2008;154:918–939.

7. Badimon L, Vilahur G. Coronary atherothrombotic disease: Progressin antiplatelet therapy. Rev Esp Cardiol 2008;61:501–513.

8. Massaro M, Scoditti E, Carluccio MA, De Caterina R.Nutraceuticals and prevention of atherosclerosis: Focus on omega-3 polyunsaturated fatty acids and Mediterranean diet polyphenols.Cardiovasc Ther 2010;28:e13–e19.

9. Willett WC. The Mediterranean diet: Science and practice. PublicHealth Nutr 2006;9:105–110.

10. Estruch R, Martinez-Gonzalez MA, Corella D, Salas-Salvado J.Effects of a Mediterranean-style diet on cardiovascular risk factors:A randomized trial. Ann Intern Med 2006;145:1–11.

11. Liu S, Manson JE, Lee IM, Cole SR. Fruit and vegetable intake andrisk of cardiovascular disease: The Women’s Health Study. Am JClin Nutr 2000;72:922–928.

12. Pierre S, Crosbie L, Duttaroy AK. Inhibitory effect of aqueousextracts of some herbs on human platelet aggregation in vitro.Platelets 2005;16:469–473.

13. Torres-Urrutia C, Guzman L, Schmeda-Hirschmann G, Moore-Carrasco R. Antiplatelet, anticoagulant, and fibrinolytic activityin vitro of extracts from selected fruits and vegetables. Blood CoagulFibrinolysis 2011;22:197–205.

14. Sabha D, Hiyasat B, Grotzinger K, Hennig L. Allium ursinum L.:Bioassay-guided isolation and identification of a galactolipid and aphytosterol exerting antiaggregatory effects. Pharmacology 2012;89:260–269.

15. Hiyasat B, Sabha D, Grotzinger K, Kempfert J. Antiplatelet activityof Allium ursinum and Allium sativum. Pharmacology 2009;83:197–204.

16. Panayiotou A, Samartzis D, Nomikos T, Fragopoulou E. Lipidfractions with aggregatory and antiaggregatory activity towardplatelets in fresh and fried cod (Gadus morhua): Correlation withplatelet-activating factor and atherogenesis. J Agric Food Chem2000;48:6372–6379.

17. Zunino SJ, Parelman MA, Freytag TL, Stephensen CB.Effects of dietary strawberry powder on blood lipids and inflam-matory markers in obese human subjects. Br J Nutr 2012;108:900–909.

18. Sesso HD, Gaziano JM, Jenkins DJ, Buring JE. Strawberry intake,lipids, C-reactive protein, and the risk of cardiovascular disease inwomen. J Am Coll Nutr 2007;26:303–310.

19. Burton-Freeman B, Linares A, Hyson D, Kappagoda T. Strawberrymodulates LDL oxidation and postprandial lipemia in response tohigh-fat meal in overweight hyperlipidemic men and women. J AmColl Nutr 2010;29:46–54.

20. Fuentes E, Castro R, Astudillo L, Carrasco G. Bioassay-guidedisolation and HPLC determination of bioactive compound that relateto the anti-platelet activity (adhesion, secretion and aggregation)from Solanum lycopersicum. Evid-Based Complement Alternat Med2012;2012:1–10.

21. Harrison P, Mackie I, Mumford A, Briggs C. Guidelines for thelaboratory investigation of heritable disorders of platelet function.Br J Haematol 2011;155:30–44.

22. Born GV, Cross MJ. The aggregation of blood platelets. J Physiol1963;168:178–195.

23. Przyklenk K, Whittaker P. Adaptation of a photochemical method toinitiate recurrent platelet-mediated thrombosis in small animals.Lasers Med Sci 2007;22:42–45.

24. Naemura A, Mitani T, Ijiri Y, Tamura Y. Anti-thrombotic effect ofstrawberries. Blood Coagul Fibrinolysis 2005;16:501–509.

25. Fuentes EJ, Astudillo LA, Gutierrez MI, Contreras SO. Fractions ofaqueous and methanolic extracts from tomato (Solanum lycopersi-cum L.) present platelet antiaggregant activity. Blood CoagulFibrinolysis 2012;23:109–117.

26. Aukrust P, Muller F, Ueland T, Berget T. Enhanced levels of solubleand membrane-bound CD40 ligand in patients with unstable angina.Possible reflection of T lymphocyte and platelet involvement in thepathogenesis of acute coronary syndromes. Circulation 1999;100:614–620.

27. Nurden AT. Platelets, inflammation and tissue regeneration. ThrombHaemost 2011;105:S13–S33.

28. Lievens D, Zernecke A, Seijkens T, Soehnlein O. Platelet CD40Lmediates thrombotic and inflammatory processes in atherosclerosis.Blood 2010;116:4317–4327.

DOI: 10.3109/09537104.2014.898747 Strawberry extract presents antiplatelet activity 5

Plat

elet

s D

ownl

oade

d fr

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form

ahea

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re.c

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emor

ial U

nive

rsity

of

New

foun

dlan

d on

07/

03/1

4Fo

r pe

rson

al u

se o

nly.

29. Koh SJ, Kim JY, Hyun YJ, Park SH. Association of serum RANTESconcentrations with established cardiovascular risk markers inmiddle-aged subjects. Int J Cardiol 2009;132:102–108.

30. Hawrylowicz CM, Howells GL, Feldmann M. Platelet-derivedinterleukin 1 induces human endothelial adhesion molecule expres-sion and cytokine production. J Exp Med 1991;174:785–790.

31. Ellis CL, Edirisinghe I, Kappagoda T, Burton-Freeman B.Attenuation of meal-induced inflammatory and thromboticresponses in overweight men and women after 6-week dailystrawberry (Fragaria) intake. A randomized placebo-controlledtrial. J Atheroscler Thromb 2011;18:318–327.

32. Angiolillo DJ, Capodanno D, Goto S. Platelet thrombinreceptor antagonism and atherothrombosis. Eur Heart J 2010;31:17–28.

33. Schror K. Aspirin and platelets: The antiplatelet action of aspirinand its role in thrombosis treatment and prophylaxis. Semin ThrombHemost 1997;23:349–356.

34. Remijn JA, Wu YP, Jeninga EH, MJ IJ. Role of ADP receptorP2Y(12) in platelet adhesion and thrombus formation in flowingblood. Arterioscler Thromb Vasc Biol 2002;22:686–691.

35. Penz SM, Reininger AJ, Toth O, Deckmyn H. Glycoprotein Ibalphainhibition and ADP receptor antagonists, but not aspirin, reduce

platelet thrombus formation in flowing blood exposed to athero-sclerotic plaques. Thromb Haemost 2007;97:435–443.

36. Fuentes E, Palomo I. Antiplatelet effects of natural bioactivecompounds by multiple targets: Food and drug interactions. J FunctFoods 2014;6:73–81.

37. Fuentes E, Fuentes F, Palomo I. Mechanism of the anti-plateleteffect of natural bioactive compounds: Role of peroxisome prolif-erator-activated receptors activation. 2013 Nov 18 [Epub ahead ofprint].

38. Violi F, Pignatelli P, Basili S. Nutrition, supplements, andvitamins in platelet function and bleeding. Circulation 2010;121:1033–1044.

39. Naemura A, Ohira H, Ikeda M, Koshikawa K. An experimentallyantithrombotic strawberry variety is also effective in humans.Pathophysiol Haemost Thromb 2006;35:398–404.

40. Raju NC, Eikelboom JW. The aspirin controversy in primaryprevention. Curr Opin Cardiol 2012;27:499–507.

41. Fuentes E, Pereira J, Alarcon M, Valenzuela C. Protective mech-anisms of S. lycopersicum aqueous fraction (Nucleosides andFlavonoids) on platelet activation and thrombus formation:In vitro, ex vivo and in vivo studies. Evid Based ComplementAlternat Med 2013;2013:1–13.

6 M. Alarcon et al. Platelets, Early Online: 1–6

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03/1

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