European Journal of Pharmaceutical Sciences 45 (2012) 552–558

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate /e jps

New peptide pENW (pGlu-Asn-Trp) inhibits platelet activation by attenuatingAkt phosphorylation

Jing Xiong a,b,1, Li Bai b,1, Wei Fang c, Jianyang Fu b, Weirong Fang b, Juan Cen b, Yi Kong d,⇑, Yunman Li b,⇑a Department of Pharmacology, Nanjing Medical University, 140 Han Zhong Rd., Nanjing, Jiangsu 210029, PR Chinab Department of Physiology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, PR Chinac Research Center of Biotechnology, School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, PR Chinad Department of Bio-Pharmaceutics, School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 August 2011Received in revised form 27 November 2011Accepted 1 December 2011Available online 21 January 2012

Keywords:Snake venomAntiplateletAkt phosphorylation

0928-0987/$ - see front matter � 2012 Elsevier B.V. Adoi:10.1016/j.ejps.2011.12.001

Abbreviations: pENW, pGlu-Asn-Trp; ADP, adenactivating factor; PI3K, phosphoinositide 3-kinasespoor plasma; PRP, platelet rich plasma; MPA, max5-hydroxytryptamine, i.e. serotonin; PT, prothroAPTT, activated partial thromboplastin time; Nguanosine monophosphate; COX-2, cyclooxygenreceptor.

⇑ Corresponding authors. Address: Department of Ptical University, Mailbox 207, Nanjing 210009, PR Chin(Y. Li), tel.: +86 25 83271080; fax: +86 25 83271020

E-mail addresses: yikong668@sohu.com (Y. Kong)(Y. Li).

1 These two authors contributed equally to this wor

Platelets play a key role in hemostasis and in the initiation and propagation of thrombus formation. Newpeptide pGlu-Asn-Trp (pENW), initially extracted from snake venom, shows a concentration-dependentantithrombotic activity, significantly attenuated thrombus formation in the arterial and venous vesselsystems. This study was designed to further reveal the mechanisms underlying its antithrombotic effectby focusing on its in vitro antiplatelet effect after precluding its influence on coagulation factors. Itshowed that pENW concentration-dependently inhibited ADP-, collagen- and platelet activating factor(PAF)-induced platelet aggregation, inversely depending upon the intensity of stimulation induced byagonists. Furthermore, data obtained by ELISA and flow cytometry presented that pENW also suppressedADP-mediated serotonin secretion and P-selectin expression in a concentration-dependent manner. Asshown by Western blot assay, ADP-induced platelet Akt phosphorylation was attenuated by the primingincubation with pENW, demonstrating the influence on platelet intracellular signaling. It provided theexplaining information for its activity of inhibiting platelet activation in vitro. These results suggestedpENW attenuated thrombus formation in part by inhibiting platelet activation instead of coagulation fac-tors, presented evidence of pENW interfering intracellular signaling system in the process of platelet acti-vation and indicated the possibility that pENW could potentially be developed as a novel therapeuticagent in the prevention and treatment of thrombotic disorders.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction let agents are still in need because of bleeding side effects and resis-

Platelets play a key role in hemostasis and in the initiation andpropagation of thrombus formation. Its role as a therapeutic targetin cardiovascular diseases has been approved by large-scalerandomized trials, in which patient outcomes in acute coronarysyndromes and percutaneous revascularization procedures wereachieved by inhibiting platelet functions. However, novel antiplate-

ll rights reserved.

osine diphosphate; PAF, platelet; PGI2, prostacyclin; PPP, plateletimal platelet aggregation; 5-HT,mbin time; TT, thrombin time;O, nitric oxide; cGMP, cyclicase-2; PAR, protease-activated

hysiology, China Pharmaceua. Tel./fax: +86 25 83271173

(Y. Kong)., yunmanlicpu@hotmail.com

k.

tance problems (Andre, 2004; Johansen, 2006; Mackman, 2008).Snake venom has been rich resources for novel antiplatelet

agent discovery since it contains a variety of proteins and polypep-tides that affect thrombosis and hemostasis (Hutton and Warrell,1993; Li et al., 2000; Zhu et al., 1997). pENW (pGlu-Asn-Trp) is anew peptide (Fig. 1) derived from Agkistrodon acutus Guenther ve-nom, and now can be synthesized chemically in a simple proce-dure, as described and characterized by Kong et al. (2009). Itattracted our attention by being stable as a small molecule (molec-ular weight 429 Da), since most snake venom-derived peptides orproteins being studied or developed are big molecules with com-plicated domains (Angulo and Lomonte, 2009; Li et al., 2000; Weiet al., 2006), which easily stimulate allergic responses as a foreignprotein. In the previous studies, pENW significantly increasedcoagulation time, but not hemorrhage time, inhibited both arterialand venous thrombi formation and protected endothelial damageinduced by thrombosis (Xiong et al., 2009). The findings are ofgreat interest by indicating an antithrombotic peptide withoutbleeding side effect. As a result, better understanding mechanismsunderlying the antithrombotic effect of pENW is an urgency since

J. Xiong et al. / European Journal of Pharmaceutical Sciences 45 (2012) 552–558 553

it might provide information for the prevention and treatment ofcardiovascular and thrombotic diseases.

Phosphoinositide 3-kinases (PI3K) play an important role inregulating a broad range of functional platelet responses, includ-ing primary platelet adhesion, cytoskeletal remodeling and plate-let aggregation (Jackson et al., 2004). Akt, a family of intracellularserine/threonine protein kinases (also called protein kinase B), isa key downstream effector of PI3K. Among its three isoforms(Akt-1, Akt-2, and Akt-3), both Akt-1 and Akt-2 are expressed inplatelets. They are important in low dose agonist-induced plateletactivation and in platelet-dependent thrombus formation in vivo(Chen et al., 2004; Woulfe et al., 2004). Following the conversionof membrane-bound PtdIns(3,4)P2 to PtdIns(3–5)P3 by activatedPI3K, Akt is anchored to the cell membrane via the interaction be-tween its N-terminal pleckstrin homology (PH) domain andPtdIns(3–5)P3, which trigger the simultaneous phosphorylationof Akt (Thr308 in the catalytic domain, and Ser473 in the C-termi-nal tail) (Alessi et al., 1996). Both translocation of Akt to cellmembranes and phosphorylation of both Thr308 and Ser473 arerequired for full enzyme activity (Hanada et al., 2004; Huanget al., 2010). Thus, Akt phosphorylation can be used as an indica-tor of PI3K pathway activation when platelets are activated (Liet al., 2003; Kroner et al., 2000).

In the present study, experiments were designed to investigatethe effects of pENW on platelet responsiveness to agonists anddownstream intracellular signaling pathway. Based on its inhibi-tory effects on platelet aggregation induced by multiple agonists,hypothesis is proposed that pENW inhibits a common signalingmolecule in platelet activation. Further, explanation of its anti-platelet and antithrombotic mechanisms will not only illuminatethe possibility of pENW being developed as a novel antiplateletagent, but also promote the discovery of new peptides for theprevention and treatment of cardiovascular and thrombosisdiseases.

2. Materials and methods

2.1. Animals

Male Sprague–Dawley rats, 250–280 g, and New Zealand whiterabbits, 2–2.5 kg (for platelet aggregation assay), were purchasedfrom Jiangsu Province Laboratory Animal Center. They werekept in a temperature-controlled environment (22 ± 2 �C), with55 ± 5% relative humidity and a 12 h light–dark cycle, and fedwith standard chow, for at least 1 week before any manipulations.All protocols were approved by the University Ethics Committee onAnimal Research and conformed to National Institutes of HealthGuideline for Care and Use of Laboratory Animals, in accordancewith international accepted principles.

Fig. 1. The chemical structure of pENW (pGlu-Asn-Trp). There are three chiralcenters (⁄), all of which are left-handed.

2.2. Reagents

pENW was synthesized and provided by the School of LifeScience and Technology, China Pharmaceutical University. Aspirinwas provided by Nanjing pharmaceutical factory; ADP and PAFwere purchased from Sigma–Aldrich Inc.; 5-HT ELISA kit for ratswas purchased from RapidBio, USA; fluorescence-conjugatedanti-human CD41 and CD62p were purchased from Jingmei Bio-tech; blotting antibodies used were anti-(phospho-Akt serine)(Ser473), purchased from Cell Signaling Technology, Anti-Akt(A444)and anti-mouse and anti-rabbit IgG HRP, purchased from BioworldTechnology, Inc. All other reagents were of analytical grade andcommercially available.

2.3. Coagulation cascade analysis

Saline, agatroban (Final concentration: 189.88 lM) or variousconcentrations of pENW were added to fresh rabbit pool plasmaand incubated at 37 �C for 5 min in cuvettes before the measure-ment using coagulation analyzer and activated partial thrombo-plastin time (APTT), prothrombin time (PT) or thrombin time(TT) test kits (Beijing Steellex Science Instrument Co. Ltd.). Briefly,PT or TT was determined by adding prothrombin or thrombin,respectively. After adding kaolin and cephalin into plasma andincubating for 3 min, CaCl2 (25 mmol/l) was added to obtain APTT.

2.4. Platelet preparation

Platelets were isolated by using the standard methods (Baenzigerand Majerus, 1974). Briefly, blood withdrawn from the carotid ar-tery was collected into 3.8% sodium citrate (1:9). Platelet-rich plas-ma (PRP) or platelet-poor plasma (PPP) were collected bycentrifugation at 100g or 800g for 15 min at room temperature,respectively. To obtain washed platelets, acid-citrate-dextrose(ACD) was added to PRP (1:9), and the suspension of plateletswas centrifuged at 800g for 15 min. The pellet obtained waswashed twice with Hepes–Tyrode’s buffer (10 mM HEPES;137 mM NaCl; 2.68 mM KCl; 0.42 mM NaH2PO4; 1.7 mM MgCl2;11.9 mM NaHCO3; 5 mM glucose, pH 7.4) and then used as washedplatelets.

2.5. Anti-platelet aggregation assay

In order to avoid individual differences in platelet preparationand aggregation, platelet aggregation was measured using rabbitPRP with an aggregometer (Beijing Steellex Science InstrumentCompany, China) according to the turbidimetry method of Bornand Cross (1963), as previously described (Xiong et al., 2009).Briefly, 250 ll PRP was incubated at 37 �C in the aggregometerwith stirring at 1000 rpm and then 10 ll saline, aspirin (Final con-centration: 205.8 lM) or various concentrations of pENW wasadded. After 5 min preincubation, platelet aggregation was in-duced by the addition of ADP (12.5 lM), collagen (1.5 mg/ml) orPAF (0.37 lg/ml).

2.6. Serotonin secretion assay

Serotonin release was measured by a rat 5-HT ELISA kitfollowing manufacturer’s instructions. In brief, to prevent the reup-take of secreted serotonin, imipramine hydrochloride (a serotoninre-uptake inhibitor, 5 lM) was added to platelet suspension.Washed rat platelets were treated with saline or various concen-trations of pENW at 37 �C for 5 min prior to addition of agonist(30 lM ADP) or saline (normal control) for 5 min. Aliquots of plate-lets suspension were centrifuged 10,000g at 4 �C for 5 min. Thesupernatant was collected and serotonin was measured using arat 5-HT ELISA kit according to manufacturer’s instructions.

Fig. 2. pENW did not show the influence on coagulation cascades. Aliquots of poolplasma were incubated with saline, agatroban or pENW at 37 �C for 5 min incuvettes before the measurement. Prothrombin time (black bars), thrombin time(light gray bars) and activated partial thromboplastin time (dark gray bars) arepresented as mean ± SEM, n = 4–6. ⁄⁄P < 0.01 vs. saline-treated group.

Fig. 3. pENW inhibited platelet aggregation induced by different agonists in aconcentration-dependent manner. Cumulative data of platelet aggregation afterincubation of PRP with saline, aspirin or pENW at 37 �C for 5 min before thechallenge with (A) ADP (12.5 lM), (B) collagen (1.5 mg/ml) and (C) PAF (0.37 lg/ml). Data are presented as mean ± SEM, n = 4. ⁄⁄⁄P < 0.001, ⁄⁄P < 0.01 and ⁄P < 0.05vs. saline-treated group.

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2.7. Flow cytometry analysis

The effect of pENW on platelet P-selectin expression was ana-lyzed using flow cytometry (BD FACScanto). Whole blood fromhealthy, drug-free donors, aging 18–22, at Nanjing Drum TowerHospital (Nanjing, Jiangsu) were collected with informed consent,in citrated tubes. PRP was prepared as described previously(Baenziger and Majerus, 1974) in accordance with the guidelinesof the institutional Human Use Review Committee. Platelets wereidentified by the phycoerythrin-conjugated anti-CD41 (CD41-PE),while P-selectin expression was assessed using a fluorescein isothi-ocyanate-conjugated anti-CD62P (CD62p-FITC). Isotype-matchedcontrol antibodies were used to set threshold and exclude nonspe-cific binding. PRP preparations divided into six aliquots were pre-treated with saline, various concentrations of pENW or aspirin at37 �C for 5 min prior to addition of agonist (20 lM ADP) or saline(normal control) for 5 min. All samples were then stained byCD41-PE and CD62p-FITC at 37 �C in dark for 20 min. Samples wereanalyzed immediately on a flow cytometry (BD FACScanto with BDFACSDiva software). On the FSC vs. SSC plot, a gate was drawnaround the platelets and 10,000 platelet events were collectedper sample. Identified platelets as CD41-PE positive, and CD62pexpression was reported as percentage of activated platelets.

2.8. Immunoblotting

Aliquots of washed rat platelets (5 � 108/ml) preincubated withsaline, aspirin (239.23 lM) or pENW (29.14–466.2 lM) for 5 minwere stimulated by agonist (ADP, 10 lM) at 37 �C for 3 min. Lysisbuffer (20 mM Tris, 2.5 mM EDTA, 150 mM NaCl, 1% Triton X-100,10% glycerol, 25 mM b-glycerol phosphate, 2 mM DTT, 1 mM so-dium orthovanadate, 1 mM PMSF, 10 mg/ml leupeptin, 20 mg/mlaprotinin) was added to obtain the cell lysates and then were centri-fuged at 12,000g for 15 min. The protein content was determined byBradford protein assay kit. An equal amount of total cell lysate(50 lg) was separated by 10% SDS–PAGE gels and then transferredonto polyvinylidene difluoride (PVDF) membranes. The nonspecificbinding sites were blocked with TBST buffer (150 mM NaCl, 10 mMTris–HCl, and 0.05% Tween-20) containing 5% nonfat dry milk for1 h. For phosphotyrosine immunoblots, membranes were initiallyincubated with Phospho-Ser473 Akt (1:1000) and Akt(A444)

(1:2000), stripped and reprobed with b-actin (1:1000, internalcontrol). Following incubation with either primary antibody,membranes were subject to four 15 min washes with TBST andincubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (1:10,000). Membranes were visualizedusing enhanced chemiluminescence. Densitometric analyses wereperformed using Scion Image program (NIH), and expressed as a ra-tio of phosphotyrosine to total Akt.

2.9. Statistical analysis

The experimental results were expressed as the mean ± SEM foreach group. Data were assessed by analysis of variance (ANOVA). Ifthis analysis indicated significant differences between the groupmeans, then each group was compared with control group by usingthe Dunnett t (2-sided). P < 0.05 was considered to be statisticallysignificant.

Fig. 4. pENW inhibited serotonin secretion induced by ADP (30 lM). (A) Standard curve of serotonin concentration vs. OD value. (B) Cumulative data of platelet serotoninsecretion after incubation with saline or pENW at 37 �C for 5 min prior to addition of ADP (30 lM). Data are expressed as mean ± SEM, n = 5. ⁄⁄P < 0.01 and ⁄P < 0.05 vs. saline-treated group.

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3. Results

Priming incubation of pENW with fresh pool plasma did not pro-long PT, TT and APTT, while positive control agatroban (189.88 lM)significantly increased PT, TT and APTT (Fig. 2, n = 4–6, P < 0.01 vs.saline-treated group).

Platelet aggregation was induced by different agonists using PRP.Before the study with stimulation of agonists, PRP was incubatedwith saline or various concentration of pENW. pENW alone didnot induce platelet aggregation and did not show any influencecompared with saline-treated group (Data not shown). The concen-tration of agonists used were determined to induce significantplatelet aggregation. Agonists displayed different intensity of stim-ulation on platelet aggregation. MPA induced by PAF (0.37 lg/ml),collagen (1.5 mg/ml), or ADP (12.5 lM) were 81.84 ± 6.31%,57.18 ± 3.61% or 39.33 ± 2.44%, respectively. Agonists-inducedplatelet aggregation was inhibited by pENW in a concentration-dependent manner (Fig. 3). For instance, 333.007 or 172.66 lMpENW significantly reduced ADP-induced platelet aggregation to23.63 ± 2.19% or 34.75 ± 1.37% (n = 4, P < 0.001, P < 0.05, respec-tively), and collagen-induced platelet aggregation to 43.05 ± 4.06%or 47.98 ± 4.46% (n = 4, P < 0.001, P < 0.05, respectively). pENWinhibiting platelet aggregation was dependent on the stimulatingintensity of agonists, in which the inhibitory effect of pENWdecreased as the agonist-induced MPA increased. Compared withsaline-treated group, pENW (333.007 lM) inhibited platelet

aggregation induced by PAF, collagen, or ADP by 16.1%, 24.7% or39.9%, respectively.

To stop serotonin reuptake after secreted from dense granule ofactivated platelets, washed rat platelet suspension was preincu-bated with imipramine (5 lM). The concentration of ADP used toinduce serotonin secretion was determined in the preliminarystudy (Data not shown). ADP (30 lM) significantly increased sero-tonin secretion from 14.48 ± 1.65 lg/l at resting to 56.27 ± 3.46 lg/l (n = 5, P < 0.01). The increases were significantly reduced to45.63 ± 3.90, 49.9 ± 1.82, and 50.72 ± 2.81 lg/l in the presence of466.2, 233.1, and 116.55 lM pENW (n = 5, P < 0.01, P < 0.01 andP < 0.05, respectively) (Fig. 4).

Influence of pENW on single platelet activation was furtherinvestigated by flow cytometry using human PRP. The concentra-tion of ADP used to induce platelet P-selectin expression wasdetermined in the preliminary study (Data not shown). ADP(20 lM) significantly increased platelet activation from12.91 ± 8.88% at resting to 51.54 ± 9.79% (n = 7, P < 0.01). The in-crease of platelet P-selectin expression induced by ADP was signif-icantly inhibited to 45.57 ± 11.88% and 46.24 ± 10.99% in thepresence of 466.2 and 279.72 lM pENW (n = 7, P < 0.05) (Fig. 5).

PI3K/Akt signaling pathway, as an important pathway regulat-ing functional platelet responses, was monitored by analyzingAkt phosphorylation. pENW significantly inhibited Akt phosphory-lation induced by ADP (10 lM). After preincubation with pENW(466.2 lM) or aspirin (239.23 lM), phosphorylation of Akt was

Fig. 5. pENW significantly inhibited platelet P-selectin expression induced by ADP (20 lM). (A) Representative plot of platelets with fluorescein isothiocyanate (FITC)-conjugated anti-CD62P. Panel 1 was normal platelets; panels 2–6 was platelets pretreated with saline, aspirin (239.23 lM) and pENW (466.2–93.24 lM) for 5 min prior to thestimulation by ADP (20 lM, 5 min) at 37 �C. (B) Cumulative data expressed as mean ± SEM from seven independent experiments. ⁄⁄P < 0.01 and ⁄P < 0.05 vs. saline-treatedgroup.

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inhibited by 36.2 ± 1.9% or 52.9 ± 4.0% (n = 3, P < 0.01) comparedwith saline-treated group. pENW inhibited the phosphorylationof Akt in a concentration-dependent way, while total Akt was notaltered within different groups (Fig. 6).

4. Discussion

This study documented that pENW, a snake venom-derived tri-peptide, had no influence on the coagulation cascade, but signifi-cantly inhibited various agonists-induced platelet aggregation,ADP-stimulated platelet serotonin secretion and P-selectin expres-sion in a concentration-dependent manner. It suppressed PI3K path-way during platelet activation, as evidenced by the reduced Aktphosphorylation (Ser473). The findings illuminated the inhibitingeffect of pENW on several aspects of platelet activation, which indi-cated a potential antithrombotic agent and could offer new knowl-edge in the prevention and treatment of thrombotic disorders.

As platelet aggregation is induced by various agonists andamplified by active substances secreted from granules, like ADP,serotonin and calcium (Rivera et al., 2009), that pENW significantlyinhibited platelet granules secretion induced by agonists would beexpected to contribute further to its antiplatelet effect (Fig. 4).Exposure of P-selectin, a content of platelet a-granule, is a markerfor platelet activation (Leytin et al., 2000). It stabilizes plateletaggregation along with initial aIIbb3–fibrinogen interactions

(Merten and Thiagarajan, 2000) as well as promotes platelet–leu-kocyte aggregates by interacting with P-selectin glycoprotein-1(PSGP-1) on leukocytes (Evangelista et al., 2007). Increased P-selectin expression has been described in cardiovascular diseases,such as myocardial infarction, atherosclerosis, atrial fibrillationand primary pulmonary hypertension (Andre, 2004; Chiu et al.,2005; Li-Saw-Hee et al., 2000). As a result, the decreased expres-sion of P-selectin due to pENW (Fig. 5) helps to indicate pENWhas a potential to be developed as a novel therapeutic agent inthe prevention of cardiovascular diseases. Compared with data ob-tained in vivo, it is noteworthy that pENW inhibiting platelet activ-ity in vitro is not as potent as when it is injected to rat, in which2 mg pENW per kg body weight significantly inhibits arterial andvenous thrombus formation (Xiong et al., 2009). The effective con-centration on antiplatelet aggregation in vitro is higher than theconcentrations in the systemic circulation reached in the rat mod-el. In this study, we used aspirin as a positive control. Contrary tothe data obtained in vivo, incubation with pENW in vitro did notdisplay a better performance than aspirin. pENW inhibiting plate-let aggregation was inversely dependent upon the intensity ofstimulation (Fig. 3), whereas aspirin inhibited platelet aggregationinduced by collagen more effectively compared with that by othertwo agonists, which was consistent with previous reports (Kariyazonoet al., 2004). In contrast, pENW inhibited P-selectin expressionmore potently than aspirin did (Fig. 5). The data suggests pENWmight not share same antiplatelet mechanisms with aspirin. In

Fig. 6. pENW significantly inhibited Akt phosphorylation induced by ADP (10 lM). Platelet suspensions pretreated with saline, aspirin or pENW at 37 �C for 5 min werestimulated by ADP (10 lM, 3 min). (A) Representative graph of blotting. (B) Quantitative data of densitometric analyses. The ratio of phosphotyrosine to total Akt areexpressed as mean ± SEM, n = 3. ⁄⁄P < 0.01 and ⁄P < 0.05 vs. saline-treated group. Lane 1, platelets pretreated with saline; lane 2, platelets pretreated with aspirin(239.23 lM); lanes 3–7, platelets pretreated with various concentrations of pENW, respectively: 466.2, 233.1, 116.55, 58.28 or 29.14 lM before the stimulation by 10 lMADP. The experiment was repeated three times.

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addition, pENW might have effect on other targets apart fromplatelets, since it showed higher efficacy in inhibiting thrombusformation when it is administrated into circulation systemin vivo (Xiong et al., 2009).

pENW inhibited platelet activation evoked by multiple agonists(Fig. 3), indicating that pENW may interfere with a common signal-ing molecule of platelet activation. Thus, the present study has, forthe first time, demonstrated the signaling events being interruptedby pENW. PI3-kinase signaling plays an important role in regulat-ing a broad range of functional platelet responses (Jackson et al.,2004; Yap et al., 2002; Yin et al., 2008). In the study, we used aspi-rin, instead of PI3K inhibitor, as a positive control to better eluci-date the inhibiting effect of pENW on Akt phosphorylationduring platelet activation, since some papers have indicated theaugmenting role of prostaglandin metabolites on PI3K/Akt signal-ing pathway (Wang et al., 2008) while inhibition of COX-2 inhibitsPI3K/Akt pathway (Uddin et al., 2010). Aspirin inhibiting Akt phos-phorylation, which was showed in our result, might be mediatedby inhibiting COX-2 activity and prostaglandin production. In addi-tion, the in vitro efficacy of pENW was compared with that of aspi-rin. Aspirin inhibited Akt phosphorylation with a lower effectiveconcentration than pENW did in vitro, which is consistent withthe data obtained from platelet aggregation and serotonin secre-tion assay (Figs. 3 and 4).

By now, the signal transduction mechanisms of Akt activation inplatelets are still less clear. Although PI3K and Akt phosphorylationare required for PAR and P2Y12-mediated platelet aggregation andplatelet activation (Wu et al., 2010; Resendiz et al., 2007), the iden-tity of kinase that phosphorylates Akt at Ser473 is complicated andcontroversial (Hanada et al., 2004). In addition, even though some

groups reported Akt activation promotes platelet NO synthesis andcGMP elevation during platelet activation and stimulates plateletsecretion and aggregation by activating the NO/cGMP pathway(Li et al., 2003; Marjanovic et al., 2005; Stojanovic et al., 2006),these results are conflicting with the inhibiting effect of NO/cGMPin platelet activation. Regarding the controversy concerning Aktdownstream molecules NO/cGMP inhibit or stimulate plateletaggregation, further experiments should be conducted to addressthe regulation of Akt signaling and its cross-talking with other sig-naling pathways. It will be of great importance for understandingplatelet physiology and reversal of thrombotic risks.

Over decades, researches on snake venom toxins have beeninspiring the design and development of therapeutic agents forthe prevention and treatment of thrombotic disorders. However,in the anticoagulant, antiplatelet or fibrinolytic peptides or pro-teins which were found or derived from snake venom, none couldinterfere hemostasis or thrombosis without promoting bleeding.The severe side effect of bleeding excludes a large number of pa-tients in clinic. As a peptide derived from snake venom, pENWinhibited platelet activation while not extending the hemorrhagetime (Xiong et al., 2009). Following work on its further mecha-nisms could raise new ideas for the development of novel anti-thrombotic peptides in the future.

Platelet activation and aggregation is essentially involved inthrombus formation, the leading cause of myocardial infarctionand stroke. Antiplatelet drugs such as ticlopidine (ADP receptorantagonist) or aspirin were shown to reduce the incidence ofstroke in high-risk patients. Nevertheless, recent reports haveshown that adequate antiplatelet effects are not achieved in5–45% of patients taking aspirin and in 4–30% patients taking

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clopidogrel (Eikelboom et al., 2002; Serebruany et al., 2005). Thesepatients are at increased risk of stent thrombosis and cardiovascu-lar complications. Consequently, agents to overcome aspirin orclopidogrel resistance and bleeding side effect are still in greatneed.

5. Conclusion

Preincubation with pENW in vitro significantly inhibited severalaspects of platelet activation by attenuating platelet Akt phosphor-ylation, which provided mechanistic information for its antithrom-botic effect in vivo. The result suggests a potential peptide forantithrombotic drug development and offers new knowledge inthe prevention and treatment of cardiovascular diseases andthrombotic disorders.

Acknowledgements

This work was supported by China National Found for FosteringTalents of Basic Science (J0630858) and Ph.D. Scientific ResearchCreative Project of Natural Science of Jiangsu 2009 (CX09B_291Z).

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