ORIGINAL ARTICLE

Tripeptide SQL Inhibits Platelet Aggregation and Thrombus Formation by Affecting PI3K/Akt Signaling Xing-li Su, MS,* Wen Su, MS,* Zhi-long He, MS,* Xin Ming, PhD,† and Yi Kong, PhD*‡

Abstract: Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatelet tripeptide SQL (H-Ser-Gln-Leu-OH) was isolated and characterized from centipede. In this study, we investigated its antithrombotic activities in vivo and underlying mechanism. It was found that SQL inhibited platelet aggregation induced by adenosine diphosphate, thrombin, epinephrine, and collagen and attenuated thrombus formation in both the ferric chloride–induced arterial thrombosis model and arteriovenous shunt thrombosis model in rats. It did not prolong the bleeding time in mice even at the dose of 10 mg/kg that showed potent antithrombosis effects. Molecular docking revealed that SQL binds PI3Kb with the binding free energy of 224.341 kcal/mol, which is close to that of cocrystallized ligand (224.220 kcal/mol). Additionally, SQL displayed inhibition on the late (180 seconds) but did not influence the early (60 seconds) Akt Ser473 phosphorylation in the immunoblot assay. These results suggest that SQL inhibits thrombus formation in vivo and that SQL inhibits PI3K-mediated signaling or even the PI3K itself in platelets. This study may help elucidate the mechanism for centipede treating cardiovascular diseases. Key Words: SQL, antiplatelet, antithrombosis, PI3K/Akt signaling, molecular docking, immunoblot assay (J Cardiovasc Pharmacol  2015;66:254–260)

INTRODUCTION Thrombosis, including acute arterial thrombosis, deep venous thrombosis, and coronary thrombosis, is the most frequent cause of morbidity and mortality in the industrialized countries.1 Platelets play a key role in thrombus formation after vascular endothelial injury.2 Platelets recruit and adhere to the injured vascular walls depending on specific interactions between platelet membrane receptors and ligands, most importantly collagen and von Willebrand factor.3 Platelet Received for publication February 3, 2015; accepted April 1, 2015. From the *Division of Biopharmaceutical Science, School of Life Science and Technology, China Pharmaceutical University, Nanjing, China; †Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, The University of North Carolina, Chapel Hill, NC; and ‡State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China. Supported by Chinese National Natural Science Foundation (81273375) and Jiangsu Provincial Qing Lan Project. The authors report no conflicts of interest. X.-l. Su and W. Su have contributed equally. Reprints: Yi Kong, PhD, Division of Biopharmaceutical Science, School of Life Science and Technology, China Pharmaceutical University, 24 Tong Jia St, Nanjing 210009, China (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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activation is initiated by exposed collagen and locally generated agonists, such as thrombin, adenosine diphosphate (ADP), and thromboxane A2 (TxA2).4 Then, the activated platelets cause conformational changes in the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor. Activated platelets finally aggregate together, and this process is mediated by interaction between the GPIIb/IIIa receptor of activated platelet and its ligands (primarily fibrinogen), which leads to platelet-rich thrombus formation.5 Antiplatelet drugs that block platelet activation and aggregation have shown clinical effects for antithrombotic therapy. However, current antiplatelet drugs are still limited by the bleeding complications. Fortunately, the latest research have defined phosphoinositide 3-kinase (PI3K) b as an important new target for antithrombotic therapy without significantly affecting primary hemostasis.6 PI3Ks are divided into 3 distinct classes (class I, II, and III) based on their primary structure, mode of regulation, and substrate specificity.7,8 The class I PI3Ks, further divided into a, b, d, and g isoforms based on their distinct p110 catalytic subunits and modes of regulation, are responsible for agonist-induced production of the second messengers phosphoinositide (PI) (3,4,5) P3 and phosphoinositide (PI) (3,4) P2 and involve the regulation of a broad range of functional platelet responses, including activation of integrin GPIIb/ IIIa.9 PI3Kb but not a, g, or d has been demonstrated to play a significant role in regulating the adhesive function of integrin GPIIb/IIIa, which is required for platelet spreading and sustained platelet aggregation, especially ADP-induced platelet responses.10,11 Recently, many platelet aggregation inhibitors have been isolated from natural sources.12–16 Centipede, a traditional Chinese medicine, which contains many bioactive components, such as 5-hydroxytryptamine, histamine, lipids, polysaccharides, peptides, and various enzymes, has been widely used for treating cardiovascular diseases in Korea, China, and other Far Eastern Asian countries for several hundred years.17 However, the mechanisms of these effects are not fully understood. Previously, we isolated and characterized an antiplatelet peptide with the sequence of H-Ser-GlnLeu-OH (SQL) from Scolopendra subspinipes mutilans.14 But, its antithrombotic effects in vivo and the mechanism had not been addressed. In this study, we investigated the in vivo antithrombotic effects of SQL using the arteriovenous (A-V) shunt thrombosis model and ferric chloride–induced arterial thrombosis model in rats. However, we explored the mechanism of its antiplatelet action by the molecular docking and immunoblot

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assay. Additionally, the bleeding time (BT) in mice was measured to evaluate the bleeding risk of SQL.

MATERIALS AND METHODS Materials Tripeptide SQL was synthesized in our laboratory with the purity of more than 98%. Epinephrine was from Harvest Pharmaceutical Co Ltd (Shanghai, China). Ferric chloride was from Sinopharm Chemical Reagent Co Ltd (Shanghai, China). Aspirin, ADP, fibrillar type I collagen from equine tendon, U46619, and thrombin were purchased from Sigma Chemical Co (St. Louis, MO). The PI3Kb inhibitor TGX-221 was purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals used in this research were of analytical grade.

Animals and Human Samples Institute of Cancer Research mice, Sprague Dawley rats, and New Zealand white rabbits were purchased from Qinglongshan Animal Center (Nanjing, Jiangsu, China). All animals were housed in a temperature-controlled (22 6 28C) environment under a 12/12-hour reversed light and dark cycle and fed with standard chow for at least 1 week before experiments. All the animal protocols were approved by the Animal Care and Use Committee at China Pharmaceutical University. Human venous blood was obtained from healthy donors in accordance with the Declaration of Helsinki and the permission from the University Ethical Committee of China Pharmaceutical University. The written informed consent was obtained from all participants.

Ferric Chloride–induced Arterial Thrombosis Model To estimate the effects of SQL on thrombus formation in vivo, a ferric chloride–induced arterial thrombosis model in rats was used according to the described method18–21 with some modification. Sprague Dawley rats (male, 180–220 g body weight) were randomly divided into 5 groups, with 8 rats in each group. Then, rats were treated with 0.9% saline, 30 mg/kg aspirin, and 3, 10, and 30 mg/kg SQL once a day for 4 days, intravenously, respectively. One hour after the last administration, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL/100 g). A cervical incision was made in the midline, then the left common carotid artery was carefully isolated, and the vessel surface was cleaned by blunt separation. The vascular injuries were induced by applying a filter paper (5 · 10 mm) saturated with 25% ferric chloride on the adventitial surface of the exposed left common carotid arteries. A small piece of plastic sheet was placed below the vascular tissue before the application of ferric chloride to separate the exposed vessel from the adjacent tissue. Fifteen minutes later, the filter paper was removed and the surface of vessel was washed with saline. Carotid blood flow was continuously monitored for 40 minutes, then the injured carotid artery segment (5 mm) was excised, and the thrombus was washed carefully with saline. After blotting the excess liquid, the thrombus was weighed immediately. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

SQL Affects PI3K/Akt Signaling

A-V Shunt Thrombosis Model In this assay, the antithrombotic activities of SQL were determined by measuring the thrombus formation in an A-V shunt tube with a modified method.22–24 The rats grouping, dosage, and administration were the same as those in ferric chloride–induced carotid thrombosis assay. Thirty minutes after the last administration, the rats were anesthetized with chloral hydrate (300 mg/kg intraperitoneally), and an A-V shunt tube was inserted between the right carotid artery and left jugular vein. The A-V shunt tube contains two 4-cm polyethylene tubes (0.6 and 0.9 mm inner and outer diameter, respectively) and a central part (12 cm long, 0.9 mm inner diameter) that contained a 10-cm cotton thread. The central tube that was filled with saline was linked to the 4-cm polyethylene tubes that were full of a heparin saline solution (25 U/mL). The extracorporeal circulation of blood through the shunt tube was maintained for 20 minutes, then both ends of the tubing were pinched and the cotton thread was taken out from the shunt tube. The wet weight of thrombus was determined by subtracting the weight of thread soaked with blood from the total weight immediately, and the dry weight was measured 6 hours later at room temperature by subtracting the weight of the dry 10-cm thread.

BT In Vivo The effects of SQL on BT in mice were measured according to a method described previously with some modification.25 Institute of Cancer Research mice (both sexes, 18–22 g body weight) were randomly divided into 5 groups, with 10 mice in each group. Three groups were injected intravenously in tail vein with 10, 30, and 100 mg/kg body weight of SQL for 4 consecutive days, respectively. The other 2 groups were injected intravenously in tail vein with normal saline and 100 mg/kg body weight of aspirin, respectively. Fifteen minutes after the last administration, the mice tail was marked with a tag in the distance of 3 mm from the tail tip and then cut in the mark. The tip of the tail was immediately dipped into saline at 378C. Blood flowing from the incision was carefully monitored, and the time interval from cutting the tail tip to stopping bleeding was recorded as BT.

Antiplatelet Aggregation Assay Inhibition of platelet aggregation induced by 4 agonists (ADP, thrombin, collagen, and epinephrine) was measured with a 4-channel aggregometer as previously described.26–29 Male New Zealand white rabbits were anesthetized by pentobarbital sodium (10 mg/kg intravenously), and the blood withdrawn from the carotid artery was collected into a centrifuge tube containing 3.2% sodium citrate (1:9 sodium citrate/ blood). The rabbit blood samples were centrifuged at 120·g for 15 minutes to prepare the platelet-rich plasma (PRP), and the residue was centrifuged at 600·g for 10 minutes to collect the platelet-poor plasma (PPP). PRP was diluted to 3 · 108/mL with PPP. Aliquots of 270 mL of PRP were incubated with 30 mL of saline, aspirin, or SQL, respectively, at 378C for 5 minutes under stirring. After incubation, platelet aggregation was induced by adding 30 mL of ADP (10 mM), thrombin (1 U/mL), collagen (1 mg/mL), and epinephrine (10 mM). The www.jcvp.org |

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maximum platelet aggregation was recorded by measuring the increase in light transmission compared with initial levels within 5 minutes. The light transmittance was calibrated with PPP.

Molecular Docking To investigate the mechanism for the inhibition of platelet aggregation by SQL, Molecular Operating Environment (MOE, version 2009.10) software was used to predict the binding mode of SQL in the active site of the PI3Kb. The x-ray crystallographic structures of the PI3Kb/GDC-0941 complex (PDB: 2Y3A) retrieved from the RSCB Protein Data Bank was applied to study the protein-ligand docking.30–32 The cocrystallized ligand in the crystalline complex was removed before docking. The binding site chosen for ligand (SQL) was the site of the cocrystallized inhibitor (GDC0941). The structure of SQL was built using the Builder module, and minimum energy conformation was determined using the Minimize module in MOE. The docking result was evaluated using Molecular Mechanics Generalized Born/Volume Integral (MM/GBVI) binding free energy.33 The docking energy of SQL was finally compared with that of cocrystallized ligand by docking the cocrystallized ligand GDC-0941 into protein receptor.

Preparation of Human Platelets Human blood was collected from the antecubital veins of healthy volunteers without any medication during the 2 weeks preceding venipuncture. Blood was drawn without stasis into the siliconized vacutainers containing a one-fifth volume of acid–citrate–dextrose (8.38 g of Tris sodium citrate, 7.35 g of citric acid, and 9.08 g of dextrose in 500 mL of H2O; pH 4.4). PRP was prepared by centrifugation at 150·g for 20 minutes at room temperature.34 Gel-filtered platelets were prepared as described by Prevost et al.35 Briefly, PRP was applied to the column that was packed with Sepharose 2B beads in Tris-HCl buffer, and platelets were eluted using Tyrode’s buffer (4.0 g of NaCl, 2.4 g of HEPES, 0.1 g of KCl, 0.2 g of NaH2PO4.H2O, 0.5 g of BSA, 0.5 g of glucose, 0.2 g of MgCl2.6H2O in 500 mL of H2O; pH 7.3) to a series of 1.5-mL tubes. The collected platelets in each tube were counted, combined, and adjusted to 2.5 · 108/mL using Tyrode’s buffer.

antibody (catalog no. 9272S) and the antiphospho-Akt (Ser473) (193H12) antibody (clone 4058S), respectively, at 48C overnight. After incubation with the corresponding secondary antibodies HRP-Conjugated Goat anti-Rabbit (catalog no. GGHL-15P-25), densitometric band scanning was performed using a Tanon infrared imaging system (chemiluminescence imaging analysis system).

Statistical Analysis The experimental results were expressed as the mean of the replicate determinations and standard deviation (SD). Statistical significance was evaluated using Student’s t test for 2-sample comparison and one-way analysis of variance for more than 2 experimental groups. Statistical significance was defined by P , 0.05. All statistic analyses were performed using GraphPad Prism (version 6; GraphPad Software, Inc, La Jolla, CA) and the Statistical Package for Social Sciences (SPSS, version 12.0). Quantitation of normalized optical density was performed using the software of Image J.

RESULTS Effects of SQL on Ferric Chloride–induced Arterial Thrombosis Ferric chloride–induced arterial thrombosis model sensitive to antiplatelet drugs is a simple and well-established model.37 In previous studies, we identified that SQL could inhibit platelet aggregation in vitro.14 To evaluate the antithrombotic activities of SQL in vivo, ferric chloride–induced arterial thrombosis model in rats was used. As shown in Figure 1, SQL could significantly and dose dependently inhibit the thrombus formation at doses of 3, 10, and 30 mg/kg by 14.0 6 9.6%, 19.5 6 5.5%, and 36.0 6 7.8%, respectively. At equal dose (30 mg/kg), the thrombus weight of aspirin-treated group decreased by 34.0 6 11.4%, which was similar to SQL.

Immunoblot Assay Immunoblotting was performed as described by Yi et al34 and Huang et al.36 Aliquots of gel-filtered platelets (200 mL, 2.5 · 108/mL) were preincubated with vehicle, TGX-221 or SQL, for 10 minutes and then stimulated by agonists (ADP, U46619, and thrombin) for 5 minutes under stirring at 378C. The reaction was stopped by adding RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM Tris, 150 mM NaCl containing protease inhibitors, and phosphatase inhibitors). After heating to 1008C for 5 minutes, samples were stored at 2208C. Proteins were separated on 10% SDS– polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a poly vinylidene difluoride (PVDF) membrane (Millipore), and subjected to Western blotting. Akt and phosphoAkt were detected through incubating with the anti-Akt (total)

FIGURE 1. Effects of SQL on ferric chloride–induced arterial thrombosis model rats. Male Sprague Dawley rats were treated with 0.9% saline, aspirin, or SQL at different doses for 4 days, respectively. *P , 0.05, **P , 0.01 versus control group.

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Effects of SQL on A-V Shunt Thrombosis To further examine in vivo antithrombotic activities of SQL, we also tested the effects of SQL on arterial thrombosis formation by evaluating the wet and dry thrombus weight in an A-V shunt thrombosis model in rats. As shown in Figure 2, SQL exhibited a dose-dependent decrease of both the wet thrombus weight and the dry thrombus weight. At the doses of 3, 10, and 30 mg/mL, the wet thrombus weight decreased by 10.4 6 4.1%, 19.5 6 7.8% and 38.4 6 13.7%, respectively, and the dry thrombus weight by 13.5 6 5.5%, 27.7 6 8.7% and 46.8 6 4.9%, respectively. Moreover, at the dose of 30 mg/kg, aspirin, a positive drug, inhibited the wet and dry thrombus growth by 40.3 6 8.2% and 48.3 6 5.4%, respectively, which was comparable with that of SQL at the same dose. In this assay, SQL showed similar effects as observed in the model of ferric chloride–induced arterial thrombosis.

Effects of SQL on BT The effects of SQL on BT in mice were measured to evaluate its bleeding risk. The SQL group (30 and 100 mg/kg) could prolong the BT (P , 0.01), but showed no significant increase of BT at the dose of 10 mg/kg. And, the BT of SQLtreated group was much shorter than that of aspirin at the same dose (100 mg/kg, P , 0.01) (Table 1), which indicated that SQL showed no bleeding risk at the effective dosage and the bleeding risk of SQL was lower than aspirin.

Effects of SQL on Antiplatelet Aggregation To investigate antiplatelet aggregation activities of SQL, we conducted platelet aggregation experiments induced by various agonists (ADP, thrombin, collagen, and epinephrine) in vitro. SQL decreased ADP-, thrombin-, collagen- and epinephrine-induced platelet aggregation from 57.6 6 4.1% to 35.7 6 0.6%, 63.2 6 2.7% to 48.4 6 3.3%, 52.8 6 5.4% to 26.7 6 4.3%, and 63.5 6 3.8% to 41.4 6 3.7%, respectively (Fig. 3). The results showed that SQL significantly inhibited ADP-, thrombin-, collagen- and epinephrineinduced platelet aggregation. Moreover, its inhibitory effects

FIGURE 2. Effects of SQL on arterial thrombosis model in rats. The tested samples or vehicle were administered intravenously for 4 days, respectively. After 30 minutes of the last administration, the rats were under the thrombogenic challenge. *P , 0.05, **P , 0.01 versus control group. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

SQL Affects PI3K/Akt Signaling

TABLE 1. Effects of SQL on BT in Mice Agent 0.9% saline Aspirin SQL

Dose, mg/kg

BT, s

— 100 10 30 100

659.3 1243.9 678.5 830.0 1115.9

6 6 6 6 6

33.7 70.3* 30.8 30.8* 21.8*

*P , 0.01 compared with control group (0.9% saline), n = 10.

on thrombin-induced aggregation were slighter than on others.

Results of Molecular Docking Previous studies have indicated that much of the inner core of the binding site of the class I PI3Ks is highly conserved.38 Recent studies have also found 2 nonconserved regions of the binding site in the PI3Kb isoform. One region (from PI3Kb 855 to 862) including a loop under the ribose pocket is very large in the PI3K. Another region (from PI3Kb 772 to 788) corresponds to the PI3K P-loop.39,40 We found that SQL binds to Ser775 and Lys776 residues of the nonconserved regions at the binding site in PI3Kb isoform (Fig. 4). In addition, hydrogen bonds are formed between Lys799, Asp807 of PI3Kb isoform, and SQL. According to the research of Zhu et al,29 residue Lys799 of PI3Kb is important to determine the PI3Kb selectivity for an inhibitor. The MM/GBVI binding free energy of SQL is 224.341 kcal/mol, whereas that of cocrystallized ligand is 224.220 kcal/mol. This docking results support our speculation that SQL inhibits the platelet by inhibiting the activities of PI3Kb.

Influence of SQL on Akt Ser473 Phosphorylation PI3K is a critical transmitter of intracellular signaling during platelet activation and aggregation. The molecular

FIGURE 3. Effects of SQL on rabbit platelet aggregation induced by ADP (10 mM), thrombin (1 U/mL), collagen (1 mg/mL), or epinephrine (10 mM). **P , 0.01 versus control group (0.9% saline). www.jcvp.org |

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In this study, we have demonstrated that SQL has antithrombus activities in vivo with low bleeding risk and inhibits collagen-, epinephrine-, ADP-, and thrombin-induced platelet aggregation. The results of molecular docking indicated that SQL may interact with PI3Kb, and the assay

of immunoblot also showed that SQL inhibited Akt phosphorylation, a downstream mediator of PI3K. Because of the antiadhesive properties of quiescent endothelial cells, platelets circulate near the endothelium and could not form stable adhesion contacts under normal physiological conditions.35,41,42 However, once tied to vascular injury, platelets will adhere to collagen and other matrix macromolecules stably, which contributes to the structure of in vivo thrombus that profoundly depends on local flow conditions.43 In the model of ferric chloride–induced arterial thrombosis, ferric chloride is applied to the topical surface of an intact vessel, inducing vascular wall injury and endothelial denudation. The A-V shunt thrombosis model is a classical assay used widely to determine the antithrombotic activities of an antiplatelet “drug,” and it can be used to measure the thrombus formation under physiological condition.22 Consequently, these 2 models were chosen in this study to observe the effects of SQL on thrombosis under physiological condition and the condition of vascular injury. The results showed that SQL significantly decreased the thrombus weight both in the ferric chloride–induced arterial thrombosis model and A-V shunt thrombosis model in a dosedependent manner, and its effects were comparable with aspirin at the same dose. These results suggest that SQL is an important contributor to the antithrombotic properties of centipede and may be a candidate for an antithrombotic agent. However, the benefit–risk balance between antithrombotic effects and the bleeding risk is extremely important for clinical application of antiplatelet drugs. So, we also evaluated the bleeding risk of SQL by tail cutting model in mice. The results indicated that SQL had little bleeding risk even at the dosage of 10 mg/kg that showed potent antithrombosis effects, and the bleeding risk of SQL was lower than that of aspirin. These results of BT and the effects on antithrombosis demonstrated that SQL may have potential to be developed as an effective and safe agent of antithrombosis. The integrin aIIbb3 is the most abundant integrin in platelets, to which soluble fibrinogen binds and bridges platelets and its activation is the common final step of platelet aggregation in response to various agonists.44 Antagonists of integrin aIIbb3, such as eptifibatide and tirofiban, could also inhibit various agonists-induced platelet aggregation by blocking fibrinogen binding to activated integrin aIIbb3.45 Our data that SQL significantly inhibited platelet aggregation induced by 4 different agonists activating platelets through different signaling pathways but not affected the binding of integrin aIIbb3 to fibrinogen (unpublished data) suggested that SQL exerted its antiplatelet effects by negatively regulating integrin aIIbb3 activation induced by agonists but not by blocking fibrinogen binding to activated integrin aIIbb3. Similar to PI3Kb inhibitor-SAA, which significantly inhibits collagen-, U46619- and ADP-induced platelet aggregation but shows a less inhibitory effect on thrombin-mediated platelet aggregation,36 SQL significantly blocked collagen-, ADP-, epinephrine-induced platelet aggregation but caused only small (15%) decrease in thrombin-mediated platelet aggregation. These results imply that SQL might function as an inhibitor of PI3Kb to prevent integrin aIIbb3 activation, leading to inhibition of platelet aggregation.

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FIGURE 4. Molecular docking results of SQL in the PI3Kb active site (PDB code 2Y3A). Green arrows represent hydrogen bonds formed with amino acid residues of receptor.

docking results suggest that SQL can bind with PI3Kb, so the effects of SQL on PI3K in platelets were further investigated using immunoblot assay. Gel-filtered human platelets were preincubated with or without SQL (378C, 10 minutes) before being challenged by ADP, thrombin, or U46619. Then, the phosphorylation of Akt, an indicator of PI3K pathway activation, was examined. The result showed that SQL markedly inhibited Akt Ser473 phosphorylation induced by all 3 tested agonists, indicating that SQL affected the PI3K signaling in platelets (Fig. 5A). To further examine if SQL acts on the PI3Kb, the PI3Kb inhibitor TGX-221 was used. SQL and TGX-221 were found to inhibit Akt Ser473 phosphorylation similarly that is appeared to inhibit ADP- and U46619-induced Akt phosphorylation potently and showed slight inhibitory effects on thrombin-induced Akt Ser473 phosphorylation (Fig. 5A). Moreover, as agonists concentration increased, the inhibitory effects of SQL and TGX-221 decreased, except with ADP stimulation (Fig. 5A). Furthermore, SQL showed no influence on the early (60 seconds) but inhibited the late (180 seconds) Akt Ser473 phosphorylation (Fig. 5B).

DISCUSSION

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SQL Affects PI3K/Akt Signaling

FIGURE 5. Influences of SQL on PI3K/Akt signal pathway in platelet. A, Western blots of protein lysates from platelets activated by ADP (A) (10 or 20 mM), thrombin (THR, T) (1 or 2 U/mL), or U46619 (U) (3 or 6 mM) for 5 minutes and probed with the indicated antibodies. A (1), A (2), and A (3) show the influence of SQL on Akt phosphorylation induced by the 3 tested agonists. B, Effects of SQL on Akt phosphorylation induced by thrombin at different time. Quantitation of normalized optical density of 4 independent experiments was performed by the software of Image J. *P , 0.05, **P , 0.01 versus that induced by corresponding agonist without treating with SQL and TGX-221. NS represents no significant difference.

To investigate possible interaction between SQL and PI3Kb, molecular docking of SQL with PI3Kb was first performed. The result that SQL interacts with PI3Kb by binding to Ser775 and Lys776 residues in the nonconserved regions at the binding site of PI3Kb suggested that SQL inhibits the platelet aggregation possibly by inhibiting PI3Kb. Akt Ser473 phosphorylation is a downstream effector of PI3K. In the assay of immunoblot, we found that SQL markedly inhibited Akt Ser473 phosphorylation induced by all 3 tested agonists, which indicated that SQL affected the PI3K pathway. Consistent with the results of platelet aggregation, SQL appeared to inhibit ADP- and U46619-induced Akt phosphorylation more potently but only showed slight inhibitory effects on thrombin-induced Akt Ser473 phosphorylation. Moreover, it is known that thrombin stimulation induces rapid Akt vphosphorylation downstream of phospholipase C activation, that is, PI3K independently and late PI3K-dependent Aktphosphorylation.46 SQL showed no influence on the early (60 seconds) but inhibited the late (180 seconds) Akt Ser473 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

phosphorylation, suggesting that SQL exerts its platelet inhibitory effects by PI3K inhibition not phospholipase C inhibition, which is consistent with the results of molecular docking. These results suggest that SQL might have the capacity to inhibit PI3K-mediated signaling and PI3Kb might be the target of SQL during the inhibition of platelet aggregation. Taken together, these results above indicate that the antithrombotic functions of SQL are related to its inhibitory effects on platelet aggregation, and SQL inhibited platelet aggregation by inhibiting PI3K-mediated signaling pathway or even interacting with PI3Kb directly. Moreover, the low bleeding risk of SQL may also be related to the fact that antithrombotic therapy targeting PI3Kb does not significantly affect primary hemostasis that has been defined in a recent study.6

CONCLUSIONS SQL effectively inhibited platelet aggregation in vitro and attenuated thrombus formation in vivo with low bleeding www.jcvp.org |

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risk. SQL binds with PI3Kb and influences the Akt phosphorylation, a key role in the PI3K/Akt signaling pathway. PI3Kb is a promising target for treatment of thrombotic disease without significant bleeding risk, and thus SQL could be developed as a candidate for an antithrombotic agent or a lead compound. In addition, this study might be helpful to understand how centipede exerts clinically beneficial effects on patients with cardiovascular diseases. REFERENCES 1. Meadows TA, Bhatt DL. Clinical aspects of platelet inhibitors and thrombus formation. Circ Res. 2007;100:1261–1275. 2. Steinhubl S, Moliterno D. The role of the platelet in the pathogenesis of atherothrombosis. Am J Cardiovasc Drugs. 2005;5:399–408. 3. Jackson SP, Schoenwaelder SM, Goncalves I, et al. PI 3-kinase p110beta: a new target for antithrombotic therapy. Nat Med. 2005;11: 507–514. 4. Vanhaesebroeck B, Leevers SJ, Panayotou G, et al. Phosphoinositide 3kinases: a conserved family of signal transducers. Trends Biochem Sci. 1997;22:267–272. 5. Anderson KE, Jackson SP. Class I phosphoinositide 3-kinases. Int J Biochem Cell Biol. 2003;35:1028–1033. 6. Jackson SP, Yap CL, Anderson KE. Phosphoinositide 3-kinases and the regulation of platelet function. Biochem Soc Trans. 2004;32:387–392. 7. Garcia A, Kim S, Bhavaraju K, et al. Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways. Biochem J. 2010;429:369–377. 8. Schoenwaelder SM, Ono A, Nesbitt WS, et al. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem. 2010;285:2886–2896. 9. Peng L, Xu X, Shen D, et al. Purification and partial characterization of a novel phosphodiesterase from the venom of Trimeresurus stejnegeri: inhibition of platelet aggregation. Biochimie. 2011;93:1601–1609. 10. Francischetti IM. Platelet aggregation inhibitors from hematophagous animals. Toxicon. 2010;56:1130–1144. 11. Hyun KW, Jeong SC, Lee DH, et al. Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus. Peptides. 2006;27:1173–1178. 12. Blue R, Murcia M, Karan C, et al. Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen. Blood. 2008;111:1248–1256. 13. Chen Z, Wu J, Zhang Y, et al. A novel platelet glycoprotein Ib-binding protein with human platelet aggregation-inhibiting activity from Trimeresurus jerdonii venom. Toxicon. 2011;57:672–679. 14. Kong Y, Huang SL, Shao Y, et al. Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans. J Ethnopharmacol. 2013;145:182–186. 15. Li YX, Sun Q, Zhang H, et al. A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation. Thromb Res. 2012;129:e217–e222. 16. Pemberton RW. Insects and other arthropods used as drugs in Korean traditional medicine. J Ethnopharmacol. 1999;65:207–216. 17. Kogushi M, Matsuoka T, Kawata T, et al. The novel and orally active thrombin receptor antagonist E5555 (Atopaxar) inhibits arterial thrombosis without affecting bleeding time in guinea pigs. Eur J Pharmacol. 2011;657:131–137. 18. Li W, McIntyre TM, Silverstein RL. Ferric chloride-induced murine carotid arterial injury: a model of redox pathology. Redox Biol. 2013; 1:50–55. 19. Moser M, Bertram U, Peter K, et al. Abciximab, eptifibatide, and tirofiban exhibit dose-dependent potencies to dissolve platelet aggregates. J Cardiovasc Pharmacol. 2003;41:586–592. 20. Tseng YL, Chiang ML, Huang TF, et al. A selective serotonin reuptake inhibitor, citalopram, inhibits collagen-induced platelet aggregation and activation. Thromb Res. 2010;126:517–523. 21. Vija H, Samel M, Siigur E, et al. Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases

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Akt Signaling.

Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatele...
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