- Email: [email protected]
Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans
Journal of Ethnopharmacology 145 (2013) 182–186
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans Yi Kong a,b,n, Shi-Long Huang a,b, Yu Shao a, Shuai Li a, Ji-Fu Wei c,nn a
School of Life Science & Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China c Research Division of Clinical Pharmacology, the First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2012 Received in revised form 22 October 2012 Accepted 25 October 2012 Available online 2 November 2012
Ethnopharmacological relevance: The centipede has been prescribed for the treatment of cardiovascular diseases in Korea, China and other Far Eastern Asian countries for several hundred years. Materials and methods: A novel antithrombotic peptide was isolated from Scolopendra subspinipes mutilans using a combination of ultrafiltration, Sephadex G-50 column, Source 15Q anion exchange column and RP-HPLC C18 column. Results: The molecular mass of the purified peptide is 346 Da measured by Electrospray Ionization Mass Spectrometry (ESI-MS). The primary structure of the peptide is Ser-Gln-Leu (SQL) determined by Edman degradation. SQL potently prolonged the activated partial thromboplastin time (aPTT), and inhibited platelet aggregation. Conclusions: These results help to clarify the mechanism of the antithrombotic activity of the centipede for effective treatment of cardiovascular and cerebrovascular diseases. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Scolopendra subspinipes mutilans Activated partial thromboplastin time (aPTT) Platelet aggregation inhibitor
1. Introduction Centipedes (Scolopendra subspinipes mutilans L. Koch) are predatory, elongated and dorsoventrally flattened arthropods, which belong to the Chilopod class (Negrea and Minelli, 1995). In China, the centipede has been used for thousands of years as a traditional Chinese medicine to treat disorders, such as strokeinduced hemiplegia, apoplexy, epilepsy, tetanus, whooping cough, tuberculosis, scald burns, and pyocutaneous disease. In addition, the centipede has been described for the treatment of cardiovascular diseases in Korea, China and other Far Eastern Asian countries for several hundred years (Pemberton, 1999). Thrombosis is caused by two main pathways. One is blood clotting, which is processed by various coagulation factors at sites of vascular injury. Because endogenous or exogenous anticoagulants interfere with coagulation factors, blood coagulation can be prolonged or stopped (Jung et al., 2007). The other thrombotic pathway is platelet aggregation, which plays an important role in hemostasis. The process of platelet aggregation includes the adhesion and activation of platelets, secretion of the granular contents, and aggregation of platelets again (Kong et al., 2009).
There are few reports about antithrombotic agents identified from the centipede. You et al. (2004) isolated a 25 kDa serine protease from Scolopendra subspinipes mutilans that demonstrated fibrinolytic activity by converting human Glu-plasminogen to activated plasmin. Wu et al. (2009) found that centipede acidic protein (CAP) significantly suppressed the development of atherosclerosis, and improved the hemorrheological disturbances and histopathological changes in the atherogenic-diet rat model. These effects may be attributed in part to reversal of dyslipidemia, inhibition of lipid peroxidation, and regulation of nitric oxide (NO) and endothelin-1 (ET-1) systems. Centipedes consist of many kinds of protein, amino acid, lipids and enzymes. Many peptides were purified or identified from Centipedes (Wang et al., 1997; Peng et al., 2010; Undheim et al., 2011; Yang et al., 2012). In the present study, we report the purification and characterization of a novel peptide (Ser-Gln-Leu) with antithrombotic activity, which may contribute to the traditional usefulness of centipede as antithrombotic medicine.
2. Materials and methods 2.1. Materials
n
Corresponding author. Tel./fax: þ 86 25 83271030. Corresponding author. Tel./fax: þ 86 25 83718836. E-mail addresses: [email protected] (Y. Kong), [email protected] (J.-F. Wei). nn
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.10.048
Centipedes were obtained from Xiansheng Pharmacy (Nanjing, Jiangsu province, China). The ultrafiltration tube was purchased from Millipore (Billerica, Massachusetts, USA). The BioLogic Duoflow
Y. Kong et al. / Journal of Ethnopharmacology 145 (2013) 182–186
system was obtained from Bio-Rad (Richmond, CA, USA). The Lichrospher C18 reverse-phase HPLC column was from Jiangsu Hanbon Science & Technology Co. Ltd (Huai’an, China). The ESI-MS System was from Waters Company (Milford, Massachusetts. USA).
183
2.5. Prothrombin time (PT) and aPTT clotting assays
Chinese Pharmacopoeia (I) provides a centipede medicinal method: centipede was extract by ethanol (50%) and the dried powder can take orally. So, Scolopendra subspinipes mutilans (n¼ 100) were ground to a powder in a mortar and then extracted by stirring for 24 h in 1000 mL of 50% ethanol solution. The homogenate was centrifuged at 8000 g for 15 min at 4 1C. The supernatant was collected. The ethanol was removed from the supernatant by rotary evaporation.
Nine parts of blood was drawn by venipuncture into one part of 3.2% sodium citrate from healthy volunteers and pooled. The blood was separately centrifuged at 2500 g for 10 min to obtain platelet poor plasma (PPP). Briefly, normal citrated PPP (80 mL) was incubated with sample solution (20 mL) for 3 min at 37 1C. For the aPTT clotting assay, 100 mL of aPTT reagent was added to the mixture (PPP and sample) and incubated at 37 1C for 10 min. Clotting time was immediately recorded after the addition of 100 mL of 20 mM CaCl2. For the PT clotting assay, PT reagent was added to the incubated mixture of PPP (80 mL) and sample (20 mL), and clotting time (s) was recorded. All coagulation assays were performed with three individual replicates (Majdoub et al., 2009). Heparin, a commercial anticoagulant, was used as positive control.
2.3. Purification of an anticoagulant peptide from Scolopendra subspinipes mutilans
2.6. Assay of anti-platelet aggregation activity
2.2. Extraction of peptides from Scolopendra subspinipes mutilans
The supernatant was filtered through a Millipore membrane filter (0.45 mm) and then applied to an ultrafiltration tube of 10,000 Da, and centrifuged at 10,000 rpm for 5 min. The low molecular weight fraction after ultrafiltration was collected and then applied to a Sephadex G-50 column (26 mm 100 cm), equilibrated with distilled water and eluted with the same buffer at a flow rate of 0.8 mL/min. The fractions were collected at 15-min intervals. The activated partial thromboplastin time (aPTT) activity was evaluated for each fraction. The fraction with the highest aPTT activity (Faction C) was collected, lyophilized, dialyzed with 50 mM Tris–HCl buffer (pH 7.4), and further separated by Source 15Q anion exchange column (12 mm 150 mm) preliminarily equilibrated with 50 mM Tris–HCl buffer (pH 7.4) in a high performance liquid chromatographic (HPLC) system (Bio-Rad, Richmond, CA, USA). A linear gradient of NaCl (0–2 M) in the same buffer was maintained at a flow rate of 3 mL/min. Absorbance was monitored at 214 nm. Each fraction was evaluated for aPTT activity. The fraction with aPTT activity (Faction C1) was separated by RP-HPLC on a C18 column (10 250 mm; Kromasil, China) under linear gradient elution conditions using methanol as the organic modifier and trifluoroacetic acid (TFA) as the volatile buffer. Eluent A consisted of 0.1% TFA in 10% methanol (v/v), eluent B of 0.1% TFA in 90% methanol (v/v). The chromatographic column was conditioned with 100% of eluent A, after which 1 mL of the peptide solution was applied on the C18 column and eluted by the remaining eluent A for 10 min and with the following increasing eluent B concentrations: 0–10 min, 0%; 10–45 min, 0–75%. The flow rate was 3 mL/min. The UV absorbance of the eluent was monitored at 214 nm. Each fraction was evaluated for aPTT activity. The active fraction (C-3) was concentrated by lyophilization and further purified by another C18 column (4.6 250 mm; Hanbon, China) to produce the final purified peptides. Eluent A consisted of 0.1% TFA in 10% methanol (v/v), eluent B of 0.1% TFA in 90% methanol (v/v). The chromatographic column was conditioned with 80% of eluent A. The second and final rechromatography involved solvent system A and B at the gradient of 20% B to 40% B in 40 mL at the flow rate of 0.8 mL/min. Elution was monitored at 214 nm. Each fraction was evaluated for aPTT activity. The faction C-2-2 showed aPTT activity.
2.4. Determination of molecular weight and peptide sequence The molecular mass of the purified peptide was determined using ESI-MS. Complete peptide sequencing was undertaken by Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491 (Carlsbad, CA, USA).
Inhibition of platelet aggregation induced by ADP (10 mM) was determined according to the reported method (An et al., 2011). Human platelet-rich plasma (PRP) provided by Jiangsu Blood Center, Jiangsu, China, was centrifuged at 500 g, for 10 min, at room temperature (RT). The pellets were suspended in Tyrode’s buffer A (137 mM NaCl, 2 mM KCl, 0.3 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose, 0.35% bovine serum albumin (BSA), 1 mM MgCl2, and 0.2 mM EGTA, pH 6.5). After successive washes and centrifugation, the pelleted platelets were resuspended in Tyrode’s buffer B (137 mM NaCl, 2 mM KCl, 0.3 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose, 0.35% BSA, and 2 mM CaCl2, pH 7.4) at the concentration of 5 108 platelets/mL, counted with a thrombocounter. Aliquots of 270 mL of PRP plus saline, acetylsalicylic acid (ASA, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL) or sample (0.2, 0.4, 0.6, 0.8 and 1 mg/mL) were placed in translucent tubes containing magnetic stir-bar and were preincubated at 37 1C for 1 min. Then the aggregating agent ADP (10 mM) was added in 30-mL amounts and aggregation (the change in light transmittance) was monitored for 300 s on a chart recorder connected to the aggregometer (Wei et al., 2006a, 2006b).
3. Results 3.1. Effect of peptides extracts The extracts of Scolopendra subspinipes mutilans prolonged aPTT but not PT in blood clotting time assays (data not shown).
3.2. Purification of an anticoagulant peptide from Scolopendra subspinipes mutilans The extract of Scolopendra subspinipes mutilans was separated into low weight fraction and high weight fraction by an ultrafiltration tube of 10,000 Da. The low molecular weight fraction showed aPTT activity, and then was separated into three factions by Sephadex G-50 column (Fig. 1A). The faction C showed aPTT activity, and then was separated into four fractions by Source 15Q anion exchange column (Fig. 1B). The faction C1 showed aPTT activity, and then was separated into four factions by RP-HPLC on a C18 column (Fig. 1C). The fraction C-3 showed aPTT activity, and then was separated into two fractions on another C18 column. The fraction C-2-2 showed aPTT activity and represented the final purified peptide (Fig. 1D).
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Y. Kong et al. / Journal of Ethnopharmacology 145 (2013) 182–186
3.3. Determination of molecular weight and peptide sequence The molecular mass of the purified peptide was determined to be 346 Da by ESI-MS (Fig. 2). Complete peptide sequencing was Ser-Gln-Leu (SQL) determined by Edman degradation.
3.4. Assay of aPTT of SQL SQL prolonged the aPTT clotting time in a dose-dependent manner. It prolonged the aPTT clotting time from 40 s to 71 s at a concentration of 1 mg/mL (Fig. 3). While, at a concentration of 6 mg/mL, heparin prolonged the aPTT clotting time from 40 s to 76 s.
3.5. In vitro platelet aggregation inhibition by SQL SQL induced human platelet aggregation in a dose-dependent manner following a 5 min incubation period. As little as a concentration of 0.2 mg/mL SQL inhibited human platelet aggregation by 5.7% (Fig. 4). SQL at a concentration of 1 mg/mL inhibited human platelet aggregation by 20.2%, similar to that of ASA (19.1%, 1 mg/mL) (Fig. 4). The molecular weights of SQL and ASA are 346 Da and 180 Da, respectively. SQL at a concentration of 2.89 mM inhibited human platelet aggregation by 20.2%, higher than that of ASA (19.1%, 5.55 mM).
Fig. 2. The molecular mass of the purified peptide determined by ESI-MS. The molecular mass of purified SQL is 345.2 Da determined by ESI-MS. The 346.2 m/z is SQL’ isotope peaks.
Fig. 1. Purification of an anticoagulant peptide from Scolopendra subspinipes mutilans. (A) the low molecular weight fraction of Scolopendra subspinipes mutilans extracts was separated by Sephadex G-50 column. (B) The faction (C) after Sephadex G-50 column was separated by Source 15Q anion exchange column. (C) The faction C1 after Source 15Q anion exchange column was separated by RP-HPLC on a C18 column. The fraction C-3 after RP-HPLC on a C18 column was separated by another C18 column. The faction C-2-2 showed aPTT activity and represented the final purified peptides.
Fig. 3. aPTT clotting time prolonging activity of purified SQL. SQL prolonged the aPTT clotting time in a dose-dependent manner. Heparin was used a positive control.
Y. Kong et al. / Journal of Ethnopharmacology 145 (2013) 182–186
Fig. 4. Platelet aggregation inhibiting activity of purified SQL. SQL induced human platelet aggregation in a dose-dependent manner following a 5-min incubation period. ASA was used as a positive control.
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mass of 365 Da. Lee and Kim (2005) purified and identified two new peptides SSGE (Ser-Ser-Gly-Glu) and DEE (Asp-Glu-Glu) from soy protein hydrolysate. Kong et al. (2009) purified and identified a new peptide AAP (pGlu-Asn-Trp), which could inhibit rabbit platelet aggregation induced by ADP, PAF-acether, collagen and thrombin, respectively. SQL is the first peptide identified from Scolopendra subspinipes mutilans that can inhibit platelet aggregation. Exploitation of antiplatelet agents and other kinds of antithrombotic drugs is one of the hot spots in medical research worldwide. Our study helps to clarify the mechanism of the antithrombotic activity of the centipede for effective treatment of cardiovascular and cerebrovascular diseases. Peptides are generally considered to be poor drug candidates because of their low oral bioavailability and propensity to be rapidly metabolized. The main reasons for the low oral bioavailability of peptide drugs are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. According to Lipinski’s ‘rule of five’ completed by Veber et al.’s analysis, peptides are poor candidates to move from the digestive tract to the circulatory system because of their physicochemical properties (Vlieghe et al., 2010). Future research will study the detailed inhibition mechanism of platelet aggregation by SQL and improve their low oral bioavailability.
4. Discussion Acknowledgement The centipede contains many bioactive components, including 5-hydroxytryptamine, histamine, lipids, polysaccharides, and various enzymes (e.g., proteinases and esterases) (Knysak et al., 1998; Gomes et al., 1983). They have been widely used as traditional folk medicine to treat thrombotic diseases in China (Pemberton, 1999) for several hundred years. In the present study, a novel tri-peptide SQL (Ser-Gln-Leu) with an apparent molecular mass of 346 Da, was isolated from Scolopendra subspinipes mutilans by a combination of gel filtration, anionic exchange chromatography, and reverse-phase HPLC. To our knowledge, this is the first report that the peptide purified from centipedes shows antithrombotic activity. The SQL peptide prolonged aPTT clotting time, but not PT clotting time. This result implies that SQL inhibits coagulation factors, such as factor XIIa, factor XIa, and tenase components (factor IXa, factor VIIIa, factor X, Ca2 þ , and phospholipids) in the intrinsic blood coagulation pathway. Moreover, SQL inhibited ADP-induced human platelet aggregation in a dose-dependent manner. Platelet aggregation plays an important role in hemostasis. This biochemical reaction includes the adhesion and activation of platelets, secretion of the granular contents, and aggregation of platelets again. All these events are induced by platelet aggregation agonists, and each of these steps may be a target of anti-aggregation agents (Fitzgerald et al., 1986). A number of anti-aggregation agents from other sources have been identified. In the rat, a novel platelet aggregation inhibitor was identified from peripheral polymorphonuclear leukocyte supernatant (Kumari et al., 1998). A novel platelet aggregation inhibitor also was purified from the gland of Rhodnius prolixus (Francischetti et al., 2000). Many snake components including phospholipase A2 (Lu et al., 2002a, 2002b), L-amino acid oxidase (Wei et al., 2007), disintegrin (Zhou et al., 2004a, 2004b), phosphodiesterase (Peng et al., 2011), and serine proteinase show specific antiplatelet activities. Platelet aggregation inhibitors also were found in the horsefly (An et al., 2011; Ma et al., 2011), wasp venom (Han et al., 2008; Yang et al., 2008) and other insects. In addition to the proteins, many peptides were identified that showed anti-aggregation activity. For example, Hyun et al. (2006) isolated and characterized a novel platelet aggregation inhibitory peptide (Trp-Gly-Cys) with a molecular
This study was sponsored by National Science and Technology Major Projects for ‘‘Major New Drugs Innovation and Development’’ (2009ZX09103-091), Chinese National Natural Science Foundation (30972822, 81273274,81273375), Jiangsu Province’s Key Provincial Talents Program (RC201170, H201108) and The innovative plan of Jiangsu provincial graduate(No. CXZZ11-0822) References An, S., Ma, D., Wei, J.F., Yang, X., Yang, H.W., Yang, H., Xu, X., He, S., Lai, R., 2011. A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies. Allergy 66, 1420–1427. Fitzgerald, D.J., Roy, L., Catella, F., FitzGerald, G.A., 1986. Platelet activation in unstable coronary disease. New England Journal of Medicine 315, 983–989. Francischetti, M.R., Ribeiro, M.C., Donald, C., John, A., 2000. Purification, cloning, expression and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus. Journal of Biological Chemistry 275, 12639–12650. Gomes, A., Datta, A., Sarangi, B., 1983. Isolation, purification, pharmacodynamics of a toxin from the venom of the centipede Scolopendra subspinipes dehaani Brand. Journal of Experimental Biology 21, 203–207. Han, J., You, D., Xu, X., Han, W., Lu, Y., Lai, R., Meng, Q., 2008. An anticoagulant serine protease from the wasp venom of Vespa magnifica. Toxicon 51, 914–922. Hyun, K.W., Jeong, S.C., Lee, D.H., Park, J.S., Lee, J.S., 2006. Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus. Peptides 27, 1173–1178. Jung, W.K., Jo, H.Y., Qian, Z.J., Jeong, Y.J., Park, S.G., Choi, I.W., Kim, S.K., 2007. A novel anticoagulant protein with high affinity to blood coagulation factor Va from Tegillarca granosa. Journal of Biochemistry and Molecular Biology 40, 832–838. Knysak, I., Martins, R., Bertim, C.R., 1998. Epidemiological aspects of centipede bites registered in greater S. Paulo, SP, Brazil. Rev Saude Publica 32, 514–518. Kong, Y., Huo, J.L., Xu, W., Xiong, J., Li, Y.M., Wu, W.T., 2009. A novel anti-platelet aggregation tripeptide from Agkistrodon acutus venom: isolation and characterization. Toxicon 54, 103–109. Kumari, R., Singh, M.P., Seth, P., Dikshit, M., 1998. Inhibition of platelet aggregation by a protein factor present in rat peripheral polymorphonuclear leukocyte supernatant. Thrombosis Research 91, 75–82. Lee, K.A., Kim, S.H., 2005. SSGE and DEE, new peptides isolated from a soy protein hydrolysate that inhibit platelet aggregation. Food Chemistry 90, 389–393. Lu, Q.M., Jin, Y., Wei, J.F., Wang, W.Y., Xiong, Y.L., 2002a. Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A2. Journal of Natural Toxins 11, 25–33. Lu, Q.M., Jin, Y., Wei, J.F., Li, D.S., Zhu, S.W., Wang, W.Y., Xiong, Y.L., 2002b. Characterization and cloning of a novel phospholipase A2 from the venom of Trimeresurus jerdonii snake. Toxicon 40, 1313–1319.
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Ma, D., Xu, X., An, S., Liu, H., Yang, X., Andersen, J.F., Wang, Y., Tokumasu, F., Ribeiro, J.M., Francischetti, I.M., Lai, R., 2011. A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets aIIbb3 or aVb3 and inhibits platelet aggregation and angiogenesis. Thrombosis and Haemostasis 105, 1032–1045. Majdoub, H., Ben Mansour, M., Chaubet, F., Roudesli, M.S., Maaroufi, R.M., 2009. Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis. Biochimica et Biophysica Acta 1790, 1377–1381. Negrea, S., Minelli, A., 1995. Chilopoda. In: Decu, V., Juberthie, C. (Eds.), Enciclopedia Bioespeologica, 1; 1995, pp. 249–254. Pemberton, R.W., 1999. Insects and other arthropods used as drugs in Korean traditional medicine. Journal of Ethnopharmacology 65, 207–216. Peng, K., Kong, Y., Zhai, L., Wu, X., Jia, P., Liu, J., Yu, H., 2010. Two novel antimicrobial peptides from centipede venoms. Toxicon 55, 274–279. Peng, L., Xu, X., Shen, D., Zhang, Y., Song, J., Yan, X., Guo, M., 2011. Purification and partial characterization of a novel phosphodiesterase from the venom of Trimeresurus stejnegeri: inhibition of platelet aggregation. Biochimie 93, 1601–1609. Undheim, E.A., King, G.F., 2011. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals. Toxicon 57, 512–524. Vlieghe, P., Lisowski, V., Martinez, J., Khrestchatisky, M., 2010. Synthetic therapeutic peptides: science and market. Drug Discovery Today 15 (1-2), 40–56. Wang, K., Fang, H., Ye, M., Chen, H., Zhu, Y., Fang, H., 1997. Investigation on the resources of medicinal centipedes and identification on their commodities. Zhong Yao Cai 20, 450–452. Wei, J.F., Li, T., Wei, X.L., Sun, Q.Y., Yang, F.M., Chen, Q.Y., Wang, W.Y., Xiong, Y.L., He, S.H., 2006a. Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A2 from the venom of Protobothrops mucrosquamatus. Biochimie 88, 1331–1342.
Wei, J.F., Wei, X.L., Chen, Q.Y., Huang, T., Qiao, L.Y., Wang, W.Y., Xiong, Y.L., He, S.H., 2006b. N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2. Biochimica et Biophysica Acta 1760, 462–471. Wei, X.L., Wei, J.F., Li, T., Qiao, L.Y., Liu, Y.L., Huang, T., He, S.H., 2007. Purification, characterization and potent lung lesion activity of an L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom. Toxicon 50, 1126–1139. Wu, Y., Li, J., Wang, J., Si, Q., Zhang, J., Jiang, Y., Chu, L., 2009. Anti-atherogenic effects of centipede acidic protein in rats fed an atherogenic diet. Journal of Ethnopharmacology 122, 509–516. Yang, H., Xu, X., Ma, D., Zhang, K., Lai, R., 2008. A phospholipase A1 platelet activator from the wasp venom of Vespa magnifica (Smith). Toxicon 51, 289–296. Yang, S., Liu, Z., Xiao, Y., Li, Y., Rong, M., Liang, S., Zhang, Z., Yu, H., King, G.F., Lai, R., 2012. Chemical Punch Packed in Venoms makes Centipedes Excellent Predators. Mol Cell Proteomics 11, 640–650. You, W.K., Sohn, Y.D., Kim, K.Y., Park, D.H., Jang, Y., Chung, K.H., 2004. Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans. Insect Biochemistry and Molecular Biology 34, 239–250. Zhou, X.D., Ding, C.H., Tai, H., Jin, Y., Chen, R.Q., Lu, Q.M., Wang, W.Y., Xiong, Y.L., 2004a. A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 139, 117–122. Zhou, X.D., Jin, Y., Lu, Q.M., Li, D.S., Zhu, S.W., Wang, W.Y., Xiong, Y.L., 2004b. Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 137, 219–224.
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans Yi Kong a,b,n, Shi-Long Huang a,b, Yu Shao a, Shuai Li a, Ji-Fu Wei c,nn a
School of Life Science & Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China c Research Division of Clinical Pharmacology, the First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2012 Received in revised form 22 October 2012 Accepted 25 October 2012 Available online 2 November 2012
Ethnopharmacological relevance: The centipede has been prescribed for the treatment of cardiovascular diseases in Korea, China and other Far Eastern Asian countries for several hundred years. Materials and methods: A novel antithrombotic peptide was isolated from Scolopendra subspinipes mutilans using a combination of ultrafiltration, Sephadex G-50 column, Source 15Q anion exchange column and RP-HPLC C18 column. Results: The molecular mass of the purified peptide is 346 Da measured by Electrospray Ionization Mass Spectrometry (ESI-MS). The primary structure of the peptide is Ser-Gln-Leu (SQL) determined by Edman degradation. SQL potently prolonged the activated partial thromboplastin time (aPTT), and inhibited platelet aggregation. Conclusions: These results help to clarify the mechanism of the antithrombotic activity of the centipede for effective treatment of cardiovascular and cerebrovascular diseases. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Scolopendra subspinipes mutilans Activated partial thromboplastin time (aPTT) Platelet aggregation inhibitor
1. Introduction Centipedes (Scolopendra subspinipes mutilans L. Koch) are predatory, elongated and dorsoventrally flattened arthropods, which belong to the Chilopod class (Negrea and Minelli, 1995). In China, the centipede has been used for thousands of years as a traditional Chinese medicine to treat disorders, such as strokeinduced hemiplegia, apoplexy, epilepsy, tetanus, whooping cough, tuberculosis, scald burns, and pyocutaneous disease. In addition, the centipede has been described for the treatment of cardiovascular diseases in Korea, China and other Far Eastern Asian countries for several hundred years (Pemberton, 1999). Thrombosis is caused by two main pathways. One is blood clotting, which is processed by various coagulation factors at sites of vascular injury. Because endogenous or exogenous anticoagulants interfere with coagulation factors, blood coagulation can be prolonged or stopped (Jung et al., 2007). The other thrombotic pathway is platelet aggregation, which plays an important role in hemostasis. The process of platelet aggregation includes the adhesion and activation of platelets, secretion of the granular contents, and aggregation of platelets again (Kong et al., 2009).
There are few reports about antithrombotic agents identified from the centipede. You et al. (2004) isolated a 25 kDa serine protease from Scolopendra subspinipes mutilans that demonstrated fibrinolytic activity by converting human Glu-plasminogen to activated plasmin. Wu et al. (2009) found that centipede acidic protein (CAP) significantly suppressed the development of atherosclerosis, and improved the hemorrheological disturbances and histopathological changes in the atherogenic-diet rat model. These effects may be attributed in part to reversal of dyslipidemia, inhibition of lipid peroxidation, and regulation of nitric oxide (NO) and endothelin-1 (ET-1) systems. Centipedes consist of many kinds of protein, amino acid, lipids and enzymes. Many peptides were purified or identified from Centipedes (Wang et al., 1997; Peng et al., 2010; Undheim et al., 2011; Yang et al., 2012). In the present study, we report the purification and characterization of a novel peptide (Ser-Gln-Leu) with antithrombotic activity, which may contribute to the traditional usefulness of centipede as antithrombotic medicine.
2. Materials and methods 2.1. Materials
n
Corresponding author. Tel./fax: þ 86 25 83271030. Corresponding author. Tel./fax: þ 86 25 83718836. E-mail addresses: [email protected] (Y. Kong), [email protected] (J.-F. Wei). nn
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.10.048
Centipedes were obtained from Xiansheng Pharmacy (Nanjing, Jiangsu province, China). The ultrafiltration tube was purchased from Millipore (Billerica, Massachusetts, USA). The BioLogic Duoflow
Y. Kong et al. / Journal of Ethnopharmacology 145 (2013) 182–186
system was obtained from Bio-Rad (Richmond, CA, USA). The Lichrospher C18 reverse-phase HPLC column was from Jiangsu Hanbon Science & Technology Co. Ltd (Huai’an, China). The ESI-MS System was from Waters Company (Milford, Massachusetts. USA).
183
2.5. Prothrombin time (PT) and aPTT clotting assays
Chinese Pharmacopoeia (I) provides a centipede medicinal method: centipede was extract by ethanol (50%) and the dried powder can take orally. So, Scolopendra subspinipes mutilans (n¼ 100) were ground to a powder in a mortar and then extracted by stirring for 24 h in 1000 mL of 50% ethanol solution. The homogenate was centrifuged at 8000 g for 15 min at 4 1C. The supernatant was collected. The ethanol was removed from the supernatant by rotary evaporation.
Nine parts of blood was drawn by venipuncture into one part of 3.2% sodium citrate from healthy volunteers and pooled. The blood was separately centrifuged at 2500 g for 10 min to obtain platelet poor plasma (PPP). Briefly, normal citrated PPP (80 mL) was incubated with sample solution (20 mL) for 3 min at 37 1C. For the aPTT clotting assay, 100 mL of aPTT reagent was added to the mixture (PPP and sample) and incubated at 37 1C for 10 min. Clotting time was immediately recorded after the addition of 100 mL of 20 mM CaCl2. For the PT clotting assay, PT reagent was added to the incubated mixture of PPP (80 mL) and sample (20 mL), and clotting time (s) was recorded. All coagulation assays were performed with three individual replicates (Majdoub et al., 2009). Heparin, a commercial anticoagulant, was used as positive control.
2.3. Purification of an anticoagulant peptide from Scolopendra subspinipes mutilans
2.6. Assay of anti-platelet aggregation activity
2.2. Extraction of peptides from Scolopendra subspinipes mutilans
The supernatant was filtered through a Millipore membrane filter (0.45 mm) and then applied to an ultrafiltration tube of 10,000 Da, and centrifuged at 10,000 rpm for 5 min. The low molecular weight fraction after ultrafiltration was collected and then applied to a Sephadex G-50 column (26 mm 100 cm), equilibrated with distilled water and eluted with the same buffer at a flow rate of 0.8 mL/min. The fractions were collected at 15-min intervals. The activated partial thromboplastin time (aPTT) activity was evaluated for each fraction. The fraction with the highest aPTT activity (Faction C) was collected, lyophilized, dialyzed with 50 mM Tris–HCl buffer (pH 7.4), and further separated by Source 15Q anion exchange column (12 mm 150 mm) preliminarily equilibrated with 50 mM Tris–HCl buffer (pH 7.4) in a high performance liquid chromatographic (HPLC) system (Bio-Rad, Richmond, CA, USA). A linear gradient of NaCl (0–2 M) in the same buffer was maintained at a flow rate of 3 mL/min. Absorbance was monitored at 214 nm. Each fraction was evaluated for aPTT activity. The fraction with aPTT activity (Faction C1) was separated by RP-HPLC on a C18 column (10 250 mm; Kromasil, China) under linear gradient elution conditions using methanol as the organic modifier and trifluoroacetic acid (TFA) as the volatile buffer. Eluent A consisted of 0.1% TFA in 10% methanol (v/v), eluent B of 0.1% TFA in 90% methanol (v/v). The chromatographic column was conditioned with 100% of eluent A, after which 1 mL of the peptide solution was applied on the C18 column and eluted by the remaining eluent A for 10 min and with the following increasing eluent B concentrations: 0–10 min, 0%; 10–45 min, 0–75%. The flow rate was 3 mL/min. The UV absorbance of the eluent was monitored at 214 nm. Each fraction was evaluated for aPTT activity. The active fraction (C-3) was concentrated by lyophilization and further purified by another C18 column (4.6 250 mm; Hanbon, China) to produce the final purified peptides. Eluent A consisted of 0.1% TFA in 10% methanol (v/v), eluent B of 0.1% TFA in 90% methanol (v/v). The chromatographic column was conditioned with 80% of eluent A. The second and final rechromatography involved solvent system A and B at the gradient of 20% B to 40% B in 40 mL at the flow rate of 0.8 mL/min. Elution was monitored at 214 nm. Each fraction was evaluated for aPTT activity. The faction C-2-2 showed aPTT activity.
2.4. Determination of molecular weight and peptide sequence The molecular mass of the purified peptide was determined using ESI-MS. Complete peptide sequencing was undertaken by Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491 (Carlsbad, CA, USA).
Inhibition of platelet aggregation induced by ADP (10 mM) was determined according to the reported method (An et al., 2011). Human platelet-rich plasma (PRP) provided by Jiangsu Blood Center, Jiangsu, China, was centrifuged at 500 g, for 10 min, at room temperature (RT). The pellets were suspended in Tyrode’s buffer A (137 mM NaCl, 2 mM KCl, 0.3 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose, 0.35% bovine serum albumin (BSA), 1 mM MgCl2, and 0.2 mM EGTA, pH 6.5). After successive washes and centrifugation, the pelleted platelets were resuspended in Tyrode’s buffer B (137 mM NaCl, 2 mM KCl, 0.3 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose, 0.35% BSA, and 2 mM CaCl2, pH 7.4) at the concentration of 5 108 platelets/mL, counted with a thrombocounter. Aliquots of 270 mL of PRP plus saline, acetylsalicylic acid (ASA, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL) or sample (0.2, 0.4, 0.6, 0.8 and 1 mg/mL) were placed in translucent tubes containing magnetic stir-bar and were preincubated at 37 1C for 1 min. Then the aggregating agent ADP (10 mM) was added in 30-mL amounts and aggregation (the change in light transmittance) was monitored for 300 s on a chart recorder connected to the aggregometer (Wei et al., 2006a, 2006b).
3. Results 3.1. Effect of peptides extracts The extracts of Scolopendra subspinipes mutilans prolonged aPTT but not PT in blood clotting time assays (data not shown).
3.2. Purification of an anticoagulant peptide from Scolopendra subspinipes mutilans The extract of Scolopendra subspinipes mutilans was separated into low weight fraction and high weight fraction by an ultrafiltration tube of 10,000 Da. The low molecular weight fraction showed aPTT activity, and then was separated into three factions by Sephadex G-50 column (Fig. 1A). The faction C showed aPTT activity, and then was separated into four fractions by Source 15Q anion exchange column (Fig. 1B). The faction C1 showed aPTT activity, and then was separated into four factions by RP-HPLC on a C18 column (Fig. 1C). The fraction C-3 showed aPTT activity, and then was separated into two fractions on another C18 column. The fraction C-2-2 showed aPTT activity and represented the final purified peptide (Fig. 1D).
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3.3. Determination of molecular weight and peptide sequence The molecular mass of the purified peptide was determined to be 346 Da by ESI-MS (Fig. 2). Complete peptide sequencing was Ser-Gln-Leu (SQL) determined by Edman degradation.
3.4. Assay of aPTT of SQL SQL prolonged the aPTT clotting time in a dose-dependent manner. It prolonged the aPTT clotting time from 40 s to 71 s at a concentration of 1 mg/mL (Fig. 3). While, at a concentration of 6 mg/mL, heparin prolonged the aPTT clotting time from 40 s to 76 s.
3.5. In vitro platelet aggregation inhibition by SQL SQL induced human platelet aggregation in a dose-dependent manner following a 5 min incubation period. As little as a concentration of 0.2 mg/mL SQL inhibited human platelet aggregation by 5.7% (Fig. 4). SQL at a concentration of 1 mg/mL inhibited human platelet aggregation by 20.2%, similar to that of ASA (19.1%, 1 mg/mL) (Fig. 4). The molecular weights of SQL and ASA are 346 Da and 180 Da, respectively. SQL at a concentration of 2.89 mM inhibited human platelet aggregation by 20.2%, higher than that of ASA (19.1%, 5.55 mM).
Fig. 2. The molecular mass of the purified peptide determined by ESI-MS. The molecular mass of purified SQL is 345.2 Da determined by ESI-MS. The 346.2 m/z is SQL’ isotope peaks.
Fig. 1. Purification of an anticoagulant peptide from Scolopendra subspinipes mutilans. (A) the low molecular weight fraction of Scolopendra subspinipes mutilans extracts was separated by Sephadex G-50 column. (B) The faction (C) after Sephadex G-50 column was separated by Source 15Q anion exchange column. (C) The faction C1 after Source 15Q anion exchange column was separated by RP-HPLC on a C18 column. The fraction C-3 after RP-HPLC on a C18 column was separated by another C18 column. The faction C-2-2 showed aPTT activity and represented the final purified peptides.
Fig. 3. aPTT clotting time prolonging activity of purified SQL. SQL prolonged the aPTT clotting time in a dose-dependent manner. Heparin was used a positive control.
Y. Kong et al. / Journal of Ethnopharmacology 145 (2013) 182–186
Fig. 4. Platelet aggregation inhibiting activity of purified SQL. SQL induced human platelet aggregation in a dose-dependent manner following a 5-min incubation period. ASA was used as a positive control.
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mass of 365 Da. Lee and Kim (2005) purified and identified two new peptides SSGE (Ser-Ser-Gly-Glu) and DEE (Asp-Glu-Glu) from soy protein hydrolysate. Kong et al. (2009) purified and identified a new peptide AAP (pGlu-Asn-Trp), which could inhibit rabbit platelet aggregation induced by ADP, PAF-acether, collagen and thrombin, respectively. SQL is the first peptide identified from Scolopendra subspinipes mutilans that can inhibit platelet aggregation. Exploitation of antiplatelet agents and other kinds of antithrombotic drugs is one of the hot spots in medical research worldwide. Our study helps to clarify the mechanism of the antithrombotic activity of the centipede for effective treatment of cardiovascular and cerebrovascular diseases. Peptides are generally considered to be poor drug candidates because of their low oral bioavailability and propensity to be rapidly metabolized. The main reasons for the low oral bioavailability of peptide drugs are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. According to Lipinski’s ‘rule of five’ completed by Veber et al.’s analysis, peptides are poor candidates to move from the digestive tract to the circulatory system because of their physicochemical properties (Vlieghe et al., 2010). Future research will study the detailed inhibition mechanism of platelet aggregation by SQL and improve their low oral bioavailability.
4. Discussion Acknowledgement The centipede contains many bioactive components, including 5-hydroxytryptamine, histamine, lipids, polysaccharides, and various enzymes (e.g., proteinases and esterases) (Knysak et al., 1998; Gomes et al., 1983). They have been widely used as traditional folk medicine to treat thrombotic diseases in China (Pemberton, 1999) for several hundred years. In the present study, a novel tri-peptide SQL (Ser-Gln-Leu) with an apparent molecular mass of 346 Da, was isolated from Scolopendra subspinipes mutilans by a combination of gel filtration, anionic exchange chromatography, and reverse-phase HPLC. To our knowledge, this is the first report that the peptide purified from centipedes shows antithrombotic activity. The SQL peptide prolonged aPTT clotting time, but not PT clotting time. This result implies that SQL inhibits coagulation factors, such as factor XIIa, factor XIa, and tenase components (factor IXa, factor VIIIa, factor X, Ca2 þ , and phospholipids) in the intrinsic blood coagulation pathway. Moreover, SQL inhibited ADP-induced human platelet aggregation in a dose-dependent manner. Platelet aggregation plays an important role in hemostasis. This biochemical reaction includes the adhesion and activation of platelets, secretion of the granular contents, and aggregation of platelets again. All these events are induced by platelet aggregation agonists, and each of these steps may be a target of anti-aggregation agents (Fitzgerald et al., 1986). A number of anti-aggregation agents from other sources have been identified. In the rat, a novel platelet aggregation inhibitor was identified from peripheral polymorphonuclear leukocyte supernatant (Kumari et al., 1998). A novel platelet aggregation inhibitor also was purified from the gland of Rhodnius prolixus (Francischetti et al., 2000). Many snake components including phospholipase A2 (Lu et al., 2002a, 2002b), L-amino acid oxidase (Wei et al., 2007), disintegrin (Zhou et al., 2004a, 2004b), phosphodiesterase (Peng et al., 2011), and serine proteinase show specific antiplatelet activities. Platelet aggregation inhibitors also were found in the horsefly (An et al., 2011; Ma et al., 2011), wasp venom (Han et al., 2008; Yang et al., 2008) and other insects. In addition to the proteins, many peptides were identified that showed anti-aggregation activity. For example, Hyun et al. (2006) isolated and characterized a novel platelet aggregation inhibitory peptide (Trp-Gly-Cys) with a molecular
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