Anticoagulant activity of Moon jellyfish (Aurelia aurita) tentacle extract

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    Chungkook-Jang soy sauce (Nikai et al., 1984; Sumi et al., attractive source of physiologically active compounds.Their extracts have been reported to exert hemolytic (Kanget al., 2009), insecticidal (Yu et al., 2005a), cardiovascular(Ramasamy et al., 2005), antioxidant (Yu et al., 2005b), andcytotoxic (Kang et al., 2009) effects. Anticoagulants frommarine organisms have rarely been isolated, except forseveral anticoagulant proteoglycans and polysaccharidesfrom marine algae (Changaff et al., 1936; Kindness et al.,

    Abbreviations: BSA, bovine serum albumin; EDTA, ethylene di-aminetetra acetic acid; JFTE, Jellysh tentacle extract; MW, molecular weight;PLA, phospholipase A; PMSF, phenyl methyl sulfonyl uoride; RBCs, redblood cells.* Corresponding author. Fax: 91 832 255 7033.

    E-mail addresses: diba27@yahoo.com, dibakarchakrabarty@bits-goa.ac.in (D. Chakrabarty).

    Contents lists available at SciVerse ScienceDirect

    Toxic

    journal homepage: www.els

    Toxicon 60 (2012) 719723provided the impetus for the development of new antico-agulants that target specic coagulation enzymes ora particular step in the clotting process.

    Potent anticoagulants have been discovered in snakevenoms, earthworm secretions, dung beetles, food-grademicroorganisms, marine creatures, herbal medicines, andfermented food products like Japanese Natto and Korean

    Marcinkiewicz, 2005; Huang and Hong, 2004; Plosker andIbbotson, 2003; Kondo and Umemura, 2002; McClellan andGoa, 1998). Ancrod/Viprinex (extracted from the venom ofthe Malayan pit viper) reduces blood brinogen levels andhas been successfully tested in a variety of ischemicconditions, including stroke (Sherman, 2002).

    In recent years, cnidarians like jellysh have become an1. Introduction

    Thrombosis has been and continuproblem leading to mortality. Anticoathe prevention and treatment of thders. Coumarins and heparin are tclinically used anticoagulants. Howmode of action of these anticoagulatherapeutic limitations in maintainithrombosis and hemostasis. The0041-0101/$ see front matter 2012 Elsevier Ltd10.1016/j.toxicon.2012.05.008be amajor healthts are pivotal foroembolic disor-ost well knownthe nonspecicccounts for theirbalance betweenimitations have

    1987, 1992; Mihara et al., 1991; Kim et al., 1996; Changet al., 2000; Choi and Sa, 2000; Jeong et al., 2001; Ahnet al., 2004).

    Anticoagulant components from snake venoms haveinspired the design and development of a number oftherapeutic agents or lead molecules. For example, inhibi-tors of platelet aggregation, such as Eptibatide and Tir-oban, were designed based on disintegrins, a large familyof platelet aggregation inhibitors found in viperid andcrotalid snake venoms (OShea and Tcheng, 2002; 2012 Elsevier Ltd. All rights reserved.

    brinogenolytic activity of the extract.Anticoagulant activity of Moon jelly

    Akriti Rastogi, Sumit Biswas, Angshuman SarDepartment of Biological Sciences, Birla Institute of Technology and Science

    a r t i c l e i n f o

    Article history:Received 18 January 2012Received in revised form 9 May 2012Accepted 17 May 2012Available online 28 May 2012

    Keywords:Moon jellyshAnticoagulantFibrinogenolytic toxin

    a b s t r a c t

    Moon jellysh (Aurin vitro. The Jellyshcleaving Aa and Bbbe stronger than sobrin clots in 24 hproteins/peptides. Tfractions of the extnolytic activity andular weight protei. All rights reserved.h (Aurelia aurita) tentacle extract

    , Dibakar Chakrabarty*

    K.K. Birla Goa Campus, Zuarinagar, Goa 403 726, India

    urita) tentacle extract was studied for its anticoagulant activityacle Extract (JFTE) showed very strong brinogenolytic activity byof brinogen molecule. The brinogenolytic activity was found toake venom derived anticoagulants. JFTE also completely liqueedE was found to contain both high and low molecular weightbrinogenolysis appears to be caused by high molecular weightt has been also noted that PMSF signicantly reduced brinoge-ing totally abolished it. Autolytic degradation of the high molec-s also noted. Autolysis slowed down, but did not abolish the

    on

    evier .com/locate/ toxicon

  • A. Rastogi et al. / Toxicon 60 (2012) 7197237201980; Maimone and Tollefsen, 1990; McLellan and Jurd,1991; Jurd et al., 1995) and ascidian tunic (Lee et al., 1998).

    In the present study, we report the presence of bri-no(geno)lytic and inhibitory activity on platelet aggrega-tion in the tentacle extract of Aurelia aurita jellysh.

    2. Materials and methods

    Moon jellysh (A. aurita) were collected fromGoa, India.All ne chemicals used were purchased from SigmaChemicals, USA. All other reagents were of analytical grade.Whole Blood Platelet Aggregometer Model 592 waspurchased from Chrono-log Corporation, USA.

    2.1. Preparation of Jellysh Tentacle Extracts (JFTE)

    The oral arm tentacles (OAT) were manually excisedwithin 1 h of collection and stored in 0.85% saline at20 C.Frozen OAT were autolyzed at 4 C in 0.85% saline (9:1 v/v)for 4 days. Resultant uid was claried by centrifugation at20,000 g for 1 h at 4 C. This claried uid was calledJellyfish Tentacle Extract (JFTE) and was used as theworking material for further investigations. It was stored at80 C in 2 ml aliquots till used.

    2.2. Protein estimation

    Protein content of JFTE was estimated by modiedBradford assay (Bradford, 1976). A standard curve wasconstructed using bovine serum albumin (BSA) as standardprotein.

    2.3. Component proteins and their molecular weightdetermination

    Different component proteins of JFTE and their molec-ular weights were assessed by electrophoresis (SDS-PAGE)on 12% separating gel with a 5% stacking gel. The proteinbands were stained with 1% Coomassie brilliant blue R250in 7.5% acetic acid and 10%methanol overnight. The gel wasthen destained by repeated washings with 7.5% acetic acidand 10% methanol.

    2.4. Assay of brinolytic activity

    Fibrinolytic activity of the JFTE or fractions was moni-tored by a modication of the brin plate method of Astrupand Mullertz (1952). Fibrinogen fraction I (3.3 mg) wasdissolved in 0.2 ml of 20 mM potassium phosphate buffer,pH 7.4. Ammonium sulfate was added to above solution toa nal concentration of 70 mM. Five microliters ofthrombin was added to the above solution and transferredimmediately to a 0.5 ml microfuge tube. The solution wasallowed to clot by incubating for 2 h at 25 C (ChandraSekhar and Chakrabarty, 2011). JFTE was applied on thesurface of the clot and incubated at 37 C for 24 h. Fibri-nolytic activity of the sample was observed by the lique-faction of the clot.Whole Russells viper venomwas used aspositive control and 0.85% saline was used as negativecontrol.2.5. Fibrinogenolytic activity

    Fibrinogenolytic activity was conrmed by incubatingbrinogen fraction I (2 mg/ml) with JFTE and 0.85% saline(control) for different time intervals and doses at 37 C. Theincubated mixtures were subjected to SDS-PAGE on 12%separating gelwith a 5% stacking gel. The protein bandswereviewed by staining with 1% Coomassie brilliant blue, R250.Fibrinogenolytic activity was monitored by comparing posi-tion and appearance of specic bandswith that of brinogenincubated with 0.85% saline only (Bos et al., 1997).

    Fibrinogenolytic activity of JFTE was also estimated aftertreatment with 2 mM EDTA (metalloprotease inhibitor),freshly prepared 1 mM PMSF (serine protease inhibitor) orexposure to 100 C for 1 min in a boiling water bath.

    2.6. Assay of hemolytic activity

    Blood was collected aseptically from male healthyvolunteers in 0.85% saline and centrifuged at 3000 rpm for3min. Supernatant was discarded and the pellet containingRBCs were washed thrice with normal saline. RBCsuspension (0.3 ml) was taken in each tube to which 0.2 ml(50 mg) of JFTE was added. Distilled water and 0.85% salinewere added to RBC suspension as positive and negativecontrol respectively. All the tubes were then incubated for1 h at 37 C and centrifuged at 3000 rpm for 5 min.Absorbance of the supernatants were measured at 540 nm.Values obtained with positive control represented 100%hemolysis (Chakrabarty et al., 2000).

    2.7. Assay of phospholipase A activity

    Presence of phospholipase A (PLA) activity was testedusing egg yolk as substrate (Neumann and Habermann,1954). Five micrograms of JFTE was added to 2 ml of eggyolk suspension, mixed well and incubated at 37 C for 1 h.Incubated samples were then placed in a boiling water bathand time required for coagulation of the samples were thennoted. 0.85% saline and 5 mg Russells viper venom wereused as negative and positive control respectively.

    2.8. Platelet aggregation studies

    Blood was freshly collected from healthy O positivehumanvolunteers.Bloodwascollected invialscontainingnineparts of blood and one part of 3.8% sodium citrate. Plateletaggregation was stimulated with 20 mmol ADP. Dose depen-dent effect of JFTEonADP inducedaggregationwasmonitoredby adding JFTE at time of incubation. Platelet aggregationwasexpressed as the change in electrical impedance and isexpressed in ohms. Aggregation curves were recorded for7 min and analyzed using AGGROLINK software. Timedependent graphs were plotted based on aggregation.

    3. Results

    3.1. SDS-PAGE of JFTE

    Protein concentration of JFTE was found to be 2.5 mg/mlby Bradfords Method. SDS-PAGE of JFTE revealed six

  • prominent protein bands between 160 and 50 kDa.Multiple low molecular weight bands were also observed(Fig. 1).

    3.2. Fibrin(ogen)olytic activity

    JFTE showed preferential digestion of Aa chain ofbrinogen, followed by Bb chain in a dose and timedependent manner. Fibrinogen solution (2 mg/ml) wasincubated with 2.5 mg of JFTE for different time periods at37 C. Immediate digestion of the Aa chain followed by Bbchainwas observed (Fig. 2a). JFTE, at a dose of 2.5 mg causedalmost complete digestion of Aa and Bb chain within 3 h.Fifteen micrograms of JFTE caused signicant digestion of gchain also (Fig. 2b).

    JFTE (15 mg) completely liqueed 200 ml brin clotsin vitro when incubated for 24 h at 37 C (Fig. 3).

    Fibrinogenolytic activity of JFTE was totally inhibited onexposure to 100 C for 1 min. Pre-treatment of JFTE with1 mM PMSF caused almost complete inhibition of brino-genolytic activity, whereas, pre-treatment with 2mM EDTAdelayed brinogenolysis.

    3.3. Hemolytic and phospholipase activity

    JFTE (15 mg) showed approximately 25.6% hemolysis onhuman RBCs compared to positive control. JFTE was foundto be devoid of phospholipase activity.

    Autodegradation of JFTE was noticed to take place intime dependent manner. It was observed that 1 mM PMSFinhibited autodegradation of high molecular weightproteins along with brinogenolytic activity. However,2 mM EDTA inhibited autodegradation without affectingbrinogenolytic activity.

    Fig. 2. a. Time-dependent brinogenolytic activity of JFTE. Fibrinogen(2 mg/ml) was incubated independently with JFTE (2.5 mg) for different time

    Fig. 3. Fibrinolytic activity of JFTE. 0.85% saline and JFTE (15 mg) wereincubated with brin clot developed in the microcentrifuge tube at 37 C.The brinolytic activity was visualized after 24 h. Fibrin clot incubated with(C) 0.85% saline, (JFTE) Jellysh Tentacle Extract.

    A. Rastogi et al. / Toxicon 60 (2012) 719723 721Fig. 1. Band pattern for 5 mg JFTE on 12% SDS-PAGE gel.3.4. Autodegradation

    intervals at 37 C. Samples were kept frozen at 80 C after their incubationperiod till run on SDS-PAGE. (F) Fibrinogen alone after 180 min incubation.Numbers at the bottom of each lane indicate minutes of incubation withJFTE. b. Dose dependent brinogenolytic activity of JFTE. Fibrinogen (2 mg/ml) was incubated independently with different concentrations of JFTE at37 C for 180 min. (F) Fibrinogen alone, (JFTE) JFTE alone. Numbers at thebottom of each lane indicate dose of JFTE in microgram.

  • molecule by Lahirin requires 24 h at a dose of 15 mg/ml(Chandra Sekhar and Chakrabarty, 2011). JFTE also

    E on A

    A. Rastogi et al. / Toxicon 60 (2012) 719723722completely liqueed brin clots in 24 h (Fig. 3). Proteolyticactivity of jellysh venom has been reported earlier by Leeet al. (2011), where one of the substrates used was brin-ogen. But it was not described whether specic brinoge-nolytic activity was observed or brinogen digestion isa reection of random proteolytic activity.

    Rapid degradation of jellysh toxins has been reported3.5. Platelet aggregation

    ADP induced platelet aggregation was inhibited by JFTEin a dose dependent manner. ADP induced platelet aggre-gation dropped by 9.4% with 10 mg of JFTE. The drop in ADPinduced platelet aggregation was as high as 82.4% with40 mg of JFTE (Fig. 4).

    4. Discussion

    Moon jellysh tentacle extract was studied for its anti-coagulant activities in vitro. JFTE was incubated with bovinebrinogen and two major chains, namely Aa and Bb werefound digested after 3 h. It may be noted here, that Lahirinpuried from Naja kaouthia (Chandra Sekhar andChakrabarty, 2011) venom digests only Aa chain in about5 h at the same dose. Complete digestion of brinogen

    Fig. 4. Effect of different doses of JFTearlier (Bloom et al., 1998). JFTE proteins were also found toundergo rapid auto-degradation. It was observed that, highmolecular weight protein bands of JFTE gradually dis-appeared with time (Fig. 2a). EDTA delayed the digestion ofBb chain, but digestion of Aa chain continued. PMSFsignicantly inhibited digestion of Aa and Bb chains byJFTE. It is possible that different toxins are involved in thedigestion of different chains of brinogen or even smallerpeptides resulted from auto-digestion also retained thebrinogenolytic activity. PMSF inhibited toxin induceddigestion of brinogen chains, but did not abolish it. It ispossible that brinogenolytic toxins of different types arepresent in the sample, i.e., serine proteases as well asmetalloproteases.

    Hemolytic activity of jellysh venom is a well docu-mented phenomenon (Nagai et al., 2000; Radwan et al.,2000; Chung et al., 2001; Gusmani et al., 1997; Rottiniet al., 1995). JFTE was also found to be hemolytic onhuman RBCs.

    Inhibition of ADP dependent platelet aggregation byJFTE was observed to follow dose dependence. However,linearity of aggregation was not observed for mid rangeconcentrations, reason for this is not yet understood. One ofthe reasons may be presence of multiple components inJFTE.

    This study shows presence of very strong brinogeno-lytic factors present in the jellysh A. aurita tentacles.Whether all of these factors are parts of the venom or someare simply present as normal tissue constituents remain tobe investigated.

    Acknowledgments

    This study was fully supported by the Birla Institute ofTechnology and Science (BITS), Pilani, K K Birla Goa campus.Akriti Rastogi is supported by a research fellowship of BITS,Pilani. The authors are grateful to Ms. Kamna Upadhyay fortechnical help and C. Chandra Sekhar for sharing theresources. The authors are particularly thankful to AbyssMarine Aquarium, Verna, Goa, India for help regardingsample collection.

    Conict of interest

    DP dependent platelet aggregation.The authors declare that there ar...

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