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Toxicon 60 (2012) 719–723

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Toxicon

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Anticoagulant activity of Moon jellyfish (Aurelia aurita) tentacle extract

Akriti Rastogi, Sumit Biswas, Angshuman Sarkar, Dibakar Chakrabarty*

Department of Biological Sciences, Birla Institute of Technology and Science–Pilani, K.K. Birla Goa Campus, Zuarinagar, Goa 403 726, India

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 jellyfishAnticoagulantFibrinogenolytic toxin

Abbreviations: BSA, bovine serum albumin; EDTAtetra acetic acid; JFTE, Jellyfish tentacle extract; MWPLA, phospholipase A; PMSF, phenyl methyl sulfonyblood cells.* Corresponding author. Fax: þ91 832 255 7033.

E-mail addresses: diba27@yahoo.com, dibakarcac.in (D. Chakrabarty).

0041-0101/$ – see front matter � 2012 Elsevier Ltd10.1016/j.toxicon.2012.05.008

a b s t r a c t

Moon jellyfish (Aurelia aurita) tentacle extract was studied for its anticoagulant activityin vitro. The Jellyfish Tentacle Extract (JFTE) showed very strong fibrinogenolytic activity bycleaving Aa and Bb chain of fibrinogen molecule. The fibrinogenolytic activity was found tobe stronger than some snake venom derived anticoagulants. JFTE also completely liquefiedfibrin clots in 24 h. JFTE was found to contain both high and low molecular weightproteins/peptides. The fibrinogenolysis appears to be caused by high molecular weightfractions of the extract. It has been also noted that PMSF significantly reduced fibrinoge-nolytic activity and heating totally abolished it. Autolytic degradation of the high molec-ular weight protein was also noted. Autolysis slowed down, but did not abolish thefibrinogenolytic activity of the extract.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Thrombosis has been and continues to be amajor healthproblem leading to mortality. Anticoagulants are pivotal forthe prevention and treatment of thromboembolic disor-ders. Coumarins and heparin are the most well knownclinically used anticoagulants. However, the nonspecificmode of action of these anticoagulants accounts for theirtherapeutic limitations in maintaining a balance betweenthrombosis and hemostasis. These limitations haveprovided the impetus for the development of new antico-agulants that target specific 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 KoreanChungkook-Jang soy sauce (Nikai et al., 1984; Sumi et al.,

, ethylene di-amine, molecular weight;l fluoride; RBCs, red

hakrabarty@bits-goa.

. All rights reserved.

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 Eptifibatide and Tir-ofiban, were designed based on disintegrins, a large familyof platelet aggregation inhibitors found in viperid andcrotalid snake venoms (O’Shea and Tcheng, 2002;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 fibrinogen levels andhas been successfully tested in a variety of ischemicconditions, including stroke (Sherman, 2002).

In recent years, cnidarians like jellyfish have become anattractive 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.,

A. Rastogi et al. / Toxicon 60 (2012) 719–723720

1980; 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 fibri-no(geno)lytic and inhibitory activity on platelet aggrega-tion in the tentacle extract of Aurelia aurita jellyfish.

2. Materials and methods

Moon jellyfish (A. aurita) were collected fromGoa, India.All fine 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 Jellyfish Tentacle Extracts (JFTE)

The oral arm tentacles (OAT) were manually excisedwithin 1 h of collection and stored in 0.85% saline at�20 �C.Frozen OAT were autolyzed at 4 �C in 0.85% saline (9:1 v/v)for 4 days. Resultant fluid was clarified by centrifugation at20,000 g for 1 h at 4 �C. This clarified fluid was calledJellyfish Tentacle Extract (JFTE) and was used as theworking material for further investigations. It was stored at�80 �C in 2 ml aliquots till used.

2.2. Protein estimation

Protein content of JFTE was estimated by modifiedBradford 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 fibrinolytic activity

Fibrinolytic activity of the JFTE or fractions was moni-tored by a modification of the fibrin 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 final 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 Russell’s viper venomwas used aspositive control and 0.85% saline was used as negativecontrol.

2.5. Fibrinogenolytic activity

Fibrinogenolytic activity was confirmed by incubatingfibrinogen 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 specific bandswith that of fibrinogenincubated 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 Russell’s 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 Bradford’s Method. SDS-PAGE of JFTE revealed six

A. Rastogi et al. / Toxicon 60 (2012) 719–723 721

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

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

3.2. Fibrin(ogen)olytic activity

JFTE showed preferential digestion of Aa chain offibrinogen, 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 significant digestion of gchain also (Fig. 2b).

JFTE (15 mg) completely liquefied 200 ml fibrin 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 fibrino-genolytic activity, whereas, pre-treatment with 2mM EDTAdelayed fibrinogenolysis.

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 fibrinogenolytic 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.

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.

Fig. 1. Band pattern for 5 mg JFTE on 12% SDS-PAGE gel.

3.4. Autodegradation

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 fibrinogenolytic activity. However,2 mM EDTA inhibited autodegradation without affectingfibrinogenolytic activity.

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

Fig. 4. Effect of different doses of JFTE on ADP dependent platelet aggregation.

A. Rastogi et al. / Toxicon 60 (2012) 719–723722

3.5. 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 jellyfish tentacle extract was studied for its anti-coagulant activities in vitro. JFTE was incubated with bovinefibrinogen and two major chains, namely Aa and Bb werefound digested after 3 h. It may be noted here, that Lahirinpurified from Naja kaouthia (Chandra Sekhar andChakrabarty, 2011) venom digests only Aa chain in about5 h at the same dose. Complete digestion of fibrinogenmolecule by Lahirin requires 24 h at a dose of 15 mg/ml(Chandra Sekhar and Chakrabarty, 2011). JFTE alsocompletely liquefied fibrin clots in 24 h (Fig. 3). Proteolyticactivity of jellyfish venom has been reported earlier by Leeet al. (2011), where one of the substrates used was fibrin-ogen. But it was not described whether specific fibrinoge-nolytic activity was observed or fibrinogen digestion isa reflection of random proteolytic activity.

Rapid degradation of jellyfish toxins has been reportedearlier (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. PMSFsignificantly inhibited digestion of Aa and Bb chains byJFTE. It is possible that different toxins are involved in thedigestion of different chains of fibrinogen or even smallerpeptides resulted from auto-digestion also retained thefibrinogenolytic activity. PMSF inhibited toxin induceddigestion of fibrinogen chains, but did not abolish it. It ispossible that fibrinogenolytic toxins of different types arepresent in the sample, i.e., serine proteases as well asmetalloproteases.

Hemolytic activity of jellyfish venom is a well docu-mented phenomenon (Nagai et al., 2000; Radwan et al.,2000; Chung et al., 2001; Gusmani et al., 1997; Rottini

et 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 fibrinogeno-lytic factors present in the jellyfish 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.

Conflict of interest

The authors declare that there are no conflicts ofinterest.

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