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Toxicon 48 (2006) 411–421 www.elsevier.com/locate/toxicon
Anticoagulant effect of Naja naja venom 50nucleotidase: Demonstration through the use of novel speciﬁc inhibitor, vanillic acid
B.L. Dhananjaya, A. Nataraju, R. Rajesh, C.D. Raghavendra Gowda, B.K. Sharath, B.S. Vishwanath, Cletus J.M. D’SouzaÃ
Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore 570 006, India Received 4 May 2006; received in revised form 17 June 2006; accepted 19 June 2006 Available online 7 July 2006
Abstract The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A2s (PLA2s), 50 nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 50 nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and speciﬁcally inhibited 50 nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 50 nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 50 nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA2. The complete inhibition of anticoagulant effect by Con-A upon recalciﬁcation time suggests the participation of both 50 nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 50 nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the ﬁrst time demonstrates the involvement of 50 nucleotidase in mediating N. naja venom anticoagulant effect. r 2006 Elsevier Ltd. All rights reserved.
Keywords: 50 nucleotidase; Naja naja; Vanillic acid; Anticoagulant; Hemostasis
1. Introduction Components acting on hemostasis are generally found in snake venoms belonging to viperidae, crotalidae and elapidae families. Interference with
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blood coagulation is one of the main causes of pathological manifestations exhibited by bite of snakes belonging to the families, crotalidae and viperidae. Even though such manifestations are not easily observed in elapidae snakebite, few reports show the involvement of venom components on blood coagulation system when studied in vitro (Lee et al., 1995; White, 2004 and references therein). A recent in vitro study has shown that phospholipase
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.06.017
Kini and Evans. and fever in traditional Chinese medicine (Li et al. USA. mechanism of action of several venom toxins/enzymes has been elucidated (Vishwanath et al... 2004). sodium arachidonate. 2005). India. prepared from the root bark of Lycium barbarum L. inhibited speciﬁcally 50 nucleotidase activity and not proteases and PLA2 that are commonly involved in hemostasis. India. Further. a neutraceutical plant. ulcers. The 50 nucleotidase puriﬁed from Trimeresurus graminus is known to inhibit platelet aggregation induced by ADP. / Toxicon 48 (2006) 411–421 A2 (PLA2) enzyme and prothrombin activator from Naja naja venom have shown effects on platelets and blood clotting factors (Sundell et al. naja venom 50 nucleotidase. Kini. India. A number of snake venom toxins that affect blood coagulation as procoagulant or anticoagulant have been puriﬁed and characterized (Hutton and Warrell. 1990).. Con-A (concanavalin-A type V). Escherichia coli (lyophilized cells of strain W (ATCC 9637) and oleic acid (C14) was obtained from Perkin Elmer Life Sciences Inc. Pithayanakul et al. Fresh human blood samples were collected from healthy volunteers of Department of Biochemistry. naja) venom was purchased from Haffkins Institute. 1997.. Materials Cobra (N. a-methly-D-glucoside. Adenosine 50 monophosphate (50 AMP).L. MA. casein and human ﬁbrinogen were purchased from Sigma Chemical Company St. Although it is widely accepted that 50 nucleotidase alters platelet function (Kini and Evans. naja venom. it has been shown that it is one of the main components of a traditional Chinese folk medicine formulation—Di-Gu-Pi decoction. 1987. Numerous studies have used speciﬁc inhibitors of enzymes as biochemical and pharmacological tool for their characterization (Lazarovici and Lelkes... Kini and Chow. Mors et al. 2001). (1951) using BSA as standard.. 2000). Vanillic acid (4-hydroxy-3-methoxy benzoic acid) is a phenolic derivative known to possess antimicrobial (Delaquis et al.. University of Mysore. 2. Thus it is now hypothesized that 50 nucleotidase probably acts synergistically with other toxins such as ADPases. 2003). Ferry et al. 1992. Vanillic acid a major chemical constituent of vanilla. 1990. 1983)... This extract is used for the treatment of diabetes. hypertension. 1999. With these inhibitors. hemorrhagic inﬂammation. USA. Erion et al. 2000). Boston. anti-ﬁlarial (Varma et al.. Markland. its action on blood coagulation cascade remains unknown. 2001). collagen. ionophore A-23187 and thrombin (Ouyang and Huang. indicating that the enzyme has not been well characterized in snake venoms. 2. Materials and methods 2. Dhananjaya et al. Boffa and Boffa (1974) demonstrated 50 nucleotidase as the most potent inhibitor of platelet aggregation from Vipera aspis. 2005).. This inhibition of platelet aggregation by 50 nucleotidase is known to consequently suppress blood coagulation without platelet lysis. a glycoprotein in exerting anticoagulant effect using novel non-nucleoside speciﬁc inhibitor—vanillic acid. 50 nucleotidase an ubiquitous enzyme of snake venoms has been shown to act as a co-factor of hemorrhagic toxins and also known to affect hemostasis by modulating platelet functions (Dimitrov and Kankonkar. This study clearly demonstrates the involvement of N. a-methyl-D-mannoside and vanillic acid were purchased from Sisco Research Laboratories.. For all the studies.1. 2005). It has been observed that a striking parallelism exists between the capability of plants and their chemical compounds in neutralizing the action of snake venom toxicity and anti-inﬂammatory properties (Mors et al. Mumbai. Soares et al.ARTICLE IN PRESS 412 B. The other pharmacological effect that is neutralized by vanillic acid is anticoagulant activity of N. 1989. 2002). 2004. Bangalore. Austria. 1979.2. Jayaraman et al.. Kini et al. 1993) and hepatoprotective activities (Singh et al. All the assays were done using double distilled water.. All other chemicals and reagents purchased were of analytical grade. venom and inhibitors were dissolved in saline. 1968.. Several snake venom toxins/enzymes were inhibited by components isolated from traditional folk medicinal plants used against snakebite (Okonogi et al. Protein estimation Protein concentration was determined according to the method of Lowry et al. 1998. . Louis. phospholipases and disintegrins to exert more pronounced anticoagulant effect (daSilva and Aird. 1993.. Cirino et al. 2004. 2005). Aird. 2005). Fat-free bovine serum albumin (BSA) fraction V was purchased from PAA Laboratories GmbH Haidmannweg. MO. The anticoagulant effects of venoms have been mainly attributed to proteases and PLA2s (Markland. 2002.
000 g for 10 min at room temperature. venom samples (100 mg) were preincubated without or with different concentration of vanillic acid (0–600 mM) or Con-A (0–6 mM) at 37 1C for 30 min. pH 7.15 M NaCl in a reaction volume of 1 ml for 2.5 ml of folin reagents (1:2. The colour developed was read at 660 nm.7. 10% ascorbic acid and water was added to the assay mixture.L. Venom samples were incubated with 50 mg of hyaluronan in 0. venom samples (30 mg) were preincubated without or with different concentrations of vanillic acid (0–600 mM) or Con-A (0–6 mM) at 37 1C for 30 min. An aliquot (140 ml) of the supernatant (containing released C14oleic acid) was mixed with scintillation cocktail and counted in a Hewlett Packard liquid scintillation analyzer TRI CARB 2100 TR. The reaction was stopped by adding 1. Venom was added in a ﬁnal volume of 320 ml containing 5 mM CaCl2 and 100 mM Tris-HCl buffer. v/v). A measure of 1 ml of ascorbic acid reagent.2 M sodium acetate buffer. A measure of 0. The reaction was terminated by adding 50 ml of potassium tetraborate buffer (pH 9.ARTICLE IN PRESS B.1) followed by 1. 2.42% ammonium molybdate in 1 N sulfuric acid. pH 8. The hydrolyzed products were analyzed on 10% SDS-PAGE and the protein pattern was visualized by staining with comassiae brilliant blue R-250. containing equal parts of 0. venom samples (10 mg) were preincubated without or with different concentrations of vanillic acid (0–600 mM) for 37 1C for 30 min and the reaction preceded as described above. 2. Activity is expressed as nmoles of fatty acid released/min/mg of protein. 10 mM KCl.5.4 M) and 0.0 and 0. In brief.5 ml of 0. The reaction was terminated by adding 100 ml of 2 N HCl followed by 100 ml of 10% fatty-acid-free BSA.6) was incubated with venom samples for 1 h at 37 1C.44 M TCA and allowed to stand for 30 min.5 ml of 1% para-dimethyl amino . 10 mM 50 AMP with an appropriate amount of venom and incubated for 30 min at 37 1C. The reaction mixture (40 ml) containing 50 mg of human ﬁbrinogen in 10 mM Tris-HCl buffer (pH 7. The tubes containing reaction mixtures were then centrifuged at 1500 g for 15 min. This was quantiﬁed by comparison with the reference curve established with KH2PO4. / Toxicon 48 (2006) 411–421 413 2. Results are expressed in nmoles of inorganic phosphate liberated/min/mg of protein. Fibrinogenolytic assay Fibrinogenolytic activity was measured according to the method of Ouyang and Teng (1976). venom samples (1 mg) were preincubated without or with different concentration of vanillic acid (0–600 mM) or Con-A (0–6 mM) at 37 oC for 30 min. Appropriate controls were carried out and the inhibition is expressed as percentage taking activity of venom alone as 100%. (1955). 1956) was used to determine the released inorganic phosphate.5) was incubated with venom sample at 37 1C for 2 h. Protease assay Protease activity was determined using casein as substrate according to the method of Murata et al. 2. The samples were then thoroughly vortexed and centrifuged at 10.01 at 660 nm/h at 37 1C. For inhibition studies. coli membrane (equivalent to 64 nmoles of fatty acid).4.5 h at 37 1C. 4% SDS and 4% b-mercaptoethanol.4.3. (1963). Appropriate controls were carried out and the inhibition is expressed as percentage taking activity of venom alone as 100%. This was left at room temperature for 30 min and absorbance at 660 nm was determined. For inhibition studies. 50 mM NaCl. pH 6. The reaction was terminated by adding 20 ml of denaturing buffer containing 1 M urea. Appropriate controls were carried out and the inhibition is expressed as percentage taking activity of venom alone as 100%. PLA2 assay PLA2 activity was assayed according to the method of Vishwanath et al.2 M Tris-HCl buffer. pH 7. incubated for 1 h in the presence of 30 ml of C14oleate labeled.. Hyaluronidase assay Hyaluronidase activity was assayed by estimating the amount of N-acetyl glucosamine released from hyaluronic acid according to the method of Reissig et al.4 ml of Casein (2% in 0.5. For inhibition studies. Dhananjaya et al. For inhibition studies. 50 nucleotidase assay Venom 50 nucleotidase activity was assayed by the procedure of Avruch and Wallach (1971) with slight modiﬁcations. The ascorbic acid method (Chen et al.5 ml of sodium carbonate (0. autoclaved E. An aliquot (1 ml) of the supernatant was mixed with 2. the reaction was carried out in a ﬁnal volume of 1 ml containing 10 mM MgCl2.6. (1993). 2. 50 mM Tris-HCl buffer. One unit of enzyme activity was deﬁned as the amount of enzyme required to give an absorbance of 0.
Appropriate controls were carried out and the inhibition is expressed as percentage taking activity of venom alone as 100%. Venom samples were added and the increase in absorbance at 420 nm was measured continuously.6 containing L-leucine (0.1%) and o-dianisidine (0. expressed as percentage taking activity of venom alone as 100%. PLA2 enzymes exerts their effect by hydrolyzing phospholipids or by inhibiting platelet aggregation and thus act as anticoagulants (Kini. To 300 ml of PPP. The stoppered tubes were boiled for 3 min in water bath and the color developed was monitored at 585 nm. 1993.006%). One unit of alkaline phosphomonoesterase activity is deﬁned as the increase of 0.ARTICLE IN PRESS 414 B. (1966). For inhibition studies. White.5 h at 37 1C. Kini et al. pH 7.8. venom samples (100 mg) were preincubated without or with different concentrations of vanillic acid (0–600 mM) at 37 1C for 30 min. For inhibition studies. 50 nucleotidase is known to affect hemostasis by inhibiting platelet aggregation (Ouyang and Huang. 2001).2 M triethanolamine buffer. Appropriate controls were carried out and the inhibition is expressed as percentage taking activity of venom alone as 100%. 2. At the end of the incubation period. naja venom (2 mg) preincubated with different concentration of Con-A (0–1 mm) or vanillic acid (0–10 mm) in 30 ml of 0. The principle proteins/ toxins that affect hemostasis are proteases and PLA2 enzymes.9. The clot formation was initiated by adding 30 ml of 0.4 ml of 0.01 M p-nitrophenyl phosphate in 0. 2005). venom sample (100 mg) were preincubated without or with different concentrations of vanillic acid (0–600 mM) at 37 1C for 30 min. Apart from proteases and PLA2s. Tris-HCl buffer (pH 7. NIH units/mg) was added to 1 ml of 0. N.05 ml of 0. Kini and Evans. 2. / Toxicon 48 (2006) 411–421 benzaldehyde (p-DMAB). Appropriate controls were carried out and the inhibition is .5. The time taken for visible clot to appear from the time of addition of calcium chloride was recorded (s).007% 250. Results and discussion Majority of the snake venom proteins affects hemostasis either by shortening or prolonging the blood coagulation time.001 absorbance. Regression analysis was used to calculate the inhibitory concentration 50 (IC50) deﬁned as the dosage of inhibitor necessary to produce a 50% inhibition of activities.5 M Tris-HCl buffer.11.. The absorbance at 440 nm was then measured. The mixture was centrifuged for 15 min at 2000 g and the supernatant obtained was used as platelet poor plasma (PPP). Generally platelet aggregation inhibitors are known to prolong clot formation indirectly (Hutton and Warrell. pH 8. 2002. pH 8. One unit of enzyme activity is deﬁned as the amount of enzyme that causes an increase of 0. Venom was added to the reaction mixture containing 0. Alkaline phosphomonoesterase assay Alkaline phosphomonoesterase activity was assayed by a modiﬁcation of the method described by Lo et al. 3.L.2 N NaOH was added and allowed to stand for 20 min. Dhananjaya et al. L-amino acid oxidase assay L-amino acid oxidase activity was determined as described in Worthington Enzyme Manual (1977) with some modiﬁcation. Proteases affect hemostasis by activating several coagulation factors or hydrolyzing ﬁbrinogen to promote clot formation (Markland. 1997). then the reaction mixture was incubated for 30 min at room temperature.4) alone was added instead of the enzyme source. 1990. Kini and Chow.001 absorbance units per min at 440 nm. Anticoagulant inhibition assay Recalciﬁcation time was determined according to the procedure described by Condrea et al. Peroxidase (0.4) was added and incubated for 1 min at 37 1C. Fresh human blood was mixed with 0. (1983). In control experiments.2 ml of 0.10 M MgSO4 and made up to 1 ml with 0. 2. Several reports also suggested that 50 nucleotidases acts synergistically with other venom toxins to induce 2.11 M trisodium citrate (9:1.5 M Tris-HCl buffer. Activity was expressed as nmoles of N-acetyl glucosamine released/2. incubated at 37 1C for 30 min. 2005). 2 ml of 0. For inhibition studies. PPP was pre-warmed at 37 1C before use. venom sample (100 mg) was preincubated without or with different concentrations of vanillic acid (0–600 mM) at 37 1C for 30 min.01 M Tris-HCl buffer (pH 7. v/v). Statistics The results are expressed as the mean7SD of at least four independent experiments.25 M CaCl2.5.10. 0. 1983.
. Dose-dependent inhibition of N. b Activity is expressed in terms of units/min/mg of protein.48a 0. daSilva and Aird. Aird. 2004). c Speciﬁc activity is expressed in terms of fatty acid released in nmoles/min/mg of protein. the exact role of venom 50 nucleotidase in inducing anticoagulant effect is not clearly understood. 2001. From these studies. it appears that vanillic acid selectively and speciﬁcally inhibits venom 50 nucleotidase among enzymes present in snake venom. Values represent mean7S.4 2. protease and PLA2 activities Enzymes Activities Vanillic acid % Inhibition 50 nucleotidase Protease PLA2 65373. russelli venoms (Unpublished observation). In this study we report vanillic acid as a speciﬁc inhibitor of 50 nucleotidase and further unravel the role of this enzyme in mediating anticoagulant activity. naja venom 50 nucleotidase activity.L. Similarly vanillic acid inhibits only 50 nucleotidase activity and not any other enzyme activity of Echis carinatus and D. ﬁbrinogenase. In order to examine the nature of crude N. One unit of activity is deﬁned as the amount of enzyme required to cause an increase in OD by 0. 1). naja venom (30 mg) was preincubated with different concentration of vanillic acid (0–600 mM) for 15 min at 37 1C. The maximum activity was observed at 25 mM 50 AMP and attained plateau upon further increase in substrate Table 1 Effect of inhibitors on N. naja venom with vanillic acid revealed that it only inhibited 50 nucleotidase activity. It failed to inhibit other venom enzymes like protease.D (n ¼ 4). The enzyme inhibition studies of N. Results are expressed as percentage inhibition of enzyme activity as compared with control. In Chinese medicine. 1. N. Further assay was carried out as described in methods section. a Activity is expressed in terms of nmoles of inorganic phosphate released/min/mg. . its inhibition was measured as a function of the substrate (50 AMP) concentration. This suggests the existence of some analogy between the mechanisms. hyaluronidase. 2002). 2000). naja venom 50 nucleotidase. However.. / Toxicon 48 (2006) 411–421 415 more pronounced anticoagulant activity (Dimitrov and Kankonkar. PLA2.7670. 1968. It has been observed that a striking parallelism exists between the capability of plants and their 100 80 % Activity 60 40 20 0 0 100 200 300 400 500 Vanillic acid (µM) Fig. naja venom 50 nucleotidase inhibition by vanillic acid.ARTICLE IN PRESS B. which govern these activities wherein plants used against inﬂammation can also be used against snake venom toxicity. Based on these observations the effect of vanillic acid against venom enzymes and their related pharmacological actions was examined. Dhananjaya et al.01 per min at 660 nm. The inhibition of 50 nucleotidase activity by vanillic acid was dose dependent and complete inhibition was achieved at 410 mM (Fig. chemical compounds in neutralizing the action of snake venoms and anti-inﬂammatory properties (Mors et al. 50 nucleotidase activity in the absence and presence of IC50 concentration (180 mM) increases almost linearly with increased substrate concentration. which is used against hemorrhagic inﬂammation (Li et al.08b 1128723c 100 0 0 IC50 (mM) 180 — — Con-A % Inhibition 100 100 0 IC50 (mM) 2. By linear regression analysis the IC50 value was found to be 180 mM (Table 1). L-amino oxidase and phosphomonoesterase activities even at very high concentration.8 — Values represent mean7SD (n ¼ 4). vanillic acid is the main constituent of Di-Gu-Pi decoction.
1992. The polyphenolic inhibitors isolated from wine grape ‘‘Koshu’’ and Areca catechu are known to possess tannic activity (Toukairin et al. Nucleocidine..4). N. The inhibition by vanillic acid at 180 mM was 50% at 10 mM substrate concentration and the inhibition percentage decreased as the substrate concentration was increased (Fig. no reports claim a speciﬁc inhibitor of 50 nucleotidase in relation to other enzymes present in snake venoms. no resemblance was observed (Fig. / Toxicon 48 (2006) 411–421 concentration (Fig.L. 1975). as these studies were carried out in different context. On examining the structures of both substrate and vanillic acid. 1985). These analogs often act as ligands for receptors where the substrate 50 AMP is involved (Ferry et al. The other mechanism proposed for anticoagulant activity of 50 nucleotidase is probably through synergistic action with other toxins such as ADPases.. Furthermore. 2). Even though several substrate analogs inhibit 50 nucleotidase (Rossomando et al. melanocidine A and melanocidine B are polysaccharide in nature. Values represent mean7SD (n ¼ 4).ARTICLE IN PRESS 416 B. Erion et al. Thus the inhibition of 50 nucleotidase by vanillic acid is substrate dependent and probably it competes with substrate for the same binding region. wherein it speciﬁcally interacts with a-D-glucose and a-D-mannose residues to bring about inhibition (Stefanovic et al. 50 mM NaCl. naja venom (30 mg) was incubated at 37 1C for 30 min with indicated concentration of 50 AMP in the absence (E) and presence (’) of vanillic acid (180 mM) in a reaction mixture containing 10 mM MgCl2.. 1979). and tannins are known to be general inhibitors of snake venom toxins (Okonogi et al. 2. its use in in vivo studies is restricted. The speciﬁc inhibitors of venom enzymes/toxins can be useful in elucidating the mechanism of action. 1983).... Citrate and EDTA inhibits venom 50 nucleotidase activity by chelating divalent metal ions (Francis et al. 50 mM Tris-HCl buffer (pH 7.. 10 mM KCl. 1992). Con-A is known to exert reversible inhibitory effect on Crotalous adamentous venom 50 nucleotidase. such inhibitors resemble the substrate molecule. 1983). . 50 nucleotidase is known to exert anticoagulant effect by inhibiting platelet aggregation and thus alters blood coagulation without platelet lysis (Ouyang and Huang. Results are expressed as percentage inhibition of enzyme activity as compared with control. 1979). Effect of substrate concentration on inhibition of venom 50 nucleotidase activity by vanillic acid. 2004. 3). 2005). 2004). Ferry et al. Thus vanillic acid is ﬁrst of its kind. Generally. PLA2 and disintegrins (daSilva and 100 % Inhibition 2000 75 50 25 0 0 1000 10 20 5' AMP (mM) 30 Pi released (nmol) 1500 500 0 0 5 10 15 5' AMP (mM) 20 25 30 Fig. The various inhibitors of venom 50 nucleotidase reported till date is dependent on protein nature and lack in speciﬁcity. Inset: inhibition of 50 nucleotidase activity by vanillic acid.. Numerous studies have employed speciﬁc inhibitors of enzymes as a biochemical or pharmacological tool to characterize and establish the mechanism of action (Lazarovici and Lelkes. 2 inset). wherein steric effects are known to play a major role in inhibition of 50 nucleotidase (Uchino et al.. 1991). which is speciﬁc for venom 50 nucleotidase and is novel type of inhibitor as it is non-nucleoside in nature in contrast to substrate analogs. a glycoprotein (Mannherz and Magener. Dhananjaya et al.
. It was interesting to observe that vanillic acid decreased the recalciﬁcation time from 387 to 283. 2005). naja venom 50 nucleotidase in mediating anticoagulant action. 3. though such symptoms are not observed in envenomated victims (Shashidharamurthy et al.ARTICLE IN PRESS B. In the control experiments.L. pH 7. In order to investigate the involvement of N. 2001). This observed result was further substantiated by Con-A inhibition studies. 1994. naja venom. Vanillic acid alone did not inﬂuence the coagulant effect at the tested dose. CaCl2 alone was used without enzyme source.. 2002. known to affect coagulation (Ouyang and Huang. Recently.25 M) was added to the preincubated mixture and time for clot formation was recorded (s).5 s in a dose-dependent manner and reaches plateau at a concentration of 8.. Control and inhibitor alone ¼ 263 s. naja venom has been reported (Jagadeesha et al. naja venom ¼ 385 s. It has been demonstrated that Con-A 400 350 Clotting time (Sec) 300 250 200 0 2 4 6 8 10 12 Vanillic acid (µM) Fig. Sundell et al.4) and incubated for 1 min at 37 1C. / Toxicon 48 (2006) 411–421 417 NH2 COOH 3N 5 2 N O O P OH OCH3 H OH Vanillic acid HO OH Adenosine Monophosphate H O CH2 O H 1 6 4 N N Fig.. N. 4). Dhananjaya et al.9 mM (Fig. 2003). the involvement of protease in mediating anticoagulant effect of N. 1983. Values represent mean7SD (n ¼ 4). The in vitro anticoagulant action of N. Dose-dependent inhibition of N. Structures of vanillic acid and adenosine monophosphate.. N. Gowda et al. Aird. naja venom has been well documented. Nikandrov et al. the addition of vanillic acid did not decrease the clotting time thus inhibiting N. 2002). Further. 50 nucleotidase is a glycoprotein present in venom along with other glycoprotein proteases. the effect of vanillic acid was tested on enzyme inhibition followed by anticoagulant activity. naja venom at a protein concentration of 2 mg increases the recalciﬁcation time from 132 to 387 s. 30 ml of CaCl2 (0. naja venom anticoagulant effect up to 40% (Table 2). . naja venom (2 mg) preincubated with different concentrations of vanillic acid (0–10 mM) for 15 min was added to 300 ml of human plasma in presence of 30 ml Tris-HCl buffer (10 mM. naja venom anticoagulant activity by vanillic acid. These data clearly indicate the partial involvement of 50 nucleotidase in mediating anticoagulant effect of N. Inhibitor at 0 concentration represents 2 mg of N. 4.
naja venom protease and 50 nucelotidase are glycoprotein in nature having glucose and mannose residues in its glycopart. the effect of Con-A on protease.L. This study indicates that N. These results are consistent with earlier reports. Bhat et al.. In addition as vanillic acid is an intermediate metabolite product of vanillin. % Inhibition 40 100 IC50 (mM) ND 0. The inability of Con-A to inhibit N.63 mM. Thus. naja whole venom.. it did not exhibit any inhibitory effect on PLA2 activity.28 100 80 % Activity 60 40 exerts reversible inhibitory effect on C.. 2005). Further. naja venom in a dose-dependent manner with an IC50 of 0. However. Rudrammaji and Gowda.D (n ¼ 4). It was observed that. 1980. protease (’) and PLA2 (m) activities. In this study.ARTICLE IN PRESS 418 B. The IC50 values for protease and 50 nucleotidase were 2. 5. naja venom 50 nucleotidase (E). Con-A completely inhibited the anticoagulant effect of N. adamentous venom 50 nucleotidase activity (Mannherz and Magener. Bhat and Gowda. naja venom anticoagulant effect was tested by recalciﬁcation time. naja venom PLA2 activity supports the observation that PLA2s in venoms are not glycoproteins. 5). pharmacological effects are known to be weak/ devoid of anticoagulant activities (Verheij et al. respectively (Table 1). Kini et al. Usually in venoms proteases and PLA2s bring about anticoagulant effect (Markland. On the other hand. 6). naja venom (Jagadeesha et al.8 and 2. In addition. Values represent mean7S. 1990). naja venom was preincubated with different concentration of Con-A (0–6 mM) for 15 min at 37 1C. It was recently shown that L-amino acid oxidase that inhibits platelet aggregation also exhibits anticoagulant effect by inhibiting factor IX (Sakurai et al. the decrease in recalciﬁcation time of N. Dhananjaya et al. It was observed that Con-A inhibited the anticoagulant effect of N. / Toxicon 48 (2006) 411–421 Table 2 Effect of inhibitors on N. 2002). further studies with puriﬁed enzyme and speciﬁc inhibitor seems interesting in order to know the anticoagulant mechanism of 50 nucleotidase. PLA2 and 50 nucleotidase activities were tested. Further. which binds speciﬁcally to Con-A reversed the inhibitory effect of Con-A on N. 1998. the effect of Con-A on the N. Con-A inhibited protease and 50 nucleotidase activities in a dosedependent manner up to 100% (Fig. as evident from decrease in recalciﬁcation time from 385 to 132 s (Table 2). naja venom anticoagulant activity. . In order to substantiate the involvement of 50 nucleotidase in N. naja venom.. the addition of a-methly-Dglucoside or a-methyl-D-mannoside.. Effect of Con-A on N. naja venom upon vanillic acid and Con-A addition suggests interaction of 50 nucleotidase directly or indirectly with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Further assay was carried out as described in methods section. that would be helpful in understanding the plausible pharmacological role of venom 50 nucleotidase.. naja whole venom anticoagulant effect by Con-A summarizes the participation of both 50 nucleotidase and protease as determined by recalciﬁcation time. the complete inhibition of N. The assay of recalciﬁcation time reﬂects the involvement of clotting factors of the intrinsic pathway in elucidating anti/pro coagulation (Stefansson et al. Thus.4 mM. a potent non-toxic anticoagulant metalloprotease NN-PF3 has been puriﬁed from N. At a concentration of 0.28 mM (Fig. this study excludes the involvement of PLA2 in mediating anticoagulant effect of N. Results are expressed as percentage inhibition of enzyme activity as compared with control. 1979). 1991. 1991. naja whole venom anticoagulant activity Inhibitors Vanillic acid Con-A ND ¼ not determined. 1998). where it has been shown that the N. N. naja venom PLA2s even though exhibit various 20 0 0 1 2 3 Con-A (µM) 4 5 6 Fig. 2002). naja venom 50 nucleotidase and protease activities (data not shown).
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