ARTICLE IN PRESS

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’Souza
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 blood coagulation is one of the main causes of

Components acting on hemostasis are generally
found in snake venoms belonging to viperidae,
crotalidae and elapidae families. Interference with
Corresponding author. Tel.: +91 821 2419621;
fax: +91 821 2415390.
E-mail address: cletus211@gmail.com (C.J.M. D’Souza).
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
 ARTICLE IN PRESS
412 B.L. Dhananjaya et al. / 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., 2003).
A number of snake venom toxins that affect
blood coagulation as procoagulant or anticoagulant
have been purified and characterized (Hutton
and Warrell, 1993; Markland, 1998; Kini et al.,
2002).
The anticoagulant effects of venoms have been
mainly attributed to proteases and PLA2s (Mark-
land, 1997; Kini, 2005). 50 nucleotidase an ubiqui-
tous 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, 1968; Aird,
2002, Kini and Evans, 1990). Boffa and Boffa
(1974) demonstrated 50 nucleotidase as the most
potent inhibitor of platelet aggregation from Vipera
aspis. The 50 nucleotidase purified from Trimeresurus
graminus is known to inhibit platelet aggregation
induced by ADP, sodium arachidonate, collagen,
ionophore A-23187 and thrombin (Ouyang and
Huang, 1983). This inhibition of platelet aggrega-
tion by 50 nucleotidase is known to consequently
suppress blood coagulation without platelet lysis.
Thus it is now hypothesized that 50 nucleotidase
probably acts synergistically with other toxins such
as ADPases, phospholipases and disintegrins to
exert more pronounced anticoagulant effect (daSil-
va and Aird, 2001). Although it is widely accepted
that 50 nucleotidase alters platelet function (Kini
and Evans, 1990; Kini and Chow, 2001), its action
on blood coagulation cascade remains unknown,
indicating that the enzyme has not been well charac-
terized in snake venoms.
Numerous studies have used specific inhibitors of
enzymes as biochemical and pharmacological tool
for their characterization (Lazarovici and Lelkes,
1992; Ferry et al., 2004; Erion et al., 2005). Several
snake venom toxins/enzymes were inhibited by
components isolated from traditional folk medicinal
plants used against snakebite (Okonogi et al., 1979;
Pithayanakul et al., 2004; Soares et al., 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., 2000). With these inhibitors, mechan-
ism of action of several venom toxins/enzymes has
been elucidated (Vishwanath et al., 1987; Cirino
et al., 1989; Jayaraman et al., 1999; Mors et al.,
2000). Vanillic acid (4-hydroxy-3-methoxy benzoic
acid) is a phenolic derivative known to possess anti-
microbial (Delaquis et al., 2005), anti-filarial (Var-
ma et al., 1993) and hepatoprotective activities
(Singh et al., 2005). Further, it has been shown that
it is one of the main components of a traditional
Chinese folk medicine formulation—Di-Gu-Pi de-
coction, prepared from the root bark of Lycium
barbarum L. This extract is used for the treatment of
diabetes, hemorrhagic inflammation, hypertension,
ulcers, and fever in traditional Chinese medicine (Li
et al., 2004). Vanillic acid a major chemical
constituent of vanilla, a neutraceutical plant,
inhibited specifically 50 nucleotidase activity and
not proteases and PLA2 that are commonly
involved in hemostasis. The other pharmacological
effect that is neutralized by vanillic acid is antic-
oagulant activity of N. naja venom. This study
clearly demonstrates the involvement of N. naja
venom 50 nucleotidase, a glycoprotein in exerting
anticoagulant effect using novel non-nucleoside
specific inhibitor—vanillic acid.
2. Materials and methods
2.1. Materials
Cobra (N. naja) venom was purchased from
Haffkins Institute, Mumbai, India. Escherichia coli
(lyophilized cells of strain W (ATCC 9637) and oleic
acid (C14) was obtained from Perkin Elmer Life
Sciences Inc., Boston, MA, USA. Fat-free bovine
serum albumin (BSA) fraction V was purchased
from PAA Laboratories GmbH Haidmannweg,
Austria. Con-A (concanavalin-A type V), casein
and human fibrinogen were purchased from Sigma
Chemical Company St. Louis, MO, USA. Adeno-
sine 50 monophosphate (50 AMP), a-methly-D-gluco-
side, a-methyl-D-mannoside and vanillic acid were
purchased from Sisco Research Laboratories, Ban-
galore, India. All other chemicals and reagents
purchased were of analytical grade. Fresh human
blood samples were collected from healthy volun-
teers of Department of Biochemistry, University of
Mysore, India. All the assays were done using
double distilled water. For all the studies, venom
and inhibitors were dissolved in saline.
2.2. Protein estimation
Protein concentration was determined according
to the method of Lowry et al. (1951) using BSA as
standard.
 ARTICLE IN PRESS
B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421
2.3. 50 nucleotidase assay
Venom 50 nucleotidase activity was assayed by the
procedure of Avruch and Wallach (1971) with slight
modifications. In brief, the reaction was carried out
in a final volume of 1 ml containing 10 mM MgCl2,
50 mM NaCl, 10 mM KCl, 50 mM Tris-HCl buffer,
pH 7.4, 10 mM 50 AMP with an appropriate amount
of venom and incubated for 30 min at 37 1C. The
ascorbic acid method (Chen et al., 1956) was used
to determine the released inorganic phosphate.
A measure of 1 ml of ascorbic acid reagent,
containing equal parts of 0.42% ammonium mo-
lybdate in 1 N sulfuric acid, 10% ascorbic acid and
water was added to the assay mixture. This was left
at room temperature for 30 min and absorbance at
660 nm was determined. This was quantified by
comparison with the reference curve established
with KH2PO4. Results are expressed in nmoles of
inorganic phosphate liberated/min/mg of protein.
For inhibition studies, venom samples (30 mg)
were preincubated without or with different con-
centrations of vanillic acid (0–600 mM) or Con-A
(0–6 mM) at 37 1C for 30 min. Appropriate controls
were carried out and the inhibition is expressed as
percentage taking activity of venom alone as 100%.
2.4. PLA2 assay
PLA2 activity was assayed according to the
method of Vishwanath et al. (1993). Venom was
added in a final volume of 320 ml containing 5 mM
CaCl2 and 100 mM Tris-HCl buffer, pH 7.5,
incubated for 1 h in the presence of 30 ml of
C14oleate labeled, autoclaved E. coli membrane
(equivalent to 64 nmoles of fatty acid). The reaction
was terminated by adding 100 ml of 2 N HCl
followed by 100 ml of 10% fatty-acid-free BSA.
The samples were then thoroughly vortexed and
centrifuged at 10,000 g for 10 min at room tempera-
ture. An aliquot (140 ml) of the supernatant (con-
taining released C14oleic acid) was mixed with
scintillation cocktail and counted in a Hewlett
Packard liquid scintillation analyzer TRI CARB
2100 TR. Activity is expressed as nmoles of fatty
acid released/min/mg of protein. For inhibition
studies, 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%.
413
2.5. Protease assay
Protease activity was determined using casein as
substrate according to the method of Murata et al.
(1963). A measure of 0.4 ml of Casein (2% in 0.2 M
Tris-HCl buffer, pH 8.5) was incubated with venom
sample at 37 1C for 2 h. The reaction was stopped by
adding 1.5 ml of 0.44 M TCA and allowed to stand
for 30 min. The tubes containing reaction mixtures
were then centrifuged at 1500 g for 15 min. An
aliquot (1 ml) of the supernatant was mixed with
2.5 ml of sodium carbonate (0.4 M) and 0.5 ml of
folin reagents (1:2, v/v). The colour developed was
read at 660 nm. One unit of enzyme activity was
defined as the amount of enzyme required to give an
absorbance of 0.01 at 660 nm/h at 37 1C. For
inhibition studies, venom samples (100 mg) were
preincubated without or with different concentra-
tion of vanillic acid (0–600 mM) or Con-A (0–6 mM)
at 37 1C for 30 min. Appropriate controls were
carried out and the inhibition is expressed as
percentage taking activity of venom alone as 100%.
2.6. Fibrinogenolytic assay
Fibrinogenolytic activity was measured according
to the method of Ouyang and Teng (1976). The
reaction mixture (40 ml) containing 50 mg of human
fibrinogen in 10 mM Tris-HCl buffer (pH 7.6) was
incubated with venom samples for 1 h at 37 1C. The
reaction was terminated by adding 20 ml of denatur-
ing buffer containing 1 M urea, 4% SDS and 4%
b-mercaptoethanol. The hydrolyzed products were
analyzed on 10% SDS-PAGE and the protein
pattern was visualized by staining with comassiae
brilliant blue R-250. For inhibition studies, 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.
2.7. 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. (1955). Venom samples were incubated with
50 mg of hyaluronan in 0.2 M sodium acetate buffer,
pH 6.0 and 0.15 M NaCl in a reaction volume of
1 ml for 2.5 h at 37 1C. The reaction was terminated
by adding 50 ml of potassium tetraborate buffer (pH
9.1) followed by 1.5 ml of 1% para-dimethyl amino
 ARTICLE IN PRESS
414 B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421
benzaldehyde (p-DMAB). The stoppered tubes were
boiled for 3 min in water bath and the color
developed was monitored at 585 nm. Activity was
expressed as nmoles of N-acetyl glucosamine
released/2.5 h at 37 1C. For inhibition studies,
venom sample (100 mg) was preincubated without
or with different concentrations of vanillic acid
(0–600 mM) at 37 1C for 30 min. Appropriate con-
trols were carried out and the inhibition is expressed
as percentage taking activity of venom alone as
100%.
2.8. L-amino acid oxidase assay
L-amino acid oxidase activity was determined as
described in Worthington Enzyme Manual (1977)
with some modification. Peroxidase (0.05 ml of
0.007% 250; NIH units/mg) was added to 1 ml of
0.2 M triethanolamine buffer, pH 7.6 containing
L-leucine (0.1%) and o-dianisidine (0.006%), then
the reaction mixture was incubated for 30 min at
room temperature. Venom samples were added and
the increase in absorbance at 420 nm was measured
continuously. One unit of enzyme activity is defined
as the amount of enzyme that causes an increase of
0.001 absorbance. For inhibition studies, venom
sample (100 mg) were preincubated without or with
different concentrations of vanillic acid (0–600 mM)
at 37 1C for 30 min. Appropriate controls were
carried out and the inhibition is expressed as
percentage taking activity of venom alone as 100%.
2.9. Alkaline phosphomonoesterase assay
Alkaline phosphomonoesterase activity was as-
sayed by a modification of the method described by
Lo et al. (1966). Venom was added to the reaction
mixture containing 0.4 ml of 0.01 M p-nitrophenyl
phosphate in 0.5 M Tris-HCl buffer, pH 8.5, 0.2 ml
of 0.10 M MgSO4 and made up to 1 ml with 0.5 M
Tris-HCl buffer, pH 8.5, incubated at 37 1C for
30 min. At the end of the incubation period, 2 ml of
0.2 N NaOH was added and allowed to stand for
20 min. The absorbance at 440 nm was then
measured. One unit of alkaline phosphomonoester-
ase activity is defined as the increase of 0.001
absorbance units per min at 440 nm. For inhibition
studies, venom samples (100 mg) were preincubated
without or with different concentrations of vanillic
acid (0–600 mM) at 37 1C for 30 min. Appropriate
controls were carried out and the inhibition is
expressed as percentage taking activity of venom
alone as 100%.
2.10. Anticoagulant inhibition assay
Recalcification time was determined according to
the procedure described by Condrea et al. (1983).
Fresh human blood was mixed with 0.11 M
trisodium citrate (9:1, v/v). The mixture was
centrifuged for 15 min at 2000 g and the supernatant
obtained was used as platelet poor plasma (PPP).
PPP was pre-warmed at 37 1C before use. To 300 ml
of PPP, N. naja venom (2 mg) preincubated with
different concentration of Con-A (0–1 mm) or
vanillic acid (0–10 mm) in 30 ml of 0.01 M Tris-HCl
buffer (pH 7.4) was added and incubated for 1 min
at 37 1C. The clot formation was initiated by adding
30 ml of 0.25 M CaCl2. The time taken for visible
clot to appear from the time of addition of calcium
chloride was recorded (s). In control experiments,
Tris-HCl buffer (pH 7.4) alone was added instead of
the enzyme source.
2.11. Statistics
The results are expressed as the mean7SD of at
least four independent experiments. Regression
analysis was used to calculate the inhibitory concen-
tration 50 (IC50) defined as the dosage of inhibitor
necessary to produce a 50% inhibition of activities.
3. Results and discussion
Majority of the snake venom proteins affects
hemostasis either by shortening or prolonging the
blood coagulation time. The principle proteins/
toxins that affect hemostasis are proteases and
PLA2 enzymes. Proteases affect hemostasis by
activating several coagulation factors or hydrolyz-
ing fibrinogen to promote clot formation (Mark-
land, 1997). PLA2 enzymes exerts their effect by
hydrolyzing phospholipids or by inhibiting platelet
aggregation and thus act as anticoagulants (Kini,
2005). Generally platelet aggregation inhibitors are
known to prolong clot formation indirectly (Hutton
and Warrell, 1993; Kini et al., 2002; White, 2005).
Apart from proteases and PLA2s, 50 nucleotidase is
known to affect hemostasis by inhibiting platelet
aggregation (Ouyang and Huang, 1983; Kini
and Evans, 1990; Kini and Chow, 2001). Several
reports also suggested that 50 nucleotidases acts
synergistically with other venom toxins to induce
 ARTICLE IN PRESS
B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421 415

more pronounced anticoagulant activity (Dimitrov
and Kankonkar, 1968; daSilva and Aird, 2001;
Aird, 2002). However, the exact role of venom
50 nucleotidase in inducing anticoagulant effect is
not clearly understood. 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.
It has been observed that a striking parallelism
exists between the capability of plants and their
100
80
60
% Activity
chemical compounds in neutralizing the action of
snake venoms and anti-inflammatory properties
(Mors et al., 2000). This suggests the existence of
some analogy between the mechanisms, which
govern these activities wherein plants used against
inflammation can also be used against snake venom
toxicity. In Chinese medicine, vanillic acid is the
main constituent of Di-Gu-Pi decoction, which is
used against hemorrhagic inflammation (Li et al.,
2004). Based on these observations the effect of
vanillic acid against venom enzymes and their
related pharmacological actions was examined.
The enzyme inhibition studies of N. naja venom
with vanillic acid revealed that it only inhibited
50 nucleotidase activity. It failed to inhibit other
venom enzymes like protease, PLA2, hyaluronidase,
fibrinogenase, L-amino oxidase and phosphomo-
noesterase activities even at very high concentra-
tion. The inhibition of 50 nucleotidase activity by
vanillic acid was dose dependent and complete

inhibition was achieved at 410 mM (Fig. 1). By linear

regression analysis the IC50 value was found to be
40 180 mM (Table 1). Similarly vanillic acid inhibits
only 50 nucleotidase activity and not any other
enzyme activity of Echis carinatus and D. russelli
20 venoms (Unpublished observation). From these
studies, it appears that vanillic acid selectively and
specifically inhibits venom 50 nucleotidase among
enzymes present in snake venom.
0
In order to examine the nature of crude N. naja
0 100 200 300 400 500
venom 50 nucleotidase inhibition by vanillic acid, its
Vanillic acid (µM)
inhibition was measured as a function of the
Fig. 1. Dose-dependent inhibition of N. naja venom substrate (50 AMP) concentration. 50 nucleotidase
50 nucleotidase activity. N. naja venom (30 mg) was preincubated activity in the absence and presence of IC50
with different concentration of vanillic acid (0–600 mM) for concentration (180 mM) increases almost linearly
15 min at 37 1C. Further assay was carried out as described in
methods section. Results are expressed as percentage inhibition of with increased substrate concentration. The max-
enzyme activity as compared with control. Values represent imum activity was observed at 25 mM 50 AMP and
mean7S.D (n ¼ 4). attained plateau upon further increase in substrate

Table 1
Effect of inhibitors on N. naja venom 50 nucleotidase, protease and PLA2 activities

Enzymes Activities Vanillic acid Con-A

% Inhibition IC50 (mM) % Inhibition IC50 (mM)

50 nucleotidase 65373.48a 100 180 100 2.4

Protease 0.7670.08b 0 — 100 2.8
PLA2 1128723c 0 — 0 —

Values represent mean7SD (n ¼ 4).

a
Activity is expressed in terms of nmoles of inorganic phosphate released/min/mg.
b
Activity is expressed in terms of units/min/mg of protein. One unit of activity is defined as the amount of enzyme required to cause an
increase in OD by 0.01 per min at 660 nm.
c
Specific activity is expressed in terms of fatty acid released in nmoles/min/mg of protein.
 ARTICLE IN PRESS
416 B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421

concentration (Fig. 2). The inhibition by vanillic
acid at 180 mM was 50% at 10 mM substrate
concentration and the inhibition percentage de-
creased as the substrate concentration was increased
(Fig. 2 inset). Thus the inhibition of 50 nucleotidase
by vanillic acid is substrate dependent and probably
it competes with substrate for the same binding
region. Generally, such inhibitors resemble the
substrate molecule. On examining the structures of
both substrate and vanillic acid, no resemblance was
observed (Fig. 3).
The various inhibitors of venom 50 nucleotidase
reported till date is dependent on protein nature and
lack in specificity. Con-A is known to exert
reversible inhibitory effect on Crotalous adamentous
venom 50 nucleotidase, a glycoprotein (Mannherz
and Magener, 1979), wherein it specifically interacts
with a-D-glucose and a-D-mannose residues to bring
about inhibition (Stefanovic et al., 1975). Citrate
and EDTA inhibits venom 50 nucleotidase activity
by chelating divalent metal ions (Francis et al.,
1992). Nucleocidine, melanocidine A and melano-
cidine B are polysaccharide in nature, wherein steric
effects are known to play a major role in inhibition
of 50 nucleotidase (Uchino et al., 1985). The poly-
phenolic inhibitors isolated from wine grape
‘‘Koshu’’ and Areca catechu are known to possess
tannic activity (Toukairin et al., 1991), and tannins
are known to be general inhibitors of snake venom
toxins (Okonogi et al., 1979). Even though several
substrate analogs inhibit 50 nucleotidase (Rossoman-
do et al., 1983), its use in in vivo studies is restricted.
These analogs often act as ligands for receptors
where the substrate 50 AMP is involved (Ferry et al.,
2004). Furthermore, as these studies were carried
out in different context, no reports claim a specific
inhibitor of 50 nucleotidase in relation to other
enzymes present in snake venoms. Thus vanillic
acid is first of its kind, 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.
The specific inhibitors of venom enzymes/toxins
can be useful in elucidating the mechanism of
action. Numerous studies have employed specific
inhibitors of enzymes as a biochemical or pharma-
cological tool to characterize and establish the
mechanism of action (Lazarovici and Lelkes, 1992;
Ferry et al., 2004; Erion et al., 2005). 50 nucleotidase
is known to exert anticoagulant effect by inhibiting
platelet aggregation and thus alters blood coagula-
tion without platelet lysis (Ouyang and Huang,
1983). The other mechanism proposed for antic-
oagulant activity of 50 nucleotidase is probably
through synergistic action with other toxins such
as ADPases, PLA2 and disintegrins (daSilva and

100

2000 75
% Inhibition

50

1500 25
Pi released (nmol)

0
0 10 20 30
1000 5' AMP (mM)

500

0
0 5 10 15 20 25 30
5' AMP (mM)

Fig. 2. Effect of substrate concentration on inhibition of venom 50 nucleotidase activity by vanillic acid. N. 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, 50 mM NaCl, 10 mM KCl, 50 mM Tris-HCl buffer (pH 7.4). Inset: inhibition of 50 nucleotidase
activity by vanillic acid. Results are expressed as percentage inhibition of enzyme activity as compared with control. Values represent
mean7SD (n ¼ 4).
 ARTICLE IN PRESS
B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421 417

NH2
COOH 4
3N N
5

2
1 6
N N

O P O CH2
OH O

H H
OCH3
H

HO OH
OH
Adenosine
Vanillic acid Monophosphate

Fig. 3. Structures of vanillic acid and adenosine monophosphate.

Aird, 2001). The in vitro anticoagulant action of 400

N. naja venom has been well documented, though
such symptoms are not observed in envenomated
victims (Shashidharamurthy et al., 2002; Sundell et
al., 2003). Recently, the involvement of protease in 350
mediating anticoagulant effect of N. naja venom has
been reported (Jagadeesha et al., 2002). In order to
Clotting time (Sec)

investigate the involvement of N. naja venom

50 nucleotidase in mediating anticoagulant action,
the effect of vanillic acid was tested on enzyme
inhibition followed by anticoagulant activity. N.
naja venom at a protein concentration of 2 mg
increases the recalcification time from 132 to 387 s.
It was interesting to observe that vanillic acid
decreased the recalcification time from 387 to
283.5 s in a dose-dependent manner and reaches
plateau at a concentration of 8.9 mM (Fig. 4).
Further, the addition of vanillic acid did not
decrease the clotting time thus inhibiting N. naja
venom anticoagulant effect up to 40% (Table 2).
Vanillic acid alone did not influence the coagulant
effect at the tested dose. These data clearly indicate
the partial involvement of 50 nucleotidase in mediat-
ing anticoagulant effect of N. naja venom. This
observed result was further substantiated by Con-A
inhibition studies. 50 nucleotidase is a glycoprotein
present in venom along with other glycoprotein
proteases, known to affect coagulation (Ouyang
and Huang, 1983; Gowda et al., 1994; Nikandrov
et al., 2005). It has been demonstrated that Con-A
300
250
200
0 2 4 6 8 10 12
Vanillic acid (µM)
Fig. 4. Dose-dependent inhibition of N. naja venom antic-
oagulant activity by vanillic acid. N. 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; pH 7.4) and incubated
for 1 min at 37 1C. 30 ml of CaCl2 (0.25 M) was added to the
preincubated mixture and time for clot formation was recorded
(s). In the control experiments, CaCl2 alone was used without
enzyme source. Control and inhibitor alone ¼ 263 s. Inhibitor at
0 concentration represents 2 mg of N. naja venom ¼ 385 s. Values
represent mean7SD (n ¼ 4).
 ARTICLE IN PRESS
418 B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421
Table 2
Effect of inhibitors on N. naja whole venom anticoagulant
activity
Inhibitors % Inhibition IC50 (mM)
Vanillic acid 40 ND
Con-A 100 0.28
ND ¼ not determined.
exerts reversible inhibitory effect on C. adamentous
venom 50 nucleotidase activity (Mannherz and Ma-
gener, 1979). Usually in venoms proteases and
PLA2s bring about anticoagulant effect (Markland,
1998; Kini et al., 2002). In order to substantiate the
involvement of 50 nucleotidase in N. naja venom
anticoagulant activity, the effect of Con-A on
protease, PLA2 and 50 nucleotidase activities were
tested. It was observed that, Con-A inhibited
protease and 50 nucleotidase activities in a dose-
dependent manner up to 100% (Fig. 5). However, it
did not exhibit any inhibitory effect on PLA2
activity. The IC50 values for protease and
50 nucleotidase were 2.8 and 2.4 mM, respectively
(Table 1). Further, the addition of a-methly-D-
glucoside or a-methyl-D-mannoside, which binds
specifically to Con-A reversed the inhibitory effect
of Con-A on N. naja venom 50 nucleotidase and
protease activities (data not shown). This study
indicates that N. naja venom protease and
50 nucelotidase are glycoprotein in nature having
glucose and mannose residues in its glycopart. The
inability of Con-A to inhibit N. naja venom PLA2
activity supports the observation that PLA2s in
venoms are not glycoproteins. Further, the effect of
Con-A on the N. naja venom anticoagulant effect
was tested by recalcification time. It was observed
that Con-A inhibited the anticoagulant effect of
N. naja venom in a dose-dependent manner with an
IC50 of 0.28 mM (Fig. 6). At a concentration of
0.63 mM, Con-A completely inhibited the antic-
oagulant effect of N. naja venom, as evident from
decrease in recalcification time from 385 to 132 s
(Table 2). Thus, the complete inhibition of N. naja
whole venom anticoagulant effect by Con-A sum-
marizes the participation of both 50 nucleotidase and
protease as determined by recalcification time. In
addition, this study excludes the involvement of
PLA2 in mediating anticoagulant effect of N. naja
whole venom. These results are consistent with
earlier reports, where it has been shown that the
N. naja venom PLA2s even though exhibit various
100
80
60
% Activity
40
20
0
0 1 2 3 4 5 6
Con-A (µM)
Fig. 5. Effect of Con-A on N. naja venom 50 nucleotidase (E),
protease (’) and PLA2 (m) activities. N. naja venom was
preincubated with different concentration of Con-A (0–6 mM) for
15 min at 37 1C. Further assay was carried out as described in
methods section. Results are expressed as percentage inhibition of
enzyme activity as compared with control. Values represent
mean7S.D (n ¼ 4).
pharmacological effects are known to be weak/
devoid of anticoagulant activities (Verheij et al.,
1980; Bhat and Gowda, 1991; Bhat et al., 1991;
Rudrammaji and Gowda, 1998). On the other hand,
a potent non-toxic anticoagulant metalloprotease
NN-PF3 has been purified from N. naja venom
(Jagadeesha et al., 2002).
The assay of recalcification time reflects the
involvement of clotting factors of the intrinsic
pathway in elucidating anti/pro coagulation (Ste-
fansson et al., 1990). In this study, the decrease in
recalcification time of 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 coagula-
tion to bring about anticoagulant effect. It was
recently shown that L-amino acid oxidase that
inhibits platelet aggregation also exhibits antic-
oagulant effect by inhibiting factor IX (Sakurai
et al., 2005). Thus, further studies with purified
enzyme and specific inhibitor seems interesting in
order to know the anticoagulant mechanism of
50 nucleotidase, that would be helpful in under-
standing the plausible pharmacological role of
venom 50 nucleotidase. In addition as vanillic acid
is an intermediate metabolite product of vanillin,

B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421
400
350
300
Clotting time (Sec)
250
200
150
100
0 0.1 0.2 0.3 0.4 0.5
Con-A (µM)
Fig. 6. Dose-dependent inhibition of N. naja venom antic-
oagulant effect by Con-A. N. naja venom (2 mg) preincubated
with different concentrations of Con-A (0–1 mM) for 15 min was
added to 300 ml of human plasma in the presence of 30 ml Tris-
HCl buffer (10 mM; pH 7.4) and incubated for 1 min at 37 1C.
About 30 ml of CaCl2 (0.25 M) was added to the preincubated
mixture and time for clot formation was recorded (s). In the
control experiments, CaCl2 alone was used without enzyme
source. Control and inhibitor alone ¼ 263 s. Zero concentration
of inhibitor represents 2 mg of N. naja venom ¼ 385 s. Values
represent mean7SD (n ¼ 4).
excreted in urine (Dirscherl and Wirtzfeld, 1964),
indicates its use as a biochemical or pharmacologi-
cal tool to decipher the role of 50 nucleotidase in
snake envenomation strategies.
In conclusion, this study for the first time
demonstrates the involvement of 50 nucleotidase, a
glycoprotein in mediating anticoagulant effect of
N. naja venom with the use of novel non-nucleoside
specific inhibitor, vanillic acid. We propose that
further studies using this specific inhibitor as
biochemical or pharmacological tool would help in
evaluating the role of 50 nucleotidase in venoms and
in particular its involvement in hemostasis.
Acknowledgments
R. Rajesh and A. Nataraju acknowledge the
Council of Scientific and Industrial Research
(CSIR), New Delhi, India, for financial assistance.
We thank Prof. T.V. Gowda for valuable sugges-
ARTICLE IN PRESS
419
tions and constant support in our work. We thank
Prof. R.M. Kini, Department of Biological Science,
Faculty of Science, National University of Singa-
pore, Singapore, and Prof. S.D. Aird, Department
of Chemistry, Norfolk State University, VA 23504,
USA for their valuable suggestions during this
study. We thank Dr. K.S. Girish, Orthopedic
Research Lab., University of Virginia, Charlottes-
ville for valuable suggestions.
References
Aird, S.D., 2002. Ophidian envenomation strategies and the role
of purines. Toxicon 40, 335–393.
Avruch, J., Wallach, D.F., 1971. Preparation and properties of
plasma membrane and endoplasmic reticulum fragments from
isolated rat fat cells. Biochim. Biophys. Acta 233, 334–347.
Bhat, M.K., Gowda, T.V., 1991. Isolation and characterization
of a lethal phospholipase A2 (NN-IVb1-PLA2) from the
0.6 0.7 Indian cobra (Naja naja naja) venom. Biochem. Int. 25,
1023–1034.
Bhat, M.K., Prasad, B.N., Gowda, T.V., 1991. Purification and
characterization of a neurotoxic phospholipase A2 from
Indian cobra (Naja naja naja) venom. Toxicon 29, 1345–1349.
Boffa, M.C., Boffa, G.A., 1974. Correlations between the
enzymatic activities and the factors active on blood coagula-
tion and platelet aggregation from the venom of Vipera aspis.
Biochim. Biophys. Acta 354, 275–290.
Chen, P.S., Toribara, T.Y., Warner, H., 1956. Microdetermina-
tion of phosphorus. Anal. Chem. 28, 1756–1759.
Cirino, G., Peers, S.H., Wallace, J.L., Flower, R.J., 1989. A study
of phospholipase A2-induced oedema in rat paw. Eur.
J. Pharmacol. 166, 505–510.
Condrea, E., Yang, C.C., Rosenberg, P., 1983. Anticoagulant
activity and plasma phophatidylserine hydrolysis by snake
venom phospholipase A2. Thromb. Haemost. 49, 151.
daSilva Jr., N.J., Aird, S.D., 2001. Prey specificity, comparative
lethality and compositional differences of coral snake venoms.
Comp. Biochem. Physiol. C 128, 425–456.
Delaquis, P., Stanich, K., Toivonen, P., 2005. Effect of pH on the
inhibition of Listeria spp. by vanillin and vanillic acid.
J. Food Prot. 68, 1472–1476.
Dimitrov, G.D., Kankonkar, R.C., 1968. Fractionation of Vipera
russelli venom by gel filtration. I. Toxicon 5, 213–221.
Dirscherl, W., Wirtzfeld, A., 1964. Vanillic acid in the human
urine. Its isolation, determination and origin. Hoppe-Seyler’s
Z. Physiol. Chem. 336, 81–90.
Erion, M.D., van Poelje, P.D., Dang, Q., Kasibhatla, S.R.,
Potter, S.C., Reddy, M.R., Reddy, K.R., Jiang, T., Lipscomb,
W.N., 2005. MB06322 (CS-917): a potent and selective
inhibitor of fructose 1, 6-bisphosphatase for controlling
gluconeogenesis in type 2 diabetes. Proc. Natl. Acad. Sci.
USA 102, 7970–7975.
Ferry, G., Ubeaud, C., Mozo, J., Pean, C., Hennig, P.,
Rodriguez, M., Scoul, C., Bonnaud, A., Nosjean, O., Galizzi,
J.P., Delagrange, P., Renard, P., Volland, J.P., Yous, S.,
Lesieur, D., Boutin, J.A., 2004. New substrate analogues of
human serotonin N-acetyltransferase produce in situ specific
and potent inhibitors. Eur. J. Biochem. 271, 418–428.
 ARTICLE IN PRESS
420 B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421
Francis, B., Seebart, C., Kaiser, I.I., 1992. Citrate is an
endogenous inhibitor of snake venom enzymes by metal-ion
chelation. Toxicon 30, 1239–1246.
Gowda, D.C., Jackson, C.M., Hensley, P., Davidson, E.A., 1994.
Factor X-activating glycoprotein of Russell’s viper venom.
J. Biol. Chem. 269, 10644–10650.
Hutton, R.A., Warrell, D.A., 1993. Action of snake venom
components on the haemostatic system. Blood Rev. 7,
176–189.
Jagadeesha, D.K., Shashidharamurthy, R., Girish, K.S., Kem-
paraju, K., 2002. A non-toxic anticoagulant metalloprotease:
purification and characterization from Indian cobra (Naja
naja naja) venom. Toxicon 40, 667–675.
Jayaraman, G., Krishnaswamy, T., Kumar, S., Yu, C., 1999.
Binding of nucleotide triphosphates to cardiotoxin analogue
II from the Taiwan cobra venom (Naja naja atra). Elucidation
of the structural interactions in the dATP-cardiotoxin
analogue ii complex. J. Biol. Chem. 274, 17869–17875.
Kini, R.M., 2005. Structure-function relationships and mechan-
ism of anticoagulant phospholipase A2 enzymes from snake
venoms. Toxicon 45, 1147–1161.
Kini, R.M., Chow, G., 2001. Exogenous inhibitors of platelet
aggregation from animal sources. Thromb. Haemost. 85,
179–181.
Kini, R.M., Evans, H.J., 1990. Effects of snake venom proteins
on blood platelets. Toxicon 28, 1387–1422.
Kini, R.M., Rao, V.S., Joseph, J.S., 2002. Procoagulant proteins
from snake venoms. Haemostasis 31, 218–224.
Lazarovici, P., Lelkes, P.I., 1992. Pardaxin induces exocytosis in
bovine adrenal medullary chromaffin cells independent of
calcium. J. Pharmacol. Exp. Ther. 263, 1317–1326.
Lee, W.-H., Zhang, Y., Wang, W.-Y., Xiong, Y.-L., Gao, R.,
1995. Isolation and properties of a blood coagulation factor X
activator from the venom of king cobra (Ophiophagus
hannah). Toxicon 33, 1263–1276.
Li, K., Chen, X., Jia, Y., Bi, K., 2004. Reverse-phase HPLC
determination and pharmacokinetic study of vanillic acid in
the plasma of rats treated with the traditional Chinese
medicinal preparation Di-Gu-Pi decoction. Yakugaku Zasshi
124, 465–468.
Lo, T.B., Chen, Y.H., Lee, C.Y., 1966. Chemical studies of
Formosan cobra (Naja naja atra) venom (I). Chromato-
graphic separation of crude venom, CM-Sephadex and
preliminary characterization of its components. J. Chin.
Chem. Soc. 13, 25–37.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193, 265–275.
Mannherz, H.G., Magener, M., 1979. Concanavalin A inhibits
the interaction of snake venom 50 nucleotidase and actin.
FEBS Lett. 103, 77–80.
Markland, F.S., 1997. Snake venoms. Drugs 54, 1–10.
Markland, F.S., 1998. Snake venoms and the hemostatic system.
Toxicon 36, 1749–1800.
Mors, W.B., Nascimento, M.C., Pereira, B.M., Pereira, N.A.,
2000. Plant natural products active against snake bite—the
molecular approach. Phytochemistry 55, 627–642.
Murata, J., Satake, M., Suzuki, T., 1963. Studies on snake venom
XII. Distribution of proteinase activities among Japanese and
Formosan snake venoms. J. Biochem. 53, 431–443.
Nikandrov, N.N., Deshimaru, M., Tani, A., Chijiwa, T., Shibata,
H., Chang, C.C., Fukumaki, Y., Ito, T., Ohno, M., 2005.
Purification, primary structures and evolution of coagulant
proteases from Deinagkistrodon actus venom. Toxicon 46,
907–917.
Okonogi, T., Hattori, Z., Ogiso, A., Mitsui, S., 1979. Detoxifica-
tion by persimmon tannin of snake venoms and bacterial
toxins. Toxicon 17, 524–527.
Ouyang, C., Huang, T.F., 1983. Inhibition of platelet aggregation
by 50 -nucleotidase purified from Trimeresurus gramineus
snake venom. Toxicon 21, 491–501.
Ouyang, C., Teng, C.M., 1976. Fibrinogenolytic enzymes of
Trimeresurus mucrosquamatus venom. Biochim. Biophys.
Acta 420, 298–308.
Pithayanakul, P., Laovachirasuwan, S., Bavovada, R., Pakma-
nee, N., Suttisri, R., 2004. Anti-venom potential of butanolic
extract of Eclipta prostrata against Malayan pit viper venom.
J. Ethnopharmacol. 90, 347–352.
Reissig, J.L., Stominger, J.L., Leloir, L.F., 1955. A modified
colorimetric method for the estimation of N-acetylamino
sugars. J. Biol. Chem. 217, 959–969.
Rossomando, E.F., Cordis, G.A., Markham, G.D., 1983.
50 -deoxy-50 -thioanalogs of adenosine and inosine 50 -mono-
phosphate: studies with 50 -nucleotidase and alkaline phos-
phatase. Arch. Biochem. Biophys. 220, 71–78.
Rudrammaji, L.M., Gowda, T.V., 1998. Purification and
characterization of three acidic cytotoxic phospholipases A2
from Indian cobra (Naja naja naja) venom. Toxicon 36,
921–932.
Sakurai, Y., Shima, M., Matsumoto, T., Takatsuka, H., Nishiya,
K., Kasuda, S., Fujimura, Y., Yoshioka, A., 2005. Antic-
oagulant activity of M-LAO, L-amino acid oxidase purified
from Agkistrodon halys blomhoffii, through selective inhibi-
tion of factor IX. Biochim. Biophys. Acta 1649, 51–57.
Shashidharamurthy, R., Jagadeesha, D.K., Girish, K.S., Kem-
paraju, K., 2002. Variation in biochemical and pharmacolo-
gical properties of Indian cobra (Naja naja naja) venom due
to geographical distribution. Mol. Cell. Biochem. 229,
93–101.
Singh, M., Tiwari, V., Jain, A., Ghoshal, S., 2005. Protective
activity of picroliv on hepatic amoebiasis associated with
carbon tetrachloride toxicity. Indian J. Med. Res. 121,
676–682.
Soares, A.M., Ticli, T.K., Marcussi, S., Lourenco, M.V.,
Januario, A.H., Sampaio, S.V., Giglio, J.R., Lomonte, B.,
Pereira, P.S., 2005. Medicinal plants with inhibitory proper-
ties against snake venoms. Curr. Med. Chem. 12, 2625–2641.
Stefanovic, V., Mandel, P., Rosenberg, A., 1975. Concanavalin A
inhibition of ecto-50 -nucleotidase of intact cultured C6 glioma
cells. J. Biol. Chem. 250, 7081–7083.
Stefansson, S., Kini, R.M., Evans, H.J., 1990. The basic
phospholipase A2 from Naja nigricollis venom inhibits the
prothrombinase complex by a novel nonenzymatic mechan-
ism. Biochemistry 29, 7742–7746.
Sundell, I.B., Ranby, M., Zuzel, M., Robinson, K.A., Theakston,
R.D., 2003. In vitro procoagulant and anticoagulant proper-
ties of Naja naja naja venom. Toxicon 42, 239–247.
Toukairin, T., Uchino, K., Iwamoto, M., Murakami, S.,
Tatebayashi, T., Ogawara, H., Tonosaki, Y., 1991. New
polyphenolic 50 -nucleotidase inhibitors isolated from the wine
grape ‘‘Koshu’’ and their biological effects. Chem. Pharm.
Bull. 39, 1480–1483.
Uchino, K., Ogawara, H., Akiyama, T., Fukuchi, A., 1985.
50 -Nucleotidase inhibitory activity of nucleoticidin,
 ARTICLE IN PRESS
B.L. Dhananjaya et al. / Toxicon 48 (2006) 411–421
melanocidin A and melanocidin. B. Structure–activity rela-
tionships. J. Antibiot. 38, 1564–1567.
Varma, R.S., Shukla, A., Chatterjee, R.K., 1993. Evaluation of
vanillic acid analogues as a new class of antifilarial agents.
Indian J. Exp. Biol. 31, 819–821.
Verheij, H.M., Boffa, M.C., Rothen, C., Bryckert, M.C., Verger,
R., De Haas, G.H., 1980. Correlation of enzymatic activity
and anticoagulant properties of phospholipase A2. Eur.
J. Biochem. 112, 25–32.
Vishwanath, B.S., Kini, R.M., Gowda, T.V., 1987. Characteriza-
tion of three edema-inducing phospholipase A2 enzymes from
421
habu (Trimeresurus flavoviridis) venom and their interaction
with the alkaloid aristolochic acid. Toxicon 25, 501–515.
Vishwanath, B.S., Frey, F.J., Bradbury, M.J., Dallman, M.F.,
Frey, B.M., 1993. Glucocorticoid deficiency increases phos-
pholipase A2 activity in rats. J. Clin. Invest. 92, 1974–1980.
White, J., 2004. Elapid snakes. In: Dart, R. (Ed.), Medical
Toxicology. Williams and Wilkins, Lippincott, pp. 1566–1578.
White, J., 2005. Snake venoms and coagulopathy. Toxicon 45,
951–967.
Worthington Enzyme Manual, 1977. L-Amino acid oxidase.
Worthington Biochemical Corporation, USA, pp. 49–50.