Post on 22-Jan-2023
Procoagulant activity of Calotropis gigantea latex associated
with fibrin(ogen)olytic activity
R. Rajesh, C.D. Raghavendra Gowda, A. Nataraju, B.L. Dhananjaya,
K. Kemparaju, B.S. Vishwanath*
Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore 570 006, Karnataka, India
Received 22 November 2004; revised 17 March 2005; accepted 18 March 2005
Abstract
The latex of Calotropis gigantea is a rich source of useful components that has medicinal properties and one of the main
applications is in controlling bleeding. The crude latex extract contained many proteins, which are highly basic in nature and
exhibited strong proteolytic activity. The crude extract hydrolyses casein, human fibrinogen and crude fibrin clot in a dose-
dependent manner. The hydrolyzing activity was completely inhibited by IAA indicating they belong to the super family,
cysteine proteases. Crude extract hydrolyses Aa, Bb and g subunits of fibrinogen. Among all the subunits the preferential
subunit to get hydrolyzed was Aa followed by Bb and g subunit is highly resistant and hydrolyzed at higher protein
concentration or over a prolonged incubation time. The crude extract hydrolysis crude fibrin clot strongly compared to trypsin
and papain. Pharmacologically the crude extract is hemorrhagic and induces skin hemorrhage at O75 mg and reduces the
coagulation time of citrated plasma from 150 to 47 s and promotes blood coagulation. Procoagulation and blood clot hydrolysis
are important in wound healing process. This is due to unique cysteine proteases of plant latex and is responsible for the
pharmacological actions observed in folk medicine.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Calotropis gigantea; Latex; Procoagulant activity; Fibrinogenolytic activity; Fibrinolytic activity; Hemostasis
1. Introduction
The presence of latex in plants is one of the characteristic
features belonging to the families Euphorbiaceae, Ascle-
piadaceae, Moraceae and Apocyanaceae. Plant latex is a
complex mixture of several hydrolytic enzymes (Yagami
et al., 1998) and also a rich source of wax, resins and lipid
0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2005.03.012
Abbreviations 8C, degree celsius; g, gram; kg, kilogram; mM,
millimolar; ml, milliliter; cm, centimeter; h, hour(s); mg, micro-
gram; %, percentage; s, second(s); SEM, standard error of the mean;
s.c, subcutaneous; nm, nanometer; i.p., intraperitoniall; TCA,
trichloroaceticacid; SDS, sodiumdodecylsulphate.* Corresponding author. Tel.: C91 821 2511218; fax: C91 821
2415390.
E-mail address: vishmy@yahoo.co.uk (B.S. Vishwanath).
like substances. The physiological role of this latex in plants
is not clearly understood. The plant resistant to variety of
infectious diseases and to the extreme harsh conditions are
partly attributed to the presence of hydrolytic enzymes of
the latex especially proteases (Boller, 1986). These latex
exhibited several pharmacological properties and are
exploited in folk medicine (Ervatamia, 1952). Latex from
several plant species has been shown to involve in
hemostatis (Bolay, 1979; Gunter et al., 2002), wound
healing and pain killing effects (Thankamma, 2003).
Calotropis gigantea commonly known as milkweed or
swallowwort is one of the latex bearing plants belongs to the
family Asclepiadaceae and is known for its medicinal
properties (Singh et al., 1996; Rastogi et al., 1991). The
latex is applied to soften the outer skin portion while
removing thorns (Pankaj, 2003). The latex from C. gigantea
Toxicon 46 (2005) 84–92
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R. Rajesh et al. / Toxicon 46 (2005) 84–92 85
and related species commonly used on fresh cuts to stop
bleeding (Ashwani, 1999) and have been used as antiin-
flammatory agent in folk medicine (Dhanukar et al., 2000).
Several tribal people used this latex for easy delivery,
abortion and for other ailments (Bhuyan, 1994).
The aqueous extract of C. gigantea latex contained
several hydrolytic enzymes (Abraham and Joshi, 1979;
Senugupta et al., 1984). Among these, proteases are found to
be relatively abundant and are probably responsible for the
various pharmacological properties exhibited by the latex.
Several proteases from mammalian system and from snake
venoms are known to interfere in hemostasis and act either
as procoagulants or as anticoagulants (Kornalik, 1990;
Markland, 1991; Siigur and Siigur, 1992). In the present
study, we report for the first time the pharmacological
properties of proteolytic activity from crude fraction of
C. gigantea latex with respect to the hydrolysis of human
fibrinogen leading to coagulation of the plasma and the
subsequent plasmin like activity of the latex.
2. Materials and methods
2.1. Materials
Trypsin, casein, and human fibrinogen, protease inhibi-
tors iodoaceticacid (IAA), and phenylmethylsulphonyl
fluoride (PMSF) were purchased from sigma chemical
company (St Louis, MO, USA). Papain, and other protease
inhibitors 1,10-phenanthroline, ethylene diaminetetraacetic-
acid (EDTA), and ethylene glycol-N, N, N 0, N 0-tetraacetic-
acid (EGTA) were purchased from SRL Chemical
Company, Bangalore, India. All other chemicals and
reagents purchased were of analytical grades. All the
solvents were re-distilled before use. Plant latex was
collected around the local area of Mysore, India. Fresh
human blood samples were collected from healthy volun-
teers. Male Swiss wistar mice weighing 20–25 g were
obtained from Central Animal House, University of Mysore,
Mysore, India.
2.1.1. Preparation of crude enzyme from C. gigantea latex
The latex was collected in a clean glass beaker by
breaking tender parts of the plant. This latex was diluted
with equal volume of 10 mM phosphate buffer (pH 7.0) and
kept overnight at 4 8C. The supernatant was decanted and
centrifuged at 12,000g for 20 min at 4 8C. The clear
supernatant was decanted and dialyzed against 10 mM
phosphate buffer (pH 7.0). The supernatant was subjected to
protein precipitation by 80% ammonium sulphate. The
precipitated pellet was dissolved in 10 mM phosphate buffer
(pH 7.0) and dialyzed against the same buffer to remove
ammonium sulphate. The protein concentration in the
supernatant was estimated according to the method of
Lowry et al. (1951) and used as crude enzyme source.
2.2. Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) was carried
out for crude latex extract on 10% polyacrylamide using
b-alanine and acetic acid buffer (pH 4.3) and Tris glycine
buffer (pH 8.8) according to the method of Davis (1964).
2.3. Caseinolytic activity
Caseinolytic activity was assayed according to the
method of Murata et al. (1963). Casein 0.4 ml (2% in
0.2 M Tris–HCl buffer, pH 8.5) was incubated with different
concentration (10–100 mg) of crude extract, trypsin and
papain at 37 8C separately 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 mixture was centrifuged at 1500g for 15 min.
An aliquot (1 ml) of the supernatant was mixed with 2.5 ml
of 0.4 M sodium carbonate and 0.5 ml of 1:2 diluted folin
reagent and the color developed was read at 660 nm. One
unit of enzyme activity was defined as the amount of
enzyme required to increase in absorbance of 0.01 at
660 nm/h at 37 8C. Activity was expressed as units/h
at 37 8C.
2.4. Human fibrinogenolytic activity
Fibrinogenolytic activity was measured according to
the method of Ouyang and Teng (1976). The reaction
mixture 40 ml contained 50 mg of human fibrinogen,
10 mM Tris–HCl buffer (pH 7.6) was incubated at 37 8C
with different concentrations of crude latex extract and
for different time intervals. The reaction was terminated
by adding 20 ml of denaturing buffer containing 1 M urea,
4% SDS and 4% b-mercaptoethanol. The hydrolyzed
products were analyzed by 7.5% SDS-PAGE and protein
pattern was visualized by staining with Coomassie
brilliant blue R-250.
2.5. Coagulant activity
Re-calcification time was determined according to the
procedure described by Condrea et al. (1983). Fresh human
blood was mixed with 0.11 M tri-sodium citrate in the ratio
of nine parts to one. The mixture was centrifuged for 15 min
at 500g. The supernatant was used as platelet poor plasma
(PPP). PPP was pre-warmed to 37 8C before use. To 300 ml
of PPP different concentration of crude latex extract in
0.01 M Tris–HCl buffer (pH 7.4) was added and incubated
for 1 min. 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.
For control experiments Tris–HCl buffer alone was added
instead of the enzyme source.
2.6. Fibrinolytic activity
Fibrinolytic activity was carried out using human blood
clot and plasma clot as substrates.
R. Rajesh et al. / Toxicon 46 (2005) 84–9286
2.6.1. Human blood clot hydrolyzing activity
One hundred microlitres of EDTA (2 mg/ml) treated
human blood was mixed with equal volume of 100 mM
CaCl2 and allowed to stand for 30 min to form clot. The hard
clot was thoroughly washed 5–6 times with phosphate
buffered saline (PBS) and suspended in 400 ml of 10 mM
Tris–HCl buffer (pH 7.6). This was incubated with different
concentrations (10–100 mg) of crude latex extract, trypsin
and papain separately for 2 h at 37 8C. The undigested blood
clot was precipitated using 0.44 M TCA and hydrolyzed
products were assayed as described in the caseinolytic
activity. For inhibition studies similar reactions were
performed after pre-incubation of enzyme source with and
without specific protease inhibitors. One unit of enzyme
activity was defined as the amount of enzyme required to
increase in OD of 0.01 at 660 nm/h at 37 8C. Activity was
expressed as units/h at 37 8C.
2.6.2. Human plasma clot hydrolyzing activity
EDTA (2 mg/ml) treated blood was centrifuged for
15 min at 500g to separate plasma. Plasma (100 ml) was
mixed with equal volume of 100 mM CaCl2 to get soft fibrin
clot. The fibrin clot was thoroughly washed with PBS (5–6
times) and suspended in 400 ml of 10 mM Tris–HCl buffer
(pH 7.6). This was incubated with different concentrations
(10–100 mg) of crude latex extract, trypsin and papain
separately for 2 h at 37 8C. Further assay and inhibition
studies were performed as explained earlier.
2.6.3. SDS-PAGE pattern of human plasma clot hydrolyzing
activity
The plasma clot was prepared and washed as explained
in Section 2.6.2. The washed clot was incubated with a
different doses (1–6 mg) of latex enzyme source in a 40 ml
reaction mixture at 37 8C for 1 h in the presence of 10 mM
Tris–HCl buffer (pH 7.6). The reaction was stopped by
adding 20 ml sample buffer containing 4% SDS, 4%
b-mercaptoethanol and 1 M urea, boiled for 3 min and
centrifuged to settle the debris of plasma clot. An aliquot
(20 ml) of the supernatant was used to analyze the
hydrolyzing pattern of plasma clot in 7.5% SDS-PAGE
according to the method of Laemmli (1970).
2.7. Hemorrhagic activity
Hemorrhagic activity was assayed as described by
Kondo et al. (1960). Groups of five mice were injected
(s.c) separately with various concentrations of crude latex
extract. After 3 h, mice were sacrificed using anesthesia
(Barbitone 30 mg/kg i.p.). The dorsal surface of the skin was
removed and the inner surface was measured for hemor-
rhagic activity. The minimum hemorrhagic dose (MHD) is
defined, as the concentration required to induce hemorrhagic
spot of 1 cm diameter from the spot of injection.
2.8. Statistics
The data of biochemical and pharmacological experi-
ments were expressed as the meanGSEM of at least five
independent experiments.
3. Results
The crude extract was obtained from C. gigantea latex
first by subjecting it to de-waxing and the clear supernatant
obtained was later precipitated for protein with 80%
ammonium sulphate. The precipitated pellet fraction was
re-suspended in 10 mM phosphate buffer (pH 7.0) and
termed as crude extract. This extract exhibited proteolytic
activity when casein was used as the substrate.
On electrophoresis of the crude extract with native gel
under acidic (pH 4.3) and basic (pH 8.8) conditions, the
proteins were resolved better in the acidic gel rather in
the basic gel (Fig. 1). This data generalizes that the crude
extract contained many proteins, which are highly basic in
nature.
Denatured casein was generally used as a substrate for
proteases and assayed as caseinolytic activity. The case-
inolytic activity of crude C. gigantea latex was compared
with that of purified trypsin, a serine protease and papain, a
cysteine protease (Fig. 2). Crude extract of C. gigantea,
papain and trypsin exhibited caseinolytic activity in a dose-
dependent manner. Trypsin exhibited the highest activity
compared to crude extract of C. gigantea and papain.
Among latex proteases, crude extract of C. gigantea showed
twice the activity compared to papain.
Since the C. gigantea latex was shown to affect the
hemostasis, its proteolytic action on pure human fibrinogen
was determined. Fig. 3 shows the electrophoretic pattern of
fibrinogenolytic activity as a function of protein concen-
tration and time. Crude extract of C. gigantea hydrolyzes
Aa, Bb and g subunits of human fibrinogen in a dose-
dependent manner (Fig. 3A). The first fibrinogen fraction to
undergo hydrolysis was Aa subunit. As the concentration of
crude extract increases the Bb chain gets hydrolyzed and at
this concentration more than 50% of the Aa subunit
hydrolysis was observed visually. At higher concentrations,
C. gigantea hydrolyzes g subunit also. Fig. 3B shows the
time-dependent hydrolysis of human fibrinogen at 1.0 mg of
crude latex extract. At this concentration hydrolysis of Aasubunit was observed as early as 5.0 min incubation time.
As the incubation time increases, hydrolysis of Bb subunit
was also observed and at 30.0 min incubation both Aa and
Bb subunits undergoes complete degradation. Compara-
tively g subunit was resistant to hydrolysis by crude latex
extract. However, prolonged incubation resulted in
the degradation of g subunit also and more than 95%
degradation was observed visually at 60.0 min incubation.
These data indicates that C. gigantea latex hydrolyzes all the
Fig. 2. Caseinolytic activity of crude C. gigantea latex, papain and
trypsin. Reaction mixture 1 ml contained 0.4 ml of 2% casein in
0.2 M Tris–HCl buffer (pH 8.5) incubated with different concen-
tration of C. gigantea latex extract (%), papain (&) and trypsin (:)
ranging from 10 to 100 mg protein for 2 h at 37 8C. Values represent
meanGSEM (nZ5).
Fig. 1. Native PAGE of Calotropis gigantea latex. Latex protein
was loaded on to the 10% Native PAGE and electrophoresis was
performed in a constant current under acidic condition using
b-alanine and acetic acid buffer. (A) 50, (B) 75, and (C) 100 mg
latex protein.
R. Rajesh et al. / Toxicon 46 (2005) 84–92 87
subunits of human fibrinogen. The hydrolysis of different
subunits is in the order of AaOBbOg.
Thrombin hydrolysis of fibrinogen results in the
formation of monomeric fibrin subunits, which undergo
polymerization to form fibrin clot. To check the clot
inducing ability by C. gigantea latex extract, the re-
calcification time of citrated plasma was determined.
Crude extract of C. gigantea latex on pre-incubation with
citrated plasma on re-calcification promote clot formation
(Fig. 4). The clot formation is dose-dependent and at 30 mg
concentration it attained plateau and clot formation was
decreased from 150 to 47 s. This data suggests that the C.
gigantea latex extract has procoagulant effect on human
plasma.
The crude extract of C. gigantea extract on prolonged
incubation or at higher protein concentration hydrolyzed g
subunit of fibrinogen. Several proteases, which hydrolyzed
g subunit of fibrinogen also hydrolyze fibrin clot. The crude
extract of C. gigantea along with trypsin and papain was
checked for the digestion of whole blood clot (Fig. 5A) and
plasma clot (Fig. 5B). All the proteases hydrolyzed these
clots in a dose-dependent manner. Maximum hydrolysis of
whole blood clot and plasma clot was observed with
C. gigantea latex compared to trypsin and papain. To
substantiate this data the hydrolyzed product of plasma clot
was analyzed on SDS-PAGE (Fig. 6). The electrophoresis of
washed clot under reducing condition showed a-polymer, b-
chains and g–g dimer. Crude latex extract hydrolysis all the
three subunits of crude fibrin of plasma clot resulted in the
generation of small molecular weight proteins. With
increasing latex protein concentration the small molecular
weight protein bands intensity increases indicating the
hydrolysis of fibrin subunits into smaller protein fragments.
The crude C. gigantea latex extract hydrolyzed sub-
strates like casein, human fibrinogen and crude fibrin clot.
The proteolytic enzyme, which hydrolyzes these substrates,
is characterized using several specific protease inhibitors
(Table 1). IAA inhibits the proteolytic hydrolysis of casein,
human fibrinogen and fibrin clot; activity is not inhibited by
other specific protease inhibitors. These data concludes that
the enzymes present in the crude latex are a family of
cysteine protease and not serine or metallo proteases.
Many proteases from snake venoms, which affect the
hemostasis, exhibit hemorrhagic activity and other deleter-
ious effects (Estevao-Costa et al., 2000; Siigur et al., 2001).
At protein concentrations of 1 and 6 mg, where it hydrolyses
completely all the subunits of fibrinogen and plasma clot,
respectively, the latex extract did not induce any hemor-
rhagic activity. The crude extract is hemorrhagic and its
MHD was O75 mg concentration (Fig. 7). The hemorrhagic
activity was completely neutralized in the presence of IAA,
which is a specific inhibitor for cysteine protease indicating
that the hemorrhagic activity of the crude latex extract is due
to the catalytic activity of proteases.
Fig. 4. Effect of C. gigantea latex on re-calcification time of human
plasma. C. gigantea latex protein ranging from 2 to 40 mg was pre-
incubated with 300 ml of human plasma in the presence of 30 ml
Tris–HCl buffer (10 mM; pH 7.4) for 1 min at 37 8C. Thirty
microlitres of CaCl2 (0.25 M) was added to the pre-incubated
mixture. The time for clot formation was recorded. In the control
experiments, Tris–HCl buffer was used instead of enzyme source.
Fig. 3. (A) Dose-dependent proteolytic hydrolysis of human
fibrinogen by C. gigantea latex protein. Crude C. gigantea latex
at different protein concentration ranging from 0.1 to 2.0 mg was
incubated with 50 mg of human fibrinogen at 37 8C for 30 min in the
presence of 10 mM Tris–HCl buffer (pH 7.6). SDS polyacrylamide
gel electrophoresis (7.5%) was performed to visualize fibrinogen
3
R. Rajesh et al. / Toxicon 46 (2005) 84–9288
4. Discussion
Interest in proteolytic enzymes is gaining importance
because of wide variety of physiological activities exhibited
by them (Mrinalini et al., 2002). Several serine proteases
and metalloproteases isolated from snake venoms are
known to affect blood coagulation pathways and acts
either as procoagulants or anticoagulants (Kornalik, 1990;
Markland, 1991; Siigur and Siigur, 1992). Some of them are
degradation products. A: Control, B: 0.1, C: 0.25, D: 0.5, E: 1.0, F:
2.0 mg of crude C. gigantea latex. (B) Time-dependent proteolytic
hydrolysis of human fibrinogen by C. gigantea latex. One
microgram of crude C. gigantea latex protein was incubated with
50 mg of human fibrinogen at 37 8C for different time intervals
(0–1 h) in the presence of 10 mM Tris–HCl buffer (pH 7.6). SDS-
PAGE (7.5%) was performed to visualize fibrinogen degradation
products. A: 0 h, B: 5, C: 10, D: 15, E: 30, F: 60 min of incubation.
(C) Inhibition of human fibrinogenolytic activity of C. gigantea
latex by specific protease inhibitors. One microgram of C. gigantea
latex was pre-incubated with and without specific protease
inhibitors for 15 min in the presence of 10 m M Tris–HCl buffer
(pH 7.6) and reaction was initiated by adding 50 mg of fibrinogen to
the pre-incubated mixture. After 30 min the reaction was terminated
by adding denaturing buffer. SDS-PAGE (7.5%) was performed to
visualize the inhibition of fibrinogen degradation. A: Fifty
micrograms of fibrinogen, B: 50 mg fibrinogenC1 mg latex extract,
C: 50 mg fibrinogenC1 mg latex extractC100 mM IAA, D: 50 mg
fibrinogenC1 mg latex extractC5 mM PMSF, E: 50 mg fibrino-
genC1 mg latex extractC5 mM EDTA, F: 50 mg fibrinogenC1 mg
latex extractC5 mM EGTA, G: 50 mg fibrinogenC1 mg latex
extractC5 mM 1–10 phenanthroline.
Fig. 6. Hydrolyzing pattern of plasma clot by the crude extract of
C. gigantea latex. Washed plasma clot was incubated with crude
C. gigantea latex protein ranging from 1 to 6 mg in Tris–HCl buffer
(10 mM; pH 7.6) for 1 h at 37 8C. SDS-PAGE (7.5%) shows plasma
clot cleavage pattern by the action of latex protease. A: Control
(plasma clot), B: plasma clotC1 mg of latex protein, C: plasma
clotC2 mg of latex protein, D: plasma clotC4 mg of latex protein,
E: plasma clotC6 mg of latex protein.
Fig. 5. (A) Dose-dependent hydrolysis of blood clot by crude
C. gigantea latex, papain and trypsin. Reaction mixture 0.5 ml
contained washed blood clot incubated with different concentration
of C. gigantea latex (%), papain (&) and trypsin (:) ranging from
10 to 100 mg of protein in the presence of Tris–HCl buffer (10 mM;
pH 7.6) for 2 h at 37 8C. Values represent meanGSEM (nZ5).
(B) Dose-dependent hydrolysis of plasma clot by crude C. gigantea
latex, papain and trypsin. Reaction mixture 0.5 ml contained
washed plasma clot incubated with different concentration of
crude C. gigantea latex (%), papain (&) and trypsin (:) ranging
from 10 to 100 mg of protein in the presence of Tris–HCl buffer
(10 mM; pH 7.6) for 2 h at 37 8C. Values represent meanGSEM
(nZ5).
Table 1
Inhibition of hemorrhagic activity and proteolytic activity on
various substrates by specific inhibitors
Protease
inhibitors
(mM)
Casein Fibrin Hemor-
rhageBlood clot Plasma clot
IAA (0.1) 97G2.3 94G3.4 92G3.0 87G3.7
PMSF (5) 10G1.7 0 0 0
Phenan-
throline (5)
0 0 0 0
EGTA (5) 0 0 0 0
EDTA (5) 0 0 0 0
Inhibition is expressed in percentage. Crude C. gigantea latex
protein was pre-incubated with indicated concentration of various
inhibitors. Hemorrhagic activity and proteolytic activity was
assayed as described in Section 2. The values represent meanG
SEM (nZ4).
R. Rajesh et al. / Toxicon 46 (2005) 84–92 89
toxic and causes deleterious effects. Mammalian metallo-
proteases are known to play a key role in ovulation,
embryogenesis, intimaproliferation, angiogenesis and ather-
osclerosis (Lijnen, 2002). In contrast serine proteases are
involved in intrinsic and extrinsic blood coagulation path-
ways (Davie et al., 1979). Another set of distinct proteolytic
enzymes was characterized from the latex of several plants
and they are classified as cysteine proteinases. However, the
pharmacological actions of this class of proteases are not
well established.
The latex of C. gigantea is applied to wounds to stop
bleeding and also for wound healing (Ashwani, 1999).
Indirectly, these pharmacological actions are attributed to the
proteolytic enzymes of the latex. Several cysteine proteases
are isolated and characterized from the latex of C. gigantea,
but their pharmacological actions are not known yet
(Abraham and Joshi, 1979; Senugupta et al., 1984).
The crude extract of the latex hydrolyses casein and other
substrates such as human fibrinogen and fibrin present in
the plasma clot. Among fibrinogen subunits Aa chain, which
contained no detectable carbohydrates, found to be degraded
preferentially. In contrast the Bb chain, which contained
carbohydrates, degraded more slowly than the earlier.
Fig. 7. Hemorrhagic action of crude C. gigantea latex protein and its inhibition by IAA. Two groups of mice were injected with crude latex
protein at the back of the skin with and with out IAA. After 3 h incubation mice were sacrificed by anesthesia. The dorsal surface of the skin was
removed and the inner surface was measured for hemorrhage. Saline was injected to the control group. (A) Control, (B) 75 mg latex protein, and
(C) 75 mg latex protein pre-incubated with 100 mM IAA.
R. Rajesh et al. / Toxicon 46 (2005) 84–9290
However, the g chain, which also contained carbohydrates,
was more resistant than the other two chains, but on
prolonged digestion it was also susceptible for the proteolytic
activity of the latex. The fibrinogenases, which attacks from
the carboxyl terminal end of a, b and g chains of fibrinogen
are known to prolong thrombin induced clot formation
(Salvatore et al., 1973; Evans, 1981; Civello et al., 1983). In
contrast, the fibrinogenases releasing fibrinopeptides A and/
or B from fibrinogen at the N-terminal disulfide knot of Aa
and Bb chain forms fibrin clots and are called thrombin-like
enzymes (Komori et al., 1985; Nolan et al., 1976; Stocker and
Barlow, 1976). Hydrolysis of fibrinogen by crude latex
proteases did not result in the formation offibrin clot, which is
observed for thrombin. However, latex induced the formation
of plasma clot during re-calcification. This re-calcification
data explains the role of latex in controlling the bleeding in
folk medicines and could be responsible for the procoagulant
property of the latex.
In addition, interestingly, the latex hydrolyzed all the
subunits of fibrinogen, the property that is unique to the
plasmin, which is a serine protease (Iwasaki et al., 1990). In
addition to fibrinogenolytic activity, it plays a vital role in
dissolving the fibrin clot. Thus the latex is also showing the
plasmin-like activity by dissolving fibrin clot.
Most of the proteases so far been isolated from
mammalian source and the venoms of snakes that interfere
in hemostasis and fibrinolysis are either serine or metallo-
proteases (Iwasaki et al., 1990; Jagadeesha et al., 2002).
Nevertheless the role of cysteine proteases in the various
pharmacological actions is not clearly understood. Recently
a cysteine protease, ficin derived from the plant latex of
Ficus carica shown to activate human factor X and induced
blood coagulation. This phenomenon has been attributed for
its observed pharmacological actions (Bolay, 1979). The
proteolytic activity from the latex of C. gigantea hydrolyzes
substrates such as casein, fibrinogen and crude fibrin clot.
R. Rajesh et al. / Toxicon 46 (2005) 84–92 91
All the activities were inhibited completely by IAA and not
by any other specific protease inhibitors indicating that the
crude latex contained proteases belong to the cysteine super
family.
Many venom proteases are highly toxic and induce
various pathological actions including hemorrhage in
various vital organs. The crude extract of C. gigantea
latex hydrolyses strongly all the subunits of fibrinogen and
crude fibrin clot even at lower concentration. However, at
higher concentrations the crude extract induces hemorrhage
at the site of injection. The reported procoagulant activity
and hemorrhagic activity were completely neutralized by
IAA indicating the possible direct involvement of cysteine
protease(s).
The clot dissolving property of the latex was much
stronger than the trypsin and papain enzymes and the
dissolution of blood clot is a pre-requisite in the process of
wound healing. In vivo plasmin enzymes in the presence of
matrix metalloproteases are responsible for such processes
(Lijnen, 2002). Thus procoagulant activity and clot dissol-
ving property are probably responsible for the observed
pharmacological actions of the latex. In vivo these clot
inducing and clot dissolving enzymes might act sequentially
and facilitate wound healing process that includes early
coagulation mechanism. These data strongly suggest that the
crude latex is abundant with cysteine protease(s) and are
therapeutically important in controlling bleeding and wound
healing.
Acknowledgements
We thank Prof. T.V. Gowda, Department of
Biochemistry, University of Mysore, Mysore, for his
valuable suggestions during this study. Rajesh. R and
Nataraju. A acknowledge the Council of Scientific and
Industrial Research (CSIR), New Delhi, India, for
financial assistance.
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