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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