Asiatic Herpetological Research

December 1993

Vol. 5, pp. 117-126

Snake Venoms on Blood Coagulation, Purified
Factors
and Synthetic Chromogenic Substrates
Coagulation

Effects of Chinese

YUN ZHANG 2 YULIANG XlONG 2 AND CASSIAN BON
1 -

1

,

'Unite des Venins, Unite associee Instilul Pasteur/INSERM 285, 25, rue du Dr. Roiix, 75724 Paris Cedex 15
France*Kunming Institute of Zoology, Academia Sinica, Kunming 650107, Yunnan, China

Abstract. -We examined the action of venoms from common Chinese Crotalidae and Elapidae snakes onblood coagulation mechanisms. Procoagulant effects were observed with venoms from Agkistrodon acutus,

Trimeresurus stejnegeri, Ophiophagus luinnah and Bungarus fasciatus, the latter two only in the presence ofAfter treatment with a serine protease inhibitor (phenylmethanesulfonyl fluoride, PMSF),
Agkistrodon acutus venom lost its ability to clot purified fibrinogen but retained its capacity to clot human2+
An anticoagulant action was obtained with venoms from Trimeresurus
plasma in the absence of Ca
mucrosquamatus, Agkistrodon halys and Naja naja atra. This action was abolished after treatment with a
specific inhibitor of PLA 2 activity (p-bromophenacyl bromide, BrPBr), revealing a procoagulant action with

Ca 2+

.

.

high concentrations of treated venoms in the cases of Trimeresurus mucrosquamatus and Agkistrodon halys.
The effects of these venoms on hemostasis have been further characterized by measuring their phospholipase

A 2 activity, their ability to hydrolyze synthetic chromogenic substrates and to activate purified blood
coagulation factors (prodirombin, factor X, protein

C and plasminogen).

These venoms showed anamidolytic activity which was mainly due to serine proteases (90 to 95% of inhibition with PMSF).
Combining the observations obtained with human plasma and purified blood coagulation factors, wei) six of the eight tested Chinese venoms {i.e.: Ophiophagus hannah, Bungarus fasciatus,
Trimeresurus mucrosquamatus, Trimeresurus stejnegeri and Naja naja atra) containacutus,Agkistrodon
2+
in a Ca-dependent manner; ii) three venoms (Agkistrodon acutus,
components which activate factor

concluded Uiat:

X

Agkistrodon halys and Trimeresurus stejnegeri) contain prothrombin activators; iii) Ophiophagus hannah
venom has a weak protein C activating activity; and iv) Trimeresurus stejnegeri venom possesses
plasminogen activating activity. In addition, several of these venoms have previously been shown tocontain thrombin-like and fibrinogenolytic enzymes, anticoagulant phospholipases A 2 (PLA 2 s) and/or non

enzymatic anticoagulant components.

Key Words: Snake venoms, blood coagulation, purified blood coagulation factors, chromogenic substrates.

Introduction

irreversibly inactivated by alkylation with

PMSF.
Snake venoms are known to be a richsource of hydrolytic enzymes, mainly
proteases and phospholipases A 2 and of
non-enzymatic proteins, which induce
disorders of blood coagulation, hemorrhage
and shock (Pirkle and Markland, 1988;
Ouyang and Teng, 1972; Teng andSeegers, 1981). Many proteases acting on
different steps of the blood coagulationcascade have been purified from snake
venoms. They cleave blood coagulation
factors, either in a specific or in a nonspecific manner, and cause acceleration orretardation of blood coagulation (Pirkle andSome of theseMarkland, 1988).
proteases, such as thrombin-like enzymes(Stocker and Meier, 1988) or protein C

Other

procoagulant

or

X

or
anticoagulant proteases, like factor
atrox
from
activators
Bothrops
prothrombin

venom (Hofmann and Bon, 1987a; 1987b)
or from Echis carinatus venom (Morita andIwanaga, 1978) are insensitive to PMSF
and have been postulated to bemetalloenzymes. PLA 2 s have also beenrecognized for their anticoagulant activity,
which has been attributed to their ability toantagonize the procoagulant action ofnegatively charged phospholipids (Ouyang
et al., 1978).

better understand thepathophysiological action of snake venoms
on haemostasis, and to examine the
In

order

to

activators (Kisiel et al.,1987), are serine

use of their procoagulant orascomponents
anticoagulant

proteases which

be rapidly

andpharmacological tools, we examined themay© 1993 by Asiatic Herpetological Researchpotential

Asiatic Herpetological ResearchVol. 5 p. 118

December 1993

effects of various snake venoms on blood(Asnieres,

coagulation mechanisms in vitro, using
human plasma, purified blood coagulation
factors (fibrinogen, prothrombin, factor X,
protein C and plasminogen), and synthetic

Phenylmethanesulfonyl fluoride (PMSF)
and /?-bromophenacyl bromide (BrPBr)
were purchased from Sigma (St. Louis,
MO, USA). All other reagents were of thechromogenic substrates. We examined indetail venoms from the eight most common

highest purity available.

Chemical modifications

venomous snakes in China; four belonging
to the Elapidae family {Ophiophagus
hannah, Naja naja atra, Bungarus fasciatus

and Bungarus multicinctus) and the otherFrance).

Inactivationof serine proteases by

PMSF was performed in 50 mM Tris-HCl,four to the Crotalidae family (TrimeresuruspH 7.8. Venom samples (2 mg-ml" ) weremucrosquamatus, Trimeresurus stejnegeri,
Agkistrodon halys and Agkistrodon

incubated at 37°C for two hours with 5
in
PMSF (stock solution:
0.1
PLA
Inactivationof2s
dimethylsulfoxid).
was carried out in the same buffer by

acutus).

Methods

1

M

mM

incubating the venom (1 mg-ml" ) at 37°C
BrPBr (stock
for one hour with 2
1

Venoms were supplied by the Kunming

solution:

0.1

mM
M in acetone).

TreatedInstitute ofZoology (Academia Sinica,
China). The venoms were collected fromvenom samples were then dialyzed for 4 to
8 hours against large volumes of the same

snakes living in the southern provinces ofbuffer.

China and stored desiccated. They were
Tris-HCl buffer, pH
dissolved in 50
7.8, at a concentration of 1 mg-ml" andwere used immediately.

mM

Chromogenic assays

1

Bovine factor X, human prothrombin
and human Glu-plasminogen were obtained
from Sigma (St. Louis, MO, USA).
Human protein C was obtained from
Diagnostica Stago (Asnieres, France).
Human fibrinogen (grade L) from Kabi
Vitrum (Stockholm, Sweden) was treatedwith diisopropylfluorophosphate according
to the instructions of the manufacturer, inorder to irreversibly inactivate traces ofthrombin or other blood coagulation
factors.

Platelet-poor human plasma was

Amidolytic activity was measured with a
Kontron spectrophotometer in 1 cm pathlength plastic cuvettes.
Assays were
Tris-HCl,performed in 500 ml of 50

mM

pH

7.8,

containing

theappropriate

chromogenic substrate (0.2 mM). The
reactions was initiated by addition of thesample to be tested (5 mg-ml" to 100
mg-ml" final concentrations) and theformation of p-nitroanilide was monitored
at 405 nm.
The amount of substratewascalculated using a molarhydrolyzed
11,extinction coefficient of 10,000 M^-cm"

1for free p-nitroanilide.

the supernatant of human blood mixed with

1/10 volume of 3.8% sodium citrate andcentrifuged at 3000 rpm for 15 min. Poolsof normal citrated plasma obtained from 510 healthy donors were stored at -20°C.

Determination ofPLA 2 activity

PLA

2

activity

was determined by thetitrimetric method described by Desnuelle et(1955), according to the procedure ofRadvanyi and Bon (1982).

al.

Chromogenic substrates H-D-Phe-PipArg-pNA (S-2238), H-D-Val-Leu-LyspNA (S-2251), H-D-Val-Leu-Arg-pNA (S2266), H-D-Pro-Phe-Arg-pNA (S-2302)
and Bz-Ile-Glu-Gly-Arg-pNA (S-2222)
were obtained from Kabi Vitrum^Stockholm, Sweden) and the chromogenic
substrate H-D-Lys(Cbo)-Pro-Arg-pNA
365-25) was from Diagnostica Stago

Effects of the venoms on blood coagulation

Citrated platelet-poor

human plasma(200 ml) was incubated at 37°C for 1 min,then a 20 ml aliquot of diluted venom
samples was added and clotting time wasrecorded.

Insome cases, 5 ml of CaCl 2

Asiatic Herpetological Research

December 1993/B.

multicinctus

6-

5""

4

i

i

1

1

i

r

"

-

0-1

.001.01

.1

1—
1

—r
10

100

.001

.01

Vol. 5 p. 119

Asiatic Herpetological ResearchVol. 5 p. 120

December 1993

TABLE 1. Phospholipase A activity of the venoms from the common Chinese venomous snakes
2

1(unmolmiir'mg' ).
native venom

Agkistrodon acutus

Agkistrodon halys
Trimeresurus mucrosquamatiis
Trimeresurus stejnegeri

Ophiophagus harmah

Bungarus fascia tus

Bungarus multicinctus
Naja naja

37±2

treated venoms

December 1993

Asiatic Herpetological Research

TABLE 2. Amidolytic activity (nmol-mirr'-mg"

Venom

1

)

of venoms from Chinese snakes.

Vol. 5 p. 121

Asiatic Herpetological ResearchVol. 5 p. 122

fibrinogen in the presence of thrombin(result not shown), indicating that the
anticoagulant action is not due tofibrinogenolysis. The anticoagulant effectof Naja naja atra venom was completely
and irreversibly prevented by treatment
with BrPBr (Figure 1) which inhibited
PLA 2 activity of the venom (Table 1). This
activity is therefore due to one or several

anticoagulant PLA 2 s, as reported in the caseof many other snake venoms.

The effects of venoms from Chinese
Crotalidae snakes on blood coagulation
mechanisms (Figure 2) appeared much
more complex than those observed with
Elapidae venoms (Figure 1). The venoms
from Agkistrodon acutus and from
Trimeresurus stejnegeri were characterized
by a procoagulant action, observed both in
the presence and in the absence of Ca2+
(Figure 2). These venoms also clotted
purified fibrinogen in a calcium-

independent manner (result not shown),
indicating that they contain potent
thrombin-like enzymes, in agreement withthe observations reported by Ouyang e t al.
(1971) and by Liu and Xiong (1990).
Treatment of Agkistrodon acutus venom
with PMSF completely abolished its ability
to clot purified fibrinogen (result notshown) and strongly reduced its
procoagulant action (Figure 2), as expected
since this effect is mainly due to thrombin-

However, PMSFtreated Agkistrodon acutus venom showed
a complex action, an anticoagulant activity
at 10 mg-ml"
and a clotting activity athigher concentrations (Figure 2),
suggesting that it contains otherprocoagulant components, in addition tothrombin-like enzymes. The anticoagulant
like serine proteases.

1,

effect of PMSF-treated Agkistrodon acutus

venom is consistent with the presence of an
anticoagulant protein of 26 kD, which isdevoid of enzymatic activity and which
prevents the formation of thrombin bybinding to the prothrombin activation

complex (Teng and Seegers, 1981).

Inaddition to this non-enzymatic component,

Agkistrodon acutus venom might contain
anticoagulant PLA 2 s, since treatment with
BrPBr (Table 1) significantly increased its
procoagulant effect (Figure 2).

December 1993

Agkistrodon halys and Trimeresurus

mucrosquamatus

venomswerecharacterized by an anticoagulant action

which was completely abolishedafter

treatment with BrPBr, but not with PMSF
(Figure 2). This indicates that they containpotent anticoagulant PLA 2 s, as previously
reported for Trimeresurus mucrosquamatus(Ouyang et al., 1978) and for Agkistrodon
In fact,
halys (Chen et al., 1987).
treatment of Agkistrodon halys andTrimeresurus mucrosquamatus venoms
with BrPBr suppressed the anticoagulant
activity of these venoms and revealed aweak procoagulant activity (Figure 2),
indicating the presence of procoagulant
components. Further, this procoagulant
action was observed only in the presence of

Ca2+, suggesting that the procoagulant
components are able to convert
prothrombin into thrombin or to activate
factor X. High concentrations (100 mg-ml"') of Agkistrodon halys venom were able toclot purified fibrinogen (result not shown).
This may be explained by the presence of athrombin-like enzyme, which has been
purified (Guan et al., 1988) but the level ofthis enzyme in the venom is low and its
activity is weak.

Action of the venoms on purified blood
coagulation factors

We determined me amidolytic activity ofthe venoms on a number of chromogenic

substrates, classically used for assaying
blood coagulation factors: thrombin (S-2238),

factorXa(S-2222),

activated

protein C (CBS65-25), kallikrein (S-2302

S-2266) and plasmin (S-2251).
Assays were performed with native,
PMSF-treated and BrPBr-treated venoms(Table 2). The venoms from Elapidae
andsnakes did not present detectable activities,
with the exception of the venom from

Ophiophagus hannah which hydrolysed
several substrates (Table 2). The venoms
from Crotalidae snakes exhibited significant
activities towards most chromogenic

but with important species
differences:the venoms fromsubstrates,

Trimeresurus snakes presented muchhigher activities than those of Agkistrodonsnakes and the venom from Agkistrodon

Asiatic Herpetological Research

December 1993

Vol. 5 p. 123

c
E
E

c

o

o

.001

.01

1

00

.001

.01

1

00

100

,001

.01

1

00

10 i

.001

.01

Venom concentration()jg/ml)

Venom concentration((ig/ml)

Figure 2. Effects of Crotalidae snake venoms on blood coagulation. Citrated platelet-poor human plasma (200ml) was incubated at 37°C for 1 min then the sample to be tested (20 ml) was added simultaneously withfinal concentration; closed symbols) or without calcium (opencalcium (5 ml of 0.45
CaCl2', 10MmM

symbols and dashed lines). Native venom (9; O); PMSF-treated venom (A; /\or p-bromophenacyl
bromide-treated venom ( I: D). The values are the mean of triplicates, standard errors being 10% of the values.

Vol. 5 p. 124

Asiatic Herpetological Research

TABLE 3:

Activation of factor X, prothrombin, protein C and plasminogen by thecommon Chinese snakes.

December 1993

venoms from

Asiatic Herpetological Research

December 1993

Vol. 5 p. 125

appeared to be due to (a) serine protease(s),since it was completely abolished byvenoms on blood coagulation mechanisms.

PMSF.

demonstrated the presence ofprothrombin activators in Agkistrodonandacutus,halys
Agkistrodon
Trimeresurus stejnegeri venoms, as well asof factor X activators in Agkistrodon
acutus, Trimeresurus mucrosquamatus,
Trimeresurus stejnegeri, Ophiophagus
hannah, Bungarus fasciatus and Naja naja
atra venoms.
These studies further
indicated that prothrombin and factor X
activators from Crotalidae (Agkistrodon
acutus, Agkistrodon halys, Trimeresurus
mucrosquamatus and Trimeresurus

We observed no correlation between theamidolytic activity of snake venoms,
measured with the chromogenic thrombin
substrate (S-2238) and their thrombin-like
activity as determined by their ability to clot
fibrinogen (result not shown). Similarly,
there was no correlation between activationof prothrombin (Table 3) and the amidolytic
activity measured with factor Xa substrate
S-2222 (Table 2). These results emphasize

the differences which exist between thesubstrate specificity of human coagulation
factors and snake venom activators, and theexistence in snake venoms of proteases

which are able to hydrolyse chromogenic
substrates without possessing the capacity
toactivate

the

blood

correspondingWe

stejnegeri) venoms are PMSF-insensitive,
and Ca2+-dependent in the case of factor

X

These activators might beandsimilar to those of Bothops atrox
venomsetrussellial.,
1976;(KisielViperaHoffman and Bon, 1987a; 1987b).
activators.

Interestingly, factor

coagulation factors.

X activators found in

Elapidae venoms (Ophiophagus hannah andBungarus fasciatus) were inactivated after

Discussion

u-eatment with PMSF, suggesting that theyIn the present study, we found that,except for Bungarus multicinctus, thevenoms from common Chinese venomous
snakes (Ophiophagus hannah, Naja najaatra, Bungarus fasciatus, Trimeresurusstejnegeri, Trimeresurus mucrosquamatus,
Agkistrodon acutus and Agkistrodon halys)possessed

procoagulant

anticoagulant activities.

An

and/orinmight be serine proteases.

We also showed the existence of aproteincombined effects of several procoagulant
Inand anticoagulant components.
nativetheeffectsofparticular, comparing
venom with those of venom in which PLA 2Agkistrodon halys venom, similar to that
descrided by Chen et al. (1987), which
effect

of

in

the

venom

of

activity
Ophiophagus hannah, although
was low compared to that found in theitsvenom of Agkistrodon contortrix contortrix(Kisiel et al., 1987; Orthneret al., 1988).

These studies also revealed the firstevidence of the existence of a plasminogenactivator. Trimeresurus stejnegeri contains
a PMSF-sensitive plasminogen activator.
Its biochemical structure and mechanism ofaction are currently under investigation.

activity has been blocked revealed thepresence of anticoagulant PLA 2 s intheactivator

vitro

analysis of these venoms indicated that theiraction on blood coagulation results from themaskedCprocoagulant

component(s). We also showed that theprocoagulant action of Agkistrodon acutus
venoms does not result only from thepreviously described thrombin-like enzyme(Ouyang et al., 1971), but also from at least
two other components, a PMSF-insensitive
prothrombin activator and a calciumThis
dependent factor X activator.
illustrates the complexity of action of the

Acknowledgments
This research was supported in part byfunds from the Direction des Recherches etEtudes Techniques. Zhang Yun is therecipient of a fellowship from the
Fondation pour la Recherche Medicale.

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