Iranian Journal of Basic Medical Sciences

ijbms.mums.ac.ir

Blood Coagulation Induced by Iranian Saw-Scaled Viper

(Echis Carinatus) Venom: Identification, Purification and
Characterization of a Prothrombin Activator
Mahdi Babaie 1, Hossein Salmanizadeh 1, Hossein Zolfagharian 2*
1 Young Researches and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute, Karaj, Iran

ARTICLE INFO ABSTRACT

Objective(s): Echis carinatus is one of the venomous snakes in Iran. The venom of Iranian Echis

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Article type:
carinatus is a rich source of protein with various factors affecting the plasma protein and blood
Original article
coagulation factor. Some of these proteins exhibit types of enzymatic activities. However, other

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Article history: items are proteins with no enzymatic activity.
Received: Feb 12, 2013 Materials and Methods: In order to study the mechanism and effect of the venom on human plasma
Accepted: Jun 14, 2013 proteins, the present study has evaluated the effect of crude venom and all fractions. A

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procoagulant factor (prothrombin activator) was isolated from the venom of the Iranian snake
Keywords: Echis carinatus with a combination of gel filtration (Sephadex G-75), ion-exchange
Blood coagulation chromatography (DEAE- Sepharose) and reverse phase HPLC. Furthermore, proteolytic activity of

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Chromatography the crude venom and all fractions on blood coagulation factors such as prothrombin time (PT) was
Iranian Echis carinatus studied.
Prothrombin time Results: In the present study, the PT test was reduced from 13.4 s to 8.6 s when human plasma was

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Protrombin activator treated with crude venom (concentraion of venom was 1 mg/ml). The purified procoagulant factor
revealed a single protein band in SDS polyacrylamide electrophoresis under reducing conditions
and its molecular weight was estimated at about 65 kDa. A single-band protein showed fragment
patterns similar to those generated by the group A prothrombin activators, which convert

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prothrombin into meizothrombin independent of the prothrombinase complex.
Conclusion: This study showed that the fraction which separated from Iranian snake Echis

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carinatus venom can be a prothrombin activators. It can be concluded that this fraction is a
procoagulant factor.
►Please cite this paper as:

Introduction
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Babaie M, Salmanizadeh H, Zolfagharian H, Blood Coagulation Induced by Iranian Saw-Scaled Viper (Echis Carinatus) Venom: Identification,
Purification and Characterization of a Prothrombin Activator. Iran J Basic Med Sci; 2013; 16: 1145-1150.
prothrombin into thrombin (6). Prothrombin is the

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protein which is broken in plasma by ecarin. In fact,
Snake venom, a complex mixture principally
this protein cleaves the bond in prothrombin and

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composed of proteins and peptides, exhibits diverse
produces meizothrombin, which is converted into
biological activities that affect several vital
α-thrombin by autolysis (7).
systems (1).

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The conversion of the prothrombin into thrombin
Echis carinatus (Saw scaled viper) is a venomous
is one of the central reactions of blood coagulation
snake found in the desert regions of Iran. The venom
(8, 9). The physiological activation of prothrombin to
of E. carinatus, a member of the Viperidae family,
affects blood coagulation due to hemostatically the serine proteinase α-thrombin is catalysed by
active enzymes with procoagulant and anticoagulant prothrombinase complex consisting of the serine
activity (2, 3). proteinase, factor Xa, cofactor Va and Ca 2+.
The venom of E. carinatus affects the blood Membranes containing anionic phospholipids are
circulation. This venom is very toxic causing severe essential for the optimal function of this enzyme
tissue and organ damage. The venom of E. carinatus complex (10, 11). However, the rate of activation is
is rich in proteins and peptides effective on the five orders of magnitude lower than the activation by
hemostatic system, i.e., its acts against some types of prothrombinase complex (12), and the mechanism of
factors involving coagulation and fibrinolysis (4, 5). cleavage proceeds through prethrombin-2 rather
E. carinatus snake venom especially contains than through meizothrombin (13).
proteins affecting the transformation of the The venom of Viperidae presents a high level of

*Corresponding author: Hossein Zolfagharian. Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute,
Karaj, Iran. Tel: +98- 2634570038; +98- 9123451699; email: [email protected]

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Blood Coagulation Induced by Echis carinatus Venom Babaie et al

haemorrhagic, coagulant and proteolytic activities The fractions exhibiting proguaolant activity in the
(14). Proteins effective on blood coagulation and previous step were pooled and dialyzed overnight at
existing in the snake venom are classified based on 4°C and applied on HPLC column, C18 (H2O, 0.1%
their ability to lengthen or shorten the clotting trifluoroacetic acid), and eluted with a concentration
process into coagulation and anticoagulation gradient of solvent B (acetonitrile, 0.1%
proteins (15). trifluoroacetic acid) from 0 to 100%, at a flow rate of
The aim of the present investigation was to study 0.3 ml/min during 55 min. The peaks were
the purification and characterization of monitored at 280 nm (17).
porothrombin activator (procoagulant factor) from
the Iranian E. carinatus venom and to evaluate the Determination of molecular weights
procoagulant activity on in vitro human plasma. Electrophoresis on 12/5% polyacrylamide gel
was performed according to the method of Laemmli
Materials and Methods (18). Samples of the crude venom and its fractions
Material were lauded and the molecular weights of protein
The lyophilized E. carinatus venom was obtained were determined under reduced conditions.
from the Department of Venomous Animals and
Antivenom Production, Razi Vaccine and Serum Prothrombin time assay
For the PT test, 200 μl of the PT reagent was

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Research Institute Karaj, Iran. Sephadex G-75, DEAE-
Sepharose, and C18 columns were purchased from added to 100 μl of citrated plasma (incubated for 1

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the Pharmacia company (Sweden). CaCl2 and PT kits min at 37°C). The time from the plasma-reagent
were purchased from the Fisher Diagnostics (USA). mixing to the clot formation was defined as the PT

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Protein markers were obtained from BioRad and clotting time was recorded (19). The PT test was
(Hercules, USA). Other reagents and chemicals were performed for different concentrations of crude
of analytical grade from Fluka and Merck. venom and its fractions.

Methods
Blood collection
Normal plasma from 20 healthy donors without
any history of bleeding or thrombosis was collected
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Coagulant activity
Normal plasma comprised mixed samples from
20 healthy donors. It was briefly incubated at 37°C
and sample aliquots containing some concentration

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from a private clinical laboratory. The citrated blood of coagulant fractions or subfraction (50 µg/ml)
was centrifuged for 15 min at 3,000 rpm, to get clear were added, mixed and shaken and PT was then

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plasma. Finally, the PT was estimated. recorded.

Protein determination Results

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The total protein of crude venom of E. carinatus, The present study showed that the crude venom
and its fractions were determined by Lowry method of E. carinatus can accelerate the blood coagulation

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(16). pathway. Our results indicated that as the
concentration of venom increases, the PT of plasma

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Purification and isolation of prothrombin decreased (Table 1).
activator According to the Table 1, when the concentrations
Purification of the prothrombin activator was of venom increased from 0.01 to 1 mg/ml, the

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performed in three steps. Lyophilized crude venom
of E. carinatus (50 mg) was dissolved in 4 ml of
starting buffer (20 mM ammonium acetate, pH 6.8)
and centrifuged at 3,000 rpm for 15 min, 4°C. The
supernatant was filtered on a 0.45 microfilter to
remove all insoluble materials. The supernatant was
then applied into a Superdex G-75 column and eluted
clotting time of plasma reduced from 13.4 to 8.6 sec.

Table 1. PT value for different concentration of E. carinatus crude

venom

Concentrate of
venom (mg/ml)
Average of PT (S) * Preamble

0.01 21 (P < 0.001) Clot is tiny

with the same buffer. (150 × 3 cm). Fractions were 0.1 12.25 (P < 0.005) increased clot size
collected at 4°C and their absorbances were 1 8.6 (P < 0.001) Clot complete
recorded at 280 nm. The fractions with proguaolant Control 13.4 (P < 0.005) Clot complete
activity were pooled, lyophilized and dialyzed *n = 8
against 50 mM Tris-HCl, pH 8.2 buffers. The dialyzed Total protein of the venom = 48300 μg/ml.
sample was centrifuged at 3000 rpm to clear the Control = 100 μl of citrated plasma + 200 μl of the PT reagent +
precipitated proteins. For further purification, the Normal saline (Instead of venom).
Test = 100 μl of citrated plasma + 200 μl of the PT reagent +
supernatant was loaded into ion exchange column different concentrate of venom.
(DEAE-Sepharose) and equilibrated with 50 mM
Tris-HCl buffer, pH 8.2 and eluted with a liner
gradient of Nacl concentration from 0.0 to 0.5 mM.

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Babaie et al Blood Coagulation Induced by Echis carinatus Venom

Table 3. PT value for sub-fractions of E. carinatus venom

Fractions PT *
Fraction F1A 14 sec
Fraction F1B 8 sec
Fraction F1C 70 sec
Fraction F1D 52 sec
Fraction F1E 90 sec
Fraction F1F 56 sec
Fraction F1G More than 300 sec
Fraction F1H 95 sec
* n=4, F1B (P-Value < 0.05)
PT: prothrombin time

Figure 1. Purification of crude venom of Echis carinatus by Table 4. Prothrombin Time for fractions obtained from HPLC
Sephadex G-75
Fractions Average of PT *
Fraction F1B1 More than 300 sec
Table 2. PT value for fractions of IEc crude venom Fraction F1B2 More than 300 sec
Fractions PT * Fraction F1B3 More than 300 sec
F1 12.3 sec Fraction F1B4 3 sec

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F2 35.5 sec Fraction F1B5 More than 300 sec
F3 More than 300 sec * n=4, F1B4 (P-Value < 0.05)

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PT: prothrombin time
* n=4, normal PT=13.4, F1 (P < 0.05) and F2 (P < 0.01)
PT: prothrombin time

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Table 5. Summerized PT value and total protein (crude venom, F 1,
F1B, F1B4)
Step Protein

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Venom 48.3 mg/ml
F1 387.77 µg/ml

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F1B 130 µg/ml
F1B4 26 µg/ml
PT: prothrombin time

v e According to the Table 1, when the

concentrations of venom increased from 0.01 to 1
mg/ml, the clotting time of plasma reduced from

h i 13.4 to 8.6 sec.

Purification, isolation and characterization of

prothrombin activator

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Figure 2. Purification of F1 by DEAE-Sepharose chromatography
As it is shown in the Figure 1, the three fractions
(F1 to F3) were obtained By Sephadex G-75.
Prothrombin time value was estimated for all the
fractions. Our observation showed that the PT value

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for F1 is less than other fractions and this fraction can
be considered as a procoagulant factor (Table 2).
Further purification was performed by ion
exchange chromatography DEAE-Sepharose. In this
step, eight fractions were separated from F 1A to F1H
(Figure 2), out of eight fractions, only F1B showed
procoagulant activity (Table 3).
The F1B was pooled, dialyzed and applied to a
C18 reversed-phase HPLC column. Our results
revealed that five peaks from F1B1 to F1B5 were
isolated (Figure 3) and out of five fractions, only F 1B4
showed coagulant activity (Table 4).
Our results summarized in the Table 5, which
showed that the PT value significantly decreased in
the F1B4 as compared with PT value of the crude
venom.
Figure 3. HPLC of F1B fraction obtained from DEAE-Sepharose
chromatography

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Blood Coagulation Induced by Echis carinatus Venom Babaie et al

Discussion
This study has investigated the venom of Iranian
E. carinatus which contains a strong procoagulant
factor enabling to activate the prothrombin. The
functional properties of the E. carinatus prothrombin
activator are similar to ecarin, the first prothrombin
activator which was recently discovered to be
present in the venom from E. carinatus (20). The
venoms of many E. species are able to convert
prothrombin into thrombin, either directly or
indirectly (21).
Under in vitro conditions, this venom also
displays coagulation properties and increases the
blood coagulation cascade. Crude venom from the
Iranian snake E. carinatus was selected and assayed
with PT test. Our results indicated that the Iranian E.

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carinatus venom has a procogulant activity and is
able to coagulate human plasma rapidly (Table 1),

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therefore it may be concluded that IEc venom
contains procoagulant factors.
The present study reports an efficient and simple
procedure for purification and isolation of
procoagulant factor from IEc venom. The fraction

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F1B4 was isolated from IEC venom by a combination
of several methods. Our results revealed that three
peaks from F1B1 to F1B5 were isolated. In addition,
out of five fractions, only F1B4 showed coagulant
activity (Table 4). The molecular weight of this

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purified fraction was approximately estimated to be
56 kDa (Figure 4C). Our observation showed that the

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molecular weight of F1B4 is similar to prothrombin

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activator enzymes which have been already reported
(20). Therefore, this coagulant factor may belong to
the intermediate-molecular-weight group of these

c h factors.
By performing the prothrombin Time test on
human plasma, the blood coagulation time on

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fraction F1 showed the least coagulation time and
fraction F3 displayed the highest coagulation time.
The total protein of crude venom is 48.3 mg/ml and

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the PT value is 8.6 s but in the hyper purified fraction
with reducing amount of total protein (26.0 µg/ml),
the PT value (3 s) also significantly decreased. It may
be suggested that with low amount of total protein
Figure 4. SDS-PAGE pattern of crude venom and its fraction:
A: Crude venom and its fractions; B: Subfractions of F1; C: Fraction
the PT value decreases.
of F1B4 Some procoagulant factors, along with its
molecular weights, have been reported by Howes JM
Purity and determination of molecular weight et al in addition to the effects of three novel metallo-
Crude venom and all fractions were analyzed by proteinases (weighting 56 kDa) from the venom of the
SDS-PAGE. As it is shown in the SDS-PAGE pattern, West African saw-scaled viper, E. ocellatus on blood
the molecular weight of crude venom and all of the coagulation and platelets (22). Daisuke Yamada et al
fractions were estimated (Figure 4A, 4B and 4C). The isolated and characterized the carinactivase, a novel
molecular weights from the snake venom ranged prothrombin activator from E. carinatus Venom with
from 6.5 to 250 kDa and the molecular weight of 62 kDa (23).
procoagulant factor was approximately 56 kDa. Mikarin is the first group of IA prothrombin
According to the Figure 4C, a single band of F1B4 activator identified in the venom of a viperidae
indicates the purity of this protein. snake. In the case of prothrombin activator, it
exhibited prothrombin activation, which was similar

1148 Iran J Basic Med Sci, Vol. 16, No. 11, Nov 2013

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Babaie et al Blood Coagulation Induced by Echis carinatus Venom

to the other group IA prothrombin activators, such 5. Fonseka CL, Jeevagan V, Gnanathasan CA. Life
as ecarin from E. Carinatus (24), aharin from threatening intracerebral haemorrhage following
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E. carinatus including the Iranian E. carinatus is one Clotting Mechanism, and it’s Function as a
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Acknowledgment
The present research project was conducted
under the supervision of Razi Vaccine and Serum
Research Institute and the results described in this
paper were part of the student thesis, which was
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and the intermediates of activation. Biochem Biophys
Res Commun 1973; 54:155-160.

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14. Maruyama M, Kamiguti AS, Tomy SC, Antonio LC,
Sugiki M, Mihara H. Prothrombin and factor X
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venom. Ann Trop Med Parasitol 1992; 86:549-556.
performed with supervision of Dr Hossein 15. Casewell NR, Harrison RA, Wüster W, Wagstaff
Zolfagharian and a scientific collaboration of Razi SC. Comparative venom gland transcriptome surveys
Institute and the University. This Research Project
was sponsored by Razi Vaccine and Serum Research
Institute, Karaj, Iran.

i v of the saw-scaled vipers (Viperidae: Echis) reveal

substantial intra-family gene diversity and novel
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Authors would like to thank all the members of the
Protein measurement with the Folin phenol reagent. J
Department of Venomous Animals and Antivenom
Biol Chem 1951; 193:265-275.

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Production. Authors would also like to thank Mr Askari 17. Ghorbanpur M, Zare Mirakabadi A, Zokaee F,
in enzyme section and Ms. Khame chiyan in Zolfagharian H, Rabiei H. Purification and partial

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Department of Serum Production for their great help. characterization of a coagulant serine protease from
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