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Introduction
Snake venoms are complex mixtures containing many
different biologically active proteins and peptides. A number
of these proteins affect the mammalian coagulation system
by cleaving limited bonds in the blood coagulation factors.
Factor V, factor X, prothrombin activators, and
thrombin-like enzymes have been separated from different snake
venoms[1_3]. Among those, snake venoms of the Viperinae
family have been reported to contain strong procoagulants.
Because of their biological activities, some of these venom
proteins are useful for basic studies of hemostasis and
thrombosis and for pharmacological and clinical
applications[16]. Daboia russelli
siamensis is widely found over southern China, central and southern Myanmar, and central Thailand.
Although we have known that the venom of Daboia russelli
siamensis also contains some hemostatic fractions, studies
about the hemostatic effect and procoagulant mechanism of
Daboia russelli siamensis (Myanmar) venom are minimal.
In the present investigation, we purified the hemostatic
fraction FIa from Daboia russelli siamensis (Myanmar)
venom, determined the hemostatic activities of FIa and
investigated the mechanism of hemostasia and the potential
application of FIa.
Materials and methods
Snake venom Daboia russelli
siamensis (Myanmar) venom was purchased from Guangzhou Medical College
(Guangzhou, China) and lyophilized and stored in a
desiccator.
Reagents CM-Sephadex C-50, Sephadex G-75, and
Superdex 75 were purchased from Pharmacia (Uppasala,
Sweden). Human fibrinogen and thrombin were purchased
from Sigma (St Louis, MO, USA). Factor X, factor Xa,
prothrombin, and the reagent packs for the activity assays
of factor Xa, prothrombin, and thrombin were purchased from
Merck (Darmstadt, Germany). All other reagents were of
analytical grade and obtained from commercial sources
(Guangzhou, China).
Purification of FIa from Daboia russelli siamensis
(Myanmar) venom
CM-Sephadex C-50 ion exchange chromatography
Daboia russelli siamensis (Myanmar) venom (1.0 g) was
dissolved in 0.5 mol/L ammonium acetate (pH 5.8), and the
supernatant was applied to a column (2.0 cm×80 cm) of
CM-Sephadex C-50. The fractions were eluted with a linear
gradient consisting of 0.5 mol/L ammonium acetate (pH 5.8) as the
starting buffer and 1 mol/L ammonium acetate (pH 8.0) as the
limit buffer. The absorbance of the eluates was measured
and then the hemostatic fraction was dialyzed and lyophilized.
Sephadex G-75 gel filtration Gel filtration was
performed on a Sephadex G-75 column (2.0 cm×100 cm)
equilibrated with 0.02 mol/L sodium phosphate buffer (pH 7.4).
The first fraction (FI, 100 mg) from CM-Sphadex C-50
chromatography was eluted with the same buffer.
Superdex 75 gel filtration Gel filtration was performed
on a Superdex 75 column (1.0 cm×80 cm) equilibrated with
0.02 mol/L sodium phosphate buffer (pH 7.4). The first
fraction (FIa, 30 mg) from Sephadex G-75 gel filtration by gel
filtration was eluted with the same buffer.
Molecular weight and isoelectric point determination
SDS-PAGE was performed according to the method of
Laemmli[17]. The molecular weight standards were
MBP-G-galactosidase (175 000), MBP-paramyosin (83 000), glutamic
dehydrogenase (62 000), aldolase (47 500), triosephosphate
isomerase (32 500), G-lactoglobulin A (25 000), lysozyme
(16 500), and aprotinin (6 500). Isoelectrofocusing_PAGE was
carried out by the Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences (Shanghai, China), with a pH gradient of
3_10 generated by ampholine (pH 3_10; Amersham Biosciences,
Uppsala, Sweden).
Measurement of hemostatic activities Three different
groups were established at various concentration intervals.
The FIa groups were given 0.5, 0.25, 0.125, 0.625, and 0.3125
mg/100 µL and each concentration was tested 6 times.
Thrombin was used as a positive control and 25, 12.5,
6.25, 3.125, 1.5625, 0.78125, and 0.390625 U/100 µL thrombin
was given. Each concentration was also tested 6 times.
Normal saline was used as a negative control.
The hemostatic activities of thrombin and FIa were
measured according to the coagulation time determined by the
method of Williams and Esnouf[4]. The citrated plasma (100
µL) was incubated with 100 µL of 0.01 mol/L Tris-HC1 buffer
(pH 7.3) containing 0.15 mol/L NaCl at 37 °C for 3 min. 100 µL
of 0.05 mol/L CaCl2 plus thrombin (100 µL) or FIa (100 µL)
were added to the pre-incubation mixture respectively. The
clotting time of the plasma was then recorded.
Effect of FIa on human blood factor X Using the method
of Gowda et al[6], the activation of purified human factor X
was followed by using the FXa differential chromogenic
substance. Purified human blood factor X (100 U) was
incubated at 37 °C in 50 mmol/L Tris-HCl (pH 7.4) containing 0.1
mol/L NaCl, 0.01 mol/L CaCl2, and 12 µg of FIa in a total
reaction volume of 500 µL. Aliquots (50 µL) were removed at
various times. 50 µL of FXa differential chromogenic
substance solution [dissolved in 50 mmol/L Tris-HCl (pH 7.4)
containing 0.1 mol/L NaCl (final concentration of 0.2
mmol/L) was added into the aliquots. The light absorbance was
recorded at 405 nm. FXa was used as a positive control and
normal saline was used as a negative control.
Effect of FIa on human prothrombin Using the method
of Hofmann and Bon[7], the activation of the purified
prothrombin was followed by using the thrombin differential
chromogenic substance. Purified human prothrombin (6.67
µg/µL) was incubated at 37 °C in 50 mmol/L Tris-HCl (pH 7.4)
containing 0.1 mmol/L NaCl and 10 mmol/L
CaCl2 with FIa. Aliquots (50 µL) were removed at various times. 450 µL of
thrombin differential chromogenic substance solution
[dissolved in 50 mmol/L Tris-HCl (pH 7.4) containing 0.1
mol/L NaCl] was added into the aliquots. The light absorbance
was recorded at 405 nm. Thrombin was used as a positive
control and normal saline was used as a negative control.
Effect of FIa on fibrinogen Using the method of Gao
et al[8], fibrinogen-clotting activity was determined by
mixing 20 µL of FIa (concentrations were 0.5, 0.25, 0.125,
0.0625, and 0.02125 mg/100 µL, respectively) with 200 µL of
human fibrinogen solution (3 mg/mL) in 50 mmol/L Tris-HCl
buffer (pH 7.4) and 0.1 mol/L NaCl (containing 10 mmol/L
CaCl2) and incubated at 37 °C. The clotting time was recorded.
Thrombin was used as a positive control and normal saline
was used as a negative control.
Results
The isolation of FIa was achieved by using 3 steps of
purification (Figure 1A_1C). Ion exchange chromatography
of crude venom using a column of CM-Sephadex C-50 yielded
11 fractions (Figure 1A). The first fraction (FI) had the
highest hemostatic activity. Its hemostatic activity, expressed
by the thrombin coagulation time, was 430±5 s/31.25 µg
(n=6). Further chromatography of FI using a column of Sephadex
G-75 resulted in 3 fractions: Fa, Fb, and Fc (Figure 1B).
The hemostatic activity of Fa was 350±5 s/31.25 µg
(n=6), which was higher than that of Fb and Fc. The collection of the
sharp peak of Fa was then applied to a Superdex 75 column.
The single peak obtained was named FIa (Figure 1C). The
hemostatic activity of FIa was 290±5 s/31.25 µg
(n=6). From 1 g of crude venom, 10.75 mg of FIa was obtained.
FIa was found to be electrophoretically homogeneous
by SDS-PAGE (Figure 2) and isoelectric focusing (Figure 3).
SDS-PAGE analysis gave a calculated molecular weight of
34 479. Isoelctric focusing studies indicated that FIa was a
neutral protein with a pI of 7.2.
The hemostatic effect of purified enzyme FIa and
thrombin was determined by coagulation time. The clotting time
of normal human plasma was 960±5 s (n=6). FIa (100 µL) at
several different dosages, 0.5, 0.25, 0.125, 0.0625, and 0.03125
mg, was applied. The clotting times of FIa were 60±5 s,
100±5 s, 142±3 s, 180±4 s, and 290±2 s, respectively
(n=6). It was shown the clotting times increased with the decreased
dosages (Figure 4A). The hemostatic effect of thrombin
(100 µL) at different dosages (5, 12.5, 6.25, 3.125, 1.5625,
0.78125, and 0.390625 u, was also determined. The clotting
times of thrombin were 6.88±0.1 s, 12.13±0.15 s, 16.52±0.08 s,
26.56±0.21 s, 64±1.2 s, 89±1.4 s, and 146±3 s, respectively
(n=6). It was shown that the clotting times also increased
with the decreased dosages of thrombin (Figure 4B).
Purified enzyme FIa from Daboia russelli
siamensis (Myanmar) venom readily cleaves a number of commercially
available chromogenic substrates that have been designed
for human blood factor X. The parameters for chromogenic
substrate conversion by the venom activator are higher than
by normal saline (Figure 5). It was shown that FIa can active
factor X to factor Xa. However, in the test of chromogenic
substrates to prothrombin, the parameters of FIa had no
change compared to the normal saline group (Figure 6). It
showed that FIa had no catalytic efficiency on protrombin.
In the test of fibrinogen-clotting activity, we found that
FIa at dosages of up to 0.5 mg/100 µL could not coagulate
human fibrinogen, which was the same with the normal
saline group. It showed that FIa had no effect on human
fibrinogen.
Discussion
Studies on Russell's viper venoms have been conducted
for a long time. It is known that fractions of Russell's viper
venom exhibit a number of hemostatic activities such as the
proteases from Russell's viper venom that activate factor X,
factor V, and prothrombin[9_12].
In this paper, we have described the purification and
characterization of the factor X-activating fraction FIa from
Daboia russelli siamensis (Myanmar) venom.
With a 3-step procedure, we obtained a purified factor
X-activating fraction FIa. Its molecular weight was 34 479 and
its pI was 7.2, showing a difference when compared with
RVV-X obtained by Esnouf and
Williams[4]. It is also different from the factor X-activating fraction obtained by Yang
et al from Thailand Daboia russelli
siamensis[5]. Judging from the above facts, FIa is a new factor X-activating protein
found from the venom of Daboia russelli
siamensis (Myanmar) venom, or these difference is a different
derivation of viper russellii. We found that FIa might shorten
clotting time, and the hemostatic activity of 0.5 mg FIa was
equal to that of 1.5625 u of thrombin.
We know that the activation of the extrinsic coagulation
pathway initiated by an expression of prothrombinase
(prothrombin, FXa) plays an important role in hemostasis.
Factor Xa, the physiological activator of prothrombin,
converts prothrombin to active thrombin. Thrombin directly
activates platelets and cleaves fibrinogen to fibrin
monomers[13,14]. In order to understand the relationship between
FIa and this process, we determined the activating activities
of FIa on factor Xa prothrombin in vitro. In light of our data,
we found that FIa could increase the activity of factor X,
which has a concentration-time relationship. However, we
found that FIa could not affect the activity of prothrombin
directly.
Fibrinogen and fibrin play essential roles in blood clotting.
During coagulation, the soluble fibrinogen is converted to
insoluble fibrin, and this process is initiated by
thrombin[15]. In this study, we found that FIa had no effect on human
fibrinogen in vitro. The results showed that FIa could not
cleave or clot fibrinogen directly.
In conclusion, FIa is a factor X-activating enzyme, which
could activate factor X to factor Xa, but has no effects on
prothrombin and fibrinogen.
These activities indicate the potential therapeutic
application of FIa for hemostasis. Therefore, the clinical
application of FIa remains to be explored and evaluated. Moreover,
because habitat, climate, age, and environment influence the
venom ingredient and toxicity at different levels, the
stability of natural venom is not ideal. It is more interesting to
produce medical venom products by molecular cloning and
expression. The N-terminus amino acid sequences of
FIa have been determined, but the data are not shown in the
present paper, which would be very useful in following
studies.
Acknowledgement
We gratefully acknowledge the Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences for technical assistance.
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