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Introduction
Studies on snake venoms have been proceeding for a
long time. It is known that fractions of snake venom exhibit
a number of biological activities, such as fibrinogenolysis
and/or fibrinolysis, and anti-platelet
aggregation[1]. Approximately 3 kinds of enzymes from snake venoms can degrade
fibrinogen, these are thrombin-like enzyme
(TLE)[2], plasminogen
activator[3], and fibrinolytic enzyme. Among them,
fibrinolytic enzymes can directly degrade not only
fibrinogen but also fibrin in vitro and
in vivo. Furthermore, they are not inhibited by proteinase inhibitors in human blood.
With their potential use for treating thrombotic disease the
fibrinolytic enzymes have been widely investigated. The
fibrinolytic enzymes have been purified from the venoms of
Agkistrodon acutus[4], A piscivorus
piscivorus[5],
A contortrix[6],
A rhodostoma[7], Bothrops
jararaca[8], Crotalus
atrox[9], Trimeresurus
mucrosquamatus[10] and Vipera
lebetina[11]. More than 70 kinds of fibrinolytic enzymes have been
isolated, and novel fibrinolytic enzymes continue to be
reported.
The fibrinolytic enzyme from Taiwanese Agkistrodon
acutus venom was first isolated by Ou-yang and
Huang[12]. In our previous work, another fibrinolytic enzyme called
FIIa was purified from Anhui Agkistrodon
acutus venom. FIIa can degrade fibrin and fibrinogen
in vitro, and solubilize thrombus
in vivo[4,13]. However, the enzymological
characteristics of FIIa have not been shown clearly. In the present
investigation, we mainly investigate the influences of
several protease inhibitors, chelating agents, and metal ions on
the fibrinogenolytic activity of FIIa. The metal content was
also determined.
Materials and methods
Snake venom Lyophilized Agkistrodon
acutus venom was collected from Qimen Snake Farm (Anhui, China).
Reagents DEAE-Sephadex A-50, Sephadex G-75,
ethylenediamine tetracetic acid (EDTA), ethyleneglycol tetraacetic
acid (EGTA), phenylmethylsulfonylfluoride (PMSF) and
soybean trypsin inhibitor (SBTi) were purchased from GE Health
Care (Little Chalfont, UK). Bovine fibrinogen and plasmin
were from Sigma (St Louis, MO, USA). Molecular weight
protein standards were from NEB (Beverly, MA, USA). All
other chemicals and solvents were of analytical grade from
commercial sources.
Purification of the
enzyme FIIa, a fibrinolytic enzyme
from Agkistrodon acutus venom, was prepared according to
the method described by Liang
et al[4].
Fibrinogenolytic activity
assay FIIa (1 g/L, 150 μL) was incubated with 450 μL of bovine fibrinogen (1 g/L) at
37 oC. Aliquots were taken at 5 min, 15 min, 30 min, 45 min, 1 h, 4 h
and 8 h intervals, and 600 μL of a denaturing solution
(10 mol/L urea, 4% sodium dodecylsulfate and 4%
b-mercaptoethanol) was added and the mixture was incubated at
100 oC for 4 min. Each sample (20 μL) was analyzed by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
using a 4% spacer gel and a 12% separation
gel[14]. Human plasmin (50 U/L) was used as positive control.
Effect of inhibitiors on fibrinogenolytic
activity The effects of EDTA (5 mmol/L), EGTA (5 mmol/L), PMSF
(5 mmol/L), SBTi (0.15 g/L), dithiothreitiol (DTT; 5 mmol/L) and
cysteine (5 mmol/L) on fibrinogenolytic
activity were examined by incubation with
FIIa (1 g/L) at
37 oC for 1 h. After adding bovine fibrinogen
(1 g/L), the mixture was incubated for a further 1 h.
Each sample (20 μL) was analyzed by SDS-PAGE.
Reactivation by metal ions on fibrinogenolytic
activity FIIa (1 g/L, 150 μL) was incubated with EDTA (final
concentration: 5 mmol/L) at
37 oC for 1 h.
MgCl2, CaCl2 and
ZnCl2 (final concentrations: 5 mmol/L) were added to the
incubation solution, and the mixture was incubated for a
further 1 h. The fibrinogenolytic activity was examined by
SDS-PAGE after a 1-h incubation with 450 μL of bovine
fibrinogen (1 g/L). The same experiment was performed with EGTA
(final concentration: 5 mmol/L) instead of EDTA.
Metal content assay Metal content was determined
using an atomic absorption spectrophotometer. The
absorbances of standard solutions were used to draw standard
graphs. The metal content of FIIa was estimated by
comparison with the standard curve[14].
Results
FIIa degraded the Aa-chain preferentially, followed
by the Bb-chain of fibrinogen, but the g-chain was the most
insusceptible to the enzyme. At a molar ration of 3:1
(fibrino-gen: FIIa), the Aa-chain was totally degraded within 5 min,
with relatively lower activity for the Bb-chain, which
disappeared within 30 min. The g-chain was only degraded
following a prolonged 8-h incubation with
FIIa (Figure 1A). Concomitant with the digestion of fibrinogen, major
fragments of Mr approximately 45 000 and 41 000 were observed.
When fibrinogen was incubated with human plasmin, the
Aa- and Bb-chains disappeared within 15 min, while the
g-chain disappeared within 1 h. The major digestion fragment
observed was at Mr 45 000, of which the cleavage pattern
was different from that of FIIa (Figure 1B).
The fibrinogenolytic activity of
FIIa was inhibited by EDTA, EGTA, DTT and cysteine, but not by PMSF or SBTi
(Table 1). The fibrinogen was still intact after incubation
with FIIa pretreated with EDTA, EGTA, DTT, and cysteine
(Figure 2). However, the fibrinogen was degraded after
incubation with FIIa pretreated with PMSF and SBTi (Figure 2).
Zn2+, Ca2+, and
Mg2+, at concentrations of 5 mmol/L, could
restore the fibrinogenolytic activity of EDTA-treated
FIIa. Only Ca2+ could restore the fibrinogenolytic activity of
EGTA-treated FIIa. Both 1 mmol/L and 5 mmol/L
Ca2+ were effective (Figure 3).
Zn2+, K+ and
Ca2+ were found in significant quantities, at
3171±25 mg/kg, 489±17 mg/kg and 319±13 mg/kg,
respec-tively. The concentrations of
Mg2+, Fe2+ and
Cu2+ were only at trace amounts (Table 2). For each mole of
FIIa, there was approximately 1 mole of
Zn2+, 0.3 mole of K+ and 0.2 mole of
Ca2+.
Discussion
FIIa is a a,b-fibrinogenase because it degraded both the
Aa-chain and the Bb-chain of
fibrinogen[15]. The Aa-chain of fibrinogen was very susceptible to
FIIa, and it was completely degraded within 5 min. Cleavage of the
g-chain of fibrinogen was observed only with a prolonged incubation
time. Thus far there have been few reports of
fibrin(ogen)olytic snake venom enzymes that cleave of the
g-chain. No enzyme reported has shown cleavage specificity directed
solely at the g-chain[16]. Because the
g-chain of fibrinogen was stable when was incubated with snake venom
fibrin(ogen)olytic enzyme, we postulated that the degradation
might occur at either an increased incubation time or at an
increased concentration. In our previous study, the
g-chain was unaffected after a 2-h incubation. However, in the
present study FIIa appeared to degrade the
g-chain after prolonged (8 h) incubation. The same phenomenon
was noticed for cerastase F-4 (from
Cerastes cerastes venom) and a fibrin(ogen)olytic enzyme from
V lebetina venom, and they appeared to degrade the
g-chain following 48-h and 24-h incubations,
respectively[17,18]. Plasmin also cleavages the
Aa-, Bb-, and g-chains of fibrinogen, but the patterns are
different from those observed when cleaved by
FIIa. It is interesting that various fibrin(ogen)olytic enzymes seem to
produce different degradation patterns for fibrinogen. For
example, FIIa mainly yields fragments of 45 kDa and 41 kDa,
while basilase produces fragments of 45 kDa, 36 kDa and
10 kDa, and atroxase gives fragments of 45 kDa and
38 kDa[19]. The studies on some fibrin(ogen)olytic enzymes reveal that
their cleavage preference is commonly directed to the
amino-terminal side of hydrophobic amino acid residues. They
display distinct and unique cleavage characteristics with
fibrinogen.
The fibrin(ogen)olytic enzymes from snake venoms can
be classified as metalloproteinases or serine proteinases.
Chelating agents (EDTA, EGTA) completely inhibited
FIIa, while serine protease inhibitors (PMSF, SBTi) were
ineffective, indicating that it belongs to the metalloproteinase
group. This was supported by data from atomic absorption
spectroscopy. For each mole of FIIa there was approximately
1 mole of Zn2+, 0.3 mole of
K+ and 0.2 mole of Ca2+. Like
many of the venom fibrinolytic enzymes,
FIIa is a zinc metalloproteinase. Besides
Zn2+, Ca2+ is another metal ion
often found in venom with fibrinolytic enzymes. Metal
analysis has indicated that the calcium content of atroxase (from
western diamondback rattlesnake
venom)[9] and lebetase (from
V. lebetina snake
venom)[20] is 0.3 mol/mol and
1 mol/mol, respectively. In adamalysin from
C. adamanteus[21] and
atrolysin c(d) from C.
atrox[22] it was found that except
Zinc-binding site, a calcium ion is bound near the
carboxy-terminus of the enzyme. Thus far, only atroxase was reported to
contain 1 mol/mol of K+, while
FIIa contains 0.3 mol/mol of
K+. The functions of calcium and potassium have not been
elucidated, but they may play a role in retaining the stability
of the protein.
Zn2+, Ca2+ and
Mg2+ were effective in restoring the
activity of EDTA-treated FIIa, while only
Ca2+ could restore the activity of EGTA-treated
FIIa. The mechanism for this is not clear. It is reported that snake venom metalloproteinases
have Zn2+-dependent activities, but some are more active in
the presence of Ca2+[23,24]. This seems probably responsible
in part for this phenomenon. The effect of
Mg2+ on the activity of FIIa needs to be elucidated.
FIIa is inhibited by DTT and cysteine, suggesting that disulfide bonds are
necessary for holding the structure.
In conclusion, like many venom fibrin(ogen)olytic
enzymes, FIIa is a metalloproteinase. Both
Zn2+ and Ca2+ play important roles in the fibrinogenolytic activity of
FIIa.
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