Thromboembolic diseases, including cardiovascular disease,
cerebrovascular disease and venous thromboembolism, are
within the leading causes of death as exposited in the global
mortality projections to 2030 by WHO [1]. Thromboembolism
often consists when the physiological equilibrium between
coagulation system and fibrinolytic system is disturbed [2].
The fundamental pathophysiological mechanism underlying
cardiovascular diseases is the formation of fibrin (blood clots),
which adheres to the unbroken wall of blood vessels. The fibrin
comprising fibrinogen by thrombin activation is lysed by plasmin,
which is activated from plasminogen by tissue plasminogen
activator [3].
The uptake of thrombolytic drugs that dissolve blood clots
for treatment is only an alternative to surgical interventions to
remove or by pass the blockage [2]. Fibrinolytic enzymes, which
are widely used in the traditional treatment of thromboembolism,
have vital disadvantages such as high cost, short half-life, high
therapeutic dose, low specificity to fibrin, bleeding complications
and allergic reactions. Therefore, it is important to investigate new fibrinolytic enzymes produced from natural sources for
low cost, no side effects and safety, specificity and efficacy of
fibrinolytic treatment [4].
A variety of fibrinolytic enzymes such as tissue-type
plasminogen activator (t-PA), urokinase (u-PA), and bacterial
plasminogen activator streptokinase have been extensively
investigated and used as thrombolytic agents [4]. Serine
proteases, which show fibrinolytic/fibrinogenolytic activity,
have been considered as therapeutic agents for thrombosis.
Notably, the genus Bacillus has been well regarded for its role
in the production of a variety of industrially important enzymes.
The fibrinolytic enzymes from Bacillus sp. such as subtilisin NAT
[5], subtilisin J [6] and subtilisin E [7], have been purified and
characterized for the development of strong thrombolytic drugs.
The recombinant subtilisin from B. subtilis strain PTTC 1023 was
identified as fibnoloytic enzyme by Ghasemi et al., [8]. Ghasemi
et al., showed that the recombinant enzyme encoded by the aprE
(Accession No. HQ699519.1) has higher subtilisin activity other
reported subtilisins and the E. coli transformants. In a later study,
proteolytic activity of subtilisin from B. subtilis strain PTTC 1023
was studied for use in detergent applications [9]. In this study,
it was carried out a comprehensive analysis of the recombinant
enzyme produced for use in pharmaceutical purposes. The aim
of the study was to investigate the potential of the enzyme for
treatment of thromboembolic diseases determining the activities
of fibrinolytic, fibrinogenolytic and proteolytic of the recombinant
subtilisin from Bacillus subtilis.
Chemicals
Unless specified, all chemicals, substrates, and reagents
with the analytical grade or the highest available purity were
purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Production of recombinant enzyme
The recombinant enzyme was produced as described in our
previous study [9]. Briefly, the coding sequence of subtilisin
was optimized for E. coli codon usage. The subtilisin gene (NCBI
accession no. HQ699519.1) from Bacillus subtilis PTTC 1023 was
directly synthesized and cloned in expression vector pD441-NH
by DNA 2.0 (Atum, Newark, CA, USA). After, recombinant vectors
were transformed into competent cells E.coli BL21 (DE3). The
C-terminal 6xHis-tagged enzyme was purified in a single-step
procedure by affinity chromatography using Ni-NTA agarose
resin (Qiagen GmbH, Hilden, Germany).
Determination of fibrinogenolytic activity
Fibrinogenolytic activity was determinated as follows:
80 mL of 1% human fibrinogen prepared in 50 mMTris/HCl
buffer containing 0.1 M NaCl was incubated with 2μg of purified
subtilisin at various periods (30, 60, 90, 120 minutes and
overnight) at 37°C [10]. Then, 20 μL samples taken from the
reaction medium were analyzed using 12% SDS-PAGE according
to Laemmli’smethod [11].
Determination of fibrinolytic activity-I
Fibrinolytic activity was determined according to Astrup and
Mullertz’s method (1952) [12] with slight modification. The fibrin
plate was prepared as follows: 5 mL bovine plasma fibrinogen
solution (0.6% in 50 mMTris-HCl buffer, pH 7.5) was mixed with
200 μL thrombin solution (10 U/mL) and 5 mL calcium chloride
solution (0.7%) in petri dish. After clot formation was observed
(about 2 hours), 25μL of recombinant enzyme and plasmin
prepared in different concentrations (2.0, 1.0, 0.5, 0.25, 0.125,
0.0625 μg/mL) was carefully placedon a plate. The enzymes were
added separately on the same plate. The plate was incubated
overnight at 37°C. The fibrinolytic enzyme activity was calculated
by measuring the area of the clear zone on the fibrin plate. The
area of the clear zone was determinated by the Image J [13], and
the number of units was calculated using the standard curve of
plasmin.
Determination of fibrinolytic activity-II
A cotton cloth was divided into 4 sections and 200 μL of
blood was dripped and allowed to dry. Then the fabric was
soaked with 2% formaldehyde for 45 minutes and the excess
formaldehyde was removed by washing with water. After drying,
different applications were made on each part of the fabric which
was divided into four parts. The fabric pieces were separately
incubated with 1 mL of the subtilisin (0.5 mg/mL), 1 mL of the
subtilisin and detergent (5% (v/v) tween 80), 1 mL of sterile
distilled water with detergent and 1 mL of sterile distilled water
at 37°C for 1 hour. Following incubation, the fabric was washed
with water, dried and the potential to remove blood was observed [14].
Substrate specificity assay
The proteolytic activity was measured according to a
Peterson’s modified method in the presence of blood clot,
casein, bovine serum albumin, lactoferrin and immunoglobulin substrates [15]. One mL of a 10 mg/mL substrate solution was
mixed with the purified enzyme (2 μL, 0.5 mg/mL), and the
reaction mixtures were incubated at 37°C for 20 min. The change
in substrate concentration was determined using a Bradford
protein assay [16]. There action was stopped by adding 1 mL
of 2% (w/v) trichloroaceticacid. 1.5 mL of 0.5 M Na2CO3 and 0.5
mL of Folin-Ciocalteu® phenol reagent was added to the 0.5 mL
supernant. The solution was incubated at 37°C for 30 min. The
absorbance was calculated at 660 nm spectrophotometrically
against a blank control. In the activity measurement, no enzyme
was added to the medium while preparing the control and all
other steps were performed according to standard protocol.
One unit of proteolytic activity was defined as the amount
of enzyme that hydrolysed the substrate and produced 1 μg
amino acid equivalent to tyrosine per min under the performed
experimental conditions. The results represent the average of
three experiments. In this assay, enzyme activity was expressed
as a relative activity, compared to that of non-enzyme treated control.
Fibrinogenolytic activity
The molecular weight of the enzyme analyzed by 10% SDSPAGE
was about 40 kDa (Figure 1). Fibrinogen degradation
of recombinant protease was analyzed 12% SDS-PAGE. The
recombinant subtilisin of B. subtilis displayed an optimum
fibrinolytic activity at pH 7.5 and 37°C (data not shown).
Recombinant protease exhibited strong fibrinogenolytic
activities. As shown in Figure 2, the ϒ-ϒ, α and β chains of
fibrinogen were totally degraded by the enzymes at 37°C for
30 min. However, the ϒ chains were more resistant to enzyme
digestion in all times.
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Figure 1: Fibrinogen degradation of recombinant protease. The
fibrinogen degradation productions were separated by 12%
SDS-PAGE. Lane 1,marker; Lane 2, control (fibrinogen without
recombinant protease); Lanes 3 to 7, fibrinogen degradations by
recombinant protease after 30, 60, 90, 120 min and overnight of
incubation, respectively at 37°C. View Figure
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Figure 2: Comparison of the fibrinolytic activity of recombinant
protease from B.subtilis and plasmin. The evaluation of fibrinolytic
activity by fibrin plate assay. The white elliptical area represents the
fibrinolytic region by B. subtilis and plasma in the fibrin plate region.
M: Marker, pS: Purified Subtilisin. In the fibrin plate assay, 25 μL of
sample solution in certain concentrations was carefully placed on
the plate and the plate was incubated for overnight at 37°C. Data are
expressed as mean±SD of three independent experiments. View Figure
Fibrinolytic activity
The fibrinolytic activity was examined by fibrin plate assay
and the fibrinolytic activities of recombinant protease from B. subtilis and plasmin was compared. The clear zones produced
by recombinant enzyme and plasmin did not significantly
differ on the fibrin plate (Figure 3). Interestingly, no significant
difference was observed in all concentrations of both enzymes.
These results indicate that recombinant protease is a plasminlike
protease which can importantly degrade the fibrin, thereby
dissolving the thrombi.
Fibrinolytic potential of the recombinant protease
The result of fibrinolytic activity is shown in Figure 4. The
degree of blood removal from the cotton fabric was found in the
order of recombinant protease with detergent >recombinant
protease > sterile distilled water with detergent > sterile distilled water.
Substrate specificity
Recombinant enzyme degraded various protein substrates,
including fibrin, casein, BSA and lactoferrin. When the relative
activity of fibrin as a substrate is considered 100%, those of
casein, BSA, lactoferrin and immunoglobulin were 78.5%, 21.8%,
12.05%, 0% (Figure 5). The caseinolytic activity of the enzyme
was relatively high; however, activity was quite low against
BSA and lactoferrin (p<0.001). Also, the enzyme did not show
activity against immunoglobulin substrate. The high specificity
and high interest of recombinant protease from B. subtilis to
fibrinas a fibrinolytic agent comparing to BSA, lactoferrin and
immunoglobulin is an important feature.
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Figure 5: Relative activity of fibrin as a substrate is considered 100%,
those of casein, BSA, lactoferrin and immunoglobulin were 78.5%,
21.8%, 12.05%, 0%. View Figure
In the present study, we report fibrinolytic potential of
about 40 kD are combinant subtilisin from culture supernatant
of Bacillus sp. strain PTTC 1023. The molecular mass of
recombinant protease was found to be higher than the molecular
mass of fibrinolytic enzymes (18-38 kDa) purified from the genus
Bacillus [17]. Themolecular weight of recombinant subtilisin was
found to be lower than KA38 (41 kDa), a fibrinolytic protease
[18], but higher than many microbial fibrinolyticserine proteases
such as nattokinase (28 kDa) [19] and TPase (27.5 kDa) [20].
Human blood plasma contains a substantial amount of
proteins (60-85 mg per mL) [20]. These include albumin
(~55%), globulin (~38%), fibrinogen (~7%), (pro) enzymes and
protease inhibitors [21,22]. As shown in Figure (4), the effects
of fibrin, casein, bovine serum albumin and lactoferrin on the
enzyme activity of recombinant protease were examined. The
recombinant enzyme showed the highest specificity against fibrin
and proteolytic activity of its had low specify to serum albumin
and lactoferrin which blood proteins. Also, the enzyme did not
show proteolytic activity against immunoglobulin. A similar
effect for serum albumin was also demonstrated by the CTSP
alkaline protease from C. tentaculata [23]. A fibrinolytic serine
protease (Brevithrombolase) Brevibacillus brevis demonstrated optimum fibrinolytic activity at pH 7.4 and 37°C, and showed
hydrolytic activity toward globulin, casein and fibrinogen [24].
The optimum temperature and pH requirement of
recombinant protease was comparable to previously reported
fibrinolytic enzymes isolated from bacteria such as Bafibrinase
[25], Nattokinase [19] and DJ-4 [26]. The optimum pH and
temperature of recombinant subtilisin and its strong fibrinolytic
activity suggested that former could be developed as an effective
thrombolytic drug capable of functioning at physiological
conditions. Optimum activity exhibition of the enzyme in
physiological conditions is an important because this feature
reinforces its future therapeutic application as a thrombolytic agent.
Fibrinogen, a fibrin precursor, is a glycoprotein (about
340 kDa) and contains two sets of disulfide-bridged α-, β- and
ϒ- chains. The recombinant subtilisin α, β and ϒ-ϒ chains
of fibrinogen were totally degraded by the enzyme at 37°C
for 30 min. The fibrinolytic and fibrinogenolytic studiesalso
confirmed the plasmin-like activity of recombinant protease.
Fibrinolytic enzymes are typically classified as α-fibrinogenases
or β-fibrinogenases on the basis of specificity towards either
the α -chain or β -chain of fibrinogen, respectively [27]. The
recombinant protease can be classified as a αβ-fibrinogenase.
The fibrinolytic pattern by recombinant subtilisin was similar to
the fibrin/fibrinogen degradation by Bafibrinase, a fibrinolytic
enzyme from Bacillus sp. strain AS-S20-I which was also
classified as a αβ-fibrinogenase [25]. In other study, although the
α and β chains of fibrinogen were degraded by Brevithrombolase
after 120 minutes, α and β chains were degraded by plasmin after
60 and 180 min, respectively. The ϒ-ϒ chains of fibrinogen were
degraded by Brevithrombolase and plasmin after 120 and 60
min, respectively [24]. Recombinant enzyme (AprEBS15) from
B. pumilus BS15 quickly degraded α and β chains of fibrinogen.
The chain was hydrolyzed in 10 min and β chain was completely
degraded in 3 h. But as in this study, the γ-chain was not degraded
even after 12 h [28]. The fibrinogenolysis pattern of recombinant protease from B. subtilis PTTC 1023 was determined to be α-
β-and γ-, which was similar to N-V protease [29], NJP [30] and
CSP [31], but absolutely different from that of FP84 [32] and
subtilisin FS33 [33], where β-chains got hydrolyzed first.
The data obtained indicates that recombinant enzyme is
subtilisin from B. subtilis, which can efficiently and directly
hydrolyze fibrinogen. Recombinant protease is a plasmin-like
fibrinolytic serine protease. Furthermore, recombinant enzyme
has advantages of possible production in large amounts, at low
cost, and with ease of purification. High purification yield and
fold displayed by purified protease using a one-step purification
procedure are quite advantageous in terms of lower costs in
fibrinolytic therapy and nutraceutical applications. It cans a
potential candidate for the development of an antithrombotic
drug and it is being considered for preclinical studies using
animal model to assess its in vivo thrombolytic potential.