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Introduction
Snake venoms are predominantly the mixtures of pharmacologically active
enzymatic and non-enzymatic protein and peptide toxins. They affect vital functions
of our system while, some of them exhibit exceptionally high degree of target
specificity. The toxins that affect hemostasis, including plasma coagulation and
platelet function are may be enzymatic or non-enzymatic in nature. Enzymes, such as
proteases are very well studied. The pro-coagulant enzymes – they are either
thrombin-like enzymes, prothrombin activators, factor X and factor V activators;
anticoagulant enzymes – they are either protein C activators, fibrin(ogen)olytic
enzymes, factor IX and X inhibitors; enzymes interfering in platelet function – they
are either activates platelets or inhibit their aggregation; fibrinolytic activators – they
are plasminogen activators and hemorrhagins [Koh et al, 2006; Larréché et al, 2008].
The fibrin(ogen)olytic metalloproteases conferring anticoagulant activity are
extensively studied, especially from the Viperid and Crotalid snake venoms but only
few reports are from the Elapid snake venoms. The Natrahagin from Naja atra [Zhu
and Wu, 1999] and Proteinase F1 from Naja nigricollis [Kini and Evans, 1991]
preferentially cleave the A chain and slowly the B chain, but Ohagin from
Ophiophagus hannah venom [Guo et al, 2007] specifically cleave the A chain of
fibrinogen. However, the serine proteases which posses only the fibrinogenolytic but
not fibrinolytic activity, cleave the fibrinogen from the N-terminal region and release
fibrinopeptides A and/or B forming the fibrin clot and are termed as thrombin-like
enzymes [Matsui et al, 2000]. A specific and potent thrombin inhibitor, Bothrojaracin
(BJC) from the Bothrops jararaca venom and hence possess anticoagulant property
[Zingali et al, 2005]. In contrast to fibrin(ogen)olytic metalloproteases, the
plasminogen activating serine proteases, indirectly cleave fibrin and as well as
fibrinogen through plasmin. In this case the observed anticoagulant property is due to
the depletion of fibrinogen concentration below the threshold level for clotting
activity. The plasminogen activators, TSV-PA from Trimeresurus stejnegeri venom
[Zhang et al, 1995] and LV-PA from Agkistrodon halys venom [Sanchez et al, 2006]
are studied extensively. “Protac” from Agkistridon contortrix contortrix venom
[Kisiel et al, 1987] is the protein C activator exhibits anticoagulant property. In the
previous study, isolation and characterization of a non-toxic high molecular weight
fibrinogenolytic metalloprotease, NN-PF3 from Naja naja venom was reported
39
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Anticoagulant activity of a..…
[Jagadeesha et al, 2002]. In the present study, the anticoagulant activity has been
investigated further using in vitro and in vivo experiments and the results are
presented.
Materials and Methods
The pooled desiccated Naja naja (Indian cobra) venom purchased from Dipak
Kumar Mitra, Hindusthan Park, Kolkata, India. CM Sephadex C-25, Collagen type-IV
(from Engelbrethe-Holme-Swarm murine sarcoma basement membrane), fibronectin
(from human plasma), collagen type-I (from rat tail tendon), fibrinogen (from human
plasma, fraction I), thrombin (from human plasma), 2-macroglobulin (2-M), were
purchased from Sigma Chemicals Company, St. Louis, USA. Molecular weight
markers were purchased from Bangalore Genei Private Limited, India. Urokinase was
purchased from Polamin Werk GmbH, Herdecke, Germany. UNIPLASTIN,
LIQUICELIN-E, and FIBROQUANT were purchased from Tulip Diagnostics Pvt.
Ltd., Goa, India. Equine polyvalent anti-venom (Batch number-ASVD 19/06)
purchased from VINS Bioproducts, Andhra Pradesh, India. Fat free casein,
iodoaceticacid (IAA), phenylmethylsulphonyl fluoride (PMSF), 1, 10-phenanthroline,
Ethylenediaminetetraaceticacid (EDTA), Pepstatin A, Ethylene glycol-N,N,N,Ntetraacetic acid (EGTA), agarose, were purchased from SRL Chemical Company,
Bangalore, India. All other chemicals and reagents used were of analytical grade,
purchased from local firms. Fresh blood sample was collected from apparently
healthy human donors. Swiss Wistar albino mice weighing 18–20 g from the central
animal house facility, Department of Zoology, University of Mysore, Mysore, India.
Animal care and handling complied with the National Regulation for Animal
Research. The animal experiments were carried out after reviewing the protocols by
the Animal Ethical Committee of the University of Mysore. Mysore, India.
Purification of NN-PF3
NNN-PF3 was purified by following the earlier protocol described by
Jagadeesha et al. [2002]. Briefly, the unbound fraction of Naja naja venom, NC-I
from the CM Sephadex C-25 column chromatography was subjected to PAGE at pH
8.3 to separate a proteolytically active protein band. The obtained proteolytically
40
Chapter-II
Anticoagulant activity of a..…
active band proteins, NN-PF2 was subjected to gel filtration on a Sephadex G-100
column to obtain the pure NN-PF3.
Proteolytic activity
The proteolytic activity was assayed using fat free casein as substrate
according to the method of Satake et al. [1963]. Fat free casein (0.4 ml, 2% in 0.2 M
Tris-HCl buffer pH 8.5) was incubated with NN-PF3 in a total volume of 1 ml for 2 h
30 min at 370C. Adding 1 ml of 0.44 M trichloroacetic acid (TCA) and left to stand
for 30 min stopped the reaction. The mixture was then centrifuged at 1500 x g for 15
min. Sodium carbonate (2.5 ml, 0.4 M) solution and 0.5 ml of Folin & Ciocalteu’s
phenol (FC) reagent (diluted to 1/3 of the original strength in water) were added
sequentially to 1 ml of the supernatant and the colour developed was read at 660 nm.
One unit of enzyme activity is defined as the amount of the enzyme required to cause
an increase in OD of 0.01 at 660 nm/h at 370C. For inhibition studies, similar reaction
was performed independently after preincubation of NN-PF3 for 15 min with 5 mM
each of EDTA, 1, 10-phenanthroline, EGTA, PMSF, pepstatin A, 0.1 mM Iodoacetic
acid and increasing molar ratio of 2-macroglobulin (2-M) and increasing amount
of polyvalent anti-venom. In all the cases appropriate controls were kept.
Mass spectrometry
Molecular mass of NN-PF3 was determined by mass spectrometry using
Bruker Daltonics Matrix Assisted Laser Desorption Ionization-Time Of Flight
(MALDI-TOF)
machine
in
positive
ionization
mode.
Alpha-Cyano-4-
hydroxycinnamic acid was used as the MALDI matrix.
Fibrinogenolytic activity
Fibrinogenolytic activity was assayed according to the method of Ouyang and
Teng [1976]. The NN-PF3 was incubated with 50 μg of human fibrinogen in a 40 μl
reaction mixture of 10 mM Tris-HCl buffer, pH 7.4 containing 10 mM NaCl at 370C.
For inhibition studies, NN-PF3 was preincubated independently with 5 mM each of 1,
10-phenanthroline, EDTA, PMSF, pepstatin A and 0.1 mM IAA for 15 min at 370C.
The NN-PF3 without any inhibitor was served as control and all the reaction mixtures
were incubated for 4 h at 370C after adding 50 μg fibrinogen. The reaction was
41
Chapter-II
Anticoagulant activity of a..…
terminated by adding 20 μl of denaturing buffer containing 1 M urea, 4% SDS and
4% β-mercaptoethanol. The reaction mixtures were analyzed using 10% SDS-PAGE
under reduced condition. The protein banding pattern was visualized by Coomassie
brilliant blue R-250 staining.
Blood / plasma clot hydrolyzing activity (Fibrinolytic activity)
Plate Method
The method described by the Gene et al. [1989] was followed with slight
modification. A mixture consisting 2 ml of healthy human plasma and 3 ml of 1.2%
molten agarose (450C) in 10 mM Tris-HCl buffer, pH 7.4 containing 0.02% sodium
azide, 70 mM (NH4)2SO4, 90 mM NaCl, 0.70 mM MgCl2 and 200 l of 0.2 M CaCl2
was poured into 10 mm x 90 mm Petri dish and allowed for 2 h at 300C. Different
concentrations of purified NN-PF3 (1–4 g) and one International Unit (IU) of
urokinase in 10 l of 10 mM Tris-HCl buffer, pH 7.4 were placed independently on
the surface of the gelly matrix of fibrin and incubated for 12 h at 300C. For inhibition
studies NN-PF3 was preincubated for 15 min with 5 mM EDTA and PMSF
independently. The diameters of the clear zones (plaque) were recorded in mm.
Colorimetric method
Blood clot/plasma clot hydrolyzing activity was assayed according to the
method described by Rajesh et al. [2005]. Briefly, 100 l of citrated human
blood/plasma was mixed with 30 l of 0.2 M CaCl2 and incubated for 2 h at 370C.
The clot obtained was washed thoroughly for 5–6 times with phosphate buffer saline
(PBS) and suspended in 400 l of 0.2 M Tris-HCl buffer, pH 8.5. The reaction was
initiated by adding varied amounts of NN-PF3 (5–25 g) in 100 l of saline and
incubated for 2 h 30 min at 370C. The undigested clot was precipitated by adding 750
l of 0.44 M trichloroacetic acid (TCA) and allowed to stand for 30 min and
centrifuged for 15 min at 1500 x g. The aliquots of 0.5 ml supernatant was transferred
to clean glass tubes and followed by the addition of 1.25 ml of 0.4 M sodium
carbonate and 0.25 ml of 1:3 diluted Folin & Ciocalteu’s phenol (FC) reagent. The
colour developed was read at 660 nm after allowed to stand for 30 min. One unit of
activity is defined as the amount of enzyme required to increase in absorbance of 0.01
at 660 nm/h at 370C.
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Anticoagulant activity of a..…
Banding pattern of fibrinopeptides in SDS-PAGE
The method described by Rajesh et al. [2005] was followed with slight
modification. The washed plasma clot obtained as described above was suspended in
40 l of 10 mM Tris-HCl buffer, pH 7.4 containing 10 mM NaCl, and 0.05% sodium
azide and was incubated with NN-BMP at 370C. For inhibition study, NN-PF3 was
preincubated with 5 mM EDTA for 15 min at 370C independently. The reaction was
terminated by adding 20 l of sample buffer containing 4% SDS, 4% mercaptoethanol and 1 M urea and boiled for 5 min and centrifuged at 5000 x g for 10
min. An aliquot of 20 l supernatant was used to analyze the cleavage pattern of
fibrin by using 10% SDS-PAGE under reduced condition. The protein banding pattern
was visualized by coomassie brilliant blue staining.
Anticoagulant activity
Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT)
Briefly, 100 l of normal citrated human plasma was preincubated with
increasing amount of NN-PF3 (1–6 g) in 10 l of 10 mM Tris-HCl buffer, pH 7.2
for 3 min at 370C for dose dependent effect, while 1 g of NN-PF3 was preincubated
for different interval of time (5, 10, 15, 30, 45 and 120 min) for the time dependent
effect. In APTT, the mixture was activated with 100 l of APTT reagent
(LIQUICELIN-E – phospholipids preparation derived from rabbit brain with ellagic
acid) for 3 min at 370C, there after 100 l of 0.02 M CaCl2 was added and the clotting
time was measured. In case of PT, the clotting time was measured after adding 200 l
of PT reagent (UNIPLASTIN – rabbit brain thromboplastin). The APTT ratio and the
international normalized ratio (INR) for PT at each point were calculated from the
values of control plasma incubated with the buffer for identical period of time.
Thrombin Clotting Time (TCT)
Thrombin clotting time was determined with the slightly modified method of
Evans [1981]. Briefly, 100 l of fibrinogen solution (2 mg/ml) or human citrated
plasma were preincubated with NN-PF3 in 10 mM Tris-HCl buffer, pH 7.4 for varied
amount or varied time intervals at a concentration of 10 g/ml of NN-PF3 at 370C.
The clotting time was then determined after adding 100 l of diluted thrombin (2.5
43
Chapter-II
Anticoagulant activity of a..…
NIH units/ml) to the 100 l of incubation sample. Aliquots of 25 l from the
incubation mixture of fibrinogen (2 mg/ml) and NN-PF3 (10 g/ml) were drawn at
different time intervals and were mixed with 20 l denaturing buffer containing 4%
SDS, 4% -mercaptoethanol and 1 M urea and boiled for 5 min and analysed by using
10% SDS-PAGE under reduced condition. For neutralization studies, the polyvalent
anti-venom was co-incubated with NN-PF3 before clotting assays.
Antithrombin activity
Antithrombin activity was assayed by the method of Daoud et al. [1986].
Briefly, the diluted thrombin (2.5 NIH units/ml) was preincubated with 20 g of NNPF3 in 1 ml of 10 mM Tris-HCl buffer, pH 7.4 at 370C. Aliquots of 100 l from the
incubating mixture of thrombin and NN-PF3 were drawn at different time intervals
(5–60 min) and added to a tube containing 100 l of fibrinogen solution (2 mg/ml in
10 mM Tris-HCl buffer, pH 7.4), then the time taken to form the visible clot is noted.
Plasminogen activation
The plasminogen activation was assayed according to the method described by
Chakrabarty et al. [2000]. Briefly, the samples of one milligram of plasminogen
contaminated fibrinogen or 20 l of citrated human plasma (a), (a) with 200 IU of
urokinase (b), (a) with 10 g of NN-PF3 (c), 10 g NN-PF3 alone (d), and 200 IU of
urokinase alone (e) in 100 l of 100 mM potassium phosphate buffer, pH 7.4 were
independently mixed with 500 l of azocasein (0.25% in 100 mM potassium
phosphate buffer, pH 7.4) and incubated for 3 h at 370C. The reaction was terminated
by adding 400 l of 25% trichloroacetic acid (TCA). It was then centrifuged at 1000 x
g for 15 min. The supernatant (600 l) was diluted with an equal volume 0.5 N NaOH
and absorbance was read at 440 nm. One unit of activity was defined as the amount
enzyme yielding an increase in absorbance of 0.01/h at 440 nm.
Degradation of human plasma proteins
The NN-PF3 (1 g) was incubated with the 100 g of human plasma proteins
for different time intervals at 370C in a reaction volume of 40 l 10 mM Tris-HCl
buffer, pH 7.4 containing 10 mM NaCl, 0.05% sodium azide.
44
Reaction was
Chapter-II
Anticoagulant activity of a..…
terminated by the addition of 20 l denaturing buffer containing 4% SDS and boiled
for 5 min. It was then analyzed on a 7.5% SDS-PAGE under non-reduced condition.
In vivo studies
Defibrinogenating activity
Defibrinogenating activity was assayed by the method of Loria et al. [2003].
Briefly the group of five mice were injected intravenously (i.v) through the caudal/tail
vein with the increasing amounts of NN-PF3 in 100 μl of PBS. Control mice received
the same volume of PBS. Animals were anaesthetized after 1, 4, 8, 12 and 24 h of
injection with diethyl ether and bled by cardiac puncture. The blood samples were
placed in a glass tube and kept at 25–300C. Tubes were tilted at regular intervals, for a
period of 2 h to observe the clotting. The minimum defibrinogenating dose (MDD)
was defined as the minimum amount of enzyme which, when injected into the mice
produced incoagulable blood 1 h later.
Estimation of fibrinogen content
NN-PF3 in 100 l PBS was injected i.v. through the tail vein in to a group of
five mice. After 1, 4, 12, 18 and 24 h of injection the animals were anaesthetized with
diethyl ether and the blood samples were collected independently by cardiac puncture
using tri-sodium citrate as anticoagulant. The blood samples were centrifuged at 3000
x g for 10 min and the plasma were used for the estimation of fibrinogen according
the protocol provided by the FIBROQUANT kit manufacturer (Tulip Diagnostics
Private Limited., Bombay, India).
Bleeding time
The bleeding time was assayed by the method of Denis et al. [1998]. Briefly,
NN-PF3 in 30 l of PBS was injected i.v through the tail vein in to a group of five
mice. After 10 min in dose dependent effect and varied time in time dependent effect,
the mice were anaesthetized using diethyl ether and a sharp cut of 3 mm length at the
tail tip of mouse was made. Immediately, the tail was vertically immersed into PBS
which is pre-warmed to 370C. Bleeding time was recorded from the time bleeding
started till it completely stopped and it was followed for 10 min.
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Anticoagulant activity of a..…
Degradation of extracellular matrix proteins
Collagens, type-I and type-IV and fibronectin were independently incubated
with NN-PF3 in the ratio of 25:1 (w/w) in a total volume of 40 l of 10 mM Tris-HCl
buffer, pH 7.4 containing 10 mM NaCl, 0.05% sodium azide at 370C for different
time intervals. Reaction was terminated by the addition of 20 l denaturing buffer
containing 4% SDS, 4% mercaptoethanol and 1 M urea and analyzed by using
7.5% SDS-PAGE under reduced condition.
Statistical analysis
The data are presented as mean ± SD. Statistical analysis was performed by
Student’s t-test. A significant difference between the groups were considered if
p<0.01.
Results
NN-PF3 was purified to homogeneity and the specific activity determined on
the fat free casein substrate was found to be 6.5±0.2/min/mg. Further, the plasma recalcification time and fibrinogenolytic activity agreed well with the earlier report
[Jagadeesha et al. 2002]. NN-PF3 revealed a sharp peak (Fig. 2.1) corresponding to
the molecular mass of 67.81 kDa in MALDI-TOF mass spectrometry. The
fibrinogenolytic activity was completely inhibited by the EDTA and 1, 10phenanthroline as evidenced by the intact A and Bchains but PMSF, IAA and
Pepstatin A did not affect as evidenced by the cleavage of both the Aand B chains
of fibrinogen (Fig. 2.2). In addition to fibrinogenolytic activity, NN-PF3 showed
fibrinolytic activity and hydrolyzed the plasma clot that was prepared by using
platelet poor plasma and agarose. The clear zones of hydrolysis against an opaque
background was found to be dose dependent with 3 ± 0.8, 6 ± 0.5, 9 ± 0.6 and 12 ±
0.5 mm diameter respectively for 1, 2, 3 and 4 g of NN-PF3 when incubated for 12
h. The parallel activity by one IU of urokinase that served as a positive control caused
a clear zone of 10 ± 0.6 mm diameter. Pretreatment with EDTA abolished the activity
of NN-PF3 while, it was insensitive to PMSF revealing clear zone of hydrolysis (Fig.
2.3A). Further, colorimetric estimation of the generated fibrinopeptides revealed
varied degree of hydrolysis with the washed plasma clot hydrolyzed readily over the
whole blood clot (Fig. 2.3B) as suggested by their respective specific activity of 3.56
46
Chapter-II
Anticoagulant activity of a..…
± 0.29 units/mg/h and 2.34 ± 0.34 units/mg/h. The polymers were preferentially
degraded over the chains while, the  chains and the  dimers remained resistant
to proteolysis throughout the incubation period. The bands corresponding to the
polymers and the chains gradually vanished in a dose (Fig. 2.4A) and time (Fig.
2.4B) dependent manner with the appearance of new low molecular mass bands
distributed in the wide range of molecular mass from 45 kDa to 14.4 kDa in SDSPAGE under reduced condition. NN-PF3 prolonged the recalcification time of citrated
human plasma [Jagadeesha et al, 2002]. It prolonged the PT, APTT and TCT, and the
effect was both dose (Table 2.1A) and time (Table 2.1B) dependent in all the three
cases. Also, the increased TCT was in parallel with the gradual disappearance of the
A chains of fibrinogen (Fig. 2.5). However, the clotting property was abolished
when plasma was incubated with the NN-PF3. But, the thrombin activity was
unaffected even when it was pretreated with NN-PF3. As shown in table 2.2, neither
NN-PF3 nor urokinase degrade azocasein when incubated independently, while only
urokinase but not NN-PF3 degraded azocasein in presence of either plasminogen
contaminated human fibrinogen or human plasma. As revealed by SDS-PAGE
pattern, it appears that NN-PF3 did not degrade any of the citrated human plasma
proteins except for fibrinogen. The intensity of fibrinogen band decreased as a
function of time but without affecting the intensity of any other plasma protein bands
(Fig. 2.6).
In in vivo experiments, following i.v injection of NN-PF3, incoagulability of
blood in mice was observed with the MDD of 2.3 µg/g body weight; but, the clotting
property was restored after 24 h. Further, fibrinogen content in plasma was also not
detectable at the MDD, while it was restored after 24 h and found to be 215 ± 12 mg
fibrinogen/dl. The PBS injected control mice revealed 227 ± 15 mg fibrinogen/dl. In
addition, NN-PF3 prolonged the bleeding time dose dependently, at MDD, the
bleeding time recorded was >600 s, p<0.01 (Fig. 2.7A) against 97 ± 7 s of PBS
injected control mice. After 24 h of injection of NN-PF3, the bleeding time comes to
normal value of 108 ± 7 (Fig. 2.7B)
The 2-M did not inhibit caseinolytic activity of NN-PF3 (Fig. 2.8A) In
contrast, the polyvalent anti-venom inhibited the caseinolytic activity of NN-PF3 dose
dependently (Fig. 2.8B). At 1: 250 ratio (NN-PF3: polyvalent anti-venom; w/w), the
caseinolytic activity was abolished and similarly the anticoagulant activity was also
47
Chapter-II
Anticoagulant activity of a..…
abolished as evidenced by the PT, APTT and TCT assays (Table 2.3). However on
exhaustive incubation for a period of 24 h, the NN-PF3 did not degrade type-I and
type-IV collagen and as well as fibronectin as evidenced by the intact banding pattern
in SDS-PAGE under reduced condition (Fig. 2.9).
Discussion
In the earlier study, purification, characterization and anticoagulant activity of
a non-toxic metalloprotease (NN-PF3) from Naja naja venom was reported
[Jagadeesha et al, 2002]. In the present investigation, both in vitro and in vivo studies
were undertaken to further investigate the anticoagulant activity of NN-PF3 in which
the possible site of action in the coagulation cascade has been addressed. The
blood/plasma coagulation is an essential acute phase response due to injury. This
occurs through the intricate net work of two pathways; the intrinsic pathway (contact
activation pathway) and the extrinsic pathway (tissue factor pathway). These
pathways act independently or together and culminate in the activation of factor-II
(prothrombin) to factor-IIa (thrombin), the common pathway activation, through
limited proteolysis. The anticoagulant activity of NN-PF3 was due to its
fibrin(ogen)olytic activity. It is likely that the degradation from the C-terminal end of
Aand or B chains of fibrinogen might have resulted in the truncated structure that
lacks polymerization property and led to anticoagulation. In addition, the fibrinolytic
activity also appears to contribute for the observed anticoagulant effect. The clot got
dissolved due to degradation of  chains and the  polymers. Increased APTT
(intrinsic pathway) and PT (extrinsic pathway) and prolonged TCT of plasma
(common pathway) supports for the interference of NN-PF3 at the common pathway
of coagulation. However, it is more likely that the anticoagulant property is not due to
inhibition of thrombin but due to the fibrin(ogen)olytic activity. Insensitivity of
thrombin to NN-PF3 supports the above said view. Further, the fact underscore that
the fibrin degradation was not by the activation of plasminogen to plasmin but by the
fibrinolytic activity of NN-PF3. Urokinase, known to activate plasminogen and thus
the formed plasmin showed activity on azocasein. Thus NN-PF3 is a
fibrin(ogen)olytic enzyme and degrade both soluble fibrinogen and as well as
less/insoluble fibrin clot. But in contrast to fibrinogen, in which the Bβ chains
although less preferentially cleaved over the Aα chains [Jagadeesha et al, 2002], the β
48
Chapter-II
Anticoagulant activity of a..…
chains remained resistant to proteolytic digestion in fibrin. It is likely that the cross
linked and less soluble fibrin structure might prevent the β chain from undergoing
proteolytic degradation [Bernardes et al, 2008].
The in vivo experiments, including defibrinogenating activity, estimation of
fibrinogen and as well as determination of the bleeding time, reinforces the
consumption of coagulation factors by NN-PF3. This could be attributed by the
proteolytic action on fibrinogen, forming fibrin monomers (unpolymerized and noncross linked fibrin), which could be removed rapidly from the circulation or depletion
of fibrinogen attaining lesser than the threshold concentration for clotting could also
be the cause [Reid and Chan, 1968]. There are few reports of fibrinogenolytic
enzymes from the Elapid venoms and there were no reports of proteases with
fibrinolytic activity except a peptide Hannahpep from the Ophiophagus hannah
[Gomes and De, 1999]. In contrast, the fibri(noge)nolytic metalloproteases from the
snake venoms, cleave preferentially the Aα chains and slowly the Bβ chains of
fibrinogen, and are found to prevent the formation of the fibrin clot [Markland, 1998a;
Laing and Moura-da-Silva, 2005]. The fibrinolytic enzymes that cause lysis of
blood/plasma clot by direct degradation of fibrin or by plasminogen activation have
been reported [Estêvão-Costa et al, 2000]. NN-PF3 being a fibrin(ogen)olytic
metalloprotease, it is similar to other snake venom metalloproteases such as fibrolase,
lebetase, atroxase, mut IIa, ammodytase and leucurolysin-a [Bello et al, 2006] which
directly acts on the fibrin. The preferential degradation of plasma fibrinogen was
conspicuous over nearly unchanged banding pattern of rest of the plasma proteins in
SDS-PAGE under non-reduced condition (Fig. 5). Hence NN-PF3 may appear to
exhibit preferential specificity for fibrinogen and or fibrin. However, this can only be
confirmed using specific synthetic chromogenic substrates or purified coagulation
factors.
The snake venom metalloproteases (SVMPs) of the metzincin family enzymes
are classified into three groups, P-I to P-III class based on their domain structure [Fox
and Serrano, 2008]. The P-I class of enzymes are low-molecular mass
metalloproteinases with only a proteinase domain. The P-II class are disintegrin
precursors and have an additional disintegrin domain carboxy to the proteinase
domain. The P-III class are a high molecular mass proteinases and have both a
disintegrin-like domain and a cysteine-rich domain carboxy to the proteinase domain.
P-II and P-III class SVMPs undergoes proteolytic processing and dimerization, hence
49
Chapter-II
Anticoagulant activity of a..…
they are again classified into subclasses. Formally called P-IV class composed of a PIII structure plus a snake C-type lectin-like (snaclec) subunit of two chains disulphide
linked to one another to the Cys-rich domain is now included in the P-III group as a
subclass (P-IIId) [Takeda et al, 2012]. In contrast to P-I class of metalloproteases that
are readily inhibited by the human serum 2-M, an endogenous protease inhibitor, the
P-III class of enzymes are not inhibited by -M [Loria et al, 2003; Estêvão-Costa et
al, 2000]. This might be due to the presence of additional domains in the structure of
these enzymes, which might hamper the interaction with the -M and thus likely to
be associated with the ability of these toxins to exert systemic bleeding and toxicity in
several cases [Loria et al, 2003]. The NN-PF3 is likely belongs to the P-III class of
SVMPs, because of its high molecular mass, lack of inhibition by the α2-M and also
the indication of the presence of disintegrin-like region, since the EDTA inactivated
NN-PF3 inhibits the collagen induced platelet aggregation in the washed platelet
suspension (Fig. 3.3A of Chapter-III). However, several P-III class SVMPs were
associated with strong hemorrhagic activity, interestingly some were completely
devoid of hemorrhagic activity [Fox and Serrano, 2005; Moura-da-Silva et al, 2008].
NN-PF3, despite being a PIII class SVMPs, it did not act on any of the extracellular
matrix proteins used for the study and thus supports its non-hemorrhagic property of
NN-PF3 [Jagadeesha et al, 2002].
In conclusion, the snake venoms being the depot of target specific molecules,
because of their high degree of specificity, several of them are extensively being used
therapeutically or they might serve as prototypes for designing better drug molecules.
The NN-PF3 being a non-toxic molecule and since this single molecule possesses the
beneficial properties, the anticoagulant activity through fibrin(ogen)olytic activity, it
may be explored for the possible therapeutic use to treat clinical conditions associated
with the cardiovascular diseases and as well as strokes.
50
Chapter-II
Anticoagulant activity of a..…
Figures
Fig. 2.1: Mass spectrometry of NN-PF3. Mass spectrometry of NN-PF3 was done
by using Bruker Daltonics Matrix-Assisted Laser Desorption Ionization Time Of
Flight (MALDI-TOF) machine in positive ionization mode. Alpha-Cyano-4hydroxycinnamic acid was used as MALDI matrix.
51
Chapter-II
Anticoagulant activity of a..…
Fig. 2.2: Inhibition of fibrinogenolytic activity by NN-PF3: Fibrinogen (50 g)
alone (1) or treated with NN-PF3 which was previously treated without any inhibitor
(2) or with specific inhibitors, PMSF (3), EDTA (4), 1, 10-phenonthroline (5),
Pepstatin A (6) and IAA (7) in 40 l 10 mM Tris-HCl buffer, pH 7.4 for 4 h at 370C.
The reaction was terminated by adding the 20 l denaturing buffer containing 1 M
urea, 4% SDS, 4% β-mercaptoethanol and boiled for 5 min. Then the samples were
subjected to 10% SDS-PAGE analysis.
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Fig. 2.3: Fibrinolytic activity of NN-PF3. (A) Plate method: A mixture of agarose
(1.2%) in 10 mM Tris-HCl buffer, pH 7.4 containing 0.15 M NaCl, 0.05% sodium
azide, 70 mM (NH4)2SO4, 90 mM NaCl, 0.70 mM MgCl2 and 200 l of 0.2 M CaCl2
along with 2 ml of platelet poor plasma was poured into 10 mm x 90 mm flat Petri
dish and left for 2 h at 300C. The samples in 10 μl of 10 mM Tris-HCl buffer, pH 7.4
were independently placed on the surface of the gel and incubated for 12 h at 300C.
The NN-PF3, 1 µg (A), 2 µg (B), 3 µg (C), 4 µg (D), 4 µg pretreated with 5 mM
PMSF (E), 4 µg pretreated with 5 mM EDTA (F), buffer alone (G) and one IU of
urokinase (U). (B) Colorimetric method: The washed, whole plasma clot (■) and the
blood clot (♦) were incubated independently with the increasing amount (5–25 g) of
NN-PF3 in 0.2 M Tris-HCl buffer, pH 8.5 at 370C for 2 h 30 min. The protein
fragments released by NN-PF3 were measured after precipitating the sample with
trichloroacetic acid.
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Fig. 2.4: Banding pattern of fibrinopeptides in SDS-PAGE. (A) The washed
plasma clot was incubated independently with the different doses of NN-PF3 for 4 h
in 40 μl of 10 mM Tris-HCl buffer pH 7.4 at 370C. The samples including, plasma
clot alone (1) and plasma clot treated with NN-PF3, 0.5 μg (2), 1 μg (3), 1.5 μg (4)
and 2 μg (5), 2.5 μg (6) and NN-PF3 pretreated with 5 mM EDTA (7) and molecular
weight markers in kDa (M). (B) The washed plasma clot was incubated independently
for different lengths of time with 1 μg of NN-PF3 in 40 μl of 10 mM Tris-HCl buffer
pH 7.4 at 370C. NN-PF3 incubated for 0 min (1), 1 h (2), 2 h (3), 3 h (4) and 24 h (5)
and NN-PF3 pretreated with 5 mM EDTA (6) and the clot incubated with buffer alone
for 24 h (7) and the standard molecular weight markers in kDa (M). In both the cases
the samples were analyzed using SDS-PAGE (10%) under reduced condition.
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Fig. 2.5: Fibrinogenolytic activity of NN-PF3 with prolonged Thrombin Clotting
Time (TCT). The incubation mixture contains 2 mg/ml human fibrinogen and 10
g/ml NN-PF3 in 10 mM Tris-HCl buffer, pH 7.4. At the indicated incubation times,
25 l aliquots were mixed with 20 l of reducing sample buffer and were analyzed by
10% SDS-PAGE under reduced condition. Also at the same incubation time, 100 l
aliquots were transferred to 12 mm x 75 mm glass tubes to determine the thrombin
clotting time (TCT). The TCT (mean ± SD for five determinations from separate
experiments) are indicated below. The lane M represents the standard molecular
weight markers in kDa.
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Fig. 2.6: Degradation of human plasma proteins by NN-PF3. Plasma proteins (100
g) was incubated with 1 g of NN-PF3 in 40 μl of 10 mM Tris-HCl buffer pH 7.4 at
370C for various intervals of time and then analyzed on 7.5% SDS-PAGE under nonreduced condition. Zero min (1), 10 min (2), 30 min (3), 1 h (4), 12 h (5), 24 h (6) 20
g of pure fibrinogen was served as control (F), and the standard molecular weight
markers in kDa (M).
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Fig. 2.7: Effect of NN-PF3 on bleeding time. Tail bleeding time was measured 10
min after i.v. administration of various doses of NN-PF3 (A) and the bleeding time
measured at 1, 3, 6, 12, 18 and 24 h after i.v. injection of NN-PF3 (2.3 g/kg) (B).
Each point represents the mean ± SD (n=5), p<0.01. Bleeding time longer than 10 min
was expressed as > 10 min.
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Fig. 2.8: Inhibition of NN-PF3 by 2-macroglobulin and polyvalent anti-venom.
The NN-PF3 was preincubated with different molar ratios of 2-macroglobulin
(mol/mol) (A) and polyvalent anti-venom (w: w) (B) in a reaction volume of 100 l of
0.2 M Tris-HCl buffer, pH 8.5 at 370C for 15 min. Then the proteolytic activity on fat
free casein was determined. The plot is representative one of three experiments (A)
and the values represents the mean ± SD (n=5) in (B).
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Fig. 2.9: Effect of NN-PF3 on the extracellular matrix proteins. The extracellular
matrix (ECM) proteins, collagen type-I (A), collagen type-IV (B) and fibronectin (C)
were incubated independently with NN-PF3 in the ratio of 25:1 (ECM proteins: NNPF3, w/w) in a reaction volume of 40 l of 10 mM Tris-HCl buffer, pH 7.4 at 370C.
The samples were analyzed on SDS-PAGE (7.5%) under reduced condition. ECM
proteins alone (1) and incubated with NN-PF3 for 2 h (2), 6 h (3), 12 h (4), 18 h (5)
and 24 h (6) and M represents the standard molecular weight markers in kDa.
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Tables
Table 2.1A: Dose dependent effect of NN-PF3 on clotting time of human
citrated plasma
NN-PF3
(µg)
PT
(INR values)
APTT ratio
TCT
(sec)
0
1.00 ± 0.01
1.00 ± 0.02
16.0 ± 1.2
1
1.26 ± 0.04
1.16 ± 0.03
20.2 ± 0.8
2
1.60 ± 0.05
1.45 ± 0.04
22.5 ± 1.3
3
2.03 ± 0.04
1.71 ± 0.01
26.4 ± 0.9
4
2.35 ± 0.02
1.90 ± 0.03
29.3 ± 1.0
5
2.90 ± 0.03
2.12 ± 0.02
32.5 ± 0.5
6
3.40 ± 0.02
2.28 ± 0.02
35.8 ± 1.0
The values are the average of the duplicate values of the three independent tests ± SD
Table 2.1B: Time dependent effect of NN-PF3 on clotting time of human
citrated plasma
Preincubation
time (min)
PT
(INR values)
APTT ratio
TCT
(Sec)
0
1.00 ± 0.01
1.00 ± 0.02
16.5 ± 1.2
5
1.46 ± 0.04
1.28 ± 0.03
25.7 ± 0.8
10
1.87 ± 0.05
1.48 ± 0.04
28.5 ± 1.3
15
2.39 ± 0.04
1.76 ± 0.01
36.6 ± 1.9
30
3.49 ± 0.02
2.90 ± 0.03
64.5 ± 4.3
45
7.90 ± 0.03
5.12 ± 0.02
157 ± 6.3
120
No clot
No clot
No clot
The values are the average of the duplicate values of the three independent tests ± SD
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Table 2.2: Plasminogen activation
Activity
(Unit)*
Samples
1) 1 mg plasminogen contaminated fibrinogen (a)/
l citrated human plasma (b)
20
NIL
2) a + 200 IU of Urokinase
6.4 ± 0.68
3) b + 200 IU of Urokinase
2.8 ± 0.50
4) a/ b + 20 g NN-PF3
NIL
5) 20 g NN-PF3
NIL
6) 200 IU of Urokinase
NIL
*One unit of activity was defined as the amount of enzyme yielding an increase in absorbance
of 0.01/h at 440 nm. The values are the mean values of three independent tests ± SD
Table 2.3: Neutralization of anticoagulant effect of NN-PF3 by polyvalent
anti-venom
PT
APTT
TCT (Sec)
(INR values)
ratio
Plasma
1.00 ± 0.01
1.00 ± 0.01
16 ± 0.8
Plasma + NN-PF3 (1 g)
1.27 ± 0.02
1.18 ± 0.03
20 ± 1.1
Plasma + NN-PF3:anti-venom (w/w,
1:1000 g)
1.16 ± 0.03
1.08 ± 0.04
18 ± 0.5
Plasma + NN-PF3:anti-venom (w/w,
1:2500 g)
1.0 ± 0.02
1.0 ± 0.03
16 ± 0.5
The values are the average of the duplicate values of the three independent tests ± SD
61