Ramachandraiah et al.
, J Blood Res Hematol Dis 2017, 2:1
Research Article
Evaluation of Anticoagulant and
Antiplatelet Activity of Pisum
sativum Pod Extract
Chethana Ramachandraiah1, Sharath Kumar M Nandish1,
Jayanna Kengaiah1, Ashwini Shivaiah1, Chandramma1,
Kesturu S.Girish1, Kempaiah Kemparaju2 and Devaraja
Sannaningaiah1*
Abstract
The current study evaluates the anticoagulant and antiplatelet
activities of Pisum sativum pod extract (PSPE). PSPE revealed
similar protein banding pattern under both reducing and non-
reducing conditions on SDS-PAGE, suggesting the presence of
monomeric proteins in the extract. PSPE showed proteolytic activity
as it hydrolyzed casein and gelatin and the specific activity was
found to be 0.16 and 0.21 units/mg/min respectively. The proteolytic
activity of PSPE was completely abolished by PMSF but not IAA,
EDTA and 1,10, Phenanthroline suggesting the presence of
serine protease. Further, PSPE was evaluated for anticoagulation
efficiency. Interestingly, PSPE increased the clotting time of citrated
human plasma from the control 220 sec to 460 sec suggesting its
anticoagulant property. Furthermore, PSPE specifically prolonged
the clotting time of APTT but not PT revealed the interference
in intrinsic pathway of coagulation cascade. In addition, PSPE
exhibited anti-aggregation activity by inhibiting agonists such
as ADP and epinephrine induced platelet aggregation in a dose
dependent manner and the detected aggregation inhibition
was found to be 58% and 82% respectively. In addition, PSPE
preferentially hydrolyzed Aα and Bβ chain of human fibrinogen and
all the chains of fibrin clot. Fibrinogenolytic and fibrinolytic activity
of PSPE was inhibited by PMSF but not IAA, EDTA and 1,10,
Phenanthroline suggesting the presence of serine protease in the
extract. Moreover, PSPE did not hydrolyze RBC cells signifying its
non-toxic property.
Keywords
Pisum Sativum pod extract; Serine protease; Anticoagulant;
Antiplatelet; Fibrinogenolysis
Abbreviations: PSPE: Pisum sativum Pod Extract; PT: Pro-
thrombin Time; APTT: Activated Partial Thromboplastin time; IAA:
Iodo Acetic Acid; PMSF: Phenyl Methyl Sulphonyl Fluoride; EDTA:
Ethylene di-Amine Tetra Acetic Acid
Introduction
Pisum sativum (commonly called pea) is the seed-pod belongs to
the family Fabaceae grown in cooler regions in all over the world.
*Corresponding author: Devaraja Sannaningaiah, Ph.D., Assistant Professor,
Department of Studies and Research in Biochemistry, Tumkur University, BH Road,
Tumkur-572103, India, Tel: +91-9902838928; E-mail: sdevbiochem@gmail.com
Received: October 19, 2016 Accepted: November 03, 2017 Published:
November 08, 2017
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Technology and Medicine
Journal of Blood Research
& Hematologic Diseases
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Peas found to possess significant quantities of macro and micro
nutrients. The key macromolecules include protein, carbohydrates
and lipids. The micro molecules include vitamins (A, B6, C, & K)
and minerals (phosphorus, magnesium, copper, iron and zinc).
In addition peas are also richest source of secondary metabolites
(flavonoids, carotenoid, phenolic acids, polyphenols). Thus, the
peas have been consuming world wide as a major dietary source
as part of routine meal. Investigation by various research groups
documented the contribution of said nutrients on various health
benefits [1]. For instance, antioxidant and anti-inflammatory effect of
peas could be due to the presence of Beta-carotene, lutein, lycopene,
selenium and phenolic acids respectively [2]. On the other hand
the presence of coumestrol and the protease inhibitors in the peas
found to exhibit anticancer activity [3]. While, the saponins present
in the peas plays a key role in reducing the risk of type II diabetes [2].
Pea contains noteworthy concentration of raffinose family and also
other galactose-containing oligosaccharides which exhibit prebiotic
effects in the large intestine. Moreover, Pisum saponins I and II
with pisomosides A and B are exclusively found in peas acts as an
anti-inflammatory phytonutrients, which helps to prevent wrinkles,
arthritis, osteoporosis, Candida and bronchitis [4]. Peas contain
niacin and ß-sitosterol help to lower the cholesterol levels and to
sustain strong bones [5]. In addition, fiber content in peas improves
constipation and also reduces blood cholesterol by declining the re-
absorption of bile acids [6]. Vitamins present in the peas found to
reduce the homocysteine level in the blood which in turn helps to
lower the risk of cardiovascular diseases [7]. Importantly, in-vitro
proteolytic generated peptides from the pea proteins found to exhibit
antihypertensive and antioxidant activities [8]. Although, nutritional
importance and the presence of hydrolytic enzymes namely amylase,
phosphatase & phytase, trypsin inhibitors, lectins have been reported
[9]. Nevertheless, the role of proteolytic enzymes of Pisum sativum
seed proteins/enzymes and their probable therapeutic potential on
coagulation disorders have been least considered. Thus, the current
study examines the anticoagulant and anti-platelet activity of Pisum
sativum pod extract.
Materials and Methods
Fat Free Casein, Gelatin, Phenyl Methyl Sulphonyl Fluoride
(PMSF), Ethylene Di-Amine Tetra Acetic acid (EDTA), Iodo-Acetic
Acid (IAA), 1, 10, Phenanthroline were purchased from Sigma
Chemicals Company (St. Louis, USA). Molecular weight markers
were from Bangalore Genie Private limited, India. APTT and PT
Reagents were purchased from AGAPPE diagnostic Pvt. Ernakulum,
Kerala, India. Human plasma fibrinogen was purchased from Sigma
Chemicals Co. St. Louis, USA. All other chemicals used were of
analytical grade. Fresh human blood was collected from healthy
donors for the platelet rich plasma (PRP).
Preparation of Pisum sativum extract and protein estimation
Pisum sativum seeds were purchased from local market Tumkur.
The seeds were washed and soaked with distilled water then the seed
coat was removed. The edible parts of the seeds were homogenized
using double distilled water and centrifuged at 8000 g for 20 min at
4°C. The supernatant was collected and proteins were precipitated
using 30% of ammonium sulphate. The precipitated protein sample
Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.
was again centrifuged at 3500 g for 20 min; pellet was dissolved and
dialyzed overnight. The protein sample obtained was stored at -20°C
until further use. This extracted protein sample was used throughout
the study and referred as Pisum sativum pod extract (PSPE). Protein
concentration was determined as described by Bradford, et al. [10]
using Bovine Serum Albumin (BSA) as standards.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) and periodic acid-Schiff (PAS) staining
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%
SDS-PAGE) was carried out according to the method of Laemmle
[11]. The crude PSPE (100 µg) prepared under reducing and non-
reducing conditions was used for SDS-PAGE. The electrophoresis
was carried out using Tris (25 mM/l), glycine (192 mM/l) and SDS
(0.1%) for 2h at room temperature. After electrophoresis, the SDS-
PAGE gels were stained with 0.1% Coomassie brilliant blue R-250
for detection of the protein bands and de-stained with 40% ethanol
in 10% acetic acid and water (40 : 10 : 50 v/v). Molecular weight
standards from 200 to 14.3 kDa were used.
PAS staining was carried out according to the method of Leach,
et al. [12] after electrophoresis; the gel was fixed in 7.5% acetic acid
solution and stored at room temperature for 1 hour. Then the gel
was washed with 1% nitric acid solution and kept in 0.2% aqueous
periodic acid solution and stored at 4°C for 45 min. After that, the gel
was placed in Schiff’s reagent at 4°C for 24h and was de-stained using
10% acetic acid to visualize a pink colour band.
Proteolytic activity
Proteolytic activity was assayed as described by Satake et al. [13].
Fat-free Casein & gelatin (0.4 ml, 2% in 0.2 M/l Tris–HCl buffer, pH
7.6) was incubated with 50 µg of crude PSPE in a total volume of 1ml
for 2 h and 30 min at 37°C. Undigested gelatin was precipitated by
adding 1.5 ml of 0.44 M/l Trichloroaceticacid (TCA) and left to
stand for 30 min. The reaction mixture was then centrifuged at
2000 g for 10 min. Sodium carbonate (2.5 ml, 0.4 M/l) and Folin-
Ciocalteu 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 the enzyme activity was
defined as the amount of the enzyme required to cause an increase
in optical density (OD) of 0.1 at 660 nm/min at 37ºC. The specific
activity was expressed as units/min/mg of protein. For inhibition
studies, a similar reaction was performed independently after pre
incubating the crude PSPE (50 µg) for 30 min with 5 mM/l each
of EDTA, 1,10-phenanthroline, PMSF and IAA. In all the cases,
appropriate controls were kept.
Zymogram
Zymogram was done as described previously [14], briefly
PSPE (50 μg) prepared under non-reduced condition was loaded
on to polymerized 2% Casein / gelatin in resolving gel. After
electrophoresis gels were washed with 10mM sodium phosphate
buffer containing 2.5% of Triton X-100 with constant agitation for 1 h
to remove SDS. The gel was incubated overnight at 37°C in Tris–HCl
buffer (50 mM) pH 7.6 containing 50 mM CaCl2 and 40mM NaCl.
Gel was then stained to observe the translucent activity bands. For
inhibition studies, a similar reaction was performed independently
after pre incubating the crude PSPE (50 µg) for 20 min with 5 mM/l
each of EDTA, 1,10-phenanthroline, PMSF and IAA. In all the cases,
appropriate controls were kept.
Volume 2 • Issue 1 • 1000109
Anticoagulant activity
Plasma re-calcification time: The plasma re-calcification time
was determined according to the method of Quick, et al. [15]. Briefly,
the crude PSPE (0-30 μg) was pre-incubated with 0.2 ml of citrated
human plasma in the presence of 10 mM Tris HCl (20 μl) buffer pH
7.4 for 1min at 37°C. 20 μl of 0.25 M CaCl2 was added to the pre-
incubated mixture and clotting time was recorded.
Activated partial thromboplastin time (APTT) and
prothrombin time (PT): Briefly, 100 μl of normal citrated human
plasma and PSPE (0–30 μg) were pre-incubated for 1 min. For APTT,
100 μl reagent (LIQUICELIN-E Phospholipids preparation derived
from Rabbit brain with ellagic acid), which was activated for 3 min at
37°C was added. The clotting was initiated by adding 100 μl of 0.02 M
CaCl2 and the clotting time was measured. For PT, the clotting was
initiated by adding 200 μl of PT reagent (UNIPLASTIN–rabbit brain
Thromboplastin). The time taken for the visible clot was recorded
in seconds. 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.
Fibrin(ogeno)lytic activity: Fibrinogenolytic activity was
determined as described previously by Ouyang and Teng et al. [16].
PSPE (0-50 μg) was incubated with the human plasma fibrinogen (50
μg) in a total volume 40 μl of 10 mM Tris–HCl buffer pH7.4 for 4
hours at 37°C. After the incubation period, reaction was terminated
by adding 20 μl denaturing buffer containing 1 M urea, 4% SDS and
4% β-mercaptoethanol. It was then analyzed by 10% SDS-PAGE. For
inhibition studies, PSPE (20 μg) was pre incubated for about 20 min
with 5 mM each of PMSF, IAA, EDTA and 1,10-Phenanathroline.
Fibrin clot-hydrolyzing activity by calorimeter
Fibrin clot-hydrolyzing activity was determined as described by
Rajesh, et al. [17]. Briefly, 100 µl of citrated human blood/plasma
was mixed with 20 µl of 0.2 M/l CaCl2 and incubated for 2h at 37°C.
The clot obtained was washed thoroughly for 5-6 times with PBS and
suspended in 400 µl of 0.2 M/l Tris–HCl buffer (pH 8.5).The reaction
was initiated by adding varied amounts of PSPE (0-50 µg) in 100 µl
of saline and incubated for 2 h and 30 min at 37°C. The undigested
clot was precipitated by adding 750 µl of 0.44M/l TCA and allowed to
stand for 30 min and centrifuged for 15 min at 1500 g. The aliquots
of 0.5ml supernatant was transferred to clean glass tubes and it was
followed by the addition of 1.25 ml of 0.4 M/l sodium carbonate and
0.25 ml of 1: 3 diluted Folin–Ciocalteu’s phenol reagent. The colour
developed was read at 660 nm after being allowed to stand for 30min.
One unit of activity is defined as the amount of enzyme required to
increase in absorbance of 0.01 at 660 nm/h at 37°C.
Fibrinolytic activity by SDS-PAGE
Fibrin clot was prepared as describe above, and the clot obtained
was incubated with the various concentrations of PSPE (0-50 µg) in
a final volume of 40 µl 10 mM/l Tris–HCl buffer (pH 7.4) at 37°C
for 6 hours. The reaction was stopped by adding 20 µl of sample
buffer containing 4% SDS, 1M/l urea and 4% β-mercaptoethanol. The
samples were kept on boiling water bath for 30min and centrifuged
to settle the debris of the plasma clot. An aliquot of 30 µl supernatant
was analyzed in 7.5% SDS-PAGE for fibrin clot degradation study. For
inhibition studies, PSPE (20 µg) was pre-incubated for about 15 min
with 5 mM/l each of PMSF, EDTA, IAA and 1, 10-phenonthroline.
The reaction was stopped by adding 20 µl of sample buffer containing
4% SDS, 1 mol/l urea and 4% β-mercaptoethanol. The samples were
• Page 2 of 8 •
Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.
kept on boiling water bath for 5 min and centrifuged to settle the
debris of plasma clot. An aliquot of 30 µl supernatant was analyzed in
7.5% SDS-PAGE for fibrin degradation.
Degradation of human plasma proteins
Degradation of human plasma protein was assayed according to
the method of Kumar, et al. [18]. The PSPE (0–50 µg) was incubated
with the 100 µg of plasma proteins for 12 h at 37°C in a reaction
volume of 40 µl 10 mM/l Tris–HCl buffer (pH 7.4) containing 10
mM/l NaCl, 0.05% sodium azide. The reaction was terminated by the
addition of 20 µl denaturing buffer containing 4% SDS and boiled for
5 min. It was then analyzed on a 7.5% SDS-PAGE under non-reduced
condition.
Preparation of platelet-rich plasma and platelet-poor
plasma
The method of Ardlie and Han [19] was employed for the
preparation of human platelet-rich plasma (PRP) and platelet-poor
plasma (PPP). The platelet concentration of PRP was adjusted to
3.1×108 platelets/ml with PPP. The PRP maintained at 37°C was used
within 2h for the aggregation process. All the above preparations
were carried out using plastic wares or siliconized glass wares.
Platelet aggregation
The turbid metric method of Born [20] was followed using a
Chronology dual channel whole blood/optical lumi aggregation
system (Model-700). Aliquots of PRP were pre-incubated with
various concentrations of PSPE (0–90 μg) in 0.25 ml reaction volume.
The aggregation was initiated independently by the addition of
agonists, such as ADP and epinephrine and followed for 3 min.
Direct haemolytic activity
Direct haemolytic activity was determined by using washed human
erythrocytes. Briefly, packed human erythrocytes and phosphate
buffered saline (PBS) (1:9 v/v) were mixed; 1ml of this suspension
was incubated independently with the various concentration of PSPE
(0-300 µg) for 1h at 37°C. The reaction was stopped by adding 9 ml
of ice cold PBS and centrifuged at 1000 g for 10 min at 37°C. The
amount of haemoglobin released in the supernatant was measured at
540 nm. Activity was expressed as percent of hemolysis against 100%
lysis of cells due to addition of water that served as positive control
and phosphate buffered saline served as negative control.
Statistical analysis
The data are presented as mean ± SD. Statistical analyses were
performed by Student’s T-test. A significant difference between the
groups were considered if P<0.01.
Results
Current study intends to identify the anticoagulant and
antiplatelet activities of Pisum Sativum Pod Extract (PSPE) and the
results are presented. In order to study the protein banding pattern in
PSPE 10% SDS-PAGE was done, it revealed similar protein banding
pattern from the range 200 to 14kDa under reduced and non-reduced
conditions suggesting the presence of monomeric proteins (Figure
1a). In addition, PSPE was examined for possible carbohydrate
moiety in the extract; fascinatingly it was positive for PAS-staining at
the region around 65 kDa to 14.3 kDa those can be compared with the
fibrinogen the positive control (Figure 1b). Furthermore, Proteolytic
Volume 2 • Issue 1 • 1000109
activity of PSPE was analyzed using casein and gelatin as substrates.
Interestingly PSPE showed proteolytic activity as it hydrolyzed both
casein and gelatin with the specific activity 0.16 and 0.21 units/
mg/min at 37°C respectively. Proteolytic activity was also further
strengthened by Zymography experiments using casein & gelatin as
the substrates. Curiously, PSPE exhibited a translucent activity band
around 200kDa region (Figure 2) in both the zymograms suggest that
presence of protease in the extract. Thus, inhibition studies were carried
out to identify the type of protease withhold in the extract, using specific
protease inhibitors such as PMSF, IAA, EDTA, 1,10, Phenanthroline.
Surprisingly, only the PMSF, a serine protease inhibitor completely
inhibited the proteolytic activity of PSPE and it was revealed in both
calorimetric assay (Table 1) and Zymography (Figure 2) experiments.
Further, the experiments was designed to identify the probable
role of PSPE in coagulation cascade, plasma re-calcification time was
performed using both human platelet rich plasma (PRP) and platelet
poor plasma (PPP). Remarkably, PSPE exhibited anticoagulant
effect by enhancing the clotting time of both PRP and PPP from
control 220 s to 460 s and 300 s to 610 s respectively. The maximaum
concentration consumed in both the cases was found be 18 µg and
remain unchanged upon increased dose to 30 µg (Figure 3).
In order to explore the exhibited anticoagulation property of
PSPE involved in which pathway of coagulation cascade, Activated
Partial Thromboplastin Time (APTT) and Prothrombin Time (PT)
were performed. Interestingly, PSPE predominantly delayed the
clotting time of only APTT but not PT, explored that the observed
anticoagulation is because of the PSPE interference in intrinsic
pathway of blood coagulation cascade (Table 2).
Further, PSPE was analyzed for fibrinogenolysis activity using
human fibrinogen. Surprisingly, PSPE degraded only Aα chain in
the dose dependent manner without affecting the Bβ and γ chains
of human fibrinogen at the maximum concentration of 50 µg for 4h
incubation time at 37°C (Figure 4a). Although, when incubation time
was extended for 24 h at the concentration of 20µg of PSPE, it degraded
both Aα and Bβ chains of human fibrinogen without disturbing the
γ chain (Figure 4b). In addition, inhibition study of fibrinogenolytic
activity was carried out; interestingly fibrinogenolytic activity was
inhibited by only PMSF, a serine protease but not by the cysteine and
metallo protease inhibitors, suggests that the role of serine protease in
the PSPE (Figure 4c).
Since, PSPE exhibited fibrinogenolytic activity; an attempt
was made on its ability to dissolve fibrin clot. Primarily fibrin clot
degradation assay was performed colorimetrically (Figure 5a) and
the obtained specific activity was found to be 2.2 units/mg/min. The
fibrin clot dissolving property of PSPE was further confirmed on SDS-
PAGE (Figure 5b). PSPE was capable to degrade only α polymer chain
in a dose dependent fashion at the concentration of 50µg at 37°C for
12h incubation time without affecting γ-γ dimer, α chain and β chain
of fibrin clot. But in time dependent manner when incubation time
was prolonged for 24 hours, PSPE degraded all the chains of fibrin
clot at the concentration of 20 µg at 37°C (Figure 5c). In addition
fibrin clot degradation was inhibited by only PMSF a serine protease
suggesting that extract withhold serine proteolytic potential (Figure
5d). Furthermore, plasma protein degradation assay was carried
out to identify the substrate specificity of PSPE on proteins. PSPE
preferably degraded only fibrinogen bands without affecting the
other plasma proteins when incubated at 37°C for 12 hours at the
concentration of 50 µg (Figure 6).
• Page 3 of 8 •
Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.

Figure 1(a): SDS-PAGE 10% (b)  Glycoprotein  staining: (a)  PSPE  as shown in SDS-PAGE (10%): PSPE  (100 µg)  under  non-reduced  (a1)  and
reduced conditions (a2), PAS staining of PSPE: positive control fibrinogen (b1) and PSPE (b2). M represents the molecular weight marker in kDa from
top to bottom: myosin-H- chain (200), BSA (66.4), ovalbumin (44.3), carbonic anhydrase (29), lactalbumin (18.4) and lysozyme (14.3) BSA: bovine serum
albumin, PSPE: Pisum sativum pod extract.

Figure 2(a): Casein Zymogram (b) Gelatin Zymogram (c) Casein Zymogram with inhibitor (d) Gelatin Zymogram with inhibitor: (a) PSPE Casein Zymogram:
PSPE 50 µg under non-reduced conditions. (b) PSPE Gelatin Zymogram: PSPE 50 µg under non-reduced conditions. (c) PSPE Casein Zymogram with
inhibitor: PSPE 50 μg alone (c1), PSPE 50 μg was pre-treated with 5 mM PMSF (c2), PSPE 50 μg was pre-treated with 5 mM IAA (c3), PSPE 50 μg was pre-
treated with 5 mM 1,10-Phenanthroline (c4), PSPE 50 μg was pre-treated with 5 mM EDTA (c5) under non-reduced conditions. (d) PSPE Gelatin Zymogram
with inhibitor: PSPE 50 μg alone (d1), PSPE 50 μg was pre-treated with 5 mM PMSF (d2), PSPE 50 μg was pre-treated with 5mM IAA (d3), PSPE 50 μg was
pre-treated with 5 mM 1,10-Phenanthroline (d4), PSPE 50 μg was pre-treated with 5mM EDTA (d5) under non-reduced conditions.

Table 1: Calorimetric assay.

Inhibitor (5mM each) Activity/ Residual Activity
None 100
EDTA 99.05
1,10-Phenanthroline 88.9
IAA 95.1
PMSF 11.71

Furthermore, PSPE was analyzed for platelet aggregation
assay using platelet rich plasma with agonists such as ADP and
epinephrine. Remarkably, PSPE inhibited both the agonists such as
ADP and epinephrine induced platelet aggregation of about 58% and
82% respectively at the concentration of 90 µg (Figures 7 and 8). The
order of inhibition observed among agonists was Epinephrine>ADP
induced aggregation. Moreover, PSPE did not hydrolyze RBC up to
the concentration of 300 µg.
Discussion
Haemostasis a physiological phenomenon is pretty much
essential event for the arrest of bleeding during injury, yet it is highly
regulated pathway [21]. Genetic disparity and environmental factors
imbalance the haemostatic pathway, which leads to thrombosis [22].
Thus, the thrombosis is a pathological phenomenon involved in the
formation of unusual clots in the arteries and vein responsible for
cerebrovascular/cardiovascular complication, which was the major
health issue worldwide [23]. Although, the usage of anticoagulant and
antiplatelet molecules has been predominant, their life threatening
side effects at higher dosage is the limiting factor. In recent time, plant
based drugs have been gaining much importance due to their least
side effects. Thus the current study examines the anticoagulant and
antiplatelet activities of Pisum sativum. PSPE showed similar banding
pattern under both reduced and non reduced conditions revealed the
presence of monomeric proteins. PSPE withholds carbhohydrate
content in the proteins as it took up PAS stain suggesting PSPE
withhold maximum number of glycoproteins. PSPE hydrolyzed
casein & gelatin suggesting its proteolytic activity. Curiously,
proteolytic activity of PSPE was completely neutralized by PMSF
suggests the presence of serine protease in the extract. Number of

Volume 2 • Issue 1 • 1000109 • Page 4 of 8 •

Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.

Figure 3: Plasma re-calcification time: (a) PSPE (0-30 µg) was pre-incubated with 0.2 ml of citrated human plasma PRP/PPP in the presence of 20 µl 10mM
Tris–HCl buffer (pH 7.4) for 1 min at 37°C. 20 µl of 0.25 M CaCl2 was added to the pre-incubated mixture and clotting time was recorded.

Table 2: PSPE interference in intrinsic pathway of blood coagulation cascade.

APTT Clotting time in
PSPE (μg) PT Clotting time in seconds PT (INR Value) APTT ratio
seconds
0 12.50 ± 0.02 0.93 ± 0.02 33.40 ± 0.05 1.22 ± 0.02
3 12.10 ± 0.03 0.91 ± 0.03 34.50 ± 0.02 1.25 ± 0.01
6 12.40 ± 0.02 0.92 ± 0.01 36.42 ± 0.07 1.37 ± 0.04
9 12.20 ± 0.01 0.90 ± 0.01 37.41 ± 0.03 1.51 ± 0.05
12 13.40 ± 0.02 1.44 ± 0.01 39.10 ± 0.01 1.78 ± 0.02
15 14.10 ± 0.01 1.50 ± 0.01 40.01 ± 0.03 1.91 ± 0.01
18 13.10 ± 0.03 1.37 ± 0.02 41.22 ± 0.05 2.15 ± 0.03
21 14.25 ± 0.02 1.50 ± 0.01 45.50 ± 0.01 2.67 ± 0.02
24 14.20 ± 0.01 1.47 ± 0.03 46.10 ± 0.04 2.72 ± 0.01
27 14.15 ± 0.04 1.40 ± 0.01 46.40 ± 0.02 2.91 ± 0.03
30 14.05 ± 0.01 1.38 ± 0.05 46.52 ± 0.01 2.95 ± 0.02

Figure 4: Fibrinogenolytic activity (a) Dose-dependent effect (b) Time-dependent effect (c) Inhibition study. (a) PSPE Dose-dependent effect: fibrinogen
alone 50 µg (a1), fibrinogen treated with 10 µg (a2), 20 µg (a3), 30 µg (a4), 40 µg (a5), 50 µg (a6) of PSPE respectively, incubated for 4 hr at 37°C and then
separated on 10% SDS-PAGE under reduced condition. (b) PSPE Time-dependent effect: PSPE 20 µg was incubated with fibrinogen 50 µg for 0 hr (b1), 4
hr (b2), 8 hr (b3), 12hr (b4), 16 hr (b5) and 24 hr (b6) respectively at 37°C. (c) PSPE Inhibition study: PSPE 20 µg was pre-incubated with protease inhibitors
for 30 min at 37°C. Further reaction was initiated by adding 50 µg of fibrinogen and incubated for 4hr, fibrinogen alone (c1), PSPE 20 µg (c2), fibrinogen 50
µg and PSPE 20 µg with 5 mM PMSF (c3), fibrinogen 50 µg and PSPE 20 µg with 5 mM IAA (c4), fibrinogen 50 µg and PSPE 20 µg with 5 mM EDTA (c5),
fibrinogen 50 µg and PSPE 20 µg with 5 mM 1,10-phenanthroline (c6).

Volume 2 • Issue 1 • 1000109 • Page 5 of 8 •

Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.

Figure 5: Fibrinolytic activity (a) Colorimetric assay (b) Dose-dependent effect (c) Time-dependent effect (d) Inhibition Study. (a) Washed plasma clot
was incubated with 0–100 µg of PSPE for 2.30hr and then the OD was measured at 660nm. (b) PSPE Dose-dependent effect; Washed plasma clot was
incubated for 12hr and then separated on SDS-PAGE (7.5%), washed plasma clot alone (b1), plasma clot treated with 10 µg (b2), 20 µg (b3), 30 µg (b4), 40
µg (b5) and 50 µg (b6) of PSPE respectively. (c) PSPE Time-dependent effect; PSPE 20 µg was incubated with fibrin clot at 37°C, fibrin clot alone (c1), 0
hr (c2), 6 hr (c3), 12 hr (c4), 18 hr (c5) and 24 hr (c6) of PSPE. (d) PSPE Inhibition study: PSPE 20 µg was pre-incubated with protease inhibitors for 30
min at 37°C. Further reaction was initiated by adding fibrin clot and incubated for 12 hr, fibrin clot alone (d1), PSPE 20 µg (d2), fibrin clot and PSPE 20 µg
with 5 mM PMSF (d3), fibrin clot and PSPE 20 µg with 5 mM IAA (d4), fibrin clot and PSPE 20 µg with 5 mM EDTA (d5), fibrin clot and PSPE 20 µg with 5
mM 1,10-phenanthroline (d6).

Figure 6: Degradation of plasma proteins: Plasma protein (100µg) was incubated with PSPE in 40 µl of 10mM Tris–HCl buffer (pH 7.4) at 37°C and then
analyzed on 7.5% SDS-PAGE under non-reduced condition. Plasma protein (100µg) alone (1), plasma protein treated with 10 µg (2), 20 µg (3), 30 µg (4),
40 µg (5), 50 µg (6) of PSPE and 20µg of fibrinogen as control (7).

serine proteases were investigated from plants (latex, seeds, leaves),
and animals (earthworms, caterpillar, snake venom, spider and
honeybees) [24]. Fascinatingly, PSPE exhibits anticoagulant property
by Prolonging the clot formation process of citrated human plasma
and it was identified that the triggered anticoagulation by PSPE is
due to its involvement in the intrinsic pathway of blood coagulation
cascade. Several anticoagulant proteases have been characterized
from seeds such as bitter gourd and jackfruit [14,18,25]. In-vivo
thrombin a serine protease generated by the action of factor Xa on
prothrombin is the one that degrade human fibrinogen from the N
terminal end and generates fibrinopeptide A and B [26]. The proteases
those hydrolyze fibrinogen from the N-terminal end and generate
fibrinopeptide A and B are thrombin like enzymes and usually
trigger procoagulation [17]. However, the proteases those degrade
fibrinogen from C-terminal end and generates truncated fibrinogen
exhibits anticoagulation [27]. Though, the PSPE hydrolyzed Aα and
Bβ chains of fibrinogen it might have hydrolyzed from the C-terminal
rather N-terminal. Thus, the fibrinogenolytic activity of PSPE further
strengthened its anticoagulant property. Numerous fibrinogenolytic
proteases have been reported from plant latex, seeds, ticks,
earthworms, caterpillar, venoms of snake, spider, and honey bees [26].
Fibrinolysis is an important event in the clot dissolution, plasmin an
activated form of plasminogen is the one involved in fibrinolysis in
the physiological condition [28]. PSPE showed fibrinolytic activity
by degrading all the chains of human fibrin. The fibrinolytic activity
was evidently abolished by PMSF, strengthening the role of serine
proteolytic activity of the extract. Thus, PSPE exhibited plasmin
like activity could be a better candidate in curing hyper coagulation
disorders. Several plasmin like proteases have been reported from
plants and venomous organisms [26]. Platelets are enucleated cells
which play an important role in hemostasis [29]. In the circulatory
system platelets are in the resting state, get activated in response to
endothelial injury up on exposure to collagen an agonist of platelets.
The activated platelets accumulate at the site of injury forming
platelet plug along with fibrin clot and prevent blood loss [30]. While,
hyper activation of platelets due to genetic/environmental factor is

Volume 2 • Issue 1 • 1000108 • Page 6 of 8 •

Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.

Figure 7: Antiplatelet activity of PSPE using ADP as agonist: Platelet aggregation was initiated by adding ADP as an agonist. (a) Traces of platelet
aggregation: Trace 1 (ADP 10 μM); Trace 2 (ADP 10 μM+30 μg of PSPE); Trace 3 (ADP 10 μM+60 μg of PSPE); Trace 4 (ADP 10 μM+90 μg of PSPE). The
values represent ± SD of three independent experiments. (b) Dose dependent platelet aggregation inhibition% (c) Dose dependent platelet aggregation%.

Figure 8: Antiplatelet activity of PSPE using Epinephrine as agonist: (a) Traces of platelet aggregation: Trace 1 (Epinephrine 5 μM); Trace 2 (Epinephrine 5
μM+30 μg of PSPE); Trace 3 (Epinephrine 5 μM+60 μg of PSPE); Trace 4 (Epinephrine 5 μM+90 μg of PSPE). The values represents of three independent
experiments. (b) Dose dependent platelet aggregation inhibition% (c) Dose dependent platelet aggregation%.

Volume 2 • Issue 1 • 1000109 • Page 7 of 8 •

Citation: Ramachandraiah C, Nandish SKM, Kengaiah J, Shivaiah A, Chandramma, et al. (2017) Evaluation of Anticoagulant and Antiplatelet Activity of Pisum
sativum Pod Extract. J Blood Res Hematol Dis 2:1.
also major contributing component in hyper coagulation disorder.
PSPE inhibited the agonists ADP and Epinephrine induced platelet
aggregation. Thus, PSPE could be a better antiplatelet agent too.
Many antiplatelet agents have been characterized from both plants
and animal sources [31].
Conclusion
In conclusion, this study reports on the anticoagulant,
fibrinogenolytic, fibrinolytic and anti-aggregation properties of
PSPE. The said activity of PSPE is due to its serine proteolytic activity.
Thus, purification and its characterization is of great interest.
Acknowledgment
Devaraja Sannaningaiah and Chethana Ramachandraiah thank the
Department of Science and Technology, Government of India, New Delhi and
Vision Group on Science and Technology, Government of Karnataka, Bangalore
for financial assistance.
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Author Affiliations Top
Department of Studies and Research in Biochemistry and Centre for
1
Bioscience and Innovation, Tumkur University, Tumkur, Karnataka, India
Department of Studies in Biochemistry, University of Mysore, Manasagangothri,
2
Mysore, Karnataka, India
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