ISSN 00062979, Biochemistry (Moscow), 2020, Vol. 85, No. 9, pp. 11131126. © Pleiades Publishing, Ltd., 2020.

Published in Russian in Biokhimiya, 2020, Vol. 85, No. 9, pp. 13051320.

Flaxseed Cysteine Protease Exhibits Strong Anticoagulant,

Antiplatelet, and ClotDissolving Properties
S. K. M. Nandish1, J. Kengaiah1, Ch. Ramachandraiah1, Chandramma1, A. Shivaiah1,
S. M. Santhosh2, Thirunavukkarasu3, and D. Sannaningaiah1,a*

1
Department of Studies and Research in Biochemistry and Centre for Bioscience and Innovation,
Tumkur University, 572103 Tumkur, India
2
Department of Medicinal Biochemistry and Microbiology (IMBM), Uppsala Biomedical Centre, 75237 Uppsala, Sweden
3
Department of Biochemistry and Molecular Biology, School of Life Sciences,
Pondicherry University, 605014 Pondicherry, Tamil Nadu, India
a
email: sdevbiochem@gmail.com
Received March 21, 2020
Revised May 26, 2020
Accepted May 29, 2020

Abstract—In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant,
antiplatelet, and clotdissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation
and ionexchange column chromatography techniques. The purity of the enzyme was evaluated by SDSPAGE, RPHPLC,
and MALDITOF. FSCP was observed as a single band of approximately 160 kDa in SDSPAGE under reducing and non
reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDITOF spectrometry. The CD
spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no betasheet structures.
FSCP hydrolyzed both casein and gelatin with a specific activity of 3.5 and 4.2 unit/mg min respectively. The proteolytic
activity of FSCP was completely abolished by iodoacetic acid (IAA), suggesting FSCP is a cysteine protease. The pH opti
mum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong antico
agulant effect in both plateletrich plasma (PRP) and plateletpoor plasma (PPP) by extending the clotting time from 222
to 1100 s and from 256 to 1210 s, respectively. FSCP degraded human fibrinogen and fibrin clots. The products of fibrino
gen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets trig
gered by ADP, epinephrine, thrombin, collagen, arachidonic acid, and platelet activating factor (PAF). FSCP was found to
be nontoxic as it did not damage the membrane of red blood cells (RBCs) and did not induce hemorrhage and edema in
experimental mice.

DOI: 10.1134/S0006297920090102

Keywords: cysteine protease, anticoagulant, antiplatelet, nontoxic

INTRODUCTION bohydrates, and secondary metabolites that attribute to

vast pharmacological applications of flaxseeds [1]. In
Flaxseeds contain a plethora of phytoconstituents plants, proteolytic enzymes are involved in various
including enzymes, nonenzymatic proteins, lipids, car processes – from germination to cell death [2]. In addi
tion, they have numerous industrial applications in food
Abbreviations: ADP, adenosine diphosphate; APTT, activated processing, brewing, cheese production, tenderization of
partial thromboplastin time; CD, circular dichroism; DEAE, meat, bread production, and manufacturing of leather
diethyl amino ethyl; EDTA, ethylenediaminetetraacetic acid; and textiles [3]. However, the effect of plant proteases on
FSCP, flaxseed cysteine protease; IAA, iodoacetic acid; OD, hemostasis is poorly explored, unlike the effects of prote
optical density; PAF, platelet activating factor; PMSF, phenyl
olytic enzymes from snake, bee, and spider venoms [4].
methylsulfonyl fluoride; PPP, plateletpoor plasma; PRP,
plateletrich plasma; PT, prothrombin time; RPHPLC, Proteolytic enzymes that exhibit the procoagulant prop
reversedphase highperformance liquid chromatography; SDS erties and degrade fibrinogen from the Nterminus (gen
PAGE, sodium dodecyl sulphate polyacrylamide gel elec erating fibrinopeptides A and B) are similar to thrombin
trophoresis; TCA, trichloroacetic acid; TFA, trifluoroacetic acid. like enzymes, prothrombin activators, and factor X and
* To whom correspondence should be addressed. factor V activators [5]. Proteases exhibiting the anticoag

1113
1114 NANDISH et al.
ulant effect and degrading fibrinogen from the Ctermi grade. Fresh human blood for plateletrich plasma (PRP)
nus (generating truncated fibrinogen that lacks a potential and plateletpoor plasma (PPP) was collected from
for polymerization) are either protein C activators or fac healthy donors. All experiments were conducted in
tor IX and X inhibitors [6]. Proteases interfering with the accordance with the ethical guidelines and approved by
platelet function can act as either activators or inhibitors the Institutional Human Ethical Committee (IHEC
of platelet aggregation [7]. Fibrinogenolytic enzymes UOM No. 47Res/201415), University of Mysore,
reported so far activate plasminogen and cause hemor Mysuru, India. Animal experiments were approved by the
rhage in experimental animals [8]. Metalloproteases and Institutional Animal Ethical Committee (UOM/IAEC/
serine proteases with fibrinogenolytic, fibrinolytic, 02/2016), University of Mysore. Animals were handled in
antiplatelet, and pro and anticoagulant properties from accordance with the guidelines of the Committee for the
the viper and crotalid snake venoms have been compre Purpose of Monitoring and Supervision of Experiments
hensively studied [9], while animal cysteine proteases on Animals (CPCSEA).
remain the least explored. On the other hand, several Preparation of flaxseed extract and protein assay.
research groups have documented the presence of cys Flaxseeds were purchased from a local market in Tumkur.
teine proteases in the latex of plants such as Asclepius The seeds were washed with doubledistilled water and
curassavica, Pergularia extensa, Calotropis gigantean, treated with 0.5 M NaHCO3 (1 : 8 w/v) with constant
Synadenium grantii, and Wrightia tinctoria [10]. Crude stirring for 1 hr. The mucilage was then removed by filtra
latex was found to interfere with hemostasis by exhibiting tion through a muslin cloth. The seeds were washed thor
procoagulant, clotinducing, or clotdissolving proper oughly, dried at room temperature for 24 h, and ground
ties. Thus, fucin, a cysteine protease from Ficus carica, into powder that was resuspended in 50 mM TrisHCl
activates coagulation factor X and induces clot formation buffer (pH 7.6) and centrifuged at 8000g for 15 min. The
[11]. Papain from the latex of Carica papaya was the first supernatant was frozen at –20°C until further use. Protein
characterized cysteine protease [12]. However, cysteine concentration was determined by the Bradford method
proteases from edible seeds are less studied. Recently, a [14] using BSA as a standard.
cysteine protease from the flaxseed extract and its antico Sephadex G100 chromatography. Sephadex G100
agulant, antiplatelet, and clotdissolving activities have column (1.0 × 62 cm) was preequilibrated with 0.1 N
been reported in [13]. Enzyme purification and charac NaCl. Flaxseed extract (100 mg in 1 ml of 0.1 M NaCl)
terization is very important for understanding the struc was loaded on the column; the fractions were eluted with
turefunction relationships in the enzyme and predicting 0.1 M NaCl at a flow rate of 0.25 ml/min; protein elution
its possible applications. The key goal behind purification was monitored at 280 nm. Every other tube was assayed for
is to achieve the maximum yield of the enzyme prepara the proteolytic activity from absorbance at 660 nm using
tion with the highest catalytic activity. Moreover, enzyme fatfree casein as a substrate. Fractions with the proteolyt
characterization provides an integrated insight in the ic activity were lyophilized and used in further assays.
allosteric effects and possibilities of protein engineering DEAESephadex A25 chromatography. The peak II
and novel drug design. In view of this, we purified and obtained by gel filtration on Sephadex G100 (20 mg in
characterized a cysteine protease from the flaxseed 1 ml of equilibrating buffer) was loaded on a DEAE
extract. Sephadex A25 column (1.0 × 15 cm) preequilibrated
with 10 mM TrisHCl buffer (pH 7.5). The proteins were
eluted stepwise with 20 mM TrisHCl (pH 9), 20 mM
MATERIALS AND METHODS TrisHCl (pH 8.5), 30 mM TrisHCl (pH 8.0), 40 mM
TrisHCl (pH 7.5), 50 mM TrisHCl pH 7.0, 75 mM Tris
Fatfree casein, gelatin, phenylmethylsulfonyl fluo HCl (pH 6.5), 100 mM TrisHCl (pH 6.0), 0.5 M NaCl
ride (PMSF), ethylenediaminetetraacetic acid (EDTA), in 50 mM TrisHCl (pH 7.5), and 1 M NaCl in 50 mM
iodoacetic acid (IAA), adenosine diphosphate (ADP), TrisHCl (pH 7.5) at 0.25 ml/min; elution was monitored
epinephrine, thrombin, arachidonic acid, platelet activat at 280 nm. The obtained fractions were assayed for the
ing factor (PAF), collagen, Sephadex G100, diethyl proteolytic activity at 660 nm using casein as a substrate.
amino ethyl (DEAE)Sephadex, human plasma fibrino Reversedphase highperformance liquid chromatog
gen, and 1,10phenanthroline were purchased from raphy (RPHPLC). The purified protease from DEAE
Sigma, USA. Molecular weight markers were from Sephadex chromatography was subjected to RPHPLC
Bangalore Genie Pvt Ltd., India. Reagents for determin on a C18 column (150 mm × 4.60 mm; particle size, 5 μm)
ing activated partial thromboplastin time (APTT) preequilibrated with 0.1% trifluoroacetic acid (TFA) in
(LIQUICELINE Phospholipids preparation derived water using a Shimadzu LC20AD Prominence HPLC
from rabbit brain with ellagic acid) and prothrombin time system equipped with a PDA detector (Japan) The pro
(PT) (UNIPLASTIN rabbit brain thromboplastin) were teins were eluted with a linear gradient (0 to 100%) ace
purchased from AGAPPE Diagnostic Pvt Ltd., tonitrile, 0.1% TFA for 40 min at a flow rate of 1 ml/min.
Ernakulum, India. All other chemicals were of analytical Protein elution was monitored at 280 nm.

BIOCHEMISTRY (Moscow) Vol. 85 No. 9 2020

 ANTICOAGULANT ACTION OF FLAXSEED CYSTEINE PROTEASE 1115
MALDITOF analysis. The molecular mass of was loaded on the 2% casein/gelatin nonreducing
flaxseed cysteine protease (FSCP) was determined by resolving gel. After electrophoresis, the gel was washed
mass spectrometry using a Bruker Daltonics MALDI with 10 mM sodium phosphate (pH 7.0), containing
TOF spectrometer (Bruker Daltonics, USA) in the posi 2.5% Triton X100 with constant agitation for 1 h to
tive ionization mode. αCyano4hydroxycinnamic acid remove SDS. The gel was incubated overnight at 37°C in
was used as a MALDI matrix. 50 mM TrisHCl buffer (pH 7.6) containing 50 mM
Circular dichroism (CD) analysis. The CD spectra of CaCl2 and 40 mM NaCl and stained to observe translu
FSCP (20 μg/ml in MilliQ Water) were obtained using a cent bands corresponding to the active enzyme.
JASCO J815 spectropolarimeter. The spectra were Effect of pH, temperature, and reaction time on
recorded at room temperature at 190250 nm in a quartz FSCP activity. To identify the pH optimum of FSCP, its
cuvette with a 1mm pathlength at a scanning speed of activity was determined within the pH 39 range by incu
100 nm/min with a bandwidth of 1 nm and response time bating 10 μg of the enzyme with 0.4 ml of fatfree casein
of 1 s. The final CD spectrum was an average of three (2% in 0.2 M TrisHCl, pH 7.6) in a total volume of 1 ml
scans. Secondary structure calculations were performed for 2.5 h at 37°C. To analyze the effect of temperature,
with the K2d software. 10 μg of FSCP was incubated with 0.4 ml of fatfree
SDSPAGE and Periodic Acid–Schiff (PAS) staining. casein (2% in 0.2 M TrisHCl, pH 7.6) in a total volume
Electrophoresis in 10% polyacrylamide gel containing of 1 ml at different temperatures from 5 to 50°C. Changes
sodium dodecyl sulphate (SDSPAGE) was carried out in the enzyme activity with time were monitored by incu
according to the Laemmli’s method [15]. Crude flaxseed bating 10 μg of FSCP with 0.4 ml of fatfree casein (2% in
extract (100 μg) and FSCP (50 μg) were loaded on the gel, 0.2 M TrisHCl, pH 7.6) in a total volume of 1 ml at 37°C
and electrophoresis was carried out under reducing and for different time intervals (30240 min). In all the exper
nonreducing conditions in 25 mM Trisglycine buffer iments, undigested casein was precipitated by adding
(pH 8.3), containing 0.1% SDS for 2 h at room tempera 1.5 ml of 0.44 M TCA for 30 min. The proteolytic activi
ture. The gels were stained with 0.1% Coomassie brilliant ty of the enzyme was determined as described above.
blue R250 for detection of protein bands and destained Plasma recalcification time. Plasma recalcification
with an ethanol/acetic acid/water mixture (40 : 10 : time was determined according to the method of Quick
50 v/v/v). Molecular weight standards from 200 kDa to et al. [19]. Briefly, FSCP (214 μg) was preincubated with
14.3 kDa were used. 0.2 ml of citrated human plasma containing 20 μl of
PAS staining was carried out as described by Leach et 10 mM TrisHCl (pH 7.4) for 1 min at 37°C. Next, 20 μl
al. [16]. After electrophoresis, the gel was fixed in 7.5% of 0.25 M CaCl2 was added to the preincubated mixture
acetic acid at room temperature for 1 h, washed with 1% and the clotting time was recorded.
nitric acid, and incubated in 0.2% aqueous periodic acid Bleeding time. The bleeding time was assayed as
at 4°C for 45 min. Next, the gel was incubated with the described in [20]. FSCP (08 μg) in 30 μl of PBS was
Schiff’s reagent at 4°C for 24 h and then destained with injected intravenously into the mouse tail vein (n = 5).
10% acetic acid to visualize pinkcolored bands. After 10 min, the mice were anaesthetized with diethyl
Proteolytic activity was assayed as described by ether, and a sharp cut 3 mm in length was made at the tail
Satake et al. [17]. FSCP (10 μg) was incubated with tip. The tail was immediately immersed vertically into
0.4 ml of fatfree casein (2% in 0.2 M TrisHCl, pH 7.6) PBS prewarmed to 37°C. The bleeding time was recorded
in a total volume of 1 ml for 2.5 h at 37°C. Undigested from the time the bleeding started till it completely
casein was precipitated by adding 1.5 ml of 0.44 M stopped.
trichloroacetic acid (TCA). After incubation for 30 min, APTT and PT tests. Normal citrated human plasma
the mixture was centrifuged at 2000g for 10 min. Sodium (100 μl) was preincubated with FSCP (010 μg) for
carbonate (2.5 ml, 0.4 M) and Folin–Ciocalteu reagent 1 min. For the APTT test, 100 μl of the reagent (activat
(1 : 2) were added sequentially to 1 ml of the supernatant, ed for 3 min at 37°C) was added, and the clotting time was
and the absorbance of the resulting colored solution was recorded after adding 100 μl of 0.02 M CaCl2. For the PT
measured at 660 nm. One unit of the enzyme activity was test, clotting was initiated by adding 200 μl of the PT
defined as the amount of the enzyme required to increase reagent, and the time taken for a visible clot to form (sec
in OD of 0.01 at 660 nm. The specific activity of the onds) was recorded. The APTT ratio and the internation
enzyme was expressed as units/min mg protein. For inhi al normalized ratio (INR) for the PT at each point were
bition studies, the reaction was carried out after FSCP calculated from the values obtained for the control plas
(10 μg) preincubation for 30 min with one of the ma samples incubated with the buffer for identical periods
inhibitors (5 mM): EDTA, 1,10phenanthroline, PMSF, of time.
or IAA. In all the experiments, appropriate controls were Fibrinogenolytic activity. Fibrinogenolytic activity
used. was determined as described in [21]. FSCP (010 μg) was
Zymogram. The zymogram of FSCP was obtained as incubated with the human plasma fibrinogen (50 μg) in a
described previously [18]. Briefly, FSCP (5 and 10 μg) total volume of 40 μl in 10 mM TrisHCl (pH 7.4) for 4 h

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1116 NANDISH et al.
at 37°C. The reaction was terminated by adding 20 μl of Acid citrate dextrose buffer (1.5 ml) was mixed with 9 ml
denaturing buffer containing 1 M urea, 4% SDS, and 4% of blood in a plastic centrifuge tube and centrifuged for
βmercaptoethanol, and the reaction products were ana 15 min at 30g. The supernatant (PRP) was transferred to
lyzed by 10% SDSPAGE. For the inhibition studies, a new plastic tube, incubated at 37°C for 15 min, and cen
FSCP (10 μg) was preincubated for 20 min with PMSF, trifuged for 20 min at 4500g. The pellet was collected,
IAA, EDTA, or 1,10phenanthroline (5 mM each). resuspended in Tyrode albumin buffer (pH 6.5), and cen
Identification of fibrinogen degradation products by trifuged at 4500g for 20 min. The formed pellet was resus
HPLC. Fibrinogen (50 μg) was treated with human pended in Tyrode albumin buffer (pH 6.5) and cen
thrombin (2.5 units) and purified FSCP (10 μg) in 50 mM trifuged again for 20 min at 4500g. The resulting pellet
TrisHCl (pH 7.8) at 37°C for 4 h. The reaction mixture was resuspended in Tyrode albumin buffer (pH 7.35) and
(20 μl) was analyzed on a C18 column using a Shimadzu used in the platelet aggregation study.
LCProminence HPLC system. The fractions were eluted Platelet aggregation was evaluated by the turbidimet
with a gradient of acetonitrile, 0.1% TFA in 0.1% aqueous ric method of Born using a dualchannel Chronolog
TFA for 10 min; elution was monitored at 280 nm. Whole Blood/Optical Lumi aggregometer. Aliquots of
Fibrinolytic activity assay. The activity of FSCP washed platelets were preincubated with various amounts
against fibrin clots was assayed as described by Rajesh et of FSCP (06 μg) in 0.25ml reaction volume. Platelet
al. [22]. Briefly, 100 μl of citrated human plasma was aggregation was initiated by adding agonists, such as
mixed with 20 μl of 0.2 M CaCl2 and incubated for 2 h at ADP, epinephrine, arachidonic acid, thrombin, collagen,
37°C. The resulting clot was washed thoroughly 56 times and PAF for 6 min. For ADP and epinephrineinduced
with PBS and resuspended in 400 μl of 0.2 M TrisHCl platelet aggregation, fibrinogen (50 μg) was added to the
(pH 8.5). The reaction was initiated by adding FSCP (0 washed platelets before the agonist addition.
10 μg) in 100 μl of PBS, and the reaction mixture was Direct hemolytic activity was determined using
incubated for 2.5 h at 37°C. The undigested clot was pre washed human red blood cells (RBCs). Briefly, packed
cipitated by adding 750 μl of 0.44 M TCA for 30 min and human RBCs were resuspended in PBS at a 1 : 9 (v/v)
removed by centrifugation for 15 min at 1500g. Aliquots ratio; 1 ml of the suspension was incubated with various
(0.5 ml) of the supernatant were transferred to clean glass amounts of FSCP (030 μg) for 1 h at 37°C. The reaction
tubes and mixed with 1.25 ml of 0.4 M sodium carbonate was stopped by adding 9 ml of icecold PBS, and the
and 0.25 ml of 0.5× Folin–Ciocalteu reagent. The reac reaction mixture was centrifuged at 1000g for 10 min at
tion mixture was incubated for 30 min, and the developed 37°C. The amount of hemoglobin in the supernatant was
color was evaluated at 660 nm. One unit of the enzyme measured at 540 nm. The hemolytic activity was
activity was defined as the amount of enzyme required to expressed as a percent of lyzed cells. RBCs in distilled
increase the absorbance of 0.01 at 660 nm. water (100% lysis) and PBS were used as positive and neg
Fibrinolytic activity assay by SDSPAGE. Fibrin clot ative controls, respectively.
prepared as described above was incubated with various Edemainducing activity was evaluated using the pro
concentrations of FSCP (010 μg) in 10 mM TrisHCl cedure suggested by Vishwanath et al. [25]. Mice (n = 5 in
(pH 7.4) in a final volume of 40 μl at 37°C for 12 h. The each group) were injected into the right foot pads with dif
reaction was stopped by adding 20 μl of denaturing sam ferent doses (10 to 200 μg) of FSCP in 20 μl of saline. Left
ple buffer containing 4% SDS, 1 M urea, and 4% βmer foot pads received 20 μl saline alone as a control. After 1 h,
captoethanol. The samples were incubated in a boiling the mice were anaesthetized by inhalation of diethyl ether.
water bath for 10 min and centrifuged to remove the Hind limbs were removed at the ankle joint and weighed.
debris. An aliquot (30 μl) of the supernatant was analyzed The extent of edema development was evaluated as the
in 7.5% SDSPAGE. For the inhibition studies, FSCP ratio of the edematous leg weight to the normal leg weight
(10 μg) was preincubated for 15 min with 5 mM inhibitor and expressed in percent (edema ratio). The minimal
(PMSF, EDTA, IAA, or 1,10phenanthroline). edemainduced dose was defined as the protein amount
Degradation of human plasma proteins was assayed as required to cause an edema ratio of 120%.
described by Kumar et al. [23]. FSCP (010 μg) was incu Hemorrhagic activity was assayed as described by
bated with 100 μg of plasma proteins for 12 h at 37°C in a Kondo et al. [26], when different amounts of FSCP (0
reaction volume of 40 μl in 10 mM TrisHCl (pH 7.4) 30 μg) in 30 μl saline were injected intradermally into
containing 10 mM NaCl and 0.05% sodium azide. The mice (n = 5 in each group). The mice receiving saline
reaction was terminated by adding 20 μl of denaturing alone or Daboia russelli venom served as the negative and
buffer containing 4% SDS and the reaction mixture was positive controls, respectively. After 3 h, the mice were
incubated in a boiling water bath for 5 min. The reaction anaesthetized by diethyl ether inhalation. A dorsal skin
products were analyzed by 7.5% SDSPAGE under patch was carefully removed and observed for hemorrhage
nonreducing condition. vs. salineinjected control mice, and the diameter of
Preparation of washed platelets. Washed platelets hemorrhagic spot on the skin inner side was measured.
were prepared according to the method of Born [24]. The minimal hemorrhageinducing dose was defined as

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 ANTICOAGULANT ACTION OF FLAXSEED CYSTEINE PROTEASE 1117
the amount of the protein producing a hemorrhagic spot Table 1. Effect of inhibitors on the proteolytic activity of
10 mm in diameter. FSCP
Statistical analysis. The data are presented as mean
Inhibitor (5 mM) Activity (%)
± SD. Statistical analysis was performed using the
Student’s ttest. The difference between the groups were
None 100
considered significant at p < 0.01.
PMSF 99
IAA 8
RESULTS EDTA 99
1,10Phenanthroline 98
Purification and characterization of FSCP. FSCP
was purified from the flaxseed extract by a combination of
Sephadex G100 and DEAEA25 Sephadex chromatog
raphy. Fractionation by gel filtration yielded two major strates with the specific activities of 3.45 and
peaks (Fig. 1a), but only peak II hydrolyzed casein, sug 4.20 unit/min mg protein, respectively. The proteolytic
gesting the presence of a proteolytic enzyme. Peak II was activity of peak IV was completely abolished by IAA (cys
fractionated by ionexchange column chromatography to teine protease inhibitor), while 1,10phenanthroline,
purify the protease. Chromatography on DEAE PMSF, and EDTA were ineffective, which suggested the
Sephadex produced peaks IVII (Fig. 1b). All seven peaks presence of cysteine residue in the enzyme’s active site
were screened for the proteolytic activity using casein and (Table 1). Therefore, we named the enzyme flaxseed cys
gelatin as substrates. Only peak IV hydrolyzed the sub teine protease (FSCP).

Fig. 1. a) Sephadex G100 column chromatography. Flaxseed extract (100 mg in 1 ml of 0.1 M NaCl) was loaded on the column (1.5 ×
108 cm) preequilibrated with 0.1 M NaCl. The proteins were eluted with 0.1 M NaCl at a flow rate of 16 ml/h; elution was monitored at
280 nm. Every other protein fraction (1.6ml) was assayed for the proteolytic activity using casein as a substrate. b) DEAESephadex chro
matography. Peak II obtained from the previous step (20 mg in 1 ml of equilibrating buffer) was loaded on the column (1.5 × 20 cm) preequil
ibrated with 10 mM TrisHCl (pH 8). The proteins were then eluted stepwise (see “Materials and Methods” Section) at a flow rate of 15 ml/h;
elution was monitored at 280 nm. Every other protein fraction was assayed for the proteolytic activity using casein as a substrate.

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1118 NANDISH et al.

a b

c d

Fig. 2. a) Electrophoresis of FSCP in 10% SDSPAG; lanes: 1) flaxseed extract (100 μg), nonreducing conditions; 2) FSCP (50 μg) under
nonreducing and (3) reducing conditions. b) PAS staining of (1) FSCP (50 μg) and (2) fibrinogen (50 μg, positive control); M, molecular
weight marker, kDa: myosin Hchain (200), βgalactosidase (120), bovine serum albumin (66), ovalbumin (43), carbonic anhydrase (29), and
lysozyme (14.3). c) MALDITOF mass spectrometry of FSCP (5 μg) in the positive ionization mode using αcyano4hydroxycinnamic acid
as a matrix. d) RPHPLC of FSCP (5 μg) on a C18 column preequilibrated with 0.1% TFA in water. The protein was eluted with an increas
ing concentration (0100%, 40 min) of acetonitrile, 0.1% TFA at a flow rate of 1 ml/min; elution was monitored at 280 nm. (Color versions
of Figs. 24, 6 and 8 are available in online version of the article and can be accessed at: https://www.springer.com/journal/10541)

FSCP was found to be a monomer, as it traveled as a The CD spectra of FSCP revealed approximately
single band with an approximate molecular mass of 25.6% helices, 25.8% turns, and 48% random coils, as
160 kDa upon SDSPAGE under both reducing and non well as the lack of betasheets, in the enzyme’s secondary
reducing conditions (Fig. 2a). However, the exact molec structure (Fig. 3a).
ular mass of FSCP determined by MALDITOF mass The extent of FSCP purification was 19fold (Table
spectrometry was 16 kDa (Fig. 2c). FSCP did not form 2). The hydrolytic activity of FSCP against casein and
Schiff bases when treated with periodic acid (unlike fib gelatin was confirmed in the zymography experiments.
rinogen), suggesting the absence of carbohydrate moieties FSCP produced transparent bands in approximately 168
(Fig. 2b). The purity of FSCP was confirmed by RPHPL kDa region in the experiments with both casein and gela
that revealed a single sharp peak with a retention time of tin (Fig. 3, b and e, respectively). The pH optimum of
7.2 min (Fig. 2d). FSCP was found to be pH 6.0 and the optimal tempera

Table 2. FSCP purification. The values are means of three independent experiments
Procedure Total protein Protein Specific activity Total Activity Purification,
(mg) recovery (%) (U/min mg protein) activity yield (%) folds

Flaxseed extract 100 100 0.18 ± 0.02 18 100 1.0

Sephadex G100 20 20 0,81 ± 0.04 16 90 4.5
DEAESephadex 1 1 3.45 ± 0.03 3 19 19

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 ANTICOAGULANT ACTION OF FLAXSEED CYSTEINE PROTEASE 1119
ture was 30°C; the highest amount of hydrolyzed substrate trationdependent manner (Fig. 5a) when incubated with
was observed after 150 min of reaction (Fig. 3, fh). fibrinogen for 4 h at 37°C. However, FSCP (5 μg) failed to
FSCP exhibits strong anticoagulant activity by inter degrade fibrinogen γchain when incubated with the sub
fering with the intrinsic pathway of blood coagulation. strate for 24 h at 37°C (Fig. 5b). The fibrinogenolytic
FSCP showed the anticoagulant activity by extending the activity of FSCP was completely abolished by IAA, but
clotting time of both citrated human PRP and PPP. not by PMSF, EDTA, or 1,10phenanthroline (Fig. 5c).
FSCP (12 μg) increased the clotting time from 222 to The substrate specificity of FSCP was evaluated using
1100 s for PRP and from 256 to 1210 s for PPP (Fig. 4a). human plasma proteins. FSCP specifically cleaved fib
The anticoagulant activity of FSCP was confirmed in rinogen without affecting other plasma proteins when
vivo using the mouse tail bleeding assay. The intravenous incubated for 12 h at a maximum dose of 10 μg at 37°C
injection of FSCP significantly prolonged the bleeding (fibrinogen alone was used as a positive control) (Fig. 5d).
time in a dosedependent manner. The bleeding time was Fibrinogen (20 μg) was used as a control.
over 800 s (p < 0.01) at the FSCP amount of 8 μg vs. 200 s FSCP dissolves fibrin clots. FSCP dissolved fibrin
in the PBStreated control (Fig. 4c). The anticoagulant clots with a specific activity of 4.16 unit/min mg protein
activity of FSCP was completely abolished by IAA both (Fig. 6a). The fibrinolytic activity was corroborated by
in vitro and in vivo, while other protease inhibitors were SDSPAGE and analysis of the fibrin degradation pat
ineffective (Fig. 4, b and d). Furthermore, FSCP pro tern. When incubated with fibrin for 12 h at 37°C, FSCP
longed only APTT but not PT, suggesting that the anti (10 μg) hydrolyzed only the αchain, while the γγ dimer,
coagulant effect of FSCP could be due to its interference αchain, and βchain were resistant to the proteolysis
with the blood coagulation cascade intrinsic pathway (Fig. 6b). Curiously, when 4 μg of FSCP was used, the
(Table 3). enzyme degraded all fibrin chains within 24 h in a time
FSCP hydrolyzes fibrinogen but no other plasma pro dependent manner (Fig. 6c). The fibrinolytic activity of
teins. FSCP (10 μg) completely hydrolyzed fibrinogen FSCP was completely abolished by IAA, but not by
Aαchain and partially hydrolyzed Bβchain in a concen PMSF, EDTA, and 1,10phenanthroline (Fig. 6d).

b c d e
f g

Fig. 3. a) CD spectra of FSCP (20 μg in doubledistilled water. b) FSCP zymogram with casein: 5 μg (1) and 10 μg (2) of FSCP. c) FSCP
(5 μg) zymogram with casein in the absence and presence of inhibitors (5 mM); lanes: 1) no inhibitors; 2) PMSF; 3) IAA; 4) 1,10phenan
throline; 5) EDTA. d) FSCP zymogram with gelatin: 5 μg (1) and 10 μg (2) of FSCP. e) FSCP (5 μg) zymogram with gelatin in the absence
and presence of inhibitors (5 mM); lanes: 1) no inhibitors; 2) PMSF; 3) IAA; 4) 1,10phenanthroline; 5) EDTA. f) Effect of pH (39) on the
proteolytic activity of FSCP. g) Effect of temperature (550°C) on the proteolytic activity of FSCP. h) Effect of incubation time (30240 min)
on the proteolytic activity of FSCP.

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1120 NANDISH et al.

a b

c d

Fig. 4. a) Effect of FSCP on plasma recalcification time. b) Effect of protease inhibitors on the anticoagulant activity of FSCP. FSCP (4 μg)
was preincubated with 5 mM inhibitor (PMSF, IAA, EDTA, and 1,10phenanthroline) for 15 min at 37°C. c) Effect of FSCP on the tail
bleeding time measured 10 min after intravenous administration of PBS or various doses of FSCP. d) Effect of inhibitors on the tail bleeding
time. FSCP (4 μg) was preincubated with 5 mM IAA for 15 min at 37°C. Each bar represents mean ± SD of three independent experiments
(p < 0.01).

FSCP degrades fibrinogen from the Cterminus. To peaks with the retention times of 1, 1.2, 1.4, 1.6, and
identify the site(s) of fibrinogen hydrolysis by FSCP, we 2 min, respectively (Fig. 7b). Although fibrinogen treat
compared the products of fibrinogen degradation by ment with FSCP also generated five peaks, their retention
thrombin and FSCP using HPLC. Fibrinogen was eluted times were 1, 1.4, 1.6, 1.8, and 2 min, respectively
as a major single peak with a retention time of 1.3 min (Fig. 7c). The elution profiles for the fibrinogen frag
(Fig. 7a). Fibrinogen cleavage with thrombin yielded five ments generated with thrombin and FSCP were different,

Table 3. Dose dependent effect of FSCP on the clotting time of normal human plasma

FSCP amount, μg PT, s PT (INR) APTT, s APTT ratio

0 11 ± 0.01 0.89 ± 0.05 34 ± 0.02 1.31 ± 0.09

2 11 ± 0.05 0.98 ± 0.02 47 ± 0.07 2.03 ± 0.01
4 12 ± 0.02 1.15 ± 0.07 71 ± 0.05 3.99 ± 0.05
6 12 ± 0.08 1.08 ± 0.03 96 ± 0.01 5.40 ± 0.01
8 11 ± 0.03 0.91 ± 0.09 127 ± 0.09 7.54 ± 0.03
10 11 ± 0.1 1.03 ± 0.04 166 ± 0.03 9.11 ± 0.02

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 ANTICOAGULANT ACTION OF FLAXSEED CYSTEINE PROTEASE 1121
suggesting that FSCP hydrolyzed fibrinogen from the C edema formation was observed in the foot pads of mice
terminus. Figure 7, d and e shows the analysis of fibrino injected with FSCP (Fig. 8d).
gen degradation products by SDSPAGE.
FSCP exhibits the antiplatelet activity. FSCP (6 μg)
inhibited platelet aggregation induced by agonists, such as DISCUSSION
ADP, epinephrine, thrombin, collagen, arachidonic acid,
and PAF in washed platelets by 62, 73, 57, 88, 70, and In this study, we purified and characterized FSCP, an
60%, respectively (Fig. 8a). Among the agonists studied, enzyme with strong anticoagulant, antiplatelet, and fibri
FSCP inhibited aggregation of washed platelet in the fol nolytic properties. FSCP was purified using gelfiltration
lowing order: collagen > epinephrine > arachidonic acid and ionexchange chromatography techniques. FSCP is a
> ADP > PAF > thrombin. monomeric protein, as it traveled as a single band in
FSCP is nontoxic. FSCP was found to be nontoxic, SDSPAGE under both reducing and nonreducing con
as it did not damage the RBC membranes (Fig. 8b). ditions and was eluted as a single peak in RPHPLC, with
FSCP also did not damage blood vessels, suggesting the suggested homogeneity and purity of the obtained
lack of hemorrhageinducing activity (Fig. 8c). No enzyme preparations. The exact molecular mass of FSCP

a b

c d

Fig. 5. Fibrinogenolytic activity of FSCP. a) Fibrinogen (50 μg) hydrolysis for 4 h at 37°C (1) in the absence of the enzyme and in the pres
ence of (2) 2 μg, (3) 4 μg, (4) 6 μg, (5) 8 μg, and (6) 10 μg of FSCP. The hydrolysis products were separated by 10% SDSPAGE under reduc
ing condition. b) Fibrinogen hydrolysis (50 μg) by FSCP (4 μg) after incubation for (1) 0 h, (2) 4 h, (3) 8 h, (4) 12 h, (5) 16 h, and (6) 24 h at
37°C. c) Effect of protease inhibitors on the fibrinogenolytic activity of FSCP. FSCP (4 μg) was preincubated with protease inhibitors (5 mM)
for 30 min at 37°C; the reaction was initiated by adding 50 μg of fibrinogen and the reaction mixture was incubated for 4 h: 1) fibrinogen alone
and in the presence of (2) FSCP; (3) FSCP and PMSF, (4) FSCP and IAA, (5) FSCP and EDTA, and (6) FSCP and 1,10phenanthroline.
M, molecular weight markers, kDa. d) Degradation of human plasma proteins. Plasma proteins (100 μg) were incubated (1) in the absence of
the enzyme or in the presence of (2) 2 μg, (3) 4 μg, (4) 6 μg, (5) 8 μg, and (6) 10 μg of FSCP in 40 μl of 10 mM TrisHCl buffer (pH 7.4) at
37°C and then analyzed by 7.5% SDSPAGE under nonreducing condition.

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1122 NANDISH et al.

a b

c d

Fig. 6. Fibrinolytic activity of FSCP. a) Colorimetric assay of FSCP fibrinolytic activity. Washed plasma clot was incubated with 010 μg of
FSCP for 2.5 h, and substrate degradation was evaluated from OD660. b) Effect of FSCP amount on the clot hydrolysis. Washed plasma clot
was incubated for 12 h with (1) in the absence of the enzyme and with (2) 2 μg, (3) 4 μg, (4) 6 μg, (5) 8 μg, and (6), and 10 μg (6) of FSCP.
The reaction products were analyzed by SDSPAGE (7.5%). c) Time dependence of clot hydrolysis. Fibrin clot was incubated at 37°C (1) in
the absence of the enzyme or in the presence of FSCP (4 μg) for (2) 0 h, (3) 6 h, (4) 12 h, (5) 18 h, and (6) 24 h (6). d) Inhibition of FSCP
activity. FSCP (4 μg) was preincubated with protease inhibitors (5 mM) for 30 min at 37°C. The reaction was initiated by adding fibrin clot;
the reaction mixture was incubated for 12 h: fibrin clot alone (1) or in the presence of (2) FSCP, (3) FSCP and PMSF, (4) FSCP and IAA, (5)
FSCP and EDTA, and (6) FSCP and with 1,10phenanthroline.

a b

c d

Fig. 7. HPLC of (a) fibrinogen (50 μg) and product of its degradation by (b) human thrombin and (c) FSCP. d, e) SDSPAGE (10%) of the
products of fibrinogen degradation by (d) thrombin and (e) FSCP end products.

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 ANTICOAGULANT ACTION OF FLAXSEED CYSTEINE PROTEASE 1123

a b

c
d

Fig. 8. a) Effect of FSCP (6 μg) on the aggregation washed platelet induced by adenosine diphosphate (10 μM ADP + 50 μg of fibrinogen),
epinephrine (5 μM epinephrine + 50 μg of fibrinogen), thrombin (2 μM), collagen (5 μg), arachidonic acid (50 μM), and platelet activating
factor (2 μM). b) Effect of FSCP on hemolytic activity: FSCP (0100 μg) was preincubated for 30 min with PBStreated packed RBCs.
Percentage of RBC lysis was calculated based on the presence of free haemoglobin in the supernatant and measured at 540 nm. c) Hemorrhagic
activity of FSCP: (cI) saline, (cII) positive control, Daboia russelli venom, (cIII) 25 μg, (cIV) 50 μg, and (cV) 100 μg of FSCP were injected
into mice in a total volume of 50 μl. d) Effect of FSCP on edema induction: positive control, MDH venom (dI); saline (dII); FSCP 50 μg
(dIII) and 100 μg (dIV).

was found to be 168 kDa by MALDITOF mass spec Hemostasis is a physiological phenomenon that plays
trometry. The secondary structure of FSCP is represented a key role in the arrest of bleeding caused by a damage to
by helices, turns, and random coils, but lacks betasheets. blood vessels [31]. It involves intrinsic pathway coagula
FSCP hydrolyzed casein and gelatin with the specific tion factors (factors IXXII), extrinsic pathway coagula
activity of 3.45 and 4.20 unit/min mg protein, respective tion factors (tissue factor and factor VII), and common
ly, at 37°C. The pH optimum for the enzyme is pH 6.0; pathway coagulation factors (factor X, prothrombin,
the temperature optimum is 30°C; the highest degree of thrombin, fibrinogen, fibrin clot, and factor XIII).
substrate hydrolysis was observed at 150 min of reaction. Activation of these factors takes place through the zymo
FSCP lacked the carbohydrate moieties, as it was nega gen activation upon exposure to unusual surfaces and/or
tive for the Schiff base staining. FSCP was inhibited by tissue injury [32]. Anticoagulants generally block specific
IAA and insensitive to metal chelators (EDTA and 1,10 factor(s) of the intrinsic, extrinsic, and/or common coag
phenanthroline) and serine protease inhibitor PMSF, ulation pathways. FSCP showed a strong anticoagulant
suggesting it belongs to cysteine proteases. Seeds are activity both in vitro and in vivo. The APTT test evaluates
known to store remarkably high amounts of proteolytic the activity of the intrinsic pathway factors in the clot for
enzymes, including serine, cysteine, and aspartate pro mation, while the PT test estimates the initiation of clot
teases and metalloproteases [27]. Serine proteases and formation through the extrinsic pathway [33]. FSCP
metalloproteases from plant latex, ticks, earthworms, delayed the clotting time only in the APTT test, but not in
caterpillars, plant seeds, and snake, spider, and honeybee the PT test, suggesting the interference of this enzyme in
venoms have been comprehensively studied [2830], the intrinsic pathway of blood coagulation. However, the
while cysteine proteases remain the least characterized exact site of FSCP action on the intrinsic pathway
enzymes in plant seeds. remains obscure. The anticoagulant effect of FSCP both

BIOCHEMISTRY (Moscow) Vol. 85 No. 9 2020

1124 NANDISH et al.
in vitro and in vivo was completely abolished by IAA, antiplatelet agents from natural sources have been char
which signifies the role of cysteine residue in catalysis by acterized [4648], including lignincapped nanoparticles
this enzyme. Proteases participating in blood coagulation [49], loxnecrogin from the venom of Loxosceles gaucho
by triggering the pro/anticoagulant activities have been brown spider [50], and serine protease from Hippasa age
identified in plant latex and snake and spider venoms [34 lenoides spider venom [34]. FSCP did not hydrolyze the
36]. Protease preparations from the snake venom RBC, as it was failed to release hemoglobin from the
(Ancrod) and Aspergillus oryzae fungus (Brinase) have RBCs. Moreover, it did not damage blood vessels due to
been successfully used to treat thrombotic disorders [37, the lack of hemorrhagic activity. In addition, FSCP did
38]. Proteases involved in blood coagulation were found not cause edema in the foot pads of experimental mice,
to degrade human fibrinogen and fibrin [39]. FSCP suggesting it is nontoxic.
cleaved Aα and Bβ chains and partially γchain of human In conclusion, highmolecularweight (168kDa)
fibrinogen. Thrombin like enzymes degrade fibrinogen FSCP was purified from the flaxseed extract. FSCP
from the Nterminus generating fibrinopeptides A and B exhibited strong anticoagulant and antiplatelet proper
and promoting coagulation [40]. Fibrinogen degradation ties. The observed anticoagulant activity of FSCP could
from the Cterminus generates truncated fibrinogen frag be due to the enzyme involvement in the blood coagula
ments dissimilar to fibrinopeptides A and B and results in tion intrinsic pathway. FSCP also specifically hydrolyzed
anticoagulation [41]. To identify the sites for FSCP in fib fibrinogen and fibrin. Further studies on the mechanism
rinogen, we compared the patterns of fibrinogen degrada of FSCP action on the coagulation cascade and platelet
tion by thrombin and FSCP, which appeared to be differ function are of significant interest.
ent for the two enzymes. Therefore, FSCP probably trig
gers anticoagulation by fibrinogen degradation from the
Cterminus. The fibrinogenolytic activity of FSCP was Acknowledgments. D. S. and S. K. thank the
completely abolished by IAA, but not by inhibitors of ser Department of Science and Technology, Government of
ine proteases and metalloproteases, suggesting that FSCP India, New Delhi and Vision Group on Science and
has cysteine in the active site. Cysteine proteases have Technology, Government of Karnataka, Bangalore for
been isolated from the latex of Asclepias curassavica L., a financial assistance.
plant from the milkweed family (Asclepiadaceae) [41]. Ethics declarations. The authors declare no conflict
Pergularain E I purified from Pergularia extensa latex of interests. This article does not contain any studies
degraded fibrinogen chains from Nterminus and exhib involving human participants or animals performed by
ited the thrombinlike activity [10]. FSCP displayed the any of the authors.
fibrinolytic activity by degrading all fibrin chains. Clot
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