Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 3 pp. 987ñ994, 2017 ISSN 0001-6837Polish Pharmaceutical Society

Venous thromboembolism (VTE) is the mostprevalent cardiovascular disease in the world aftermyocardial infarction and stroke (1). VTE is silentbut still potentially fatal disease which includes,deep vein thrombosis (DVT) and pulmonaryembolism (PE) (2). The risk of VTE in Indian pop-ulation (53.6%) is nearly similar to that of the glob-al population (51.8%) (3).

Unfractionated heparin (UFH) and low molec-ular weight heparins (LMWHs) are the cornerstonesin treatment and prophylaxis of thrombosis for morethan 65 years (4). Prophylaxis for VTE by usingpharmacologic agents like heparin (UFH), LMWHsand direct thrombin inhibitors (DTIs) reduces therelative risk of VTE by 45-63% and 60% in hospi-talized medical and general surgery patients, respec-

tively, as compared with no prophylaxis (4-8). As itrequires long-term therapy of heparin, for the treat-ment of VTE, it causes a number of side effects,such as thrombocytopenia, bleeding, changes inlipid metabolism and osteoporosis (9).

To overcome these problems, newer anticoag-ulant drugs are required. Sulfated polysaccharides(SPS) are abundantly present in some of the marineorganisms which exhibit anticoagulant andantithrombotic activities (10, 11). Green marinealgae Codium dwarkense Børgesen is one of themwhich is commonly found on Indian waters (12).Bioassay-guided purification of sulfated polysac-charides from Codium dwarkense Børgesen, yieldedtwo products, which contained arabinan sulfate andarabinogalactan sulfate. An in vitro study showed

EVALUATION OF ANTICOAGULANT EFFECT OF SULFATED POLYSACCHARIDE (SPS) FROM CODIUM DWARKENSE BØRGESEN

IN κñCARRAGEENAN INDUCED HYPERCOAGULABLE STATE IN WISTAR ALBINO RATS

HIRAL N. GOLAKIYA1, VIREN N. NAIK1*, HIREN N. HIRAPARA2, KALPANABEN H. MODY3,ALPESH P. GOSWAMI4 and CHANDRABHANU TRIPATHI1

1Department of Pharmacology, 4Department of Pathology, Government Medical College, Bhavnagar 364001, Gujarat, India

2Department of Pharmacology, Gujarat Medical Education and Research Society, Junagadh 362001, Gujarat, India

3Central Salt and Marine Chemical Research Institution (CSMCRI), Bhavnagar 364001, Gujarat, India

Abstract: Our aim of the present study was to evaluate the anticoagulant effect of sulfated polysaccharides(SPS) from Codium dwarkense Børgesen in κ-carrageenan induced hypercoagulable state in Wistar albino rats.48 Wistar albino rats of either sex were randomly divided into 6 groups ñ disease control, active control, treat-ment low and high dose and preventive low and high dose. κ-Carrageenan (3 mg/kg) single dose intravenous-ly was given in rat tail vein at pre-decided time to produce hypercoagulable state. Baseline, 24, 48 and 72 hoursduration blood samples were collected for PT, INR, aPTT, platelet count and fibrinogen level, FDP and D-Dimer measurements. At the end of experiment, animals were sacrificed for histopathology analysis of lung,liver and mesentery. Sulfated polysaccharide (SPS) significantly restored altered coagulation parameters (PT,INR, aPTT, platelet count and fibrinogen level) without affecting fibrinolytic parameters (FDP and D-Dimer).Administration of SPS both as a treatment and preventive therapy reduced the number of microthrombi alongwith less structural damage in histopathology of lung, liver and mesentery. Heparin served as active control andits administration significantly prolonged aPTT and restored PT. This study shows, anticoagulant activity ofSPS extracted from Codium dwarkense Børgesen in Wistar albino rats.

Keywords: hypercoagulable state, κ-carrageenan, anticoagulant, heparin, sulfated polysaccharide, Codiumdwarkense Børgesen

987

* Corresponding author: e-mail: naikviren2001@yahoo.com; hiralgolakiya@gmail.com; phone: +919825447370; fax: +912782422011

988 HIRAL N. GOLAKIYA et al.

that the product containing arabinan sulfate exhibit-ed stronger anticoagulant activity than product con-taining arabinogalactan sulfate (13). However, up toour best knowledge, there is no in vivo study to sup-port anticoagulant and antithrombotic activities ofSPS obtained from Codium dwarkense Børgesen.So, the present in vivo study was planned to evalu-ate an anticoagulant effect of sulfated polysaccha-rides, obtain from Codium dwarkense Børgesen inWistar rats.

MATERIALS AND METHODS

Materials

Sulfated polysaccharides from Codiumdwarkense Børgesen was provided by the CentralSalt and Marine Research Institute (CSMCRI),Bhavnagar, Gujarat. SPS was dissolved in distilledwater and 50 mg/mL solution was prepared beforedosing. κ-Carrageenan was purchased from SigmaChemical Company, St. Louis, MO, USA; etherfrom Rachana Ether India Pvt. Ltd., Nasik,Maharastra and heparin from Gland Pharma Ltd.,Hyderabad, India. The main equipments used in thisstudy included ELISA reader for FDP and D-DimerELISA kit (Washington St., Concordia, KS 66901,USA) and fibrinogen kit (Peary Court, Novi, MI48377, USA).

Animals

Wistar albino rats (250 ± 50 g) of either sexand Swiss albino mice (25 to 30 g) of females wereprocured from central animal house of the institute.They were housed in standard transparentpolypropylene cages and kept under controlled roomtemperature (25 ± 2OC) in a 12 h light-dark cycle.The animals were given standard laboratory diet andwater ad libitum. The animals were allowed to accli-matize to the laboratory conditions at least 7 daysbefore starting of the experiments. The animal han-dling was performed according to Good LaboratoryPractice (GLP) guidelines. Present study was startedafter approval from Institutional Animal EthicsCommittee (IAEC), of Government MedicalCollege, Bhavnagar, Gujarat, India [Approval No:IAEC 34/2014, dated 08/02/2014)].

Study design

Before starting the study, an acute toxicitystudy was performed according to, ìorganization foreconomic co-operative development (OECD)îguidelines no. 423. Gradually increasing doses of 5,50, 300, 1000 and 2000 mg/kg of sulfated polysac-charide was administered orally in female mice (3animals for each dose). Mice were closely observedfor development of signs of toxicity and mortalityfor 14 days. Sulfated polysaccharide was found safe

Figure 1. Gross examination of rat dorsal tail vein thrombosis in various study groups; Arrows indicate cut off margin of thrombosis. (1)disease control (2) active control (3) treatment low dose (4) treatment high dose (5) preventive low dose (6) preventive high dose

Evaluation of anticoagulant effect of sulfated polysaccharide... 989

up to 2000 mg/kg. Two different doses 200 and 400mg/kg were selected for evaluation of anticoagulantactivity.

After that, forty eight Wistar albino rats (250 ±50 g) were randomly divided (Computer generatedsoftware ñ Rando software) into six groups (n = 8for each group). A single dose of κ-carrageenanintravenously in rat tail vein was given at pre-decid-ed time in all the study groups for producing hyper-coagulable state. The study groups were as follows:Group 1 (Disease control): rats received 1 mL dis-tilled water orally for 3 days after injecting κ-car-rageenan. Group 2 (Active control): received 300units/kg heparin subcutaneously for 3 days afterinjecting κ-carrageenan. Group 3 (Treatment lowdose): received 200 mg/kg sulfated polysaccharideorally for 3 days after injecting κ-carrageenan.Group 4 (Treatment high dose): received 400 mg/kgsulfated polysaccharide orally for 3 days after inject-ing κ-carrageenan. Group 5 (Preventive low dose):received 200 mg/kg sulfated polysaccharide orallyfor 3 days followed by single dose of κ-carrageenanon last day. Group 6 (Preventive high dose):received 400 mg/kg sulfated polysaccharide orallyfor 3 days followed by single dose of κ-carrageenanon last day.

Study procedure and outcome measures

Induction of thrombosisκ-Carrageenan (dissolved in normal saline)

(14) was injected intravenously in the dose of 3

mg/kg body weight of rat to produce thrombosis. Inrestrained rat, wax thread was tied at root of tail.Xylene was applied over the site to dilate the vein.After 10 min of injection, thread was removed.Animals were observed for gross finding of throm-bosis in rat tail from 5 minutes to 6 hours of postinjection period. Length of tail thrombosis wasmeasured.

Serological parametersRats were anesthetized with inhalational ether

anaesthesia. Blood samples were collected at 0(baseline), 24, 48 and 72 h after κ-carrageenaninjection alternatively from right and left retro-orbital plexus. All the samples collected into citratevaccute and EDTA vaccute. They were sent topathology laboratory for Prothrombin Time (PT),International Normalized Ratio (INR), activatedPartial Thromboplastin Time (aPTT) and plateletscount analysis. ELISA was used to determine thelevels of the fibrinogen, fibrin/fibrinogen degrada-tion products (FDPs) and D-dimer, which are sen-sitive indices of fibrinolytic activity in microbiolo-gy laboratory. The standard curve for FDP, FIBand D-Dimer was created by using curve expertprofessional software version 2.2 and from thestandard curve, concentration of FDP, FIB and D-Dimer were calculated. As samples were diluted upto 1 : 2000, the concentration reading from thestandard curve was multiplied by dilution factor(2000).

Figure 2. Histopathology images of lung sections after hematoxylin-eosin staining under light microscope (x10) (a) disease control (b)active control (c) treatment low dose (d) treatment high dose (e) preventive low dose (f) preventive high dose. Arrow 1 indicatesmicrothrombi and arrow 2 indicates neutrophil infiltration, arrows 3 indicate normal lung parenchyma

990 HIRAL N. GOLAKIYA et al.

Histopathological analysis

After 72 h, rats were sacrificed after givinginjection of ketamine (75 mg/kg; intraperitoneally)and injection of xylazine (10 mg/kg; intraperitoneal-ly). Lung, liver, mesentery were removed and keptin formaldehyde (10% v/v) solution for 24 h. Fivemm sections were taken and processed in a series ofgraded acetone and xylene, embedded in paraffinwax. Five µm thin sections were taken and stainedwith hematoxylin-eosin (H & E) and evaluatedunder optical light microscope for formation ofmicrothrombi in lung, liver and mesentery. Allslides were coded and blinded. Analysis was doneblindly by the pathologist from our institute withoutknowledge of treatment plan.

Histological results were graded as following:0 - no lesions; 1 - minimal lesions (individual

or a few necrotic cells); 2 - mild lesions (10ñ25%necrotic cells or mild diffuse degenerative lesions);3 - moderate lesions (25ñ40% necrotic or degenera-tive cells); 4 - marked lesions (40ñ50% necrotic ordegenerative cells); 5 - severe lesions (more than50% necrotic or degenerative cells). The secondscale is based on the first scale, but simply considerssections with scores = 3 as significant liver lesions(15).

For lung, leukocyte infiltration was evaluatedto determinate the severity of alveolar inflammation.0. no extravascular leukocytes; 1. < 10 leukocytes;2. 10ñ45 leukocytes; 3. > 45 leukocytes. The aver-age of the numbers was used for comparison (16).

Microthrombi were calculated in 10 low powermicroscopic fields in liver and lung and in 5 lowpower microscopic fields in mesentery.

Statistical analysis

All the parameters were expressed as the mean± standard error of mean (S.E.M.). Data werechecked for normal distribution using the Kolmo-gorovñSmirnov test.

For intragroup comparison, parametric datawere analyzed by repeated measures ANOVA fol-lowed by Tukeyís multiple comparison test and non-

parametric data were analyzed by Friedman test fol-lowed by Dunnís multiple comparison test. Forintergroup comparison, parametric data were ana-lyzed by one way ANOVA followed by Tukeyísmultiple comparison test and non-parametric datawere analyzed by Kruskal Wallis test followed byDunnís multiple comparison test using GraphPadInStat (version 3.00, GraphPad Software, CaliforniaUSA). A p value < 0.05 was considered as statisti-cally significant.

RESULTS

Sulfated polysaccharide was found safe up to2000 mg/kg. Two different doses 200 and 400mg/kg were selected for evaluation of anticoagulantactivity. One animal in preventive high dose groupwas expired due to study related procedure.Injection of κ-carrageenan single dose in dorsal rattail vein produced visible thrombosis maximum upto 11 cm. Figure 1 shows production of gross throm-bosis in dorsal rat tail vein. As shown in Table 1,administration of SPS in high dose as a treatmentand in low as well as high dose as a preventive ther-apy significantly reduced the gross thrombosis sizein dorsal rat tail vein as compared to disease controlgroup after 72 hour of injecting κ-carrageenan.

As shown in Table 2, κ-carrageenan signifi-cantly decreased PT at 48 h in disease control group(p < 0.05). In both the treatment groups SPS signif-icantly restored the altered PT caused by κ-car-rageenan, at 72 h (p < 0.05). Whereas in prophylax-is groups SPS significantly prevented the alterationof PT as compared to the baseline. At 72 h, PT wasfound higher in all the study groups as compared todisease control group. However, the statistical sig-nificant difference was found between disease con-trol group and active control group.

International Normalised Ratio (INR) of allstudy groups was comparable at baseline (Table 2).INR was significantly decreased at 24 h in diseasecontrol group after injecting κ-carrageenan (p <0.05). Altered INR was significantly restored in boththe SPS treatment groups at 72 h. Whereas, in the

Table 1. Comparison of thrombosis size after 72 h in dorsal rat tail vein between experimental groups.

GroupsGroup 1 Group 2 Group 3 Group 4 Group 5 Group 6 p

DC (n = 8) AC (n = 8) TLD (n = 8) THD (n = 8) PLD (n = 8) PHD (n = 7) value

Thrombosis (mm)

18.3 ± 8.4 8.4 ± 7.4 22.1 ± 13.4 7.4 ± 6.8@ 1.6 ± 1.5@ 1.4 ± 1.0@ 0.01

All values are expressed as the mean ± SEM. @p < 0.05 as compared with DC group by Kruskal-Wallis test followed by Dunn's multiplecomparison test; (DC - Disease control, AC - Active control, TLD - Treatment low dose, THD - Treatment high dose, PLD - Preventivelow dose, PHD - Preventive high dose).

Evaluation of anticoagulant effect of sulfated polysaccharide... 991T

able

2. C

ompa

riso

n of

coa

gula

tion

and

fibr

inol

ytic

par

amet

ers

in e

xper

imen

tal g

roup

s.

Stud

y D

urat

ion

PTaP

TT

Plat

elet

cou

nts

Fibr

inog

enFD

PD

-Dim

ergr

oups

(h

ours

)(s

econ

ds)

INR

(s

econ

ds)

(lac

s/cu

mm

) (n

g/m

L)

(µg/

mL

) (n

g/m

L)

Gro

up 1

028

.91

± 1.

012.

32 ±

0.0

960

.50

± 1.

287.

91 ±

0.3

080

.49

± 4.

405.

10 ±

0.8

51.

34 ±

0.1

3

DC

2426

.41

± 0.

732.

06 ±

0.0

6*54

.03

± 1.

72*

9.14

± 0

.61

94.7

9 ±

18.2

71.

29 ±

0.6

72.

52 ±

0.0

7*

(n =

8)

4824

.25

± 0.

68*

1.87

± 0

.07*

52.0

0 ±

1.66

*9.

46 ±

0.8

966

.12

± 4.

645.

77 ±

0.9

22.

52 ±

0.1

2*

7224

.48

± 0.

78*

1.91

± 0

.07*

49.6

3 ±

1.31

*11

.87

± 0.

96*

114.

18 ±

15.

006.

97 ±

0.2

72.

44 ±

0.0

7*

028

.03

± 0.

752.

16 ±

0.0

854

.00

± 1.

277.

85 ±

0.2

610

3.31

± 1

5.46

5.86

± 0

.47

1.10

± 0

.13

Gro

up 2

24

27.1

5 ±

1.41

2.14

± 0

.13

50.5

3 ±

2.17

7.42

± 0

.39^

99.2

7 ±

17.1

25.

02 ±

0.5

3*@1.

87 ±

0.1

6^

AC

4828

.16

± 1.

092.

24 ±

0.1

061

.85

± 2.

09*@

6.24

± 0

.58

98.1

6 ±

15.8

44.

05 ±

0.4

7*1.

00 ±

0.1

4@

(n =

8)

7230

.89

± 1.

36@

2.50

± 0

.13#@

64.0

5 ±

2.69

*@5.

05 ±

0.7

3#^62

.56

± 3.

84^

2.64

± 0

.50*^

2.17

± 0

.14#

025

.53

± 0.

931.

98 ±

0.0

755

.09

± 2.

326.

55 ±

0.2

4 11

8.51

± 2

3.43

6.48

± 0

.58

1.15

± 0

.18

Gro

up 3

2425

.03

± 1.

221.

99 ±

0.1

157

.23

± 2.

643.

51 ±

0.3

8*^96

.30

± 11

.15

6.59

± 0

.62@

2.22

± 0

.07

TL

D48

27.1

1 ±

0.97

2.14

± 0

.09

58.7

8 ±

3.62

3.79

± 0

.36*@

10

2.93

± 1

5.03

6.61

± 0

.43

2.21

± 0

.09

(n =

8)

7228

.76

± 0.

95*

2.30

± 0

.08*@

59.9

9 ±

3.01

@4.

40 ±

0.2

3*^56

.40

± 9.

65^

6.56

± 0

.29

2.27

± 0

.11*

026

.04

± 1.

172.

06 ±

0.1

152

.68

± 1.

176.

53 ±

0.3

814

1.63

± 1

3.88

4.90

± 0

.44

1.19

± 0

.10

Gro

up 4

2425

.78

± 0.

872.

03 ±

0.0

857

.01

± 1.

594.

31 ±

0.2

7*^76

.05

± 5.

53*

5.40

± 0

.27@

1.33

± 0

.11^

TH

D48

27.6

1 ±

0.91

2.19

± 0

.08

58.1

9 ±

0.77

#4.

56 ±

0.3

6*@82

.75

± 6.

18*

5.81

± 0

.60

2.62

± 0

.09*

(n=8

)72

28.6

5 ±

1.76

*2.

27 ±

0.1

660

.30

± 1.

38#@

4.96

± 0

.38*^

84.7

5 ±

12.7

6*5.

86 ±

0.3

52.

55 ±

0.8

6*

025

.55

± 0.

862.

00 ±

0.0

852

.95

± 2.

747.

13 ±

0.5

211

6.04

± 1

1.19

4.53

± 0

.66

1.25

± 0

.16

Gro

up 5

2426

.75

± 0.

972.

11 ±

0.0

853

.65

± 1.

636.

47 ±

0.4

0^84

.32

± 5.

403.

65 ±

0.4

42.

14 ±

0.1

0*

PLD

4823

.85

± 0.

631.

84 ±

0.0

655

.09

± 1.

715.

77 ±

0.3

5*45

.43

± 8.

59#

4.70

± 0

.59

2.54

± 0

.09*

(n =

8)

7226

.34

± 0.

622.

08 ±

0.0

548

.96

± 2.

115.

68 ±

0.3

8*^10

1.74

± 1

0.16

6.47

± 0

.69*

1.36

± 0

.17^

025

.96

± 0.

612.

00 ±

0.0

553

.74

± 2.

806.

71 ±

0.3

612

7.37

± 1

9.75

4.73

± 0

.40

1.42

± 0

.26

Gro

up 6

2424

.93

± 1.

351.

86 ±

0.0

755

.60

± 2.

756.

07 ±

0.5

2^13

8.6

± 16

.46

4.45

± 0

.38

1.50

± 0

.31^

PHD

4826

.03

± 1.

161.

95 ±

0.0

758

.64

± 2.

22*

5.83

± 0

.37

95.7

4 ±

11.0

34.

24 ±

0.5

51.

21 ±

0.2

5(n

= 7

)72

25.8

6 ±

1.26

1.93

± 0

.09

55.5

4 ±

1.84

6.40

± 0

.59^

109.

86 ±

11.

084.

65 ±

0.2

0^0.

87 ±

0.0

3^

All

valu

es a

re e

xpre

ssed

as

the

mea

n ±

SEM

.* p <

0.0

5 as

com

pare

d w

ith 0

hou

r va

lue

by r

epea

ted

mea

sure

s A

NO

VA

fol

low

ed b

y D

unne

tt m

ultip

le c

ompa

riso

n te

sts;

# p <

0.0

5 as

com

pare

d w

ith 0

hou

r va

lue

byFr

iedm

an te

st f

ollo

wed

by

Dun

nís

mul

tiple

com

pari

son

test

s; **

p va

lue

for

repe

ated

mea

sure

s A

NO

VA

; ##p

valu

e fo

r Fr

iedm

an te

st; ^ p

< 0

.05

as c

ompa

red

with

DC

gro

up b

y on

e w

ay A

NO

VA

fol

low

ed b

y D

unne

ttm

ultip

le c

ompa

riso

n te

st; @

p <

0.05

as

com

pare

d w

ith D

C g

roup

by

Kru

skal

-Wal

lis te

st fo

llow

ed b

y D

unní

s m

ultip

le c

ompa

riso

n te

st; ^^

p va

lue

for o

ne w

ay A

NO

VA

;@@P

valu

e fo

r Kru

skal

-Wal

lis te

st; (

DC

- D

isea

seco

ntro

l, A

C -

Act

ive

cont

rol,

TL

D -

Tre

atm

ent l

ow d

ose,

TH

D -

Tre

atm

ent h

igh

dose

, PL

D -

Pre

vent

ive

low

dos

e, P

HD

- P

reve

ntiv

e hi

gh d

ose)

.

992 HIRAL N. GOLAKIYA et al.

preventive groups, INR was not significantly alteredat 72 h as compared to baseline.

After injecting κ-carrageenan intravenously,aPTT was significantly decreasing in disease controlgroup from 24 to 72 h as compared to baseline value(p < 0.05; Table 2). At 72 h, in both doses of treat-ment group and high dose preventive group thealtered aPTT was restored. However, statistical sig-nificance was achieved in high dose treatment withSPS (p < 0.05).

Single dose of intravenous injection of κ-car-rageenan produced statistically significant increasein platelet counts at 72 h as compared to 0 h (p <0.05; Table 2). At 72 h, two different doses of SPSas treatment and preventive therapy restored thealtered platelet counts caused by κ-carrageenan.

Fibrinogen level was non-significantlyincreased in disease control group at 72 h afterinjecting κ-carrageenan in rat tail vein (Table 2).SPS and heparin administration prevented the

Figure 3. Histopathological images of liver sections after hematoxylin-eosin staining under light microscope (10◊); (a) disease control (b)active control (c) treatment low dose (d) treatment high dose (e) preventive- low dose (f) preventive- high dose. In a: black arrow indicatesnecrosis and white arrow indicates degeneration and microthrombi. Other arrows (in b-f) indicate normal hepatocyte arrangement

Figure 4. Histopathological images of mesentery sections after haematoxylin-eosin staining under light microscope (x10); (a) disease con-trol (b) active control (c) treatment low dose (d) treatment high dose (e) preventive- low dose (f) preventive- high dose. In a: black arrowindicates microthrombi and white arrow indicate capillary hemorrhage. Grey arrows indicate normal mesenteric structure

Evaluation of anticoagulant effect of sulfated polysaccharide... 993

change in fibrinogen level caused by κ-car-rageenan.

κ-Carrageenan non-significantly increased theFDP and significantly increased the D-Dimer levelin disease control group (Table 2). Treatment andpreventive therapy of SPS did not show any effecton FDP and D-Dimer level. Effect of SPS on fib-rinogen and D-Dimer levels are shown in Table 2.

As shown in Table 3, number of microthombiin lung, liver and mesentery were significantlyreduced due to SPS administration as compared todisease control group. Figures 2, 3 and 4 show thehistopathological changes in lung, liver and mesen-tery, respectively, in various study groups.

DISCUSSION

The present study showed anticoagulant effectof SPS from Codium dwarkense Børgesen in hyper-coagulable Wistar albino rats.

Carrageenan is straight chained sulfur contain-ing macromolecular polysaccharides composed ofrepeating units of D-galactose and 3, 6-anhydro-D-galactose. κ-Carrageenan is commonly used for pro-ducing tissue inflammation and tail thrombosis inexperimental models. κ-Carrageenan can cause localvascular inflammation and endothelial cell injurythrough releasing inflammatory factors, which maylead to the formation of thrombus (17). Anothermechanism for the thrombotic activity of κ-car-rageenan is an activation of Hageman factor (factorXII) that is followed by intravascular coagulation (18,19). In present study, intravenous injection of κ-car-regeenan produced significant thrombosis evidencedby gross visible thrombosis, altered coagulationparameters along with histopathological findings.

In present study, SPS significantly restoredaltered coagulation parameters (PT, INR, aPTT,Platelet count and Fibrinogen level) without affect-ing fibrinolytic parameters (FDP and D-Dimer). PTis a sensitive screening test for the extrinsic coagu-lation pathway while aPTT reflects the integrity ofthe intrinsic pathways of coagulation. Extrinsiccoagulation pathway gets activated on tissue traumathat liberates tissue factor (factor III) into the bloodwhich in turn activates factor VII. Activated factorVII ultimately activates factor X and common path-way of coagulation is reached. Intrinsic pathway isactivated through damaged endothelial cells thatresults in activation of factor XII, XI, IX and X insequential manner to reach the common pathway ofcoagulation (20). Interference in extrinsic pathwayprolongs the PT whereas, disturbances in intrinsicpathway prolongs the aPTT. When common path-way is affected, both PT and aPTT get prolonged. Inour study, administration of SPS resulted into therestoration of both PT and aPTT that suggests SPSmay have its anticoagulant effect through a commonpathway of coagulation. During the coagulationprocess, activation and recruitment of platelets leadsto increased platelet count. However, administrationof SPS either reduced or restored the increasedplatelet count caused by κ-carrageenan. This findingsuggests that SPS may have its effect on activationand recruitment of platelets during the coagulationprocess. Effect of SPS on fibrinogen level and otherfibrinolytic parameters also suggests its anticoagu-lant effect through a common pathway of coagula-tion. Our in vivo study findings are similar to the invitro findings of Shanmugam et al. (21). Admini-stration of SPS both as a treatment and preventivetherapy reduced the number of microthrombi along

Table 3. Inter-group comparison of number of microthrombi in lung, liver and mesentery.

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 valueDC AC TLD THD PLD PHD F

(n = 6) (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) (df)

Lung < 0.0001(per 10 LPF) 7.83 ± 0.54 4.50 ± 0.67^ 3.83 ± 0.60^ 2.00 ± 0.58^ 3.33 ± 0.71^ 0.67 ± 0.33^ 17.45

(5, 30)

Liver < 0.0001(per 10 LPF) 7.00 ± 0.37 3.67 ± 0.80^ 1.33 ± 0.33^ 1.5 ± 0.67^ 3.33 ± 0.42^ 1.17 ± 0.48^ 15.59

(5, 30)

Mesentery(per 5 LPF) 4.17 ± 0.31 0.83 ± 0.48@ 0.50 ± 0.22@ 0.83 ± 0.17@ 0.50 ± 0.34@ 0.17 ± 0.17@ < 0.0001

All values are expressed as the mean ± SEM. ^p < 0.05 as compared with DC group by one way ANOVA followed by Dunnett multiplecomparison tests; @p < 0.05 as compared with DC group by Kruskal-Wallis test followed by Dunnís multiple comparison test (LPF - Lowpower field; DC - Disease control, AC - Active control, TLD - Treatment low dose, THD - Treatment high dose, PLD - Preventive lowdose, PHD - Preventive high dose).

994 HIRAL N. GOLAKIYA et al.

with less histopathology damage in lung, liver andmesentery. Findings of histopathology parametersfurther support the anticoagulant effect of SPS.

We used heparin as an active control drugwhich is a highly sulfated mucopolysaccharide. Itproduce the anticoagulant effect by catalyzingantithrombin mediated inhibition of coagulation fac-tors thrombin (factor IIa) and activated factors Xa,IXa, XIa and XIIa (22). In present study, heparin hassignificantly prolonged aPTT and restored PT. Inlow concentration, heparin prolongs aPTT withoutaffecting PT, whereas, in high concentration it pro-longs both. In present study, we used two differentdoses of SPS (200 mg/kg and 400 mg/kg) and wedid not find significant differences of these twoselected doses on coagulation parametes. It suggeststhat there is no dose dependent anticoagulant effectof SPS.

In present study, the extract control group wasnot performed due to limited amount of SPS avail-able. Due to limited resources available, plasma lev-els of clotting factors were not measured.

CONCLUSION

This study shows that sulfated polysaccharidesextracted from Codium dwarkense Børgesen indoses of 200 mg/kg and 400 mg/kg showed an anti-coagulant activity in hypercoagulable Wistar albinorats. The anticoagulant activity was evidenced byrestoration of altered coagulation parameters and byreduction in the number of micro-thrombi alongwith less structural damage in histopathology oflung, liver and mesentery. Further research isrequired to gain closer insights into the exact mech-anism that support an anticoagulant effect of SPSand its dose dependent action before conductingresearches in human beings.

Conflict of Interest: None declared.Funding: No funding.

REFERENCES

1. Naess I.A., Christiansen S.C., Romundstad P.,Cannegieter S.C., Rosendaal F.R. et al..: J.Thromb. Haemost. 5, 692 (2007).

2. Parakh R., Kakkar V.V., Kakkar A.K: JAPI 55,49 (2007).

3. Cohen A.T., Tapson V.F., Bergmann J.F.,Goldhaber S.Z., Kakkar A.K. et al.: Lancet 371,387 (2008).

4. Mulloy B., Gray E., Barrowcliffe T.W.:Thromb. Haemost. 84, 1052 (2000).

5. Geerts W.H., Bergqvist D., Pineo G.F., HeitJ.A., Samama C.M. et al.: Chest 133, 381S(2008).

6. Samama M.M., Cohen A.T., Darmon J.Y.,Desjardins L., Eldor A. et al.: N. Engl. J. Med.341, 793 (1999).

7. Spyropoulos A.C.: Chest 128, 958 (2005). 8. Leizorovicz A., Cohen A.T., Turpie A.G.,

Olsson C.G., Vaitkus P.T., Goldhaber S.Z.:Circulation 110, 874 (2004).

9. Alban S.: Handb. Exp. Pharmacol. 207, 211(2012).

10. Glauser B.F., Pereira M.S., Monteiro R.Q.,Mourão P.A.S.: Thromb. Haemost. 100, 420(2008).

11. Pomin V.H., Mourão P.A.S.: Glycobiology 18,1016 (2008).

12. Shanmugam M., Ph. D. thesis, BhavnagarUniversity, 1998.

13. Siddhanta A.K., Shanmugam M., Mody K.H.,Goswami A.M., Ramavat B.K.: Int. J. Biol.Macromol. 26, 151 (1999).

14. Kamiya S., Ogasawara M., Arakawa M.,Hagimori M.: Biosci. Microbiota Food Health32, 101 (2013).

15. Manautou J.E., Silva V.M., Hennig G.E.,Whiteley H.E.: Toxicology 172, 1 (1998).

16. Calikoglu M., Tamer L., Sucu N., Coskun B.,Ercan B., Gul A.: Pharmacol. Res. 48, 397(2003).

17. Begum R., Sharma M., Pillai K.K., Aeri V.,Sheliya M.A.: Pharm. Biol. 53, 437 (2015).

18. Bekemeier H., Hirschelmann R., Giessler A.J.:Biomed. Biochim. Acta 43, S347 (1984).

19. Bekemeier H., Hirschelmann R., Giessler A.J.:Agents Actions 16, 449 (1985).

20. Weitz J.I.: Goodman and Gilmanís ThePharmacological Basis of Therapeutics, 12th

edn., p. 849, Mc Graw Hills, New Delhi 2011. 21. Shanmugam M., Mody K.H.: Curr. Sci. India

79, 1672 (2000).22. Garcia D.A., Baglin Y.P., Weitz J.I., Samama

M.M.: Chest 141 (2 Suppl.), e24S (2012).

Received: 23. 12. 2016