Influence of red algal sulfated polysaccharides on blood coagulation and platelets activation in vitro

Influence of red algal sulfated polysaccharides on blood coagulationand platelets activation in vitro

Ekaterina V. Sokolova,1* Anna O. Byankina,1 Alexandra A. Kalitnik,1 Yong H. Kim,2

Larisa N. Bogdanovich,3 Tamara F. Solov’eva,1 Irina M. Yermak1

1Department of Molecular Immunology, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch of the Russian

Academy of Sciences, Prospect 100-let Vladivostoku, 159, Vladivostok 690022, Russia2Department of Food Science and Biotechnology, Kyonggi University, Suwon 442-760, Republic of Korea3Laboratory of Innovation Medical and Biological Researches and Technologies, Medical association of the Far East Branch

of the Russian Academy of Sciences, Vladivostok, st. Kirova, 95, 690022, Russia

Received 24 January 2013; revised 16 May 2013; accepted 31 May 2013

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34827

Abstract: The influence of sulfated polysaccharides (k-, j-, and

j/b-carrageenan and porphyran) – on platelet activation was

studied. Carrageenans were much weaker inhibitors of a coag-

ulation process than heparin, while porphyran had not that

effect. Results of the aPTT and PT assays suppose that carra-

geenans affected mostly intrinsic pathway of coagulation,

while their effect on the extrinsic pathway is extremely low (kand j/b) or absent (j, LMW derivative of j-carrageenan). k-

Carrageenan was the most potent anticoagulant agent in TT,

aPTT, PT, and anti-factor Xa activity. This sample was also the

strongest inhibitor of collagen-induced platelet aggregation in

PRP. Generally, the correlation of anticoagulant and antithrom-

botic action in PRP is preserved for carrageenans but not for

heparin. Carrageenans and porphyran affected platelet adhe-

sion to collagen by influencing glycoprotein VI. Low molecular

weight j-carrageenan had a similar effect on platelet adhesion

mediated with both major collagen receptors: integrin a2b1

and glycoprotein VI as native polysaccharide had. Carra-

geenans resulted in activation of platelets under platelet adhe-

sion mediated by integrin aIIbb3 with less degree than heparin.

The least sulfated j/b-carrageenan that possessed an inhibi-

ting effect on thrombin- and collagen-induced aggregation of

washed platelets and on the PT test but it had no significant

effect on TT was the weakest promoter of integrin aIIbb3 medi-

ated platelet activation. In summary, our study showed that

the polysaccharide action was complex, since it depended on

its molecular mass, sulfation degree, and monosaccharide

contents (3,6-anhydrogalactose). VC 2013 Wiley Periodicals, Inc. J

Biomed Mater Res Part A: 00A: 000–000, 2013.

Key Words: carrageenan, porphyran, platelet adhesion/aggre-

gation, coagulation, red blood cells

How to cite this article: Sokolova EV, Byankina AO, Kalitnik AA, Kim YH, Bogdanovich LN, Solov’eva TF, Yermak IM. 2013.Influence of red algal sulfated polysaccharides on blood coagulation and platelets activation in vitro. J Biomed Mater Res PartA 2013: 00A: 000–000.

INTRODUCTION

Sulfated polysaccharides containing sulfate groups in theirresidues are commonly found in three major groups ofalgae: red algae (Rhodophyta), brown algae (Phaeophyta)and green algae (Chlorophyta).1 Sulfated polysaccharides ofred algae are galactans known commercially as agar, carra-geenan, and porphyran. Production and applications of sul-fated polysaccharides as therapeutic agents have beenincreasingly important topics of intensive research.

Carrageenans are soluble sulfated linear galactans of redseaweeds. Repeating a(1-3) and b (1-4)-linked units of D-galactose underlie the structure of these polysaccharides.Structural diversity of carrageenans is provided by modifica-tion of a b (1-4) unit into 3,6-anhyrogalactose, and by varia-tions in the amount and position of sulfated groups along a

polymer chain.2 Porphyra species contain sulfated polysac-charides called porphyran, as a complex galactan. Porphyranis a dietary fiber of high quality and chemically resemblesagar.3 There are few data on the influence of the polysaccha-ride structure and their molecular mass on hemostasis com-ponents to date.4

It is well known that sulfated polysaccharides possessanticoagulant activity. Studies of sulfated polysaccharides iso-lated from different algae clearly demonstrated thatstructure-activity dependence is not a function of sulfate con-tent.5 Of sulfated polysaccharides, heparin has the most abun-dant application in clinical practice. Heparin is widely usedfor treatment of thromboembolic disorders as an inhibitor ofanti-thrombin, but, because it is a negatively charged polysac-charide, it binds platelet integrin aIIbb3.

6 As a result of this

Correspondence to: E. V. Sokolova, e-mail: [email protected] grant sponsor: Programs for Fundamental Research of the Presidium of Russian Academy of Sciences “Molecular-cell biology”, Presid-

ium of Far-East Branch of the Russian Academy of Sciences

VC 2013 WILEY PERIODICALS, INC. 1

binding, heparin not only reduces the platelets’ activationthreshold by other agonists, but also induces platelet spread-ing.7 In this connection, a directed search for substances witha strong antithrombotic effect and not causing bleeding con-tinues to be of importance.8 For algal sulfated polysaccharidesand extracts enriched with them is known that they exert astrongly marked dual effect on hemostasis: anticoagulant andproaggregating activity increase simultaneously.8–11 Accord-ing to some literature data, the mechanism of action ofheparin-like compounds including sulfated polysaccharidesfrom marine seaweeds differs from that for heparin. Themajor factor for the anticoagulant action of heparin-like com-pound is thought to be not so much the polyanion nature ofthese compounds as their configuration, molecular mass, andspecific fine structural features.12–14

Consequently, the current study is designed to investi-gate some various types of carrageenan, porphyran and lowmolecular weight (LMW) j-carrageenan on the activation ofhuman platelets and clot formation processes in comparisonto unfractionated heparin in vitro.

EXPERIMENTAL

Carrageenans were isolated from seaweeds of Chondrusarmatus (Gigartinaceae) and Tichocarpus crinitus (Tichocar-paceae) harvested at the Sea of Japan of the Russian Coast,and their structures were described elsewhere.15,16 Porhyranwas isolated from Porphyra tenera (Bangiaceae) harvested inKwangcheon, Chungcheongnambo, Korea, its characteristicswere obtained according to the methods described ear-lier.15,16 Commercial unfractionated heparin as sodium salt(Cat No: 101931, Lot No: 2024H, St. Louis, Sigma, USA) wasused for comparison. Average molecular mass of all investi-gated polysaccharides was in the range of 150–300 kDa (thischaracteristic for heparin was unknown). LMW j-carrageenansample (1.6 kDa - about 4 disaccharide repeating units) fromChondrus armatus (��l, 0.1N 24 h 37��) was prepared asdescribed elsewhere.17 Thrombin, collagen (predominantlytype 1), fibrinogen were purchased from Renam (Russia).Bovine and human serum albumin (BSA, HSA), p-nitrophenylphosphate were purchased from Sigma (USA).

Porphyran extractionDried algae (50 g) were milled and immersed in distilledwater (1.5 L) and heated at 90�C for 2 h in a boiling waterbath. After cooling down to room temperature, the obtainedsuspension was centrifuged (6 5003g, 30 min, 4�C). Thesupernatant was separated, and the pellet consisting ofinsoluble algal residues were re-extracted twice as previ-ously. The supernatants were collected, heated at 45�C inwater bath and polysaccharides were precipitated by 3 folds95% ethanol and kept overnight at 4�C. Then precipitatewas resolved by hot water at 45�C and dialyzed for over-night. After that suspension was centrifugation (6 5003g,30 min, at 4�C), and supernatant was lyophilized.

Ethical approvalThe study protocol was approved by the medical ethicalcommittee of the local hospital (Vladivostok, Russian

Federation). Informed consent was obtained from all sub-jects who participated in the study. All donors were free ofsalicylic acid administration for 14 days before blood taking.Blood was drawn from the antecubital vein of normalhealthy human volunteers and anticoagulated in plastictubes Vacuette (Greiner Bio-One International AG, Krems-muenster, Austria) with ACD (85 mM trisodium citrate; 71mM citric acid; 111 mM dextrose) in a 6:1 (blood:ACD) asan anticoagulant or with 3.8% trisodium citrate in 1:9,citrate/blood (the exact anticoagulant for each assay wasindicated below).

Red blood cells hemolysisRed blood cells were washed three times with PBS. Then250 mL 10 % (v/v) red blood cell suspension was added to5 mL sample (1 mg 3 mL21 in PBS). Triton X-100 (1%) wasused as a positive control, and PBS – as a negative control.The mixture was incubated for 1 h at ambient temperatureand shaking and then centrifuged 5 min 10 0003g. Absorb-ance was measured at 550 nm.18 Hemolysis percent wascalculated according to the following formula:

%5ððAsample2APBSÞ=AtritonÞ3100: (1)

Hemagglutination assayA hemagglutination assay was carried out using a microtitermethod in a 96-well microtiter V-plate.19 First, 25 lLamounts of serially two-fold dilutions of a test solutionwere prepared in saline on a microtiter V-plate. Sample con-centration range was similar to concentrations used forother assays. To each well, 25 lL of a 2 % red blood cellsuspension was added, and the mixtures were incubatedand gently shaken at room temperature for 2 h. Hemaggluti-nation was observed macroscopically and judged as positivein the case that more than 50% of red blood cells in thewell were agglutinated. The assay was carried out in tripli-cated each test solution.

Thrombin timeThe thrombin time was performed according to the methoddescribed earlier.20 Sample aliquot (20 mL, 100 mg 3 mL21

in PBS) was added to 200 mL citric poor platelet plasma(PPP). Heparin used as a positive control (20 mL, 10 mg 3

mL21 in PBS), and PBS – as negative control. Mixtures wereincubated at 37�C for 2 min, then thrombin solution (200mL, 0.5 U 3 mL21) was added, and registration of thethrombin time was performed. The influence on the throm-bin time was calculated according to the following equation:

%5ððTsample2TPBSÞ=TPBSÞ3100: (2)

Prothrombin timePolysaccharides from red algae and unfractionated heparinin final concentrations of 10 and 1 lg/mL, respectively,were incubated for 1 min at 37�C with 0.1 mL of humanplasma followed by the addition of 0.2 mL of solution of a

2 SOKOLOVA ET AL. INFLUENCE OF RED ALGAL SULFATED POLYSACCHARIDES

thromboplastin calcium mixture (Tekhnologiya-Standart,Russia) preheated at 37�C, and then the time of clot forma-tion was registered. The influence on PT was calculatedaccording to the following equation:


Activated partial thromboplastin timePolysaccharides from red algae and unfractionated heparinin final concentrations of 10 and 1 lg/mL, respectively,were incubated for 2 min at 37�C with 90 lL of humanplasma followed by the addition of 90 lL aPTT reagent(ellagic acid phospholipid reagent) (Tekhnologiya-Standart,Russia). After incubation at 37�C for 3 min, 40 lL of CaCl2of 0.025M, and the timer was started. The influence onaPTT was calculated according to the following equation:


Anti-factor Xa activityThe effect on activity of factor Xa was investigated by thereagent kit Reaclot Heparin test (Renam, Russia). The reac-tion mixture consisted of 10 mL of 15 mM Tris-HCl buffer,pH 8.4, with polysaccharides from red algae and unfractio-nated heparin in final concentrations of 10 and 1 lg/mL,respectively, or 10 mL of buffer as a control and 90 mL ofsolution of human substrate plasma as a source of ATIII,fibrinogen and factor V. It was incubated in a water bath at37�C for 1 min followed by addition of 40 mL of a mixtureof factor Xa (activity of 0.05 U/mL) and phospholipids. Afterincubation at 37�C for 2 min, 40 lL of 0.035M CaCl2 wasadded to the mixtures, and the clotting time was recorded.The influence on factor Xa was calculated according to thefollowing equation:

%5ððTsample2TbufferÞ=TbufferÞ3100: (5)

Washed platelet preparationBlood was drawn into tubes with ACD with the ratio 6:1(blood:ACD). Suspension of human platelets was preparedaccording to the modified method of double washingdescribed earlier.21 To prepare platelet rich plasma (PRP),supernatants obtained after centrifugation at 1203g for 12min were collected. PRP was centrifuged 22003g for 15min to prepare platelet pellet. The platelet pellet werewashed by suspending in Tyrode’s albumin buffer (145 mMNaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM Na2HPO4, 1 mMMgCl2, 2 mM CaCl2, 6 mM glucose, and 0.35% bovine serumalbumin) containing 10 U 3 mL21 heparin and 0.02 U 3

mL21 apyrase at pH 6.5, 37�C. After 10 min incubation at37�C platelets were centrifuged again at 19003g for 8 min.Then platelet pellet was again resuspended with the samebuffer but without heparin and after 10 min of incubationat 37�C, the platelets were again washed using Tyrode’salbumin buffer. Finally, the platelets were resuspended in

the Tyrode’s albumin buffer, pH 7.4 containing 0.02 U 3

mL21 apyrase. Platelets were counted using a Biola aggreg-ometer (Moscow, Russia) provided with counter 230LA andadjusted to required number of platelets.

Platelet aggregationPlatelet aggregation speed was measured using a dual chan-nel laser analyzer of platelet aggregometer Biola (Moscow,Russia) based on the turbidimetric method of Born andCross.22 Platelet suspension or PRP (300 mL, 3003103 permL) was incubated with sample solution (30 mL, 100 mg 3

mL21 in Tyrode’s albumin buffer) of saline solution (a nega-tive control) for 2 min at 37�C in a siliconized glass cuvette.Aggregation was initiated by addition of such agonist as col-lagen (washed platelets and PRP) and thrombin (washedplatelets). Thrombin (1 U 3 mL21) and collagen (2 mg 3

mL21) were used independently for induction of plateletaggregation. To investigate a lytic effect of polysaccharides,aggregation of platelets in PRP without any agonist additionwas used. Aggregation was then followed for 5 min withconstant stirring at the speed of 800 rpm. In each case,aggregation induced by an agonist alone was considered as100% aggregation speed. The sample aggregation speedchanging was calculated according to the following formula:

%5ððAsample2AsalineÞ=AsalineÞ3100: (6)

Platelet adhesionInfluence of polysaccharides on platelet adhesion wasassessed according to modified method of Bellavite et al.23

The basic of that assay is a colorimetric measurement ofactivity by “end point” of acid phosphatase containing inplatelets adhered to immobilized proteins. Briefly, 100 mLfibrinogen solution (20 mg 3 mL21 in PBS) or collagen ofpredominantly type 1 (20 mg 3 mL21 in acetic acid 0.01 M)and were added to the wells of a 96-well plate (MaxiSorp,Nunc) and incubated overnight at 4�C. The plates werewashed twice with PBS. Then the wells were blocked byadding 200 mL 1 % BSA in PBS and incubated for 1 h at37�C, and then washed twice with PBS. Preparation ofwashed platelets with concentration of 60–803103 per mLwas performed according to the method described beyond.

Tyrode’s buffer used for various glycoproteins:

1. For glycoprotein IIb/IIIa (integrin aIIbb3), the concentra-tion of calcium chloride was of 1 mM value with employ-ment of the plate coated with fibrinogen.

2. For glycoprotein Ia/IIa (integrin a2b1), the concentrationof calcium chloride was of 15 mM with employment ofthe plate coated with collagen.

3. For glycoprotein VI, calcium chloride was absent, andethyleneglycoltetraacetic acid (EGTA) was added withfinal concentration of 1 mM.

Washed platelets (50 mL) and sample solution (50 mL, 50 ng3 mL21 in Tyrode’s buffer) or buffer as a negative controlwere added to the plate wells and incubated for 30 min at

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00 3

37˚�. Then the plates were washed three times with PBS,and 100 mL of substrate buffer (0.1M citrate buffer pH 5.5containing 5 mM p-nitrophenyl phosphate and 0.1% TritonX-100) was added. Triton X-100 causes instantaneous lysis ofplatelets without affecting the acid phosphatase activity.23

After incubation at room temperature for 120 min with agentle rocking motion, the reaction was stopped and thecolor was developed by addition of NaOH (20 mL, 1M). Subse-quently, the reaction product, p-nitrophenol, at acidic condi-tions forms quinoid-type compound, and the absorbance wasmeasured at 410 nm. Platelet adhesion was expressed as per-cent adhesion, considering PBS-treated platelets suspensionas 100%.

Statistical analysisAll data were expressed as mean 6 standard deviation. Sta-tistical analysis was done by one-way Anova. Differenceswere considered to be statistically significant if p < 0.05.

RESULTS

The following carrageenan structural types were used duringthe study: j-, j/b-, and k-types of carrageenans (Table I).Carrageenans differ by number and position of sulfatedgroups and by the presence or absence of a 3,6-anhydroga-lactose unit. Porphyran and LMW j-carrageenan were alsoincluded in the study, which characteristics are illustrated inTable I.

Red blood cells hemolysisPolysaccharides of carrageenans and porphyran were testedon hemolytic activity under the final polysaccharide concen-tration of about 20 mg 3 mL21 compared with the sampleconcentration for a platelet aggregation assay. According tothe data obtained hemolysis of red blood cells by sulfatedpolysaccharides was about 1%.

Hemagglutination assaySince some molecules with sugar content may interferewith platelets, hemagglutinating property of polysaccharideswas also investigated. This assay did not reveal an

agglutinating property of investigated samples as regards tohuman red blood cells.

Thrombin time (TT)The thrombin time assay was used to investigate the influ-ence of polysaccharides on the common mechanism ofplasma/secondary hemostasis. All carrageenans exceptingLMW j-carrageenan and j/b-carrageenan retarded the timeof clot formation, but their activity was considerably lessthan that of heparin. Of all carrageenans, k-carrageenan wasthe strongest anticoagulant increasing the thrombin time by50% compared with the negative control. K/b- and j-carrageenans retarded the coagulation process by 30 and37%, respectively. Porphyran and LMW j-carrageenan hadno significant effect compared to the control (Fig. 1).

Prothrombin timeEffect on the extrinsic pathway of blood coagulation wasestimated by the prothrombin time (PT). This assay showedthat j/b- and k-carrageenans were the only polysaccharideaffected extrinsic pathway of blood coagulation (about 40%)(Fig. 1).

Activated partial thromboplastin timeThe activated partial thromboplastin time (aPTT) was usedto estimate the effect of the samples on the intrinsic

TABLE I. The Structures of Carrageenans From Algae of Gigartinaceae and Tichocarpaceae Families and Porphyran

Structure of DisaccharideRepeating Unit

Molar Ratio Gal :Algal Species Sample 3-Linked 4-Linked 3,6-AnGal: SO4

22

C. armatus k-Carrageenan G2S D2S, 6S 1 : 0.02 : 0.97C. armatus j-Carrageenan G4S DA 1 : 0.9 : 0.73C. armatus j-Carrageenan (LMW) G4S DA 1 : 0.83 : 0.78T. crinitus j/b-Carrageenan G4S/G DA/DA 1 : 0.75 : 0.5Porphyra tenera Porphyran nd nd 1 : 0.15 : 0.09

FIGURE 1. Coagulation time change in the presence of red algal poly-

saccharides (C 5 10 lg 3 mL21, final value): 1, control (PBS); 2, hepa-

rin (C 5 1 lg 3 mL21, final value); 3, k-carrageenan; 4, j-carrageenan;

5, j-carrageenan (LMW); 6, j/b-carrageenan; 7, porphyran. The results

are expressed as % change in thrombin time relative to the negative

control (100 %). – thrombin time; – prothrombin time; – acti-

vated partial thromboplastin time; – anti-Factor Xa.


pathway of blood coagulation. The heparin effect was notmuch impress comparing to carrageenans because of the10-fold difference in polysaccharide concentration. The mostactive of carrageenans was k-type (increase in clotting timeby 130%) while the others (j, j-LMW, and j/b) had a simi-lar action retarding on the average by 30%. Influence ofporphyran was insignificant (Fig. 1).

Anti-factor Xa activityFactor Xa is the upstream common point for intrinsic andextrinsic coagulation pathways. k-Carrageenan was the onlyactive sample of the red algal polysaccharides towards fac-tor Xa (increase by 25%). Heparin as expected had a higheffect (Fig. 1).

Platelet aggregationAggregation of platelets is known to be caused by cross link-ing of integrin aIIbb3 at various platelets by means of fibrino-gen, and von Willebrand factor is a final step of a plateletactivation process.24 This type of aggregation is consideredto take place at physiological conditions with low shear rates(<1000 s21) and in an aggregometer.24,25 Platelet aggrega-tion in PRP with polysaccharide alone did not occur for 5min indicating on the absence of a lytic effect for investigatedpolysaccharides (heparin induced low degree of plateletaggregation). The presence of carrageenans was accompaniedby a decrease in washed platelets aggregation speed underthe influence of thrombin and collagen (Fig. 2). j/b-Carra-geenan and porphyran reduced the speed of thrombin-induced platelet aggregation by 22 and 24%, respectively,whereas heparin reduced aggregation by only 19.5% (Fig. 2).

When collagen was used as an agonist, all polysaccha-ride samples inhibited aggregation of washed platelets moreintensively than for thrombin. Heparin reduced collagen-induced platelet aggregation by 77%, j/b-carrageenan – by54%, and the other carrageenans by on average 45%. How-ever, porphyran had no significant effect.

On the other hand, influence of the same polysaccha-rides on aggregation induced by collagen in platelet richplasma showed inhibition by 68% for k-carrageenan andabout 20% for j- and j/b-carrageenans when heparin asporphyran were ineffective (Fig. 2). LMW derivative of j-carrageenan was ineffective (data not shown).

Platelet adhesionThe polysaccharide influence on adhesion of platelets toimmobilized fibrinogen and collagen was studied. The pres-ence of the polysaccharides increased adhesion of plateletsto immobilized fibrinogen by means of integrin aIIbb3 ondifferent value.26 Heparin increased platelet adhesion by85% compared to a control, j- and k-carrageenan by about50%. The effect of j/b-carrageenan and LMW j-carrageenan was to increase platelet adhesion to fibrinogenby on average 23%, but porphyran had no effect on plateletadhesion to fibrinogen (Fig. 3).

The investigated red algal polysaccharides slightlychanged platelet adhesion by means of integrin a2b1 toimmobilized collagen. Carrageenans exhibited a tendency toinhibit platelet adhesion with less potency than that of hep-arin (Fig. 4). Significant inhibition was observed for j-carrageenan by 10%, and the LMW derivative of that carra-geenan type was more effective (decrease by 18%). j/b-Carrageenan and porphyran did not affect that process.

To investigate the adhesion that was strongly dependenton another collagen receptor (glycoprotein VI), an experi-ment was conducted with EGTA (1 mM) as a chelating agentof calcium ions which results in blocking both integrinaIIbb3 and integrin a2b1.

27,28 Platelet adhesion to collagenmediated by glycoprotein VI was inhibited by all the investi-gated polysaccharides by 20% on average (Fig. 4). j-Carrageenan inhibited adhesion by 20%, but its LMW deriv-ative was more effective than native polysaccharide, as forintegrin a2b1.

DISCUSSION

The anticoagulant effect of substances was investigated bytheir capacity to inhibit the clotting process. According tothe TT assay, all carrageenans retarded fibrin clot formationtime, but their effect was less than that of heparin.

FIGURE 2. Platelet aggregation speed change under the influence of

some agonists in the presence of red algal polysaccharides (C 5 10

lg 3 mL21, final value). 1, control (PBS); 2, heparin (C 5 10 lg 3

mL21, final value); 3, k-carrageenan; 4, j-carrageenan; 5, j/b-carra-

geenan; 6, porphyran. The results are expressed as % of change in

aggregation speed relative to the negative control (100 %). – throm-

bin, washed platelets; – collagen, washed platelets; – collagen,

PRP.

FIGURE 3. Platelet adhesion to fibrinogen by means of integrin aIIbb3

in the presence of red algal polysaccharides (C 5 25 ng 3 mL21, final

value): 1, control (PBS); 2, heparin; 3, k-carrageenan; 4, j-

carrageenan; 5, j-carrageenan (LMW); 6, j/b-carrageenan; 7, por-

phyran. The results are expressed as % of change in adhesion relative

to the negative control (100%).

ORIGINAL ARTICLE


Moreover, the direct dependence of the thrombin time onsulfate content was observed. k-Carrageenan was the mosteffective anticoagulant of investigated samples, and por-phyran with low sulfate and 3,6-anhydrogalactose contenthad not any inhibiting effect. A similar dependency on sul-fate contents with respect to carrageenans was observedearlier.1,29,30 For example, Opoku et al. showed additionalchemical sulfation of j-carrageenan resulted in significantincrease in anticoagulant activity in TT test.30 Our resultsalso showed that besides sulfation degree and the presenceof 3,6-anhydrogalactose influencing the anticoagulant effectin TT, decrease in molecular weight of j-carrageenanresulted in the absence of any effect on the coagulation pro-cess (Fig. 1). Previous analogues data were observed forLMW derivates of j-, k-, and i-carrageenans.1,31,32 Thiseffect according to Shanmugam may be caused by the factthat undegraded carrageenans form insoluble complexeswith fibrinogen when low molecular carrageenans formsoluble ones.1

Our data showed that k-carrageenan was the only oneactive towards factor Xa activity (a component of commoncoagulation pathways), when j-type, also statistically signifi-cant sample in TT, was inert in anti-factor Xa assay (Fig. 1).k-Carrageenan, the most potent anticoagulant agent in theTT assay, expresses its action at least partly by affectingactivity of factor Xa.

The mechanism of the anticoagulant activity of carra-geenans can be shown via aPTT and PT assays. The resultsobtained in an aPTT assay showed that carrageenans hadvery low anticoagulant activity when compared to heparin.Of carrageenans, the most sulfated k-type was the strongestinhibitor of coagulation (Fig. 1). It is in accord with datareported by Silva et al.,29 where k-carrageenan displayedelevated anticoagulant activity (240 s at 20 mg), this value ismuch lower than that of heparin (250 s at 2.5 mg). Further,j-carrageenan containing 3,6-DA and less sulfated was alsoless effective anticoagulant than k-type according to both

our results (Fig. 1) that is in line with literature data.29 Thefact that LMW derivative of j-carrageenan preserved theeffect of the native polysaccharide supposing that molecularmass was not important for action of carrageenans in aPTTassay. This result is interesting because supposes existenceof specific carrageenan action which does not depend onthe structure of the j-carrageenan chain. It is interestingassumption because it is known that some sulfated polysac-charides act as anticoagulants by complex mechanisms. Hep-arin is a bright example of it, as it accelerates theantithrombin–protease reaction by a combined bridging andconformational activation mechanisms. The latter effectrequires a specific pentasaccharide sequence inducing aconformational activation of the antithrombin, whereas theformer resulting from bridging the protease and the antith-rombin molecules is determined by the size and bulk struc-ture of the heparin chain.33 In the case of carrageenans itwas shown earlier that antithrombin played a minor or norole in the anticoagulant activity of carrageenans.34

Another assay used for investigating anticoagulant mech-anism is PT test indicating the influence on the extrinsiccoagulation pathway. It is important that PT is affected notonly by highly sulfated k-carrageenan and also low sulfatedj/b-carrageenans (Fig. 1). It is known that sulfation degreeis an important factor for this test because the oversulfatedkappa-carrageenan showed 30 times higher anticoagulantactivity in doubling PT in comparison with a native com-pound.30 Analogous data were observed according to over-sulfated fucoidans.35,36 Nevertheless, the fact that it is alsoaffected by low sulfated j/b-carrageenan supposes thathigh sulfate contents is not the single important structuralfeatures in that assay. So, some studies suggested the influ-ence of impact of stereospecifity on anticoagulant action ofsulfated polysaccharides.12,13 Earlier, we showed that in lowpolysaccharide concentrations (10 mg/mL) both k- and j/b-carrageenans formed unordered polymorph structure con-trary to j-carrageenan,37 and their even effect may be con-nected with their similar macromolecular organization.

Hence, results of the aPTT and PT assays suppose thatcarrageenans affected mostly intrinsic pathway of coagula-tion, while their effect on the extrinsic pathway is extremelylow (k and j/b) or absent (j, LMW derivative of j-carra-geenan). Our results practically correspond to conclusionsmade by Silva and coauthors who also observed that carra-geenans affected intrinsic coagulation pathway.29

The influence of polysaccharides on platelet activationprocess was investigated by their aggregation induced bythrombin or collagen and adhesion to fibrinogen or collagen.For washed platelet aggregation induced by thrombin orcollagen, polysaccharides generally diminished it. The analy-sis of the current data showed that there was no obviouscorrelation between thrombin-induced washed plateletaggregation and clot formation by thrombin. So, k-carrageenan increasing the thrombin time by 50% had nosignificant effect on the speed of platelet aggregationinduced by thrombin. Analogous effect was observed byCumashi et al.38 investigating an antithrombotic effect offucoidans earlier.

FIGURE 4. Platelet adhesion to collagen by means of – integrin

a2b1 and – glycoprotein VI in the presence of red algal polysaccha-

rides (C 5 25 ng 3 mL21, final value): 1, control (PBS); 2, heparin; 3,

k-carrageenan; 4, j-carrageenan; 5, j-carrageenan (LMW); 6, j/b-carra-

geenan; 7, porphyran. The results are expressed as % of change in

adhesion relative to the negative control (100%).


When collagen was used as an inductor of washed plate-let aggregation, all the polysaccharides had a moreimpressed inhibiting effect than for thrombin-induced plate-let aggregation. Carrageenans were less effective than hepa-rin, and had similar inhibiting action in comparison to thecontrol. Nevertheless, it is worth noting that k-carrageenan,being an ineffective sample for platelet aggregation withthrombin, expressed a high inhibiting activity (up to 50%)with respect to collagen-induced platelet aggregation.

On the contrary, results obtained by platelet aggregationin plasma allow us to conclude that some plasma substan-ces are not only involved in antithrombotic action of carra-geenans but also significantly change it. For example,heparin was not active at all, at the same time k-carrageenan had extremely high inhibitory effect on PRPaggregation induced by collagen, while the others wereeither inactive or moderately effective (Fig. 2). Generally, thecorrelation of anticoagulant and antithrombotic action inPRP is preserved for carrageenans but not for heparin (Figs.1 and 2). It can be connected with some different mecha-nism of action of carrageenans and heparin in respect toplatelets.

As is known integrin aIIbb3 besides taking part in plate-let aggregation is also involved in platelets adhesion to vonWillebrand factor, fibrin and/or fibrinogen.39 For currentstudy, influence of the red algal polysaccharides on thereceptor was investigated by means of platelet adhesion toimmobilized fibrinogen. We showed that all the carrageenantypes activated platelets under platelet adhesion mediatedby integrin aIIbb3, with less potency than heparin did. More-over, not only sulfation degree but also the presence of 3,6-anhydrogalactose (porphyran was ineffective) forced thepolysaccharides action on hemostasis (Table I, Figs. 2 and3). It is quite interesting that an activating effect of carra-geenans on platelets may result in thrombogenic activity. Itis worth noting that, of major commercial carrageenans (k,j, and i), only j-carrageenan possessed thrombogenic effectfor carrageenan-induced thrombosis in Wistar rats accord-ing to the literature data.40 In our case, the least sulfated j/b-carrageenan that possessed an inhibiting effect on throm-bin- and collagen-induced aggregation of washed plateletsand on the PT test but it had no significant effect on TTwas the weakest promoter of integrin aIIbb3 mediated plate-let activation. The LMW j-carrageenan exhibited less effectthan native polysaccharide, which was one of the strongestactivators of platelet adhesion mediated by that receptor(Fig. 3). Hence, molecular mass also influence on that typeof platelet adhesion.

It is known that platelets have two major receptors spe-cific to collagen: integrin a2b1 and glycoprotein VI, and theboth exert a synergistic effect on platelet activation.25 Ourresults showed that heparin inhibited adhesion to collagenwith participation of the both receptors, which are in accordwith collagen-induced aggregation of washed plateletsresults (decrease by 77% in comparison to the control). Aswas showed earlier, humans and mice deficient by glycopro-tein VI did not tend to notable bleeding, and platelets wereable to adhere to collagen but could not become active.39,41

Investigated carrageenans in contrast to heparin affectedpredominantly platelet adhesion to collagen mediated byglycoprotein VI. Dependence of structural peculiarities ofcarrageenans on platelet adhesion to collagen was not eli-cited. LMW derivative of j-carrageenan preserved had asimilar effect on platelet adhesion mediated with both majorcollagen receptors: integrin a2b1 and glycoprotein VI asnative polysaccharide had (Fig. 4).

In summary, our study showed that the polysaccharideaction was complex, since it depended on its molecularmass, sulfation degree, and monosaccharide contents (3,6-anhydrogalactose). k-Carrageenan was the most potent anti-coagulant agent in TT, aPTT, PT, and anti-factor Xa activity.This sample was also the strongest inhibitor of collagen-induced platelet aggregation in PRP. So, it can be concludedthat sulfation degree and monosaccharide content areimportant structural features of these polysaccharides foranticoagulant activity, and generally, the correlation of anti-coagulant and antithrombotic action in PRP is preserved forcarrageenans but not for heparin. Carrageenans alsoreduced aggregation of washed platelets induced by throm-bin and collagen, and, in the latter case, the effect of theinvestigated substances expressed at expense of predomi-nantly platelet activation mediated by glycoprotein VI. How-ever, additional investigations are required to find out theprecise mechanism of carrageenans in primary and second-ary hemostasis.

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Influence of red algal sulfated polysaccharides on blood coagulation and platelets activation in vitro