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Acta Scientiarum 


ISSN printed: 1679-9283 
ISSN on-line: 1807-863X 
Doi: 10.4025/actascibiolsci.v39i3.32095 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 

In vitro inactivation of thrombin generation by polysulfated 

fractions isolated from the tropical coenocytic green seaweed 
Caulerpa racemosa (Caulerpaceae, Bryopsidales)  

José Ariévilo Gurgel Rodrigues


, Norma Maria Barros Benevides


, Ana Maria Freire Tovar



Paulo Antônio de Sousa Mourão




Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 

21941-590, Rio de Janeiro, Brazil. 


Laboratório de Carboidratos e Lectinas, Departamento de Bioquímica e Biologia Molecular, Universidade 

Federal do Ceará, Fortaleza, Ceará, Brazil. *Authors for correspondence. E-mails: arieviloengpesca@yahoo.com.br,



ABSTRACT. Pharmacological efficacy of Caulerpa racemosa (Chlorophyta) sulfated polysaccharidic (SPs) 
fractions (F I→III) on models of coagulation and inflammation has been demonstrated, but not their 
effects on thrombin generation (TG). This study examined fractions for composition and physical-
chemical characteristics and in vitro inactivation of TG by F I and F II in 60-fold diluted human plasma 
using continuous method. Papain-extraction yield of 0.7% revealed F I→III by DEAE-cellulose 
chromatography, with differences among the relative proportions of sulfate (17.37-24.00%), total sugars 
(30.03-48.34%) and absence of proteins. Charge density patterns and molecular sizes > 100 kDa of the 
fractions were verified by both agarose/polyacrylamide analyses, respectively. These electrophoreses 
combined with toluidine blue/Stains-All also indicated nonSPs. Anticoagulant effects of 4.76 (F I), 12.00 (F 
II) and 2.32 (F II) IU mg


 by activated partial thromboplastin time test were recorded against heparin (193 

IU mg


), without changes in prothrombin time. Diluted plasma treated with F I and F II reduced 

concentration-dependent and sulfation pattern TG by both intrinsic and extrinsic pathways, with 50% 
inactivation by intrinsic pathway of F II even at 4.1 μg. Heparin abolished TG at least 4-fold lower. 
Therefore, C. racemosa produces SPs with TG inhibition.


Keywords: Chlorophyta, complex glycans, sulfation pattern, clot. 

Inativação in vitro da geração de trombina por frações polissulfatadas isoladas da 

macroalga verde cenocítica tropical Caulerpa racemosa (Caulerpaceae; Bryopsidales) 

RESUMO. Eficácia farmacológica de frações (F I→III) polissacarídicas sulfatadas (PSs) da Chlorophyta 

Caulerpa racemosa sobre modelos de coagulação e inflamação tem sido demonstrada, exceto seus efeitos 
sobre geração de trombina (GT). Examinaram-se frações quanto à composição, características físico-
químicas e inativação in vitro de GT por F I e F II, em plasma humano diluído 60 vezes usando método 
contínuo. Rendimento de extração-papaína (0,7%) revelou, por cromatografia de DEAE-celulose, F I→III 
com diferenças entre as proporções relativas de sulfato (17,37-24,00%), açúcares totais (30,03-48,34%) e 
ausência de proteínas. Foram verificados, por ambas as análises agarose/poliacrilamida, graus de densidade 
de carga e tamanhos moleculares > 100 kDa das frações, respectivamente. Também essas eletroforeses, 
combinadas com azul de toluidina/Stains-All, indicaram polissacarídeos não sulfatados. Foram registrados, 
pelo teste do tempo de tromboplastina parcial ativada, efeitos anticoagulantes de 4,76 (F I), 12,00 (F II) e 
2,32 (F II) UI mg


 contra heparina (193 UI mg


), porém não modificando tempo de protrombina. Plasma 

diluído tratado com F I e F II reduziu GT por ambas as vias intrinsíca/extrínsica, dependente de 
concentração e grau de sulfatação, com F II em 4,1 μg apresentando eficácia de 50% pela via intrínsica. 
Heparina, quatro vezes menos, aboliu GT. Portanto, C. racemosa produz PSs com inibição de GT. 

Palavras-chave: Chlorophyta, glicanos complexos, grau de sulfatação, coágulo. 


The coagulation system comprises a series of 

complex proteolytic reactions that lead to the 

formation of thrombin, which then form a fibrin 

clot. Two pathways are recognized: 1) intrinsic 

(initiated  by  contact  activation  with   a   negatively 

charged surface); and 2) extrinsic (initiated by 
exposure of tissue factor to blood components at the 
site of injury), converging them to a common 
pathway to display the activation of factor X. They 
are classically analyzed using the activated partial 
thromboplastin time (APTT) and  the  prothrombin 

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Rodrigues et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 

time (PT), respectively, although emphasizing that 
these two in vitro tests do not reflect the hemostasis 

(Ferreira, Sousa, Dusse, & Carvalho, 2010). Given 

these limitations and the complexity of the 

coagulation process, thrombin generation (TG)-

based assays would offer a better understanding of 

the in vivo event (Wu et al., 2014) to measure TG in 

a blood plasma sample (Castoldi & Rosing, 2011; 

Xin, Chang, & Ovanesov, 2016) and analyze 

anticoagulants (Wu et al., 2014; Zavyalova & 

Kopylov, 2016). 

Circulatory dysfunctions are usually associated 

with the sedentary lifestyle, poor eating habits and 

stress that potentially lead to venous 

thromboembolism, heart attack and pulmonary 

embolism as the most prevalent cardiovascular 

diseases worldwide, leading causing to patient death 

or else partial or total disability. Routinely, 

unfractionated heparin (UHEP) as anticoagulant 

drug has been primarily employed in clotting 

disorders, which inhibits thrombin and activated 

factor X mediated by antithrombin (AT) due to its 

specific pentasaccharide sequence (Mourão et al., 

2015). Although effective, UHEP develops 

thrombocytopenia and bleeding being necessary to 

monitor its anticoagulation using the APTT test 

(Mourão et al., 2015) since UHEP significantly 

decreases TG (Wu et al., 2014). This therapeutic 

agent is mainly extracted from pig and bovine 

intestine and its potential risks of chemical 

contamination with other glycosaminoglycans for 
use in cardiac surgeries also justify the current 

efforts to develop alternatives to UHEP (Mourão & 

Pereira, 1999; Pomin, 2012; Rodrigues et al., 2016). 

Seaweeds have structurally diverse unique 

chemicals (e.g., proteins, carbohydrates and lipids) 

(Carneiro, Rodrigues, Teles, Calvacante, & 

Benevides, 2014) with industrial relevance and 

benefits to human and animal health (Costa-Lotufo 

et al., 2006) as potentially useful ingredients in 

cosmetics (Cardozo et al., 2007), aquaculture 

(Rodrigues, Júnior, Lourenço, Lima & Farias, 2009), 

nutrition (Carneiro et al., 2014) and biomedicine 

(Pomin, 2012; Mourão, 2015). Of all algae products, 

extracellular matrix sulfated polysaccharides (SPs) 

comprise a diverse group of biomaterials exhibiting 

high degree of sulfation (S=O, sulfate radicals) and 

molecular distribution of more than 100 kDa, which 

are capable of displaying anticoagulant, 

antithrombotic and anti-inflammatory effects 

(Pomin, 2012; Mourão, 2015; Rodrigues et al., 

2012a; Rodrigues et al., 2016). Their functionalities 

as gelling and stabilizing agents make them also 

economically attractive for hydrocolloid industry 

(Cardozo et al., 2007). Depending on the chemical 

class, sulfated galactans (Rhodophyta) (Kloareg & 
Quatrano, 1988; Mourão, 2015), fucans or fucoidan 

(Ochrophyta) (Athukorala, Jung, Vasanthan, & Jeon, 

2006) and sulfated heteropolysaccharides 

(Chlorophyta) (Wang, Wang, Wu & Liu, 2014) are 

the main polymeric identities of the SPs playing 

regulatory roles of the algae (Kloareg & Quatrano, 

1988). On a basis of abundance, seaweeds produce 

larger amounts of SPs than other natural sources 

(Aquino, Landeira-Fernandez, Valente, Andrade, & 

Mourão, 2005; Mourão & Pereira, 1999; Dantas-

Santos et al., 2012; Chang, Lur, Lu, & Cheng, 2013). 

There are very few reports concerning the effects 

of SPs isolated from aquatic organisms on in vitro 

TG assays. For example, SPs derived from the sea 

cucumber Ludwigothurea grisea (Mourão et al., 2001) 

and from the seaweeds Ecklonia kurome 

(Ochrophyta) (Nishiro, Fukuda, Nagumo, Fujihara 

& Kaji, 1999) and Botryocladia occidentalis 

(Rhodophyta) (Glauser et al., 2009) inactivated TG 

after induction by both contact-activated and 

thromboplastin-activated systems and/or by the 

prothrombinase complex. Zhang et al. (2014) 

demonstrated that the fucoidan derived from the 

brown seaweed Fucus vesiculosus showed both pro- 

and anticoagulant effects using calibrated automated 

thrombography. Studies by Rodrigues et al. (2016) 

revealed that a native SPs fraction and its various 

chemically (HCl treated)-modified products from 

the red seaweed Acanthophora muscoides attenuated 

coagulation status activated by the use of cephalin in 
an  in vitro TG continuous system. Recently, SPs 

isolated from Brazilian samples of Gracilaria birdiae 

(Gracilariales, Rhodophyceae) exerted in vitro 

inhibitory potential on a chromogenic TG assay in 

diluted human plasma (Rodrigues et al., 2017a). The 

skin of Nile tilapia (Oreochromis niloticus) also 

contained SPs (dermatan sulfate-type 

glycosaminoglycans) with in vitro TG inhibition 

(Salles et al., 2017). To the best of our knowledge, 

there is only one report concerning the in vitro 

inhibitory effects of SPs isolated from Chlorophyta 

species on TG tests (Rodrigues et al., 2017b). 

Studies on the order Bryopsidales of green 

seaweeds of the Caulerpa genus Lamouroux (1809), 

which is distributed in tropical areas to warm-

temperate zones, revealed compositional complexity 

(galactose, glucose, arabinose, sulfate, xylose and 

uronic acid and traces of fucose residues) and high 

structural heterogeneity of their SPs exhibiting 

bioactivities (Hayakawa et al., 2000; Ji, Shao, Zhang, 

Hong & Xiong, 2008; Maeda, Ida, Ihara & 

Sakamoto, 2012; Rodrigues, Vanderlei, Quinderé, 

Fontes, & Benevides, 2010; Rodrigues et al., 2012a; 

Wang et al., 2014). Focusing on the C. racemosa 

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Chlorophyceae contains heteropolysaccharides with anti-clotting effect 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 

(Forsskal) J. Agardh species, a crude SP was shown 
to be anticoagulant and antiviral in vitro (Ghosh et 

al., 2004; Ghosh et al., 2009). Subsequently, three 

SPs fractions (F I, F II and F III) from this algal 

species have been isolated (Rodrigues et al., 2010), 

of which F II had anticoagulant (Rodrigues et al., 

2012b), antinociceptive and anti-inflammatory 

effects devoid of toxicity in mice (Ribeiro et al., 

2014). Fractions F I and F II modestly altered the 

normal APTT method compared with UHEP 

(Rodrigues et al., 2010; Rodrigues et al., 2012b).  

Considering the above and the invasive biology 

of the Caulerpa species (Verlaque, Durand, 

Huisman, Boudouresque, & Parco, 2003), SPs from 

C. racemosa were examined for their physical-

chemical characteristics using a combination of 

agarose/polyacrylamide gel electrophoresis and 

sequential staining with toluidine blue/Stains-All. 

Analyses of the fractions F I and F II on an in vitro 

TG system in 60-fold diluted human plasma using 

chromogenic method in a continuous measurement 

system were also conducted to explore their 

anticoagulant dynamics. 

Material and methods 

Marine alga and analysis of SPs 

The  C. racemosa

  (Forsskal) J. Agardh coenocytic 

seaweed was manually collected in August 2011 
from the rocky shore located at Pacheco beach 

(Caucaia, Ceará state, Brazil). Algal samples were 

placed in plastic bags and then transported to the 

Carbohydrates and Lectins laboratory (CarboLec), 

Universidade Federal do Ceará, Brazil, where 

macroscopic epiphytes attached to material were 

removed, the material was washed with distillated 

water to eliminate salt and sand, followed by 

dehydration of the algal tissue at room temperature 

prior to crude SP extraction. A voucher specimen  

(# 52418) was deposited in the Herbarium Prisco 

Bezerra of the Department of Biological Sciences, 

Universidade Federal do Ceará, Brazil. The analyses of 

the  C. racemosa

  SPs were conducted at Connective 

Tissue laboratory, Universidade Federal do Rio de 

Janeiro (UFRJ), Brazil. 


Essentially, two grams of dehydrated algal tissue 

cut in small pieces were subjected to papain 

digestion (60°C, 24 hours) as previously described 

(Rodrigues et al., 2010), followed by fractionation of 

a sample of extract (20 mg) dissolved in 10 mL of 50 

mM sodium acetate buffer (pH 5.0) by anion-

exchange chromatography (DEAE-cellulose), where 

the column (1.2 × 12 cm)-bound SPs were eluted 

with NaCl (from 0 to 2 M, with 0.25 M of intervals) 

added to buffer of equilibrium and the collected 

fractions (2.5 mL) checked for SPs using 
metachromatic assay containing dimethymethylene 

blue in an Amersham Bioscience Ultrospec 3100 

spectrosphotometer at 525 nm (Farndale, Buttle, & 

Barrett, 1986). The metachromatic fractions (F I, F 

II and F III) were further freeze-dried and examined 

for their contents of sulfate (Dogdson & Price, 

1976), total sugars (Dubois, Gilles, Hamilton, 

Rebers, & Smith, 1956) and proteins (Bradford, 

1976), while their purity and molecular mass were 

revealed by agarose (Dietrich & Dietrich, 1976) and 

polyacrylamide (Rodrigues et al., 2016) analyses, 

respectively, using toluidine blue/Stains-All as 

staining reagents (Volpi & Maccari, 2002) by 

comparison with the electrophoretic mobility of 

standards dextran sulfate ( 


 8 kDa), chondroitin-4-

sulfate ( 


 40 kDa) and chondroitin-6-sulfate ( 



kDa) (Rodrigues et al., 2016; Salles et al., 2017).


Since F I and F II were available in higher quantities, 

more refined in vitro coagulation experiments 

focused only on these fractions.  

In vitro coagulation experiments 

Blood samples 

Coagulation analyses were conducted using 

venous blood samples collected in citrated 

vacutainer tubes containing 3.2% sodium citrate 

from 10 different donors (University Hospital 

Clementino Fraga Filho, UFRJ), followed by 

centrifugation at 2000 × g for 15 min prior to tests. 

Normal citrated human plasma aliquots of 1 mL 

were frozen and stored at - 70°C as described 

elsewhere (Rodrigues et al., 2017a). 

APTT and PT tests 

Fractions were assessed by both in vitro APTT 

and PT tests according to the manufacturers’ 
specifications, for measure anti-clotting effect in a 
coagulometer Amelung KC4A before the in vitro TG 
assay. For APTT assay, a mixture of 100 μL plasma 
and concentrations of SPs (0-1 mg mL


) was 

incubated with 100 μL APTT reagent (kaolin bovine 
phospholipid reagent). After 2 min incubation at 
37°C, 100 μL 25 mM CaCl


 was added to the 

mixtures, and the clotting time was recorded. 
Regarding the PT assay, a mixture of 100 μL plasma 
and concentration of SPs (1 mg mL


) was incubated 

for 1 min at 37°C. After that, 100 μL PT reagent was 
added to the mixtures, and the clotting time was 
recorded using the same coagulation equipment. 
UHEP with 193 international units per mg (IU mg


of  polysaccharide  was  used  as  the  standard  in  both 
tests. All the tests were done in triplicate and the 
data were expressed as mean ± S.E.M. 

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Rodrigues et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 

TG assay 

This assay was based on Rodrigues et al. (2016) 

in a microplate format, containing: 10 μL cephalin 
(contact-activator system) or thromboplastin (830 μg 


, factor tissue-activator system) + 30 μL 

0.02 M Tris HCl/PEG-buffer (pH 7.4) + 10 μL SPs 
(C. racemosa fractions (F I and F II): 0, 4.1, 41.6 or 
83.3 μg well-plate


 or UHEP: 2 or 4 μg well-plate


+ 60 μL of 20 mM CaCl


:0.33 mM specific 

chromogenic substrate S2238 (10:50 ratio, v:v). The 
in vitro reaction was triggered at 37°C by addition of 
plasma (diluted 60-fold well-plate


) (10 μL), and the 

absorbance (405 nm) was recorded for 60 min (Plate 
reader Thermo-max, America Devices). The in 
  inhibitory  response  of  TG  by  SPs  was 
determined by peak thrombin (PTh) and time to 
peak (TPeak). 

Results and discussion 

In this study, 24 h after papain-assisted 

extraction, a total yield only of 0.7% crude SP from 
the dehydrated raw matter was achieved against 2.2 
and 4% obtained by Rodrigues et al. (2010) and by 
Ribeiro et al. (2014), respectively, who formerly 
analyzed the C. racemosa crude SP extraction yield 
using protease treatment. This lowest yield result 
was consistent with those of other studied Caulerpa 
species that revealed total yields varying from 0.6 to 
20% (Ghosh et al., 2004; Ji et al., 2008; Rodrigues et 
al., 2012b; Wang et al., 2014). Although presenting a 
yield of about 5.7-fold lower than those previously 
published, the DEAE-cellulose column-bound 
coenocytic tissue structure SPs were separated into 
three SPs fractions (F I, F II and F III) at 0.5, 0.75 
and 1 M NaCl, respectively, as assessed by 
metachromasy due to the complex-binding capacity 
of SPs (Farndale et al., 1986). On the basis of these 
results, the total amount of material (76.5%) 
recovered from the column (Table 1) was also as 
expected (Rodrigues et al., 2010). Thus, regarding 
the chemical analyses, the 0.75 M NaCl fraction (F 
II) confirmed its highest levels of sulfate (24%), total 
sugars (48.34%) and absence of SPs-bound proteins 
among the fractions (Table 1), as accordingly 
published on the heterogeneous composition of the 
C. racemosa SPs from the total sugars/sulfate ratio 
(Ribeiro et al., 2014) by using the DEAE-cellulose 
algal crude SP separation (Rodrigues et al., 2010). 

Since the chemical profiles of the C. racemosa SPs 

were similar to those reported, our studies were 

further extended to characterize complex glycans-

containing fractions by a combination of 

electrophoreses (agarose/polyacrylamide) and 
sequential toluidine blue/Stains-All staining. 

Furthermore, the anticoagulant dynamics of the 

fractions F I and F II (Table 1) on an in vitro TG 

system by continuous measuring (60 min, 37°C) of 

the amidolytic activity of thrombin using the specific 

chromogenic substrate in 60-fold diluted normal 

human plasma samples was also examined in this 

current investigation. 

Table 1. Yield and composition of the SPs fractions obtained by 

ion-exchange chromatography (DEAE-cellulose) from the green 
seaweed Caulerpa recemosa

Fraction NaCl (M)


Yield (%)


Chemical analyses (%) 









F I  


















a - NaCl concentration;




- Yield calculated as the percentage from a sample of extract 

applied on DEAE-cellulose column; c - Dosage by Dodgson and Price (1976)  method 
using NaSO


 as standard; d - Dosage by Dubois et al. (1956) method using D-galactose 

as standard; e - Sulfate/hexose ratio; f - Dosage by Bradford (1976) method using 
bovine serum albumin (- not detected); * Non-detected. 

Agarose/polyacrylamide analyses combined with toluidine 
blue/Stains-All reveal Caulerpa racemosa nonSPs in 
complex mixtures 

To evaluate the physical-chemical characteristics 

of the C. racemosa SPs (extract/fractions) and 
standards (glycosaminoglycans chondrotin sulfate, 
UHEP or dextran sulfate) in complex mixtures, 
agarose/polyacrylamide techniques associated with


sequential toluidine blue/Stains-All staining were 
carried out as a function of charge density and 
molecular size, respectively, as already reported for 
glycosaminoglycans extracted from animal tissues 
(Volpi & Maccari, 2002; Salles et al., 2017) (Figure 1). 

Conventional treatment with the cationic dye 

toluidine blue confirmed SPs in extract and fractions 
exhibiting polydispersion (Rodrigues et al., 2012b; 
Ribeiro et al., 2014) co-migrating close to UHEP on 
agarose gel (Figure 1Aa). Additionally, F I and F III 
had lowest metachromasy between extract and F II 
as supported the sulfate analysis (Table 1) due to a 
relatively lower degree of sulfation (ester sulfate 
groups, S=O) of the SPs (Rodrigues et al., 2010).    
F I had highest mobility on agarose gel 
electrophoresis, suggesting a different structural 
conformation and charge/mass ratio of the C. 
 polysaccharides during comparisons with 
other studies (Dietrich & Dietrich, 1976; Fidelis et 
al., 2014). Polyacrylamide gel electrophoresis 
(Figure 1Ba) revealed large molecular sizes SPs (> 
100 kDa) since they remained close to the origin of 
the gel, typical for algal SPs (Pomin, 2012; Fidelis et 
al., 2014; Mourão, 2015; Rodrigues et al., 2017a). 










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Chlorophyceae contains heteropolysaccharides with anti-clotting effect 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 





60 kDa

8 kDa

40 kDa

> 100 kDa















E    F I  F II F III



E     F I  F II  F III




















60 kDa

8 kDa

40 kDa

> 100 kDa


Figure 1. Electrophoreses in agarose gel (A) and polyacrylamide (B) gel of the SPs isolated from the green seaweed Caulerpa racemosa

Extract (E) and fractions (F I, F II and F III) and standard glycosaminoglycan chondroitin-4-sulfate (C-4-S, 40 kDa), chondroitin-6-
sulfate (C-6-S, 60 kDa), dextran sulfate (DexS, 8 kDa) and/or unfractionated heparin (UHEP, 14 kDa) present on gels stained with 0.1% 
toluidine blue (a) or Stains-All (b).


Such observations contrasted with those found 

for two SPs fractions from the green seaweed C. 

cupressoides var. lycopodium, where average molecular 

masses varying from 8 to > 100 kDa were observed 

by polyacrylamide gel electrophoresis (Rodrigues et 

al., 2013; Rodrigues et al., 2017b). Ghosh et al. 

(2004) obtained a hot water crude SP from the red 

seaweed  C. cupressoides and found apparent 

molecular weights of two major peaks (F 1 and F 2) 

to be 120 and 70 kDa, respectively, when a size 

exclusion chromatography (Sephacryl S-100 

column) was used. For the SPs from Caulerpa 

lentillifera by treating with water, Maeda et al. (2012) 

obtained a xylogalactan with a molecular size of 

more than 100 kDa using a Superdex 200 HR 10/30 

column. Therefore, the revealed molecular 

characteristics by Caulerpa SPs can also vary 

according to algal species and the analytical 

procedure used (Wang et al., 2014). 

Staining of the C. racemosa SPs after separation by 

agarose gel electrophoresis procedure with toluidine 

blue/Stains-All is illustrated in figure 1Ab. Extract 

and fractions became more visible on gel compared 

with the toluidine blue alone (Figure 1Aa) 

(Rodrigues et al., 2017a) as previously demonstrated 

for glycosaminoglycans (Volpi & Maccari, 2002; 

Salles et al., 2017), postulating non negatively charged 
polysaccharides in preparations since the fractions F II 

and F III appeared an additional second band with 

higher mobility as CS, which is composed of alternate 

disaccharide sequences of differently sulfated residues 

of D-glucuronic acid and of N-acetyl-D-galactosamine 

(Volpi & Maccari, 2002). 

The degree of staining among the fractions 

compared with extract and standards was also 

individualized with the stepwise of NaCl, where 

0.75 M NaCl-eluted SPs (F II) showed staining 

pattern similar to that of the extract; therefore, 

suggesting compositional variability of the C. 

racemosa polysaccharides of high molecular sizes 

(Figure 1Bb) (Rodrigues et al., 2017a) based on the 

proportions of its sugar residues (Ghosh et al., 2004; 

Ji et al., 2008). The combined method of 

electrophoreses and toluidine blue/Stains-All 

staining to reveal Caulerpa nonSPs was also 

important for distinguishing other glycans present in 

the fractions, when the polysaccharides were co-

eluted from the DEAE-cellulose column (Rodrigues 

et al., 2017a, b), given the difficult in the 

determination of sugar composition after 

chemically-modified polysaccharides (Rodrigues 


et al., 2016).  

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Rodrigues et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 

Collectively, the use of this methodology could 

also be useful for the co-detection of acidic 

components naturally occurring in coenocytic 

structures of green seaweeds, as already reported for 

SPs isolated from other natural sources (Volpi & 

Maccari, 2002; Rodrigues et al., 2017a, b). Further 

studies on the sensitivity to heavy metal–acid 

polysaccharide complexes are also needed since the 

concentrations of glycans are regulated in response 

to environmental stresses (Cardozo et al., 2007; 

Wang et al., 2014).  

Higher levels of anticoagulation by Caulerpa racemosa SPs 
are measured on a TG system than in traditional APTT 

and PT tests 

Anticoagulant effects of the C. racemosa SPs were 

initially checked by routine coagulation screening 

tests (APTT and PT) using a 193 IU mg



standard (Table 2). Algal SPs modified the normal 

intrinsic APTT values (31.80 ± 0.1 s) in the orders 

of 4.76 (250 μg mL


, 38.15 ± 0.35 s), 12.00 (100 μg 



, 38.60 ± 0.30 s), and 2.32 IU mg


(500 μg mL



, 37.35 ± 0.35 s) for the fractions F I, F II and F III, 

respectively. Fraction F II had effect on APTT at 

least 2.52 IU mg


-fold higher than F I and F III and 

doubling the APTT at 500 μg mL


 of the test 


This anticoagulant profile of the fractions was 

positively correlated with the sulfation pattern of the 

complex SPs (Table 1 and Figure 1) to prolong the 

APTT (Rodrigues et al., 2010; Pomin, 2012; 

Mourão, 2015). UHEP prolonged the intrinsic 

pathway by APTT model (Mourão et al., 2015) up 

to 2.5 μg mL


 (42.15 ± 0.60 s) (Rodrigues et al., 

2017a). SPs from C. racemosa had no effect on PT (1 

mg mL


) compared with UHEP (100 μg mL


20.30 ± 0.70 s) vs. control without SPs (11.70 ± 

0.50 s), reflecting the disability of the algal SPs on 

the extrinsic pathway factors inhibition based on 

other published studies (Rodrigues et al., 2010; 

Dantas-Santos et al., 2012; Rodrigues et al., 2017a). 

SPs from the Caulerpa genus act on the 

coagulation by blocking the production of thrombin 

(which degrade fibrinogen into fibrin) depending on 

their sulfate content and charge density (Ghosh  

et al., 2004; Rodrigues et al., 2010; Rodrigues et al., 

2012b; Wang et al., 2014), as also confirmed in the 
present study (Figure 1 and Table 2). The action of 

these compounds would be mediated by serpins 

(antithrombin and heparin II cofactor) (Rodrigues et 

al., 2013; Wang et al., 2014) because galactose-rich 

polysaccharides reveal as inhibitors of thrombin 

(Hayakawa et al., 2000; Ghosh et al., 2004), but the 

relationship between structure and anticoagulation 

still lacks in-depth details since the existence of 

complexity and heterogeneity of the algae glycans 

make comparison difficult with UHEP at molecular 

level and bioactivity (Athukorala et al., 2006; Pomin, 

2012; Fidelis et al., 2014;  Mourão, 2015; Rodrigues 

et al., 2016). TG assays may measure several 

feedback reactions to evaluate diverse classes of SPs 

regarding their anticoagulant dynamics (Nishino  

et al., 1999; Mourão et al., 2001; Glauser et al., 2009; 

Zhang et al., 2014; Rodrigues et al., 2017b; Salles  

et al., 2017).  

The current study demonstrated that 60-fold 

diluted human plasma treated with concentrations 
ranging from 4.1 to 83.3 μg well-plate


 of the C. 

racemosa SPs (F I and F II) reduced dependently TG 
induced by cephalin or thromboplastin (data not 
shown). This was in conformity to the PTh and 
TPeak parameters (Nishino et al., 1999; Rodrigues 
et al., 2016; Rodrigues et al., 2017a; Rodrigues et al., 
2017b; Salles et al., 2017), based on the amidolytic 
activity of thrombin that decayed immediately until 
a plateau was reached at 28 and the negative control 
without activators min (Mourão et al., 2001; Salles 
et al., 2017). The anticoagulant dynamic of the 
fractions was monitored for 60 min at 37°C in 
parallel with the control curve of UHEP used 
(Figure 2) (Rodrigues et al., 2017a; Rodrigues et al., 
2017b; Salles et al., 2017). 

As the effects of the fractions F I and F II on TG 

examined in diluted plasma were of concentration-
dependent manner and sulfation, the in vitro 
inhibitory reactions of the C. racemosa SPs also 
reflected as coherent responses with those of 
classical APTT assays (Table 2), but not with PT 
results (data not shown) (Mourão et al., 2001; 
Rodrigues et al., 2016; Rodrigues et al., 2017a; Salles 
et al., 2017). 

Table 2. Anticoagulant effect of fractions obtained by anion-exchange chromatography (DEAE-cellulose) from the green seaweed 

Caulerpa racemosa compared to UHEP. 

Fractions NaCl 


APTT test (s)






μg mL






μg mL






μg mL






μg mL






μg mL











F I 









0.75  38.60±0.30s 50.75±0.75s 77.35±0.305s  105.2±0.105s 158.1±1.05s 2.43 









NaCl – Sodium chloride; 


Activated partial thromboplastin time (APTT); 


SPs concentration to prolong the APTT in seconds; 


Ratio for double the APTT; 


Anticoagulant effect 

expressed in international units (IU) per mg of SPs (IU mg


); UHEP (193.00 IU mg


: 2.5 μg mL


 for APTT: 42.15 ± 0.60 s); Plasma control: 31.80 ± 0.10 s (n = 3) 

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Chlorophyceae contains heteropolysaccharides with anti-clotting effect 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 


Figure 2. Effect of different concentrations of F I (A) and F II (B) from the red green seaweed Caulerpa racemosa on cephalin-triggered TG 

in 60-fold diluted human plasma using chromogenic method in a continuous detection system after 60 min at 37°C.  

Our TG system had higher more accuracy at a 

level of concentration 24.4 and 243.9-fold lower 

than respective APTT and PT assays, and 50% 

inhibition of PTh by intrinsic pathway was observed 

even at 4.1 μg well-plate


, given the limited results 

proved by classical tests (Castoldi & Rosing, 2011; 

Rodrigues et al., 2017a; Salles et al., 2017). 

Complete suppression of TG in intrinsic 

pathway-activated diluted human plasma was 

achieved in all the F II concentrations, except at 4.1 

μg well-plate


 that still had an inactivation at 31 min 

(49.34% PTh inhibition) (Figure 2B) (Rodrigues et 

al., 2017b) compared with those of F I, which 

abolished TG at 41.6 and 83.3 μg well-plate



2A). These combined observations led to an 

intrinsic inhibition level of 10-fold higher than F II, 

reinforcing F I to be less potent in preventing in vitro 

clot formation due to its comparatively lower sulfate 

composition (Table 1) as previously confirmed the 

APTT assay (Table 2).  

This distinct inhibitory profile of the C. racemosa 

fractions on TG by the coagulation reaction of the 

intrinsic pathway stimulated by cephalin (contact-

activation) could perhaps be revealed as serpins-

dependent thrombin inhibitors (Rodrigues et al., 

2013; Wang et al., 2014) at differential inactivation 

patterns to modulate thrombosis in vitro (Nishino et 

al., 1999; Mourão et al., 2001) because 

antithrombin-dependent anticoagulants do not 

enhance TG in plasma and have ability to attenuate 

thrombin activity (Furugohri, Sugiyama, Morishima 

& Shibano, 2011). On the contrary, the impact of 

sulfation on the extrinsic-pathway induced TG did 

not appear using both fractions F I and F II, showing 

that the sulfation level was not relevant in this 

process because the algal SPs had only a discrete 

inhibitory action from the positive control 

(thromboplastine); therefore, a significant response 

was not found in plasma treated with the polymers 

(data not shown).  

Overall, these data indicated that the SPs from 

C. racemosa do not have important action in the 

inhibition of thrombus by extrinsic pathway. 

Considering this question, it was postulated that the 

SPs from this algal species revealed with their most 

inhibitory effects to be on intrinsic pathway-
activated coagulation than extrinsic one. Similar to 

the SPs isolated from the red seaweed G. birdiae 

(Rodrigues et al., 2017a) and from the skin of Nile 

tilapia (O. niloticus) (Salles et al., 2017) that displayed 

in vitro TG inhibition.  

Although no correlation with the molecular 

masses of the SPs fractions (C. racemosa) was 

attributed on the TG inactivation because the 

polysaccharide chains were not affected after 

protease treatment as visualized by polyacrylamide 

gel electrophoresis (Figure 1) (Athukorala et al., 

2006; Rodrigues et al., 2013; Fidelis et al., 2014), our 

study clearly evidenced SPs as blockers of TG 

dependently of charge, contrasting with the SPs 

from the red seaweed G. birdiae that suggested 

stereospecific mechanisms (Rodrigues et al., 2017a) 

to modulate the active coagulation factors converted 

by thrombin in plasma (Rau et al., 2007).   

 In these connections, decrease in TG by C. 

racemosa SPs reflected a different mode of action 

from the other classes of polysulfated that have 

diverse structures and mechanisms of thrombin 

inhibition (Nishino et al., 1999; Mourão et al., 2001; 

Glauser et al., 2009; Zhang et al., 2014; Rodrigues et 

al., 2016) distinct from that of UHEP, which 

abolished TG at 2 (intrinsic pathway) (Figure 2) 

(Rodrigues et al., 2016; Rodrigues et al., 2017b) or 4 

(extrinsic pathway) (data not shown) μg well-plate


(Rodrigues et al., 2017a; Salles et al., 2017) because 

of its specific pentasaccharide sequence with high 

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Rodrigues et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 

antithrombin affinity displaying thrombin inhibition 
(Mourão, 2015). These results allowed us to 

postulate that the modulation of TG by intrinsic 

pathway in the presence of UHEP were in 

concordance with the automated method (Jun et al., 

2014), but occurred at concentration 5-fold lower 

than that of Glauser et al. (2009), who reported no 

inhibition of TG by UHEP up to 10 μg.  

As our TG system would be sensitive to analyze 

anticoagulants (Rodrigues et al., 2016; Rodrigues et 
al., 2017a; Salles et al., 2017), inactivation of TG by 
C. racemosa SPs could interfere with their anti-
inflammatory actions (Ribeiro et al., 2014), because 
the concentration of SPs was about 2.43-fold lower 
in the anticoagulation. Increased TG induces 
thrombosis associated with neutrophil adhesion 
during the inflammatory response initiated when 
injury to a vessel wall exposes the blood to tissue 
factor in the subendothelium (Rau et al., 2007).  

By contrast, TG inhibition by intrinsic pathway 

in plasma treated with C. racemosa SPs was correlated 
with the antiviral property based on another study 
(Ghosh et al., 2004). Observation suggested that our 
system could also constitute as an useful tool to 
guide complementary analyses for antivirally active 
algal SPs development (Cardozo et al., 2007) in 
parallel to prevention of thrombosis in vitro (Nishiro 
et al., 1999; Mourão et al., 2001; Rodrigues et al. 
2016, 2017b) as support to predict the risk of 
bleeding or coagulopathy (Castoldi & Rosing, 2011; 
Zavyalova & Kopylov, 2016) on the focus of patients 
subjected to antiviral treatment (Ghosh et al., 2004; 
Ghosh et al., 2009). 

In summary, C. racemosa features SPs have 

potential applicability as novel, biomaterial to 
prevent thrombosis in vitro  as  demonstrated  in  the 
TG assay and test parameters could indicate to 
studies of their underlying mechanisms (Glauser et 
al., 2009; Rodrigues et al., 2016) when an increase of 
plasma prothrombin’s referential values increase the 
TG after activation by both intrinsic and extrinsic 
pathways (Rao et al., 2007; Castoldi & Rosing, 


The green seaweed Caulerpa racemosa reveals large 

molecular sizes complex glycans displaying in vitro 
diluted human plasma thrombin generation 
inactivation, with their most inhibitory effects to be 
on intrinsic pathway-activated coagulation than 
extrinsic one dependent on the concentration and 
charge density, when in a continuous system, 
although less potent than unfractionated heparin.



We thank to the funding from CAPES/PNPD, 

FAPERJ, CNPq and MCTIC. Benevides, N. M. B 

and Mourão, P. A. S. are senior investigators of 



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Received on May 29, 2016. 
Accepted on March 21, 2017. 



License information: This is an open-access article distributed under the terms of the 
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