A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and
antiadhesive activities of nine different fucoidans from brown seaweeds
Albana Cumashi2, Natalia A. Ushakova3, Marina
E. Preobrazhenskaya3, Armida D’Incecco2,
Antonio Piccoli4, Licia Totani4, Nicola Tinari2, Galina
E. Morozevich3, Albert E. Berman3, Maria I. Bilan5,
Anatolii I. Usov5, Nadezhda E. Ustyuzhanina6, Alexey
A. Grachev6, Craig J. Sanderson7, Maeve Kelly7, Gabriel
A. Rabinovich8, Stefano Iacobelli2, and Nikolay
E. Nifantiev1,6and on behalf of the Consorzio
Interuniversitario Nazionale per la Bio-Oncologia
2Department of Oncology and Neurosciences, University G. D’Annunzio
Medical School & Foundation, 66013 Chieti, Italy;3V.N. Orekhovich
Research Institute of Biomedical Chemistry, Russian Academy of Medical
Sciences, Pogodinskaya str. 10, 119121 Moscow, Russian Federation;
4Consorzio Mario Negri Sud, Laboratory of Vascular Biology and
Pharmacology, Santa Maria Imbaro, Chieti 66030, Italy;5Laboratory of plant
polysaccharides,6Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky
Prospect, 119991 Moscow, Russia;7Scottish Association for Marine Sciences,
Oban, Argyll, Scotland, PA37 1QA, UK; and8Division of Immunogenetics,
Hospital de Clı ´nicas “Jose ´ de San Martı ´n,” Faculty of Medicine, University of
Buenos Aires, Buenos Aires, Argentina
Received on May 24, 2006; revised on January 14, 2007; accepted on
February 2, 2007
The anti-inflammatory, antiangiogenic, anticoagulant, and
antiadhesive properties of fucoidans obtained from nine
species of brown algae were studied in order to examine
the influence of fucoidan origin and composition on their
biological activities. All fucoidans inhibited leucocyte
recruitment in an inflammation model in rats, and
neither the content of fucose and sulfate nor other struc-
tural features of their polysaccharide backbones signifi-
cantly affected the efficacy of fucoidans in this model. In
vitro evaluation of P-selectin-mediated neutrophil adhesion
to platelets under flow conditions revealed that only poly-
saccharides from Laminaria saccharina, L. digitata, Fucus
evanescens, F. serratus, F. distichus, F. spiralis, and
Ascophyllum nodosum could serve as P-selectin inhibitors.
All fucoidans, except that from Cladosiphon okamuranus
carrying substantial levels of 2-O-a-D-glucuronopyranosyl
branches in the linear (1!3)-linked poly-a-fucopyranoside
chain, exhibited anticoagulant activity as measured by acti-
vated partial thromboplastin time whereas only fucoidans
from L. saccharina, L. digitata, F. serratus, F. distichus,
and F. evanescens displayed strong antithrombin activity
in a platelet aggregation test. The last fucoidans potently
inhibited human umbilical vein endothelial cell (HUVEC)
tubulogenesis in vitro and this property correlated with
decreased levels of plasminogen-activator inhibitor-1 in
HUVEC supernatants, suggesting a possible mechanism
of fucoidan-induced inhibition of tubulogenesis. Finally,
fucoidans from L. saccharina, L. digitata, F. serratus,
F. distichus, and F. vesiculosus strongly blocked MDA-
MB-231 breast carcinoma cell adhesion to platelets, an
effect which might have critical implications in tumor
metastasis. The data presented herein provide a new
rationale for the development of potential drugs for
thrombosis, inflammation, and tumor progression.
Key words: fucoidan/brown algae/blood coagulation/
Fucoidans represent a class of fucose-enriched sulfated poly-
saccharides found in the extracellular matrix of brown algae.
Similar fucan sulfates were isolated also from marine invert-
ebrates (the jelly coat of sea urchin eggs and the body wall
of sea cucumbers) (Berteau and Mulloy 2003). Several bio-
logical properties of fucoidans have been investigated in
different experimental models. Fucoidans, like heparin, may
inhibit thrombin activity by directly acting on the enzyme
(Grauffel et al. 1989) or through the activation of thrombin
inhibitors, including antithrombin III and heparin cofactor II.
Some fucoidans activate antithrombin only, whereas others
interact with both inhibitors (Mauray et al. 1995; Pereira
et al. 1999; Kuznetsova et al. 2003). Based on these obser-
vations and other previous findings (Nardella et al. 1996;
Pomin et al. 2005), it has been proposed that fucoidans may
represent promising candidates as anticoagulant agents. In
addition, fucoidans have also been shown to possess antiproli-
ferative and antiadhesive activities (McCaffrey et al. 1992) and
can also protect cells from infection by viruses (Boisson-Vidal
et al. 1995; Damonte et al. 2004).
A growing body of experimental evidence indicates that
fucoidans may function as anti-inflammatory agents in
several experimental murine models. In this regard, perfusion
of fucoidans into the myocardium can suppress the infiltration
of neutrophils and injury after ischemia-reperfusion of this
organ (Kubes et al. 1995; Omata et al. 1997). Also, fucoidans
can decrease the extravasation of leucocytes to the cerebrosp-
inal fluid during meningitis (Granert et al. 1999). Possible
mechanisms have been postulated by which fucoidans may
affect leukocyte recruitment. The ability of these agents to
prevent selectin-mediated cell–cell interactions is supported
by in vitro experiments showing that fucoidans may indeed
bind to purified and membrane-exposed P- and L-selectins
(Foxall et al. 1992) but not to E-selectin (Game et al. 1998).
1To whom correspondence should be addressed; Tel/Fax: þ7-495-1358784;
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Glycobiology vol. 17 no. 5 pp. 541–552, 2007
Advance Access publication on February 12, 2007
A role of fucoidans as antineoplastic agents has also been
suggested. Different studies have reported the antitumor and
antimetastatic activity of fucoidans in xenograft mouse
models (Yamamoto et al. 1984; Coombe et al. 1987; Riou
et al. 1996). Moreover, fucoidans have been shown to induce
apoptosis in cancer cell lines and promote macrophage-
induced tumor cell death. In addition, these compounds have
been shown to block the interactions between cancer cells
and the basement membrane. Finally, some fucoidans, particu-
larly those extracted from Fucus vesiculosus, have been found
to inhibit angiogenesis by interfering with the binding of
vascular endothelial growth factor (Koyanagi et al. 2003)
and basic fibroblast growth factor (bFGF) (Soeda et al. 2000)
to their respective receptors.
Due to the difficulties in identifying the chemical structure
of algal fucoidans, the relation between their structure and bio-
logical activities remain largely unknown. For this reason,
these compounds have become commercially available
mainly as noncharacterized structurally crude preparations,
for example, of the fucoidan from F. vesiculosus. This prep-
aration may contain the impurities of other types of polysac-
charides and non-carbohydrate concomitants. Therefore some
important correlations between structure and biological
activity came out by investigating the anticoagulant activity
of fucan sulfates isolated from invertebrates, which are avail-
able in very limited amounts, but possess, unlike brown
algal fucoidans, a regular structure. The fucan chains of
animal origin are made of repeating oligosaccharide units dif-
fering in the number and arrangement of sulfate groups
(Pereira et al. 1999). Their anticoagulant activity has been
shown to depend not only on their molecular weight and sulfa-
tion degree, but also on the distribution of sulfate groups in the
repeating units and on the structure of the polymeric backbone
(Pereira et al. 1999).
Due to the wide variety of biological effects elicited by
fucoidans, a current challenge is to investigate whether there
could be any difference or similarity in the structural features
of fucoidans which may account for certain biological effects
of these compounds, including their ability to regulate
selectin-mediated inflammation, blood coagulation, angiogen-
esis, and cell adhesion. Our results show several noteworthy
differences in the activities of fucoidans from different
species of algae, which are likely connected to differences in
the chemical structure of these compounds. This work was
directed to the selection of the most active fucoidan samples
to be studied further as potential novel drugs for thrombosis,
inflammation, and cancer therapy.
Structural motifs of the brown seaweed fucoidans
The chemical composition of the fucoidans under investigation
is summarized in Table I. Contrary to regular animal fucan sul-
fates, the seaweed fucoidans are heterogenic and represent the
mixtures of structurally related polysaccharides with certain
variations of the content of carbohydrate units (L-fucopyranose
and non-fucose ones) and non-carbohydrate substituents
(mainly sulfate and acetyl groups). Due to this circumstance,
the precise assessment of their structures and determination
of the exact location of minor structural elements is not
practically possible in all cases. Therefore, the investigation
of their backbones and branches which form essential struc-
tural motifs are the main targets of structural analysis of
According to our data, the polysaccharide backbones in
studied fucoidans are similar to that of type (I) or (II) chains
(Figure 1). The type (I) chains are organized in repeating
(1!3)-linked a-L-fucopyranose residues, whereas type (II)
chains contain alternating (1!3)- and (1!4)-linked a-L-fuco-
pyranose residues. These two backbones could carry carbo-
hydrate [first of all L-fucopyranose (Fuc) and D-glucuronic
acid (GlcA)] and non-carbohydrate (sulfate and acetyl
groups) substituents R as depicted in Figure 1. Location of
minor monosaccharide constituents, also found in known
seaweed fucoidan samples [galactose (Gal), mannose (Man),
xylose (Xyl), glucose (Glc)], remains unknown.
Table I. Composition of seaweed fucoidans studied (in %, w/w)
Fuc XylMan GlcGal Uronic
36.7 1.21.02.2 4.6 4.8 29.6
30.11.9 1.71.4 6.3 7.027.5
30.9 0.7— 2.2— 23.4 15.1
22.214.171.124 2.25.0 10.323.6
24.8 126.96.36.199 4.88.2 29.2
40.8 0.8—— 0.8
33.0 2.8 1.4 1.23.08.2 25.9
26.64.4 2.6 1.1 4.79.4 24.4
Fig. 1. Two types of homofucose backbone chains in brown seaweed
fucoidans. Chains (I) are constructed only of repeating (1!3)-linked
a-L-fucopyranose residues whereas chains (II) contain alternating (1!3)-
and (1!4)-linked a-L-fucopyranose residues. R depicts the places of potential
attachment of carbohydrate (a-L-fucopyranose, a-D-glucuronic acid) and
non-carbohydrate (sulfate and acetyl groups) substituents.
A Cumashi et al.
The polysaccharide chain of the fucoidan from Laminaria
saccharina (Usov et al. 1998) belongs to type (I) and is built
up mainly of 4-sulfated (1!3)-linked a-L-fucopyranose resi-
dues A (Figure 2), some of which are additionally 2-sulfated
(B) or carry 2-O-a-L-fucopyranosyl substituent (C). The
ratio of units A–C is approximately 5:1:1 as evaluated by
The same (1!3)-linked backbone of type (I) was discov-
ered for Cladosiphon okamuranus fucoidan and found to
contain non-sulfated (A), 4-sulfated (D) units and also a
branch (E) carrying a-D-glucuronyl residues linked through
the (1!2) bond (Figure 2). The ratio Fuc:GlcA:sulfate in
this polysaccharide was reported as 6.1:1:2.9 (Nagaoka et al.
1999) or 4:1:2 (Sakai et al. 2003). The main chain of a
similar type (I) is in the fucoidan derived from L. digitata
(Usov et al. unpublished results), but the location of the substi-
tuents along the backbone chain remains to be investigated.
Other preparations isolated from algae of the genus Fucus
and Ascophyllum nodosum contain the backbone of type (II)
with alternating (1!3) and (1!4) linkages. In particular,
the fucoidan from F. evanescens is built up of di-(F) and tri-
sulfated (G) disaccharide repeating units in the ratio of about
3:1 (Bilan et al. 2002), whereas units G form the main frag-
ment of the fucoidan from F. distichus (Bilan et al. 2004),
which is comparatively much more regular than other brown
The chain of the fucoidan from F. vesiculosus is built up
mainly of units H (Chevolot et al. 2001), which are also
present in the fucoidans from F. spiralis (Usov et al. unpub-
lished results) and from A. nodosum as the major repeating
units, together with minor units G and probably some units
containing other substituents (xylose, fucose or sulfated
fucose) at O-4 (Chevolot et al. 1999; 2001). The backbone
of the fucoidan from F. serratus is built up mainly of the
repeating units F (Bilan et al. 2006), whereas about half of
the 3-linked residues are substituted at O-4 by trifucoside
side chains I.
Gel-permeation chromatography investigation of fucoidan
samples demonstrated their comparable elution profile and
domination of the polysaccharide fractions with molecular
weight of 200–500 kDa.
Anti-inflammatory activity of the brown seaweed fucoidans
We have previously shown (Preobrazhenskaya et al. 1997;
Ushakova et al. 1999) that a fucoidan from L. saccharina
reacts with L- and P-selectins and decreases the escape of
neutrophils [polymorphonuclear leucocytes (PMNs)] to the
abdominal cavity and the induction of acute peritonitis. The
present study showed that all fucoidans at a dose of about
4 mg/kg inhibited at different extents neutrophil extravasation
into peritoneal cavity in an acute peritonitis rat model
(Table II). The most active inhibitors were fucoidans from
L. saccharina and F. evanescens, which inhibited neutrophil
extravasation by more than 90% as compared to controls.
The least active compounds were fucoidans from F. distichus
and F. spiralis, which inhibited the neutrophil transmigration
by approximately 60%. In all cases, the differences between
the groups of control and treated animals were statistically sig-
nificant (P, 0.05). It is noticeable that in this assay the fucoi-
dan from C. okamuranus demonstrated a very potent inhibitory
activity, whereas in other assays it was remarkably less active
when compared with other fucoidans.
The comparison of the data presented in Tables I and II and
the available information regarding the structure of fucoidans
Fig. 2. Reported structural motifs for fucoidans isolated from the brown
seaweeds L. saccharina (A–C) (Usov et al. 1998), C. okamuranus (A, D, E)
(Nagaoka et al. 1999; Sakai et al. 2003), F. evanescens (F, G) (Bilan et al.
2002), F. distichus (G) (Bilan et al. 2004), F. vesiculosus (F, H) (Chevolot
et al. 2001), A. nodosum (F major, H, G minor) (Chevolot et al. 1999; 2001),
and F. serratus (F, G, I) (Bilan et al. 2006).
Table II. Anti-inflammatory and anticoagulant activities of brown seaweed
neutrophils per rat
4 3.8+1.8 91.024.2+1.2
7 4.8+1.5 88.60.5+0.1
4 3.0+0.2 92.9 15.1+0.9
4 5.4+3.6 87.29.4+1.2
aThe anti-inflammatory activity was determined as the effect on neutrophil
extravasation to the peritoneal cavity of rats (for details see the Materials
and methods section). Fucoidans were injected intravenously in a dose of
4.0 mg/kg of rat weight. Data are presented as mean+SEM; n, number of
rats in a group.
bAnticoagulant activity was measured as the activated partial thromboplastin
time (APTT) related to the heparin standard (Fluka) with an activity of
140 U/mg. Data are shown as mean+SEM; n ¼ 4.
cAnimals received 0.9% NaCl instead of fucoidan.
Biological effect of the fucoidans
did not show any direct relation between the anti-inflammatory
activity of fucoidans and the content of monosaccharides and
sulfate, as well as other parameters of their main chains,
such as the presence of branching points. Nevertheless,
taking into account that the interaction of P- and L-selectins
with their receptors could be inhibited by relatively small
natural ligand, namely the tetrasaccharide sialyl Lewis X
(SLeX), we might expect that SLeX could be mimicked by
some structural motifs presented on the fucoidans belonging
both to groups (I) and (II). This conclusion could be confirmed
in the future by studying the activity of the fragments of the
fucoidans of both types, whose systematic synthesis and
conformational analysis are currently in progress (recent com-
munications from the series: Grachev et al. 2006; Ustuzhanina
et al. 2006).
Effect of fucoidans on PMNs adhesion to platelet-coated
glass surface under flow conditions
In order to elucidate the mechanisms underlying the efficacy of
fucoidans in reducing PMNs extravasation into the peritoneum
in the rat inflammatory model, we examined the effects of
fucoidans on P-selectin-dependent adhesion of PMNs to
adherent platelets under flow. In this model, P-selectin block-
age by a specific anti-P-selectin antibody (WAPS) almost com-
pletely prevented PMNs adhesion (Figure 3). These results
demonstrated that PMNs recruitment in this model involves
a P-selectin-dependent mechanism. In the presence of
fucoidans from L. saccharina, L. digitata, F. evanescens,
F. serratus, F. distichus, F. spiralis, and A. nodosum,
(100 mg/mL), PMNs adhesion to platelets monolayers was
reduced by 50–60% (P, 0.05) as compared to controls.
In contrast, the same concentration of other fucoidans did
not significantly affect this interaction.
Anticoagulant activities of fucoidans
The study of anticoagulant activities in activated partial throm-
boplastin time (APTT) model showed considerable differences
among fucoidans obtained from different seaweeds (Table II).
The fucoidans tested can be conventionally divided into three
main groups according to their anticoagulant activity. The
most active anticoagulants were fucoidans from L. saccharina,
L. digitata, F. distichus, and F. serratus, whose activities
exceeded 19 arbitrary heparin U/mg. Fucoidans of the
second group, such as fucoidan from F. evanescens, F. spiralis,
A. nodosum, and F. vesiculosus, exhibited an approximately
halved activity, being 9–15 U/mg. It is remarkable that the
fucoidan from C. okamuranus was the least active among all
the fucoidans and had virtually no anticoagulant effect
(Table II). The absence of its anticoagulant activity could be
explained by the fact that this preparation contains the lowest
amount of sulfate in its polysaccharide backbone. Another
important structural feature distinguishing this polysaccharide
from the others is the presence of vicinal 2,3-branching point
formed by 2-O-a-D-glucuronyl substituents (Nagaoka et al.
1999; Sakai et al. 2003).
Since thrombin is a primary inducer of platelet activation
and coagulation, we also examined the effects of 100 mg/
mL fucoidans on thrombin-induced platelet aggregation.
After exposure to fucoidans from L. saccharina (Figure 4C),
Fig. 3. Effect of fucoidans in polymorphonuclear leucocyte (PMN) adhesion
to P-selectin expressed on platelet-coated surface under flow conditions.
Fucoidans at 100 mg/mL final concentration were added to platelet-coated
surface and incubated for 10 min at room temperature. The same concentration
of fucoidans was also added to PMN suspensions before PMN addition to
platelets. Under flow conditions, the migration of PMNs was followed and
photographs were taken using a camera. The number of attached PMNs per
field were counted. The mean percentage+SEM with respect to control of at
least three independent experiments are represented. *P , 0.05.
Fig. 4. Effect of fucoidans on human platelet aggregation. (A) Washed platelets
were preincubated in the absence or presence of 100 mg/mL of fucoidans and the
ability to prevent thrombin-induced platelet aggregation was evaluated.
Percentages of thrombin-induced activation of platelets, in absence or in presence
of different fucoidans, were reported. Data are collected from at least three
independent experiments. ***P, 0.001; **P, 0.01; and *P, 0.05. (B, C)
Representative aggregation curves are shown. (B) Aggregation in response of
0.5 U/mL thrombin was recorded. (C) The addition of L. saccharina caused no
aggregation of human washed platelets. Subsequently, thrombin was added and
light transmittance was measured for at least 3 min.
A Cumashi et al.
or others such as L. digitata, F. evanescens, F. serratus, and
F. distichus (Figure 4A), the platelets showed no or little
response after additional exposure to 0.5 U/mL thrombin,
but aggregated in response to thrombin receptor activating
peptide (TRAP) or collagen (Figure 4C). Lower concentrations
(10 mg/mL) of the same fucoidans did not prevent thrombin-
induced aggregation of platelets (data not shown). Under the
same conditions, fucoidan from A. nodosum induced a lower
effect on the inhibition of aggregation, reducing by 50%
the effects of thrombin, whereas others from C. okamuranus,
F. vesiculosus, and F. spiralis could not prevent thrombin-
induced platelet aggregation (Figure 4A).
Fucoidan effects on the formation of human umbilical vein
endothelial cell (HUVEC) capillary-like structures in vitro
To determine whether the difference in fucoidan structures
might have an impact on their ability to modulate angiogen-
esis, we analyzed their properties in an in vitro assay of
HUVEC tubulogenesis. HUVECs have been shown to form
capillary-like structures (tubes) when plated on matrigel, and
this phenomenon is known as in vitro tubulogenesis. As seen
in Figure 5, in the presence of serum, HUVECs reorganize
into tube-like structures, and this effect is blocked (99% of
inhibition, P, 0.0001) by the addition of 100 mg/mL of
fucoidans from L. saccharina, L. digitata, F. evanescens,
F. serratus, and F. distichus. To ensure that the suppression
of in vitro HUVEC tubulogenesis was not due to toxic
effects, cells were analyzed by trypan-blue exclusion test
after 18 h of culture in the presence of fucoidans and then
compared to controls. None of fucoidans caused significant
cell death (data not shown).
The fucoidans from F. spiralis and A. nodosum were less
active (Figure 5B), since tubulogenesis was only partially
inhibited when they were used at a concentration of 100 mg/
mL. Under the same conditions, addition of 100 mg/mL of
fucoidans from C. okamuranus and F. vesiculosus was not
able to impair the formation of tubes. These results confirmed
the specificity and the selectivity of each polysaccharide in the
regulation of angiogenesis. Particularly, from the comparison
of the structures of the fucoidans from L. saccharina and
C. okamuranus, one might speculate that the lack of antiangio-
genic activity in the latter could be connected with the lower
content of sulfates and/or the presence of 2-O-a-D-glucuronyl
substituents along the linear polysaccharide backbone (Table I,
Effects of fucoidan on plasminogen-activator inhibitor-1
(PAI-1) release from HUVEC
In order to identify a potential mechanism responsible for the
antiangiogenic activity described above, we measured the
levels of PAI-1 in the conditioned medium (CM) of HUVEC
cultured in the absence or presence of fucoidans. As shown
in Figure 6, the levels of PAI-1 were markedly reduced
when cells were exposed to fucoidans from L. saccharina,
L. digitata, F. serratus, and F. distichus. Other fucoidans did
not affect the release of PAI-1. The addition of fetal bovine
serum (FBS) significantly enhanced the levels of PAI-1
present in CM of HUVEC (174 ng/mL in CM versus
258.7 ng/mL in the presence of 10% of FBS, P, 0.01).
Effect of fucoidans on the adhesion of breast cancer
cells to immobilized platelets
Several studies have established a key role of tumor cell–plate-
let interaction as one of the earliest processes favoring tumor
metastasis (Hejna et al. 1999). Therefore, we aimed to deter-
mine the effect of fucoidans on the adhesion of a highly meta-
static breast cancer cell line MDA-MB-231 to a platelet-coated
surface under static conditions. Adherent platelets are known
to have partially released their a-granule content (Lahav and
Hynes 1981), becoming activated and expressing different gly-
coproteins (GPs) or adhesion molecules, such as P-selectin and
integrins. Breast cancer cells were added on a platelet-covered
surface, under static condition. Fucoidans (added at a final con-
centration of 100 mg/mL) were preincubated for 10 min at
room temperature with tumor cells (1 ? 105cells). Cells
were left to adhere to platelet-coated plates for 1 h. After
washing, adherent cells were fixed and stained with hematox-
ylin/eosin. The number of cells present in different fields was
counted. As shown in Figure 7, fucoidans from L. saccharina,
L. digitata, F. vesiculosus, F. serratus, and F. distichus signifi-
cantly reduced by approximately 80% (P, 0.01) the tumor
cell adhesion to human platelets. On the other hand, fucoidans
from F. evanescens and A. nodosum were less active and
showed 78% and 66% inhibition, respectively (P , 0.05),
whereas the fucoidans from C. okamuranus and F. spiralis
did not significantly inhibit heterotypic cell adhesion of
tumor cells to human platelets.
Fucoidans represent an intriguing group of naturally occurring
polysaccharides that might have promising therapeutic appli-
cations in various clinical settings. Because algal fucoidans
are characterized by a wide variety of biological activities
and by a highly complex and heterogeneous structure, which
obviously vary with algal species, we currently aimed to
determine whether fucoidans from various sources might
differentially affect inflammation, coagulation, and some
Our finding show that the i.v. administration of each fucoi-
dan results in remarkable decrease of leucocytes recruitment in
an experimental model of peritonitis in rat. One of the possible
mechanisms by which fucoidans could successfully prevent
PMNs accumulation is by interfering with P-selectin binding
activity (Ushakova et al. 1999). In fact, by analyzing the
activities of fucoidans in a flow model of P-selectin-mediated
PMNs adhesion to platelets, we found that only fucoidans
from L. saccharina, L. digitata, F. evanescens, F. serratus,
F. distichus, F. spiralis, and A. nodosum could serve as more
efficient P-selectin inhibitors than fucoidan from C. okamura-
nus. The high content of 2-O-a-D-glucuronyl substituent in the
polysaccharide chain of fucoidan from C. okamuranus
suggests that these lateral branches may impair the antiadhe-
sive effect of this polysaccharide.
The observed inhibitory effect of fucoidans on PMNs
adhesion on platelets is consistent with the hypothesis that
fucoidans may inhibit PMNs recruitment in the peritonitis
inflammatory model, at least in part, by interfering with
P-selectin adhesive function. However, the interaction of
fucoidans with other leukocyte adhesion receptors cannot be
Biological effect of the fucoidans
excluded; in fact it has been reported that fucoidans can also
bind L-selectin (Ley et al. 1993). Moreover, other putative
selectin-independent mechanisms that can mediate fucoidans
activity in vivo might also be taken into consideration. For
instance, studies in selectin-deficient mice suggest that
selectins are not required for the observed effects of sulfated
polysaccharides in mobilization of stem cells in vivo
(Sweeney et al. 2000).
Many studies have long reported that fucoidans are active
modulators of the coagulation, and represent potential
Fig. 5. Differential inhibitory effects of fucoidans on human umbilical vein endothelial cell (HUVEC) tubulogenesis. (A) Representative pictures of HUVEC
on matrigel in presence of fetal bovine serum (FBS) along with 100 mg/mL of each of the indicated fucoidans. (B) Quantitative analysis of tube formation
was performed by counting of closed areas (tubes) in four different fields. Data are collected from at least three independent experiments. ***P, 0.001 and
A Cumashi et al.
therapeutic compounds as an alternative to heparin (Moura ˜o
2004). In this regard, we confirmed here that, like heparin,
all fucoidans were able to prolong the clotting time of
human plasma, with the exception of C. okamuranus.
Interestingly, a higher specificity regarding fucoidan’s activity
was observed in a thrombin-induced platelet aggregation test.
We documented here that only five fucoidans derived from
L. saccharina, L. digitata, F. distichus, F. serratus, and F. eva-
nescens could strongly inhibit thrombin activity on human
platelet aggregation. An antithrombin effect for some fucoi-
dans was previously described in a study of rabbit platelet
aggregation (Trento et al. 2001). The most plausible hypoth-
esis to explain the specific inhibitory effect of fucoidans on
thrombin-induced platelet activation could be their ability to
inhibit the catalytic activity of thrombin at the concentrations
used in the present study. Supporting this theory, chromato-
graphic studies have demonstrated that thrombin can indeed
bind to fucoidans (Minix and Doctor 1997). In addition,
other authors have shown that some highly branched sulfated
fucoidans from brown algae
(Pereira et al. 1999). However the possibility that, like
heparin, fucoidans may inhibit thrombin-induced platelet
aggregation by blocking the interaction between thrombin
and its two main receptors on human platelets, protease-
activated receptor-1 and GP-1b (De Candia et al. 1999;
2001) should not be completely excluded. Additionally, we
suggest that the specific antithrombin effects of fucoidans
might be determined by specific features of their chemical
structure. In particular, we hypothesize that the high presence
of glucuronic acid branches is the most probable feature
responsible for the lack of anticoagulant activity, as seen by
C. okamuranus fucoidan-induced effect on APPT assay
(Figure 2; Tables I and II). Nevertheless, the absence of this
chemical group was thought to be important but might not rep-
resent the only factor determining the antithrombin properties
of fucoidans (Figure 4).
Anticoagulants have historically been proposed as a comp-
lementary treatment in cancer, especially for their ability to
negatively affect hemostasis and angiogenesis (Carmeliet
2003). Therefore, we have further explored the antiangiogenic
properties of fucoidans in vitro. Interestingly, our data show
that fucoidans displaying strong antithrombin properties,
such as those from L. saccharina, L. digitata, F. evanescens,
F. serratus, and F. distichus, are also potent inhibitors of tubu-
logenesis. On the contrary, fucoidans from C. okamuranus and
F. vesiculosus (Table I) lack any inhibitory activity on
tubulogenesis. In our opinion, these variable effects on tube
formation might be related to differences in chemical
composition of the different fucoidans. Particularly, the less
active compounds are characterized by a low degree of sulfa-
tion and a high presence of 2-O-a-D-glucuronyl substituents
along the linear polysaccharide backbone (see for example,
C. okamuranus) . On the other hand, we also observed that
the inhibition of tubulogenesis correlated well with a reduction
of PAI-1 levels found in the HUVEC supernatants, suggesting
a potential mechanism responsible for inhibition of angiogen-
esis. Indeed, our results concur with previous studies that
reported several pathways by which PAI-1 could exert proan-
giogenic activity (Bajou et al. 2001; Kim 2003). Reduction
of PAI-1 levels may be explained by the ability of fucoidan
to hijack PAI-1 antigen, which might generate the formation
of a fucoidan-PAI-1 complex, thus reducing PAI-1 availability
as reported previously (Minix and Doctor 1997). However,
other PAI-1-independent mechanisms responsible for the
inhibitory activity observed on tubulogenesis should not be
ruled out, since a strong inhibitor of tubulogenesis in vitro,
F. evanescens did not cause a significant reduction on PAI-1
antigen levels found in HUVEC supernatants (Figures 5 and
6). Moreover, preliminary data from our group documented
that fucoidan from L. saccharina could completely abolish
bFGF-induced tubulogenesis (data not shown). Collectively
these data indicate that several fucoidans may serve as
potent antiangiogenic agents in vitro.
Another effect investigated in the present study is related to
the activity of fucoidans on platelet–tumor cell interactions. A
growing body of experimental evidence has indicated that the
formation of platelet–tumor aggregates in the bloodstream is
important in facilitating the metastasis process (Hejna et al.
1999), and the blockade of platelet adhesion molecules, such
as P-selectin (Kim et al. 1998), or the combined inhibition
of GPIIb/IIIa and alpha V beta 3 integrin could provide
significant tools for the inhibition of tumor metastasis
(Trikha et al. 2002; Gomes et al. 2004). In this regard, we
investigated the ability of fucoidans to affect the adhesion of
MDA-MB-231 (breast cancer cell) to platelets. Our results
demonstrate that specific fucoidans may significantly reduce
the number of breast cancer cells adhered to immobilized
platelets under static conditions, whereas the polysaccharides
derived from C. okamuranus, enriched with glucuronic acid
substituents, and also those from F. spiralis, failed to prevent
this type of interaction. Although we have not identified
specific cell adhesion molecules involved in cell–cell inter-
actions, we hypothesize that, similarly to heparin (Borsig
et al. 2001), fucoidans could initially block P-selectin-
mediated cell adhesion. Furthermore, the contribution of
other adhesion molecules is assured, since the use of only
WAPS (anti-P-selectin antibody) was not sufficient to comple-
tely block cell–cell interactions (data not shown). One of the
possible candidate molecules involved in this process might
be thrombospondin, a heparin-binding GP present in the plate-
let granules (Lawler et al. 1978). It has been shown that once
released, thrombospondin is capable of binding to the surface
of resting and activated platelets (Nelson et al. 1993). On the
Fig. 6. Effect of fucoidans in plasminogen-activator inhibitor-1 (PAI-1)
(ng/mL) released from HUVECs. HUVECs were plated onto matrigel in
the presence or absence of fucoidans. After 18 h, HUVEC supernatants
were collected and PAI-1 levels were measured using a commercially
available enzyme-linked immunosorbent assay (ELISA) kit.
Biological effect of the fucoidans
Fig. 7. Effect of fucoidans on breast cancer cell adhesion to platelets. (A) The MDA-MB-231 cells were preincubated with fucoidans prior to exposure to platelet-
coated plates. Photographs are representative of at least three independent experiments. (B) Quantification of cell adhesion was performed by counting cells adhered
to at least three different fields. The results were expressed as % of treated sample in respect to control **P , 0.01 and *P , 0.05.
A Cumashi et al.
other hand, Incardona et al. (1996) indicated that similarly to
heparin, a natural fucoidan was capable of blocking the
binding of thrombospondin to MDA-MB-231 breast cancer
cells. However, the possibility that fucoidans might also block
other important candidate molecules involved in tumor cell
adhesion to platelets, such as integrins (Haroun-Bouhedja
et al. 2002; Liu et al. 2005), should not be ruled out.
Materials and methods
Extraction and purification of fucoidans
The fucoidans used in the present work are listed in Table I.
The procedure for isolating fucoidans from L. saccharina
has been described earlier (Usov et al. 1998). The procedure
includes the extraction of the dry defatted algal biomass with
a dilute solution of calcium chloride, precipitation of acidic
polysaccharides with Cetavlon, transformation of Cetavlonic
salts into calcium salts, and an alkaline treatment to remove
acetyl groups (if any in the native polysaccharide) and to trans-
form the fucoidan into the sodium salt. The same procedure
was used to obtain preparations L. digitata, F. vesiculosus,
F. spiralis, and A. nodosum from the corresponding algae
(Table I). Three preparations were isolated by similar extrac-
tion procedures, but the alkaline treatment was omitted and
an ion-exchange chromatography of the native polysaccharide
was performed to obtain the most sulfated fraction. This
method produces polysaccharides from F. evanescens (Bilan
et al. 2002), F. serratus (Bilan et al. 2006), and F. distichus
(Bilan et al. 2004) (for detailed isolation procedures, see
cited works). Fucoidan from C. okamuranus was a gift from
Dr M. Iha (South Product Co. Ltd, Suzaki, Japan). The
structure of the polysaccharide from C. okamuranus has been
investigated previously (Nagaoka et al. 1999; Sakai et al.
2003), and this preparation is currently under study as a cell
adhesion inhibitor of Helicobacter pylori (Shibata et al.
2003) but also exhibits other biological properties (Shibata
et al. 2000; Matsumoto et al. 2004). The monosaccharide
and sulfate contents of fucoidans were determined as pre-
viously described (Usov et al. 1998; Bilan et al. 2002; 2004).
Gel-permeation chromatography characterization of fucoidans
The molecular-weight distribution of fucoidan samples were
column with TSK-HW-65(S) gel (2.7 ? 60 cm, separation
range of 10–1000 kDa) by elution with water (2 mL/min)
and detection with differential refractometer (Knauer, Berlin,
Germany). Standard dextran samples (50, 75, 150, 250, and
500 kDa) were used for column calibration.
Rat peritoneal inflammation model
A rat model of acute peritonitis was used as described earlier
(Ushakova et al. 1999) with some modifications. A 9.0% sol-
ution of peptone (7 mL) in 0.9% NaCl was injected intraperi-
toneally into female Wistar rats (about 250 g) under ethereal
anesthesia. Fucoidans were injected into the femoral vein of
rats in sterile 0.9% NaCl (0.3 mL) 15 min after peptone injec-
tion. The same volume of 0.9% NaCl was injected to control
animals. After 3 h, the animals were anesthetized, sacrificed,
and their peritoneal cavities were washed with phosphate buf-
fered saline (PBS) (30 mL) containing heparin (60 U/mL),
0.02% ethylenediaminetetraacetic acid, and 0.03% bovine
serum with vigorous peritoneum massage for 1 min. The cell
number in the lavage fluid was counted and the cell suspension
was concentrated by centrifugation at 400g for 10 min. The
cell pellet was then diluted 1:1 with bovine serum;
the smears were prepared and stained according to the
Pappenheim method. The number of PMNs was determined
in two parallel smears, each containing 600 cells.
Isolation of platelets and PMNs from human blood
Blood was collected from healthy volunteers who had not
received any medication for at least 2 weeks. Nine parts of
blood were anticoagulated with one part of 3.8% trisodium
citrate. Human platelets were prepared by differential centrifu-
gations as described (Cumashi et al. 2001). After removing
the platelet-rich plasma, PMNs were isolated by dextran sedi-
mentation followed by Ficoll–Hypaque gradient and hypotonic
lysis of erythrocytes. PMNs were washed and resuspended in
HEPES–Tyrode’s buffer (pH 7.4) containing 129 mmol/L
NaCl, 9.9 mmol/L NaHCO3, 2.8 mmol/L KCl, 0.8 mmol/L
KH2PO4, 0.8 mmol/L MgCl2.6H2O, 5.6 mmol/L glucose,
10 mmol/L HEPES, and 1 mmol/L CaCl2.
Glass coverslips were coated with 4% 3-aminopropyl-triethox-
ysilane (APES) in acetone. Platelet suspension of 0.5 mL in
1 mol/L Ca2þcontaining 3.5 ? 107PLT/mL was stratified
on APES-coated glass-slide, and platelets allowed to adhere
for 3 h at room temperature. Density and confluence of platelet
layers were examined by light microscopy.
Flow adhesion assay
PMNs adhesion under physiologic flow was investigated in a
parallel plate flow chamber. Platelet-coated slides were
mounted in a flow chamber and placed in a thermoregulated
plexiglass box maintained at 378 by an electric heating
element. Platelet surface was perfused with 5 mL of PMNs
suspension [106mL21in 0.1% bovine serum albumin-
Dulbecco’s modified eagle (BSA-DME) medium], at a wall
shear stress of 2 dynes/cm2for 2 min, followed by perfusion
with medium without cells at wall shear stress of 10 dyne/
cm2for 2 min, in order to remove nonadherent PMNs. The
interaction of PMNs with platelets was observed by phase
contrast video microscopy with a 10 ? objective (Olympus,
Hamburg, Germany) and images were continuously recorded
for playback analysis (Pro-Series video camera, High
Spring, MD). Adherent PMNs were counted at the end of
the perfusion, in four randomized fields, by using an ad hoc
software for image analysis (Image Pro-Plus for Windows,
Media Cybernetics, Silver Spring, MD), and reported as
mean+SEM. P-selectin was immunologically blocked by
incubating platelets with the monoclonal antibody WAPS
12.2 (20 mg/mL) for 10 min at room temperature. Fucoidans
were added to platelet surfaces for 15 min, at a concentration
of 100 mg/mL, and then exposed to PMNs suspension.
Clotting time assay
The anticoagulant action of fucoidans was measured within an
APTT clotting assay according to Anderson et al. (1976).
Normal pooled human plasma (80 mL) was mixed with a
Biological effect of the fucoidans
solution (20 mL) of fucoidan (0–5 mg) in 0.9% NaCl, and
the mixture was incubated for 1 min at 378C. Thereafter, a
solution (100 mL) containing a mixture of phospholipids and
an activator was added, the resulting mixture was incubated
for 2 min at 37 8C. Finally, a solution of 0.025 M CaCl2
(100 mL) preheated at 37 8C was added to the mixture. The
time of clot formation was detected. The activity of fucoidans
was expressed as heparin U/mg, using a parallel curve
obtained with the use of heparin standard (Fluka, Buchs,
Switzerland; 140 IU/mg activity).
Platelet aggregation test
Platelet aggregation test was performed as described (Cumashi
et al. 2001). Briefly, 500 mL of 108/mL washed platelets in
1 mmol/L Ca2þ-containing HEPES-Tyrode’s, were incubated
with continuous stirring at 378C in silanized glass tubes placed
in the aggregometer. Platelet aggregation was expressed as the
increase in light transmission observed after thrombin (0.5 U/
mL) addition. Fucoidans, 100 mg/mL, were added to platelets
before the thrombin. Five minutes after thrombin, a different
stimulus, TRAP (50 mg/mL) or collagen (30 mg/mL) was
added to evaluate whether platelets retained the ability to
HUVECs were isolated by collagenase digestion as described
(Gimbrone, 1976). The endothelial cells were grown on
gelatin-coated dishes in 199 medium containing 10% FBS
(Gibco-Invitrogen, Carlsbad, CA), supplemented with 12 U/
mL heparin and 50 mg/mL bovine crude endothelial cell
growth factor (ECGF) at 37 8C under 5% CO2. HUVECs
from passage 1 to 5, were used for experiments. MDA-MB-
231 breast cancer cells were grown in DME medium sup-
plemented with 10% heat-inactivated FBS.
The ability of fucoidans to modulate angiogenesis in vitro was
evaluated in a capillary tube formation (tubulogenesis assay)
as previously described (Rabinovich et al. 2006). Briefly,
chamber slides were coated with growth factor-depleted
Matrigel (Becton Dickinson, Bedford, MA) for 1 h at 37 8C.
HUVECs resuspended in M199 containing 10% FBS were
seeded on Matrigel (5 ? 104/perwell). Fucoidans were added
at the final concentration of 100 mg/mL. After 18–20 h incu-
bation at 37 8C and 5% CO2, the cultures were photographed.
For each individual well, three digitized photographs were
taken from different locations. Photographs were analyzed by
ImagePro Plussoftware (Media Cybernetics, Silver Spring,
MD), and the closed areas (tube-like structures) were
counted. The extension of tube formation was expressed as
the portion of tubes (%) found on fucoidan-treated samples
versus controls. The final results were pooled from at least
three independent experiments.
PAI-1 levels were measured in CM from HUVECs plated on
Matrigel for the tubulogenesis assay, in the presence or
absence of fucoidans. After 20 h incubation, CM were col-
lected, centrifuged, and stored. Five-microliter aliquots of
CM wereassayed usinga specific enzyme-linked
GmbH, Pfungstadt, Germany).
assay (ELISA; American Diagnostica
In vitro platelet–tumor cell adhesion assay
For tumor cell adhesion assays, platelet-coated surfaces were
generated as described (Karpatkin et al. 1988) with modifi-
cations. Platelet suspension of 0.1 ml containing 3 ? 107plate-
lets in HEPES Tyrode’s buffer was added to flat-bottomed
plastic micro-titter wells. Plates were incubated for 1 h at
room temperature. Afterwards, the same volume of HEPES–
Tyrode’s buffer enriched with 2 mM Ca2þwas added to plate-
lets. The plate was then left at 48C overnight. The day after,
nonadherent platelets were removed by washing with PBS
plus 1% of BSA. Two-hundred microliters of this solution
was added and incubated for 1 h at 37 8C in order to block
“free adherent” sites on the plastic. Tumor cells were detached
and resuspended in PBS enriched with Ca2þand Mg2þ. After
counting, 1 ? 105cells were added to each platelet-coated
well, in the presence or absence of 100 mg/mL fucoidans
and incubated for 1 h at 378C. Plates were then washed
twice and fixed with methanol. Adherent cells were stained
by using a hematoxylin/eosin solution in order to make
visible the cancer cell nuclei.
Statistical significance among different experimental groups
was determined using the Student t test. P values less than
0.05 were considered statistically significant.
Our data demonstrate that fucoidans obtained from brown algal
species different from the traditionally studied F. vesiculosus
and A. nodosum may act as inhibitors of inflammation, angio-
genesis, and heterotypic tumor cell adhesion. The results
described herein might suggest the importance of 2-O-a-D-glu-
curonyl branch in decreasing several biological activities if
weaker inhibitory potential of C. okamuranus fucoidan is not
due to lower sulfate content. On the contrary, the structure
of the polysaccharide backbone (type I versus type II) seems
to be less critical. This could be explained by the presence
of biological relevant structural elements on both polysacchar-
ide backbones or by nonspecific mechanisms, including their
poly-anionic structure. Thus, the decreased inhibitory activity
of 2-O-a-D-glucuronylated polysaccharides could be associ-
ated with the corresponding conformational changes of the
linear backbone influenced by vicinal branching which we
could observe by using the corresponding synthetic oligosac-
charide models (Gerbst et al., unpublished results).
Among thestudied compounds,
L. saccharina seems to be a more powerful inhibitor of angio-
genesis and tumor cell adhesion to platelets. Further investi-
gation of different sulfated fractions of this polysaccharide
(obtained by ion-exchange chromatography) and of synthetic
fragments is in progress to delineate the structural motifs
responsible for distinct biological activities. This information
could open the perspective for the development of novel
low-toxic agents for the treatment of thrombosis, inflam-
mation, and tumor progression.
A Cumashi et al.
We thank Rachel Miller (Loch Duart Ltd, Badcall Salmon
House, Scourie, Lairg, Sutherland, IV27 4TH, UK) and
Angus Morrison (Biolitec Pharma Ltd, Breasclete, Isle of
Lewis, Scotland, HS2 9ED, UK) for kind help in harvesting
and drying of the seaweeds, and Dr Patrizia Di Gregorio and
Gabriele Merciaro of “Transfusion Center” SS Annunziata
Hospital, Chieti, Italy, for helpful collaboration. We are in
debt with Dr Virgilio Evangelista for review of the manuscript.
This work was supported in part by GlycoSense AG (Jena,
Germany) and the Russian Foundation for Basic Research
(grants 04-04-49464a and 06-03-33080).
Conflict of interest statement
APES, 3-aminopropyl-triethoxysilane; APTT, activated partial
thromboplastin time; bFGF, basic fibroblast growth factor;
BSA, bovine serum albumin; CM, conditioned medium;
DME, Dulbecco’s modified eagle; ELISA, Enzyme-linked
immunosorbent assay; FBS, fetal bovine serum; GP, glyco-
protein; HUVEC, human umbilical vein endothelial cell;
PAI-1, plasminogen-activator inhibitor-1; PBS, phosphate buf-
fered saline; PMN, polymorphonuclear leucocytes; SLeX,
sialyl Lewis X; TRAP, thrombin receptor activating peptide
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