Journal of Thrombosis and Haemostasis, 12: 43–53 DOI: 10.1111/jth.

12448

ORIGINAL ARTICLE

Is the antithrombotic effect of sulfated galactans independent

of serpin?
A . - L . G . Q U I N D E R !E, * G . R . C . S A N T O S , † ‡ S . - N . M . C . G . O L I V E I R A , † ‡ B . F . G L A U S E R , † ‡
B . P . F O N T E S , * I . N . L . Q U E I R O Z , * N . M . B . B E N E V I D E S , * V . H . P O M I N † ‡ and P . A . S . M O U R AO~ †‡
*Departamento de Bioqu!ımica e Biologia Molecular, Universidade Federal do Cear!a, Fortaleza; †Laborato ! rio de Tecido Conjuntivo, Hospital
!rio Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Cidade Universit!aria; and ‡Instituto de Bioqu!ımica M!edica,
Universita
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

To cite this article: Quinder!e A-LG, Santos GRC, Oliveira S-NMCG, Glauser BF, Fontes BP, Queiroz INL, Benevides NMB, Pomin VH, Mour~ao
PAS. Is the antithrombotic effect of sulfated galactans independent of serpin? J Thromb Haemost 2014; 12: 43–53.

pendent anticoagulation and FXIIa-dependent procoagu-

Summary. Background: Sulfated galactans are polysaccha-
rides with heterogeneous structures that frequently show
anticoagulant activity. Their anticoagulant mechanisms
are complex and distinct from those observed for heparin.
Sulfated galactans act through a combination of effects
involving serpin-dependent and serpin-independent mech-
anisms. Interestingly, these polymers can also induce
blood coagulation due to activation of factor XII (FXII).
Objectives: The structure of a complex sulfated galactan
from the red alga Acanthophora muscoides was character-
ized by solution nuclear magnetic resonance. This poly-
saccharide and another previously characterized algal
sulfated galactan from Botryocladia occidentalis were each
used in in vitro and in vivo anticoagulant and antithrom-
botic assays to understand the possible structural determi-
nants of their functional effects. Results and Conclusions:
The serpin-dependent anticoagulant effects and FXII-
related procoagulant effects of the sulfated galactans
decreased in parallel with the molecular size. The serpin-
independent anticoagulation also correlated with the
chemical structure of the sulfated galactans. The sulfated
galactan from A. muscoides, which showed mostly serpin-
independent anticoagulant activity and reduced activation
of FXII, drastically reduced arterial thrombus formation.
However, the sulfated galactans produced opposite effects
on venous thrombosis; this difference appears to result
from the tenuous balance between the various effects on
coagulation, including serpin-dependent and serpin-inde-
Correspondence: Paulo A. S. Mour~ao, Laborat! orio de Tecido Con-
juntivo, Instituto de Bioqu!ımica M!edica, Universidade Federal do
Rio de Janeiro, Cidade Universit!aria, Caixa Postal 68041, Rio de
Janeiro, RJ, 21941-590, Brazil.
Tel.: +55 21 2562 2090; fax: +55 21 2562 2090.
E-mail: pmourao@hucff.ufrj.br
Received 5 June 2013
Manuscript handled by: R. Camire
Final decision: P. H. Reitsma, 31 October 2013
lation. This study of novel sulfated polysaccharides with
distinct effects on coagulation and thrombosis helps to
establish the minimal structural-function relationship
required for the development of antithrombotic drugs.
Keywords: anticoagulant drugs; antithrombotic agents;
bleeding; coagulation factor XII; heparin sodium.
Introduction
In the past 20 years, innumerable sulfated polysaccharides
with anticoagulant activity have been described, particu-
larly from marine organisms [1–11]. Sulfated galactans
are noteworthy examples of anticoagulant polysaccharides
that have been attracting interest in glycomics [11]. These
sulfated polysaccharides are structurally heterogeneous in
terms of sulfation patterns, side-chain substitutions, and
the occurrence of methyl ethers or anhydro sugars. Nev-
ertheless, the sulfated galactans of marine red algae are
homogeneous in terms of their backbone constitution,
which is composed of alternating 4-linked a-galactopyr-
anosyl and 3-linked b-galactopyranosyl units.
Sulfated galactans do not contain the antithrombin-bind-
ing pentasaccharide required for anticoagulation in heparin
[5,6]. The anticoagulant activity in these polysaccharides has
been primarily attributed to a catalytic effect rather than the
formation of blood serpin–protease complexes [12–15].
More recently, it has been reported that sulfated galactans
can inhibit tenase and prothrombinase complexes [16]. Simi-
lar to major anticoagulants, sulfated galactans also have
undesirable effects. In contrast to heparin, which induces
bleeding, sulfated galactans display procoagulant and pro-
thrombotic effects, which are mainly due to their ability to
activate factor XII (FXII) [14,15,17]. The detailed mecha-
nism underlying these undesired effects is still unknown.
To further study the complex effects of sulfated galactans
in the coagulation system, we attempted to elucidate the

© 2013 International Society on Thrombosis and Haemostasis

44 A.-L. G. Quinder!e et al
α-Galp units β-Galp units
CH2OH
CH2OH
O
O O
R1O O
OR2
O vious studies [15–17].
OR3
OR1
R1 = H or SO3–
R2 = H (~ 33%) or SO3–(~ 66%)
R3 = H (~ 66%) or SO3–(~ 33%)
Fig. 1. Structure of the sulfated galactan from the red alga Botryocl-
adia occidentalis. This polysaccharide has a repeating structure (-4-a-
Gal-1?3-b-Gal-1?) with a variable sulfation pattern. Approxi-
mately one-third of the total a-units are 2,3-disulfated, and another
third are 2-sulfated. The sulfation esters are highlighted by red-
shaded rectangles. (For interpretation of the references to color in
this figure legend, the reader is referred to the online version of this
paper).
relationship between the structure and the effects on coagu-
lation. Our previous work involved a systematic and com-
parative study using sulfated polysaccharides from more
than 50 species of marine algae for screening as well as for
preliminary selection. Subsequently, we focused on the sul-
fated galactan from Botryocladia occidentalis; this galactan
demonstrated the most potent activity of all previously
tested polysaccharides. Detailed structural analysis of this
sulfated galactan revealed a relatively simple structure,
composed of alternating a(1?3) and b(1?4) galactose
units with heterogeneous sulfation substitutions (Fig. 1).
Our studies indicated that the presence of 2,3-disulfated
units accounted for the high anticoagulant activity [12].
In view of the serpin-independent effects of the sulfated
galactans, we decided to follow a different approach to
determine the mechanisms that contribute to their actions
on coagulation and thrombosis. Instead of selecting sul-
fated galactans with potent activity, we chose a sulfated ga-
lactan with reduced serpin-dependent activity. This new
sulfated galactan was found in the red algal species
Acanthophora muscoides. Initially, the structure of this sul-
fated galactan was investigated using nuclear magnetic res-
onance (NMR) spectroscopy, which revealed the presence
of anhydrogalactose units, methyl ethers, and heteroge-
neous sulfation sites. This sulfated galactan was tested in
animal models of experimental thrombosis, and the results
revealed a major serpin-independent antithrombotic effect.
Based on our findings, this effect is likely a consequence of
a tenuous balance between the procoagulant and anticoag-
ulant effects of the polysaccharide.
Materials and methods
Sulfated galactans were purified by anion exchange
chromatography and analyzed by NMR spectroscopy, as
previously described [12,14]. The molecular size of the sul-
fated galactan from B. occidentalis was reduced using
mild acid hydrolysis [17]. The measurements of anticoagu-
lant activity, antithrombotic effects in animal models and
bleeding tendency were performed as reported in our pre-
Results
The sulfated galactan from A. muscoides (fraction AM-2)
has a very complex and heterogeneous structure
The purification of the sulfated galactan from A. musco-
ides (fraction AM-2) is described in the Supplementary
Material (Table S1). Solution NMR experiments were
used in attempt to characterize fraction AM-2, including
the backbone and the distribution of substituents
(Figs. 2, S1, and S2). One-dimensional (1D) 1H NMR
spectrum of fraction AM-2 showed several unresolved
signals (Fig. 2A), indicative of a polysaccharide with a
high degree of structural heterogeneity. Two-dimensional
(2D) edited 1H/13C-HSQC (Supplementary Material, Fig.
S1A) showed three sets of signals ascribed to a-anomers
(named A1, B1, and C1) and four to b-anomers (termed
D1, E1, F1, and G1) (Table S2). The carbohydrate back-
bone of red algal sulfated galactans is usually composed
of alternating 4-linked a-galactose and 3-linked b-galac-
tose [10]. The structural heterogeneity of these polysac-
charides mostly arises from variable sulfate ester and/or
methyl ether substitutions, occasional occurrence of 3,6-
anhydro-a-galactosyl units, and pyruvate [10]. The inter-
pretations of the 1D and 2D NMR spectra were based
on the rationale described in several of our previous pub-
lications [10,12,14,18,19].
When structural heterogeneity arises exclusively from
multiple sulfation sites, the desulfated derivative might
show a simple NMR spectrum, as previously reported for
several sulfated galactans [12,18]. Conversely, if the heter-
ogeneity results from additional 3,6-anhydro-a-galactose
units in the polysaccharide chain, the alkaline-treated
derivative should show a simpler NMR spectrum than
that of the native polymer. This reaction converts 6-sul-
fated a-galactopyranosyl units into their 3,6- anhydro-a-
galacto-derivatives, leading to a more homogeneous sac-
charide chain in terms of a-units. This procedure has pre-
viously been reported for the sulfated galactan from
Gracilaria cornea [10].
The desulfated derivative of the sulfated galactan from
A. muscoides (represented by fraction AM-2) still has a
complex NMR spectrum (Figs. 2B and S1B). Simpler
spectra, however, were obtained from the alkaline-treated
polysaccharide (Figs. 2C and S1C). Clearly, these obser-
vations indicate that the heterogeneity of fraction AM-2
arises from both a random distribution of substituents
and a complex saccharide backbone. Despite this com-
plexity, the 1D and 2D NMR spectra of the native and
© 2013 International Society on Thrombosis and Haemostasis
 Antithrombotic effect of sulfated galactans 45

E1/F1 G1
A B1

c1
1/
C
D1
A1
E4
CH3

2-O-CH3
5.3 4.9 4.5

5.3 4.9 4.5 4.1 3.7 3.3 2.9 2.5 2.1 1.7 1.3 0.9

B G1
A1
b1
F1

c1
1/
CH3

C
2-O-CH3
5.3 4.9 4.5

5.3 4.9 4.5 4.1 3.7 3.3 2.9 2.5 2.1 1.7 1.3 0.9

D1

2-O-CH3 C1/c1

CH3

5.3 4.9 4.5

5.3 4.9 4.5 4.1 3.7 3.3 2.9 2.5 2.1 1.7 1.3 0.9

1H – chemical shift (ppm)

Fig. 2. One-dimensional (1D) 1H NMR spectra of the native sulfated galactan from the red alga Acanthophora muscoides (A) and its desulfated (B) and
alkali-treated, anhydro-enriched (C) derivatives. All 1H-signals are displayed from 5.7 to 0.7 ppm in expansions shown on the left. The anomeric region,
5.3-4.4 ppm, is expanded for each spectrum in an inserted panel on the right. The a-anomeric protons, designated as A1, B1, C1 and c1, resonate in the
downfield regions and belong to the 4-linked a-units, whereas the b-anomerics, designated as D1, E1, F1, and G1, resonate in the upfield region and
belong to the 3-linked b-units. The labels 2-O-CH3 and CH3 represent the 1H-signals from the protons of the methyl-esterified C2 position the methyl
protons, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this paper).

© 2013 International Society on Thrombosis and Haemostasis

46 A.-L. G. Quinder!e et al

α-Galp units β-Galp units of residue B (unit B in Fig. 3) was the only one among
the 4-linked a-anomeric protons that showed displace-
CH2OH CH2OSO3–
ment from the original position upon the desulfation
O O
HO reaction (dH/C = 5.24/101.73 of peak B1 at Fig. S1A ver-
O O
sus 5.28/98.41 ppm of peak b1 in Fig. S1B). The connec-
OH
O tivities in the 1H/1H-TOCSY spectrum of the native
O
polysaccharide (red line in Fig. S2A) and the 1H/13C-
Unit A (~ 32%) OCH3 Unit D (~ 22%) OH HSQC spectrum suggest a 6-sulfated unit.
The NMR spectra were assigned for the four spin sys-
tems of the 3-linked b-galactoses (denoted with the letters
D–G). The 1H/13C anomeric pairs of both the D and E
CH2OSO3– CH2OSO3– units (Fig. 3) were shifted upfield on desulfation (Fig. S1A
O –O SO O
O 3 O
versus S1B, see also Table S2). Residue E is 4,6-disulfated,
whereas residue D is exclusively 6-sulfated (see Fig. 3).
OH
O The other two 3-linked b-galactose residues, denoted as
O
F and G (Fig. 3), are not sulfated because their reso-
Unit B (~ 43%) OH Unit E (~ 30%) OH nances did not change after desulfation. Unit F is
6-methyl etherified, as indicated by the assignments for
the TOCSY spectrum of the native polysaccharide (trace
CH2OCH3 in beige lines in Fig. S2A). Unit G is an unsubstituted
O O b-galactose, as indicated by its chemical shift values
O HO
O (Table S2), and resistance to desulfation.
O The signal at ~ 1.5 ppm (Fig. 2, A–C) comes from car-
O O
bon-bound methyl groups, likely those from pyruvate ace-
OR tyls. Due to the highly complex nature of this
Unit F (~ 22%) OH
Unit C, R = H polysaccharide’s structure, pyruvate groups were hard to
(~ 25%) assign, even performing the approach to observe this spe-
Unit c, R = CH3 (minor)
CH2OH cific group as reported [19].
O In conclusion, the sulfated galactan from the red algae
HO O A. muscoides has a complex structure due to variable sul-
O
fate ester and methyl ether substitutions along with the
occurrence of 3,6-anhydro-a-galactose. The integral of the
anomeric peaks from the 1H/13C-HSQC spectrum allowed
Unit G (~ 26%) OH
an estimation of the relative proportion of the chemical
Fig. 3. Major units found in the sulfated galactans from the red alga modifications that occur in the a- and b-residues, as
Acanthophora muscoides. The backbone of this polysaccharide is shown in Fig. 3. More significantly, a comparison
composed of alternating 4-linked a-galactose and 3-linked b-galac- between the structural components found in the sulfated
tose units. The complexity of this polysaccharide arises from substi- galactans from B. occidentalis and A. muscoides revealed
tutions with methyl ethers (blue-shaded ellipses) and sulfate esters
a marked contrast (Figs. 1 versus 3, respectively). The
(red-shaded rectangles). Furthermore, some a-units also occur as an-
hydro-galactose residues. The percentages of the various units are sulfated galactan from B. occidentalis has a relatively sim-
indicated in the panel as percent of the total a- or b-residues. The ple structure. Variations among the units are exclusively
letters used for structural assignments are also indicated per unit. due to the occurrence of O-sulfation (Fig. 1). In contrast,
(For interpretation of the references to color in this figure legend, the sulfated galactan from A. muscoides has a high degree
the reader is referred to the online version of this paper).
of chemical modifications (Fig. 3).

chemically modified sulfated galactan derivatives enabled

us to determine the assignments of most signals.
Residue A (unit A in Fig. 3), a 4-linked a-galactopyr-
anosyl unit, is methyl etherified at the C2 position
(see spin-system notation for the blue traces in the
1
H/1H-TOCSY spectrum in Fig. S2; the 1H/13C-HSQC
spectrum in Fig. S1A; and Table S2). The unit labeled C
(unit C in Fig. 3) is a 3,6-anhydro-a-galactose unit [14]
(see Table S2). The characterization of this type of unit
was confirmed by the 1H/13C-HSQC spectrum of the
alkaline-treated sample (Fig. S1C). The anomeric signal
Molecular size of the sulfated galactans
The molecular size of the sulfated polysaccharides is rele-
vant to their biological activities [17]. Therefore, we com-
pared the average molecular size of the sulfated galactans
from A. muscoides (fraction AM-2) and B. occidentalis
using gel permeation chromatography (Fig. 4A) and poly-
acrylamide gel electrophoresis (PAGE) (Fig. 4B). Both
methods revealed marked differences between these two
polysaccharides. The sulfated galactan from B. occidental-
is has a very high molecular weight (average mass

© 2013 International Society on Thrombosis and Haemostasis

 Antithrombotic effect of sulfated galactans 47

A hydrolysis. This method yielded a derivative of ~ 8 kDa

6
8
60
40

0.
0

4.
V
(red versus blue lines in Fig. 4A). No desulfation
1.0 occurred during the mild acid hydrolysis of the sulfated
galactan from B. occidentalis, as indicated by NMR spec-
0.8 troscopy (not shown; see also Ref. 20). Unfortunately, it
Refractive index

was difficult to control the mild acid hydrolysis and

0.6
obtain a derivative with the same dispersion as the native
0.4
polysaccharide from A. muscoides.

0.2 Sulfated galactans have serpin-independent anticoagulant

activity
0.0
The intact sulfated galactan from B. occidentalis has high
20 30 40 50 60 70 anticoagulant activity, as indicated by three clotting tests
Retention time - min (red triangles versus open black squares in A–C of Fig. 5)
and by assays using purified serpins and thrombin (D–F
of Fig. 5). The sulfated galactan has significantly lower
is
tal

activity than heparin on the assays using purified FXa

en
cid

(F), whose inhibition by antithrombin relies mostly on

W ide
oc

LM sco

B the conformation-dependent activation of serpin. The

rin
B.

H
mu
pa

absence of action by the sulfated galactan is expected

He

– +
A.

– Origin because it lacks the pentasaccharide that has a high affin-

60
40
20
8 can, was also lower in the assays shown in Fig. 5 (green
+
Fig. 4. Gel permeation chromatography (A) and polyacrylamide gel
electrophoresis (B) of the sulfated galactans from Acanthophora mus-
coides (green in A) and Botryocladia occidentalis, before (red in A, – in
B) and after (blue in A, + in B) mild acid hydrolysis. (A) Sulfated ga-
lactans (200 lg of each) were applied to a TSK 3000 column-linked
TSK 4000 column coupled to an HPLC system. The columns were
eluted with 0.1 mol L!1 ammonium acetate at ~ 20 °C with a flow rate
of 0.3 mL min!1. The eluent was monitored by the differential refrac-
tive index. The numbers in the panel indicate elutions of shark chon-
droitin 6-sulfate (~ 60 kDa), fucosylated chondroitin sulfate from sea
cucumber (~ 40 kDa), a hexadecasaccharide (4.8 kDa), and a disac-
charide (0.6 kDa), both derived from heparin. The Vo of the column
was determined from the elution of ~ 500 kDa dextran sulfate. (B)
Sulfated galactans (10 lg of each) were applied to a 6% polyacryl-
amide gel slab. After electrophoresis, the gel was stained with 0.1%
toluidine blue. The electrophoretic mobility of molecular mass mark-
ers is indicated at the left side of B. LMWH, low molecular weight
heparin. (For interpretation of the references to color in this figure
legend, the reader is referred to the online version of this paper).
> 500 kDa; red line in Fig. 4A), while the sulfated galac-
tan from A. muscoides has a naturally occurring low
molecular size (~ 20 kDa; green line). Of course, any
attempt to compare the biological activity of these two
sulfated polysaccharides requires the preparation of simi-
larly sized derivatives. The molecular size of the sulfated
galactan from B. occidentalis was reduced using mild acid
ity for antithrombin.
As the molecular size of the sulfated galactan from B. oc-
cidentalis decreases, the anticoagulant activity also mark-
edly decreases (red versus blue symbols in Fig. 5). The
anticoagulant activity of native sulfated galactan from
A. muscoides, which is a natural low molecular weight gly-
symbols). The two sulfated galactans with reduced molecu-
lar size are almost devoid of activity in the assays using
purified serpins and coagulation proteases. Modest inhibi-
tion of the proteases was only observed at a very high con-
centration (> 100 lg mL!1). For heparin, total inhibition
occurs at a concentration close to 0.05 lg mL!1.
The residual anticoagulant activity of the low molecular
weight sulfated galactans in plasma (Fig. 5, A–C) and the
almost complete absence of effects on the purified serpins
and coagulation proteases (D–E) may be explained by the
preponderance of the serpin-independent effect. In fact,
when the activate partial thromboplastin test (APTT) assay
was performed using serpin-free plasma, the anticoagulant
activity of the sulfated galactans remained unchanged but
was abolished for heparin (compare green and blue versus
black symbols in Fig. 6). The native sulfated galactan from
B. occidentalis, with a high molecular weight, has a combi-
nation of serpin-independent and serpin-dependent antico-
agulant effects. The serpin-dependent effect was evaluated
by assays with purified serpins and coagulation proteases
(red symbols in Fig. 5, D–F), while the serpin-independent
effect was estimated by APTT tests using serpin-free
plasma (open red symbols in Fig. 6A). Interestingly, when
antithrombin was added to the serpin-depleted plasma, the
anticoagulant activity of heparin was markedly restored,
whereas the activities of the sulfated galactans were only
modestly affected (Fig. 6C).

© 2013 International Society on Thrombosis and Haemostasis

48 A.-L. G. Quinder!e et al

A D 125
5
100
4
APTT - T1/T

75 ∗
3
50
2
25
1

0 ∗

0 1 2 3 4 5 0 100 200

B E
30 125

100

Residual activity
20
75
TT - T1/T

∗
50
10

25

NS
0 0

0 0.1 0.2 0 1.25 2.5 x103

C F 125

100
10
∗
75
PT - T1/T

50
5

25

0
0
0 0.6 1.2 0 40 80 120 0.001 0.1 10 1000
µg mL–1 µg mL–1

Fig. 5. Concentration dependence of heparin (open black squares), the native (red triangles) and low molecular weight derivative (blue circles)
of the sulfated galactan from Botryocladia occidentalis and the native sulfated galactan from Acanthophora muscoides (open green circles) in the
APTT (A), PT (B), TT (C) assays and the inactivation of thrombin (D and E) or factor Xa (F) by antithrombin (D and F) or heparin cofactor
II (E). (A–C) Clotting assays were performed using citrated human plasma and the appropriate reagents, as specified by manufacturers. The
results are expressed as ratios of clotting time in the presence (T1) or absence (T) of sulfated polysaccharide. The results in the panel are typical
results out of three experiments. (D–F) Antithrombin (10 nmol L!1) or heparin cofactor II (15 nmol L!1) was incubated with thrombin
(2 nmol L!1) or factor Xa (2 nmol L!1) in the presence of various concentrations of sulfated polysaccharide. After 60 s, the remaining throm-
bin or factor Xa was measured with a chromogenic substrate (A405nm min!1). No inhibition was observed when thrombin or factor Xa was
incubated with only the sulfated polysaccharide over the range of concentrations tested. The curves obtained for the clotting assays (A–C) were
fitted using exponential growth. The curves in D–E were fitted using sigmoidal Hill plots. In D–F, F-tests were used for comparison between
datasets. * Indicates P < 0.05; NS, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to
the online version of this paper).

© 2013 International Society on Thrombosis and Haemostasis

 Antithrombotic effect of sulfated galactans 49

A 5 The structure of the polysaccharide appears to be relevant

because the sulfated galactans from B. occidentalis are
more active than those from A. muscoides (Fig. 6A versus
4
6B) and also have a lower molecular weight (~ 8 versus
~ 20 kDa) (Fig. 4).
3

Antithrombotic effects of sulfated galactans devoid of the

2 serpin-dependent effect
The observation that sulfated galactans act on coagula-
1
tion through serpin-independent and serpin-dependent
0 1 2 3 4 5
mechanisms raises an interesting question: Which of these
two effects is preponderant in the antithrombotic action
B 3 of the polysaccharide? Of course, it is difficult to clarify
this question using the native sulfated galactan from
B. occidentalis because this polysaccharide combines the
two effects. The low molecular weight sulfated galactans
described in the present study enable investigation of this
T1/T

2
1 black symbols in Fig. 7A), whereas both the native and
0 50 100 150 200 250 300
µg mL–1
C 3
2
1
AT: – + – + – + – +
Fig. 6. Anticoagulant activity of heparin (black symbols), the native
(red) and low molecular weight sulfated galactans from Botryocladia oc-
cidentalis (blue) and native sulfated galactan from Acanthophora musco-
ides (green), as measured using normal human plasma (closed symbols)
and antithrombin + heparin cofactor II–free plasma (open symbols).
The assays were performed as described in the legend to Fig. 5A. The
results shown are the means " SD, n = 3. The curves for heparin and
for the low molecular weight sulfated galactan from B. occidentalis fit a
sigmoidal plot. The curves for the native sulfated galactans from B. oc-
cidentalis and A. muscoides were fitted to second-order polynomials.
For C, assays were performed using concentrations of polysaccharides
that double the APTT values found for normal plasma; serpin-depleted
plasma was used before (!) and after (+) the addition of 100 nmol L!1
antithrombin (AT). The panel shows representative results from three
different assays. (For interpretation of the references to color in this fig-
ure legend, the reader is referred to the online version of this paper).
In conclusion, low molecular weight sulfated galactans,
which lack the high-affinity antithrombin-binding site,
mostly show serpin-independent anticoagulant activity.
topic because their actions on coagulation occur almost
exclusively through the serpin-independent effect.
The sulfated galactan from A. muscoides is as active in
an arterial model of thrombosis as heparin (green versus
low molecular weight derivative of the sulfated galactan
from B. occidentalis are inactive (red and blue symbols).
Doses of native sulfated galactan from B. occidentalis >
1.0 mg kg body weight!1 were not tested because they
produce a sharp drop in blood pressure, likely due to
activation of FXII. The reasons for the modest anti-
thrombotic effect that is observed with the low molecular
weight derivative from B. occidentalis galactan at
1.0 mg kg body weight!1 are still unknown.
The intact sulfated galactan from B. occidentalis pre-
vents thrombosis at low doses in a venous model, but the
effect is abolished at high doses (red symbols in Fig. 7B).
In fact, the polysaccharide is prothrombotic at high doses
because the weight of the formed thrombus is higher than
that from control, untreated animals. The two low molec-
ular weight sulfated galactans have similar dual effects on
venous thrombosis, but the effects differ in magnitude
and range. Almost total inhibition was achieved with the
sulfated galactan from A. muscoides, and the inhibitory
curve was slightly shifted to the right (green symbols in
Fig. 7B). The low molecular weight glycan from B. occi-
dentalis inhibits only ~ 70% of the thrombus formation
(blue symbols in Fig. 7B).
The dual effects of sulfated galactans on venous throm-
bosis are associated with a balance between the anticoag-
ulant and procoagulant activities. At low doses, the
anticoagulant activity predominates, and at high doses,
this action is overcome by FXII activation [17]. For the
experiment shown in Fig. 8, we compared the effects of
the various sulfated galactans on the activation of FXII.
Clearly, the native sulfated galactan from B. occidentalis
activates FXII, and smaller galactans show less of this
undesirable effect. Heparin is inactive in this assay.

© 2013 International Society on Thrombosis and Haemostasis

50 A.-L. G. Quinder!e et al

A 70 Fig. 7. Effects of the sulfated polysaccharides on arterial (A) and

Occlusion time (min) venous (B) thrombosis and on the bleeding tendency (C). Assays
60
were conducted in rats using different doses of heparin (black sym-
50 bols), the native (red) and low molecular weight (blue) derivatives of
the sulfated galactan from Botryocladia occidentalis and the native
40 sulfated galactan from Acanthophora muscoides (green). (A) Arterial
30 thrombosis model induced in carotid artery by FeCl3. Mean time for
complete occlusion is the average time for each dose. (B) Stasis
20 thrombosis model in vena cava of rats. The mean thrombus weight
10 is the average weight for each dose, expressed as percentage of the
weight in the absence of the polysaccharide. The broken black line
0.0 0.4 0.8 1.2 1.6 2.0 2.4 in B shows the thrombus weight of the control (saline-treated ani-
mals). (C) The sulfated polysaccharide was infused into rats, and
B
after 5 min, the rat’s tail was cut 3 mm from the tip and immersed
120 in 40 mL of distilled water at room temperature. The blood loss was
Thrombus weight (%)

determined 60 min later by measuring the hemoglobin in the water.

80 The results were expressed in lL (means " SE, n = 5). In A, the
curves for heparin and for the native sulfated galactan from B. occi-
dentalis.were fitted with a polynominal function. The curves for the
40 low molecular weight galactans from B. occidentalis and A. musco-
ides were fitted with an amplitude version of Gaussian peak and lin-
0 ear fit, respectively. In B, the curve for heparin was fitted using a
exponential function. The data obtained for the sulfated galactans
did not fit any function and they were connected using broken line.
0.0 0.25 0.50 0.75 1.00 The maximum value in B, indicated by the broken black line, is the
mg kg–1 thrombus weight of the control, saline-treated animals. (For interpre-
tation of the references to color in this figure legend, the reader is
C referred to the online version of this paper).
100
Blood loss - µL

80

60

40

5 10 5 10 5 10 5 10 (mg kg–1)
l
tro

in

es
–
on

ar

is

is

id
C

ep

al

al

co
nt

nt
H

us
de

de

m
ci

ci
oc

oc

A.
B.

B.

Finally, in the experiments shown in Fig. 7C, we mea-

sured the effect of the sulfated polysaccharides on the
bleeding time. In this model, heparin increases bleeding,
but the various sulfated galactans do not, regardless of their
molecular weights or chemical structures. The experimental
model that we used to assess the bleeding tendency involves
a well-known and standard procedure [21]. The observed
absence of bleeding tendency for the sulfated galactans may
be due to absence of inhibitory effects on platelet aggrega-
tion [17] or to the activation of FXII (Fig. 8).
Thrombosis is not a single entity and differences are
observed between episodes in veins and arteries. Arterial
thrombosis occurs in regions of high shear stress while
venous thrombosis is mostly associated with blood coagu-
lation in regions of stasis or low shear stress. Thus, it is
conceivable that the effect of sulfated galactans may vary
from arterial to venous experimental thrombosis, as
reported in A and B of Fig. 7.
Fig. 8. Factor XII activation in human plasma incubated with hepa-
rin, the sulfated galactans from Acanthophora muscoides and Botry-
ocladia occidentalis, before (!) and after mild acid hydrolysis (+).
After 60 s of incubation at 37 °C, 0.3 mmol L!1 chromogenic sub-
strate for plasma kallikrein was added. The increase in absorbance
at 405 nm was expressed as milli–optical density units min!1
(means " SE, n = 3). The insert shows typical curves for the native
sulfated galactan from B. occidentalis (red triangles) and heparin
(open black squares). The main panel compares the results for two
different doses of the various polysaccharides. (For interpretation of
the references to color in this figure legend, the reader is referred to
the online version of this paper).
In conclusion, our results suggest that the effects of the
sulfated galactans on experimental thrombosis may result
from a tenuous balance among the different effects on the
coagulation system, including their actions as an antico-
agulant by distinct mechanisms and as a procoagulant

© 2013 International Society on Thrombosis and Haemostasis

 Antithrombotic effect of sulfated galactans 51

through activation of FXII. Each sulfated galactan has a and antithrombotic sulfated polysaccharide to this ser-
particular effect. The antithrombotic effect of the low pin-unrelated phenomenon.
molecular weight sulfated galactans possibly relies on the 3 Procoagulant effect due to activation of FXII [17]. This
balance between their serpin-independent anticoagulant effect may be quantified using assays with a chromo-
action and their procoagulant effect. genic substrate for plasma kallikrein (Fig. 8).
The three sulfated galactans used in the current study
Discussion differ in their effects and mechanisms of action. A decrease
in molecular size results in a dramatic reduction in the
Sulfated galactans are abundant polysaccharides and are
anticoagulant effect, as measured by the serpin-dependent
found in marine red algae, sea urchins (Echinoidea) [22],
inhibition of the coagulation proteases (Fig. 5), and also in
ascidians (Tunicates) [10,23], and higher plants (Angio-
their activation of FXII (procoagulant effect, Fig. 8). The
spermae) [24] that grow in high-salt environments [25].
two sulfated galactans with low molecular weights (the
Sulfated galactans account for > 40% of the dry weight
intact glycan or that obtained by mild acid hydrolysis) dif-
of marine algae. Certain sulfated galactans are potent an-
fer in their serpin-independent anticoagulant effects
ticoagulants and antithrombotics. However, the mecha-
(Fig. 6), possibly due to their distinct structures (Figs. 1
nisms underlying their actions on the coagulation system
and 3). However, this unusual effect is mainly governed by
are complex. It is not possible to explain their actions
the absence of the high-affinity antithrombin-binding
solely using analogies to heparin.
motif. A balance between these various effects will result
In the present study, we compared the effects of two sul-
in antithrombotic or prothrombotic effects in experimental
fated galactans on coagulation and thrombosis. The struc-
animals. Compared with the two sulfated galactans from
ture and the molecular weights of the polysaccharides were
B. occidentalis, the sulfated galactan from A. muscoides is
well characterized. The sulfated galactans from B. occiden-
less able to activate FXII (Fig. 8); this may favor an anti-
talis (a native galactan with a high molecular weight and a
thrombotic effect on arterial thrombosis (Fig. 7A). The
derivative with a low molecular weight) possess a simple
potent effect of the native sulfated galactan from B. occi-
structure, and their heterogeneity arises exclusively from
dentalis on FXII activation results in a prothrombotic
their sulfation patterns (Fig. 1). Another sulfated galactan
action of this polysaccharide at higher doses (Fig. 7B).
was obtained from the red alga A. muscoides. This polysac-
Interpreting the results for the venous thrombosis is
charide has a naturally occurring low molecular weight and
more challenging. The antithrombotic effect preponder-
a very heterogeneous structure due to substitution with sul-
ates at lower doses but disappears or even becomes pro-
fate esters, methyl ethers, pyruvate and partial conversion
thrombotic at higher doses. The structure of the polysac-
of a-galactose units into 3,6-anhydro-a-galactose (Fig. 3).
charide also influences the response in the experimental
Our proposal was to test sulfated galactans with known
thrombosis assay. The sulfated galactan from A. musco-
structures in a variety of coagulation tests using purified pro-
ides achieves almost total inhibition of thrombus forma-
teins and to compare their effects on native and serpin-free
tion, whereas only ~ 70% inhibition is obtained with the
plasma. The sulfated galactans were also evaluated for their
sulfated galactan from B. occidentalis.
procoagulant effect through activation of FXII. Finally,
In conclusion, our results indicate that sulfated galactans
these sulfated galactans were tested on models of arterial
prevent experimental thrombosis though a less commonly
and venous thrombosis using experimental animals.
known mechanism, which is independent of serpin. The
Our results suggest that the final effect of sulfated ga-
effects of the sulfated galactans rely on a tenuous balance
lactans on arterial and venous thrombosis relies on a ten-
among their various actions on the coagulation system; these
uous balance between the multiple actions of the
actions either favor or inhibit coagulation. The molecular
galactans on a variety of events, such as the following:
size is critical in modulating these effects, but the chemical
1 Serpin-dependent inhibition of the coagulation prote-
structure of the polysaccharide is also relevant. All these
ases, which is analogous to the effect of heparin. How-
observations indicate the difficulty in future attempts to
ever, we previously demonstrated that sulfated galactan
develop therapeutic antithrombotic drugs based on sulfated
interacts with antithrombin through a mechanism that
galactans from marine organisms. Here, we clearly extended
is distinct from that of heparin. Sulfated galactan does
the background knowledge about the mechanisms underly-
not induce the activating conformational change that
ing the anticoagulant and antithrombotic effects of sulfated
has been reported for heparin [20].
galactans. In-depth elucidation of mechanisms of action is
2 Serpin-independent anticoagulation due to inhibition of
certainly a key requirement for drug development.
the tenase and prothrombinase systems. These effects
were previously demonstrated using assays of purified
proteins [16] and may be evaluated using APTT in ser-
Addendum
pin-free plasma (Fig. 6). The absence of the high-affin-
ity antithrombin-binding motif that is observed for the A.L.G. Quinder!e and I.N.L. Queiroz performed the
heparin pentasaccharide may direct the anticoagulant experiments related with extraction and purification of

© 2013 International Society on Thrombosis and Haemostasis

52 A.-L. G. Quinder!e et al
the polysaccharides. G.R.C. Santos, S.N.M.G.G. Oliveira,
and B.P. Fontes performed the anticoagulant and anti-
thrombotic assays. B.F. Glauser contributed with reagents
and performed the assays with the serpin-free plasmas.
N.M.B. Benevides provided the material and analysis
tools. V.H. Pomin performed and interpreted the NMR
analysis. P.A.S. Mour~ao wrote the paper.
Acknowledgements
This work was supported by grants from Conselho Nacional
de Desenvolvimento Cientifico e Tecnologico (CNPq),
Coordenac!~ao de Aperfeic!oamento do Pessoal de N!ıvel
Superior (CAPES), and Fundac!~ ao de Amparo " a Pesquisa
do Estado do Rio de Janeiro (FAPERJ). We would like to
thank Adriana A. Piquet for technical assistance and Dr.
Mauro S.G. Pav~ao for critical reading of the manuscript.
Disclosure Conflicts of Interests
The authors state that they have no conflict of interest.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. 2D 1H/13C-edited-HSQC spectra of the native
sulfated galactan from the red alga A. muscoides (A), the
desulfated derivative (B), and the alkali-treated anhydro-
enriched derivative (C).
Fig. S2. 2D 1H/1H-TOCSY spectra of the native sulfated
galactan from the red alga A. muscoides (A), and the the
alkali-treated anhydro-enriched derivative (B).
Table S1. Chemical composition and concentrations of
NaCl necessary to elute the different sulfated galactan
fractions of the red alga A. muscoides from a DEAE cell-
ulosecolumn.
Table S2. 1H and 13C chemical shifts (ppm) of the com-
posing units of the sulfated galactan from the red alga
A. muscoides, and from comparative galactans from other
red algal species previously assigned in reference works.
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