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Vitor H. Pomin
Fucanomics & Galactanomics: marine Fucanomics & Galactanomics: marine glycans with differential actions in glycans with differential actions in
coagulation and thrombosis coagulation and thrombosis
Universidade Federal Universidade Federal do do
Rio de JaneiroRio de Janeiro
Laboratório de Coagulação & Trombose
September, 2013
OMICSOMICS - English-language neologism that refers to a field of studyfield of study in biology.
The related suffix-OME OME is usually used to address the objects of study of such fields, referring thus to international research projects.projects.
GenomicsGenomics (if not the first, one of the first projects to be launched into biology: Human Genome Project at mid-´80s, sheep Dolly from Cloning project at mid-´90s):
- Cognitive Genomics / - Comparative Genomics / - Functional Genomics / - Metagenomics / - Nutritional Genomics /
- Pharmacogenomics / - Toxicogenomics / - Psychogenomics / - Stem cell genomics / - Epigenomics
TranscriptomicsTranscriptomics Proteomics:Proteomics:
- Immunoproteomics / - Nutriproteomics / - Proteogenomics / - Structural Genomics / - Pharmacoproteomics
MetabolomicsMetabolomics MetabonomicsMetabonomics PharmacomicrobiomicsPharmacomicrobiomics LipidomicsLipidomics GlycomicsGlycomics (one of the latest projects to be launched into biology, 21st century, very challenging)
INTRODUCTION:INTRODUCTION: “ “OMICS” studies and “OME” projectsOMICS” studies and “OME” projects
GLYCOMICS:GLYCOMICS: overview overview CHALLENGES: 1) Structural characterization is difficult. Structural diversity and variety are both high, probably higher than any other biomacromolecule. Experimentation and data interpretation are laborious. In addition, previous technology was very limited.
2) Multiple biological functions. For a long time, carbohydrates were considered just a mere class of energetic and structural molecules. Biological roles have been discovered and reported just recently. Innumerous functions have been reported.
Glycomics turned out to be a very extensive project. This is leading to a tendency of Glycomics turned out to be a very extensive project. This is leading to a tendency of subdivision or sectorization to allow the natural flow of the project development. In fact, subdivision or sectorization to allow the natural flow of the project development. In fact, subprojects, and new terminologies using the suffix-OME are emerging:subprojects, and new terminologies using the suffix-OME are emerging:
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: overview overview Sulfated fucans Sulfated fucans (SFs): marine polysaccharides composed predominantly or essentially (SFs): marine polysaccharides composed predominantly or essentially of of αα-L-fucopyranosyl units. Usually highly sulfated. Example: Fucoidan-L-fucopyranosyl units. Usually highly sulfated. Example: Fucoidan
Occurrence:Occurrence:
- cell wall of brown macroalgae- cell wall of brown macroalgae- egg jelly coat of sea urchins- egg jelly coat of sea urchins- body wall of sea cucumber- body wall of sea cucumber
Function:Function:
- structural component of the extracellular matrices of the above-mentioned - structural component of the extracellular matrices of the above-mentioned tissuestissues- triggers the acrosome reaction in sea urchins through a specie-specific way - triggers the acrosome reaction in sea urchins through a specie-specific way
Sulfated galactans Sulfated galactans (SGs): marine polysaccharides composed essentially of (SGs): marine polysaccharides composed essentially of αα-L-, -L-, αα-D-, -D-, ββ--D-galactopyranoses. Usually highly sulfated. Examples: Carrageenans and AgaransD-galactopyranoses. Usually highly sulfated. Examples: Carrageenans and Agarans
Occurrence: Occurrence:
- cell wall of green/red macroalgae- cell wall of green/red macroalgae- egg jelly coat of sea urchins- egg jelly coat of sea urchins- body wall of ascidians- body wall of ascidians
Function:Function:
- structural component of the extracellular matrices of the above-mentioned - structural component of the extracellular matrices of the above-mentioned tissuestissues- triggers the acrosome reaction in sea urchins through a specie-specific way- triggers the acrosome reaction in sea urchins through a specie-specific way
Laboratories Worldwide: Brazil (CE, RN, RJ, SP, RS), Argentina, USA, Russia, China, Italy, France and Japan. Laboratories Worldwide: Brazil (CE, RN, RJ, SP, RS), Argentina, USA, Russia, China, Italy, France and Japan.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Chemistry vs PhylogenyChemistry vs Phylogeny Heterogeneous and branched structures of brown algal SFs
Pomin V H , and Mourão P A S Glycobiology 2008;18:1016-1027
© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Chemistry vs PhylogenyChemistry vs Phylogeny Regular, homogeneous and well-defined structures of invertebrate SFs
Pomin V H , and Mourão P A S Glycobiology 2008;18:1016-1027
© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
Structures of the repeating units of the α-L-SFs from the cell wall of the sea cucumber (A) and from the egg jelly coats of sea urchins (B-H)
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Chemistry vs PhylogenyChemistry vs Phylogeny Algal and invertebrate structures of SGs: heterogeneity vs regularity
Pomin V H , and Mourão P A S Glycobiology 2008;18:1016-1027
© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
Structures of the SGs from (A) red algae, (B) green algae, (C) sea grass (marine angiosperm), and marine invertebrates, such as (D) ascidians (also known as tunicates), and (E) sea urchins.
Sulfated fucans & Sulfated galactans
MOLECULAR INTERACTIONS IN FUCANOMICS AND GALACTANOMICS: DRUG DEVELOPMENT IN DIFFERENT CLINICAL AREAS
Serpins: antithrombinand heparin cofactor II
P- and L-selectins
Vascular endothelialgrowth factor (VEGF)
Chemokines Factors IIaand Xa
basic FibroblastGrowth Factor (FGF2)
VEGF receptor FGF receptor
InflammationCoagulation
and Thrombosis
Tumor angiogenesis
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Medical actions Medical actionsTherapeutic actions in diverse systems: inflammation; hemostasis and vascular biology; angiogensis; tumor growth, progression and spreading; oxidative stress; infections; and nociception.
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Medical actions Medical actions Molecular and cell mechanisms in coagulation/thrombosis
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Medical actions Medical actions Molecular mechanisms of actions of SFs and SGs in anticoagulation/antithrombosis
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Medical actions Medical actions Molecular mechanisms of actions of SFs and SGs in anticoagulation/antithrombosis
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Chemistry Chemistry Regular structures enable accurate and advanced structure-function relationships
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisean
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in � 66% and � 33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in � 60% and � 15%, respectively)
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
O
CH2OH
R3O
OOR4
O
O
CH2OH
R1O
OR2O
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: Medical Actions Medical ActionsRegular structures enable accurate and advanced structure-function relationships
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION STRUCTURE vs FUNCTION
Advanced structure-function relationships: SUGAR TYPE-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisean
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in � 66% and � 33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in � 60% and � 15%, respectively)
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
O
CH2OH
R3O
OOR4
O
O
CH2OH
R1O
OR2O
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisean
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in � 66% and � 33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in � 60% and � 15%, respectively)
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
O
CH2OH
R3O
OOR4
O
O
CH2OH
R1O
OR2O
VSVS
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTIONSTRUCTURE vs FUNCTION
Advanced structure-function relationships: SUGAR TYPE-DEPENDENT ANTICOAGULANT ACTION
Structures of the complexes between different SP (red, yellow) and AT (blue). (A) ternary complex between AT, thrombin (gold) and a heparin derivative (PDB ID 1TB6); (B) AT bonded to the synthetic pentasaccharide (PDB ID 1E03); (C) final structure from a 5 ns MD of AT complexed to a SF decasaccharide with pyranose rings; (D) final structure from a 5 ns MD of AT complexed to a SG decasaccharide with pyranose rings. For (B)–(D), two orientations of the complexes are presented.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTIONSTRUCTURE vs FUNCTION
Advanced structure-function relationships: CONFORMATION-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisean
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in ±66% and ±33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in ±60% and ±15%, respectively)
O
CH2OH
R3O
O
OR4O
O
CH2OH
R1O
OR2O
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION STRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION PATTERN-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION STRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION PATTERN-DEPENDENT ANTICOAGULANT ACTION
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTIONSTRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION TYPE-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisean
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in � 66% and � 33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in � 60% and � 15%, respectively)
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
O
CH2OH
R3O
OOR4
O
O
CH2OH
R1O
OR2O
2SO3-
> 2SO3-
4SO3-
4SO3- 2SO3
-
> 2SO3-
[2SO3-] =
[4SO3-]
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTIONSTRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION TYPE-DEPENDENT ANTICOAGULANT ACTION
4SO3-
[2SO3-] = [4SO3
-]
2SO3-
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: 11STST CONCLUSIONS CONCLUSIONS
SFs and SGs are essentially found in marine organisms such as macroalgae, sea urchins and sea cucumbers, and ascidians.
Unlike algal SFs and SGs, the invertebrate molecules have well-defined chemical structures which allow accurate and advanced structure-function relationships.
Although SFs and SGs may exhibit many clinical activities, the mechanisms of actions are known just for inflammation, coagulation/thrombosis and tumoral angiogenesis.
In inflammation, SGs and SFs can block chemokines, P- and L-selectins. In hemostasis, they block the factors IIa and Xa. In tumoral angiogenesis, they inhibit VEGF and bFGF.
2-sulfated 3-linked α-galactan is an anticoagulant polysaccharides, whereas 2-sulfated 3-linked α-fucan is not. Their conformational binding preference to serpins are different.
2,4-di-sulfation and maybe 4-sulfation in 3-linked α-SFs have shown a benefitial effect on the anticoagulation, whereas 2-sulfation alone is certainly deleterious. Ongoing NMR relaxation studies will facilitate further explanations.
Our dataset strongly support the the fact that the clinical actions of SFs and SGs are not a mere consequence of sulfation degrees, but intimately correlated to specific features: anomeric configuration, monosaccharide type, sulfation positions or patterns, glycosylation position, molecular weights, and so forth.
1H-chemical shift (ppm)
0.91.31.72.12.52.93.33.74.14.54.95.3
A
B
C
CH3
CH3
CH3
2-O-CH3
2-O-CH3
2-O-CH3
4.54.95.3
B1
E4
D1A1
b1
C1/c1
C1/c1
D1
C1/c1
A1
4.54.95.3
4.54.95.3
E1/F1
G1
G1
F1
0.91.31.72.12.52.93.33.74.14.54.95.3
0.91.31.72.12.52.93.33.74.14.54.95.3
1H-chemical shift (ppm)
1H-chemical shift (ppm)
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE DETERMINATIONSTRUCTURE DETERMINATION
NMR structure determination of a new red algal SG (Acanthophora muscoides)
E1/F1
3.43.63.84.04.24.44.64.85.05.25.4 3.2
1H – chemical shift (ppm)
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2c1
D1
A
B
13C
– c
he
mic
al s
hift
(p
pm)
102
98
94
90
86
82
78
74
70
66
62
58
102
98
94
90
86
82
78
74
70
66
62
58
A1
B1
C1D1 E1
F1G1
F1G1
C5
C4C3
C1
A1
b1
C3
C5
C2
C2
6-unsubstituted 6-SO3
-
E4
F6 F6´
C6 C6´
F6 F6´
C6C6´
6-unsubstituted
2-O-CH3
2-O-CH3
c1
c1
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
A1H
– che
mica
l shift (ppm
)
B
A1
B1
C
1H – chemical shift (ppm)
3.33.53.73.94.14.34.54.74.95.15.3
102
98
94
90
86
82
78
74
70
66
62
58
C1
C5
C4C3
D1
C2
6-unsubstituted
2-O-CH3
c1
C6C6´
F1
E1
E6´E6 D6/D6´
D6/D6´E6´E6
B6/B6´
6-SO3-
E1
F1
E4
E6
c2-O-CH3
D6
F6
B6F6´
A2-O-CH3
E6´
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: STRUCTURE PROPOSITIONSTRUCTURE PROPOSITION
Structures of SGs from Acanthophora muscoides and Botriocladia occidentalis
α-Galp units β-Galp units
O
CH2OH
OCH3
OHO
OO
CH2OSO3-
OH
HO O
O
O
CH2OSO3-
OH
OHO
OO
CH2OSO3-
OH
-O3SO O
O
O
OR
OO
OO
CH2OCH3
OH
HO O
O
O
CH2OH
OH
HO O
O
Unit A (~ 32 %) Unit D (~ 22%)
Unit B (~ 43 %) Unit E (~ 30 %)
Unit C, R = H Unit c, R= CH3 (minor)
(~ 25%)Unit F (~ 22%)
Unit G (~ 26%)
O
CH2OH
OR1
R1O
O
CH2OH
OR3
OR2O
OO
α-Galp units β-Galp units
R1 = H or SO3-
R2 = H (~ 33%) or SO3- (~ 66%)
R3 = H (~ 66%) or SO3- (~ 33%)
VS
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: MW DIFFERENCESMW DIFFERENCES
Structures of Acanthophora muscoides and Botriocladia occidentalis
_ +Origin
+
B
60
40
20
8
20 30 40 50 60 70
0.0
0.2
0.4
0.6
0.8
1.0
Ref
ract
ive
Inde
x
Retention Time - min
A
A mB occ (LMW)B occ
nativ
e B. o
ccid
enta
lis
LMW
B. o
ccid
enta
lis
nativ
e A. m
usco
ides
0 50 100 150 200 250
1
2
3
4
5
T1/T
0
25
50
75
100
125
Resid
ual a
ctivity
0.001 0.1 10 1000
0
25
50
75
100
125
g mL-1
Resi
dual act
ivit
y
g mL -1
0
25
50
75
100
125
0.001 0.1 10 1000
A B
C D
A m UFH
B occ (LMW) B occ
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: ANTICOAGULATIONANTICOAGULATION
SERPIN-DEPENDENT ANTICOAGULANT ACTIVITIES
AT/IIa
APTT in serpin-containing plasma
HCII/IIaAT/Xa
0.0 0.4 0.8 1.2 1.6 2.0 2.4
10
20
30
40
50
60
70A
40
60
80
100
Blo
od loss
-
L
Hep
ari
n
A. m
usco
ides
B. o
ccid
enta
lis -
B. o
ccid
enta
lis
+
Cont
rol 5 10 5 10 5 10 5 10 (mg kg-1)
C
Occ
lusi
on t
ime (
min
)
0.0 0.25 0.50 0.75 1.00
0
40
80
120
Thro
mb
us
weig
ht
(%)
mg kg-1
B
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: ANTITHROMBOSISANTITHROMBOSIS
ARTERIAL AND VENOUS MODELS, AND BLEEDING EFFECTS
A m UFH
B occ (LMW) B occ
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: PROTHROMBOTIC EFFECTSPROTHROMBOTIC EFFECTS
Activation of factor XII
0
5
10
15
20
25
30V
max
–m
OD
min
-1
1 50 1 50 1 50 1 50 (g mL-1)
HeparinB.occidentalis
+_A. muscoides
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: 22rdrd CONCLUSIONS CONCLUSIONS
Unlike the SG from B. occidentalis, the recently characterized SG from A. muscoides is structurally very complex and heterogeneous. Besides sulfation at different positions, anhydro sugars and methyl ethers are also observed;
The MW of B. occidentalis was reduced to allow a comparative study in terms of structure-function relationships in anticoagulation and antithrombosis with the natural low-MW of A. muscoides;
The native SG from B. occidentalis has high anticoagulant activity due to its significant activity on serpins (AT and HCII) and proteases (mostly thrombin). It also shows high serpin-independent anticoagulant activity. The low-MW form lost both activities. SG from A. muscoides has just no anticoagulant activity regardless the plasma type.
SG from A. muscoides is curiously a potent antithrombotic agent at the arterial model, whereas the one from B. occidentalis is not. Both red algal SGs are antithrombotic at the venous model, however, at only small doses. SG from B. occidentalis is prothrombotic at higher doses due to its capacity in activate factor XII.
As opposed to heparin which is highly hemorragic, neither one of the red algal SGs have shown this side effect.
Through this comparative study using red algal SGs, we have dissociated the anticoagulant (both serpin-dependent and serpin-independent), antithrombotic (both arterial and venous) and hemorragic effects, as compared with the standard heparin.
FUCANOMICS & GALACTANOMICS:FUCANOMICS & GALACTANOMICS: ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
Universidade Federal Universidade Federal do do
Rio de JaneiroRio de Janeiro
Prof. Paulo A.S Mourão Prof. Norma Maria Barros Benevides Prof. Ana Cristina Vilela-Silva Prof. Ana Paula Valente Prof. Fabio Ceneviva Almeida Stephan Nicollas de Oliveira Gustavo Ramalho dos Santos Bianca Fernandes Glauser Ismael Lino Nilo de Queiroz Bruno Pedrosa Fontes Ana Luiza Quinderé Eros Falcão Gabriel Baptista de Sá
