RESUMO
An algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor (F)Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and FXa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for FXa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes FXa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of FXa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with FXa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate FXa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the gamma-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator Xa/metabolismo , Galactanos/farmacologia , Anticoagulantes/química , Sítios de Ligação/genética , Cálcio/farmacologia , Cisteína Endopeptidases/metabolismo , Fator V/antagonistas & inibidores , Fator V/química , Fator V/metabolismo , Fator Xa/química , Fator Xa/genética , Inibidores do Fator Xa , Galactanos/química , Heparina/farmacologia , Humanos , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/farmacologia , Serpinas/sangueRESUMO
Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates alpha-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on alpha-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for alpha-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of alpha-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin-alpha-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.
Assuntos
Antitrombinas/farmacologia , Galactanos/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Trombina/metabolismo , Sítios de Ligação , Catálise/efeitos dos fármacos , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Galactanos/química , Galactanos/farmacologia , Hirudinas/farmacologia , Humanos , Fragmentos de Peptídeos/farmacologia , Ésteres do Ácido Sulfúrico/química , Ésteres do Ácido Sulfúrico/farmacologiaRESUMO
We compared sulfated galactans (SGs) from two species of red algae using specific coagulation assays and experimental models of thrombosis. These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns. As a consequence of these differences, the two SGs differ in their anticoagulant and venous antithrombotic activities. SG from G. crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (>1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, SG from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (>1.0 mg/kg) the SG from B. occidentalis inhibits arterial thrombosis. As with heparin, SG from G. crinale does not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Both SGs do not modify bleeding time in rats. These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fibrinolíticos/farmacologia , Galactanos/farmacologia , Rodófitas/química , Sulfatos/farmacologia , Trombose/prevenção & controle , Animais , Anticoagulantes/efeitos adversos , Anticoagulantes/isolamento & purificação , Tempo de Sangramento , Testes de Coagulação Sanguínea , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fator XIIa/metabolismo , Feminino , Fibrinolíticos/efeitos adversos , Fibrinolíticos/isolamento & purificação , Galactanos/efeitos adversos , Galactanos/isolamento & purificação , Hemorragia/induzido quimicamente , Heparina/farmacologia , Humanos , Masculino , Estrutura Molecular , Peso Molecular , Agregação Plaquetária/efeitos dos fármacos , Ratos , Ratos Wistar , Sulfatos/efeitos adversos , Sulfatos/isolamento & purificação , Trombose/sangue , Trombose Venosa/sangue , Trombose Venosa/prevenção & controleRESUMO
Sulfated galactan from the red alga Botryocladia occidentalis has a potent anticoagulant activity, due to its ability to enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. It is less active than unfractionated heparin in arterial thrombosis, but in a venous thrombosis presents a dual effect, inhibiting thrombosis in low but not in high doses. This dual effect on venous thrombosis is a consequence of two actions, one that inhibits thrombin and factor Xa and one that induces factor XII activation. In order to dissociate these effects, we prepared derivatives of the sulfated galactan with low molecular weights. Two fractions that were similar in size to unfractionated heparin and low-molecular-weight heparin were obtained. As the molecular weight decreased, the ability to activate factor XII and to promote inhibition of coagulation proteases in the presence of antithrombin and heparin cofactor II diminished. At approximately 5 kDa, the sulfated galactan fragment had no effect on factor XII activation, and showed the same effect as unfractionated heparin in a venous thrombosis model. The approximately 5-kDa fragment is an antithrombotic with several advantages: i) It is as active as unfractionated heparin in venous thrombosis, but it has little activity in arterial thrombosis; ii) It inhibits venous thrombosis with very little anticoagulant effect; iii) It does not cause bleeding; and iv) It is not obtained from mammals.
Assuntos
Anticoagulantes/farmacologia , Fator XIIa/metabolismo , Inibidores do Fator Xa , Fibrinolíticos/farmacologia , Galactanos/farmacologia , Rodófitas/química , Sulfatos/farmacologia , Trombina/antagonistas & inibidores , Trombose/prevenção & controle , Animais , Anticoagulantes/efeitos adversos , Anticoagulantes/isolamento & purificação , Antitrombinas/metabolismo , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fator Xa/metabolismo , Feminino , Fibrinolíticos/efeitos adversos , Fibrinolíticos/isolamento & purificação , Galactanos/efeitos adversos , Galactanos/isolamento & purificação , Hemorragia/induzido quimicamente , Heparina/farmacologia , Cofator II da Heparina/metabolismo , Humanos , Masculino , Peso Molecular , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Sulfatos/efeitos adversos , Sulfatos/isolamento & purificação , Trombina/metabolismo , Trombose/sangue , Trombose Venosa/sangue , Trombose Venosa/prevenção & controleRESUMO
We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Dermatan Sulfato/química , Dermatan Sulfato/farmacologia , Electrophorus , Pele/química , Animais , Anticoagulantes/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Dermatan Sulfato/isolamento & purificação , Humanos , Ratos , SuínosRESUMO
Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Galactanos/química , Galactanos/farmacologia , Rodófitas/química , Sequência de Carboidratos , Inibidores do Fator Xa , Cofator II da Heparina/farmacologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Tempo de Tromboplastina Parcial , Trombina/antagonistas & inibidoresRESUMO
We compared the disaccharide composition of dermatan sulfate (DS) purified from the ventral skin of three species of rays from the Brazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habits the Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. However, DS from the skin of P. motoro presented a very low content of the disulfated disaccharides. The anticoagulant actions of ray skin DS, measured by both APTT clotting and HCII-mediated inhibition of thrombin assays, were compared to that of mammalian DS. DS from D. americana had both high APTT and HCII activities, whereas DS from D. gutatta showed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity in the APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of the disulfated disaccharide units within their polysaccharide chains.
Assuntos
Anticoagulantes/química , Coagulação Sanguínea/efeitos dos fármacos , Dermatan Sulfato/química , Rajidae , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Dermatan Sulfato/isolamento & purificação , Dermatan Sulfato/farmacologia , Estrutura Molecular , Especificidade da EspécieRESUMO
We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.
Assuntos
Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Heparina/farmacologia , Polissacarídeos/farmacologia , Ácidos Sulfúricos/farmacologia , Animais , Anticoagulantes/química , Sítios de Ligação , Cromatografia por Troca Iônica , Galactanos/química , Galactanos/isolamento & purificação , Galactanos/farmacologia , Heparina/química , Heparina/isolamento & purificação , Espectroscopia de Ressonância Magnética , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Rodófitas , Ácidos Sulfúricos/química , Ácidos Sulfúricos/isolamento & purificação , Trombina/metabolismoRESUMO
We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.