RESUMEN
Heparin is a sulfated polysaccharide of animal origin showing excellent anticoagulant properties. Although it strongly inhibits the coagulation cascade, its interaction with multiple sites results in several side effects. An ideal alternative compound should not only possess anticoagulant and antithrombotic activities, but also provide specific binding to components of the coagulation cascade to decrease side effects and facilitate the control of pharmacologic actions in patient's body. In this work, we performed a scan of potential targets for chemically sulfated pectin from Citrus sinensis (SCP) that shows an efficient anticoagulant activity by combining proteomics and molecular docking techniques. Defining the interaction partners of SCP is fundamental to evaluate if its pharmacological side effects can be as harmful as those from heparin. SCP interacts directly with heparin cofactor II, probably favoring its interaction with thrombin. SCP interaction with antithrombin depends likely on its association with thrombin or factor Xa. In addition to the interaction with factors related to homeostasis, SCP may also act on the renin-angiotensin and on the complement systems. BIOLOGICAL SIGNIFICANCE: The knowledge of potential molecular targets of SCP provides clues to understand its mechanism of action in order to guide molecular changes in this compound to increase its specificity.
Asunto(s)
Anticoagulantes/química , Citrus sinensis/química , Pectinas/química , Antitrombinas/metabolismo , Cofactor II de Heparina/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Pectinas/metabolismo , Pectinas/uso terapéutico , Unión Proteica , Proteómica , Sulfatos/química , Trombina/química , Trombina/metabolismoRESUMEN
HD1, a DNA aptamer, binds exosite 1 on thrombin and blocks its clotting activity. Because HD1 also binds prothrombin and inhibits its activation by prothrombinase, we hypothesised that HD1 would be a more potent inhibitor of coagulation than other exosite 1-directed ligands, such as Hir(54-65)(SO(3)(-)). Supporting this concept, the effect of HD1 on the prothrombin time and activated partial thromboplastin time was two-fold greater than that of Hir(54-65)(SO(3)(-)) even though both agents inhibited thrombin-mediated factor (F) V and FVIII activation to a similar extent. In thrombin generation assays, HD1 (a) delayed the lag time, (b) reduced peak thrombin concentration, and (c) decreased endogenous thrombin potential to a greater extent than Hir54-65(SO(3)(-)). To eliminate thrombin feedback, studies were repeated in FV- and/or FVIII-deficient plasma supplemented with FVa and/or FVIIIa. Only HD1 prolonged the lag time in FV- and FVIII-deficient plasma supplemented with FVa and FVIIIa. In contrast, HD1 and Hir54-65(SO(3)(-)) inhibited the lag time in FVIII-deficient plasma supplemented with FVIIIa and in normal plasma. The more potent anticoagulant properties of HD1, therefore, reflect its capacity to attenuate FV activation by thrombin and inhibit prothrombinase assembly. These findings identify prothrombin as a potential target for new anticoagulants.
Asunto(s)
Anticoagulantes/farmacología , Aptámeros de Nucleótidos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Hirudinas/farmacología , Oligopéptidos/farmacología , Protrombina/metabolismo , Sulfatos/farmacología , Trombina/metabolismo , Anticoagulantes/metabolismo , Antitrombinas/metabolismo , Aptámeros de Nucleótidos/metabolismo , Sitios de Unión , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Factor VIIIa/metabolismo , Factor Va/metabolismo , Retroalimentación Fisiológica , Cofactor II de Heparina/metabolismo , Hirudinas/metabolismo , Humanos , Cinética , Oligopéptidos/metabolismo , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Sulfatos/metabolismo , Tromboplastina/metabolismoRESUMEN
The antithrombotic activity of dermatan sulfate from avian crown with the mean molecular weight of 38000 was compared to those from bovine intestine with the mean molecular weight of 16000 in vivo and in vitro. In an in vitro test, bovine intestine dermatan sulfate exhibited stronger effects on stimulation of heparin cofactor II and activation of Glu-plasminogen by tissue plasminogen activator. In vivo, avian crown dermatan sulfate more effectively prevented the development of thrombus in a rat deep vein thrombosis model. The measurement of plasma levels of these two kinds of dermatan sulfate revealed that avian crown dermatan sulfate circulated in higher concentration and longer duration than bovine intestine dermatan sulfate after intravenous administration to rats.
Asunto(s)
Anticoagulantes/química , Anticoagulantes/uso terapéutico , Dermatán Sulfato/química , Dermatán Sulfato/uso terapéutico , Trombosis de la Vena/tratamiento farmacológico , Animales , Anticoagulantes/sangre , Bovinos , Pollos , Condroitina ABC Liasa/metabolismo , Cromatografía en Gel , Dermatán Sulfato/sangre , Disacáridos/análisis , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Semivida , Cofactor II de Heparina/metabolismo , Cofactor II de Heparina/farmacología , Masculino , Peso Molecular , Plasminógeno/metabolismo , Ratas , Especificidad de la Especie , Sulfatasas/metabolismo , Trombina/antagonistas & inhibidores , Activador de Tejido Plasminógeno/metabolismo , Trombosis de la Vena/prevención & controlRESUMEN
A sulfated polysaccharide (AFE-HCD) purified from the leaves of Artemisia princeps Pamp selectively accelerated the rate of thrombin inhibition by heparin cofactor II (HCII). By using plasma derived HCII and bacterial expressed recombinant HCII molecules, the interaction between each HCII molecule and AFE-HCD was analyzed. AFE-HCD accelerated thrombin inhibition by plasma derived HCII or bacterial expressed wild type HCII to the same extent (IC50: 0.056 micrograms/ml for plasma derived HCII and 0.066 micrograms/ml for recombinant HCII under the experimental condition). The recombinant HCII (rHCII) molecule with Lys173-->Leu or Arg189-->His substitution, which is defective in interactions with heparin and dermatan sulfate, respectively, is activated by AFE-HCD to inhibit thrombin in a manner similar to wild type rHCII. These results suggested that activation of HCII was independent of its Lys173 or Arg189 residue. Although AFE-HCD is a selective activator of HCII like dermatan sulfate, the amino acid residue required for the activation of HCII was distinct form that of dermatan sulfate as well as heparin.
Asunto(s)
Artemisia/metabolismo , Cofactor II de Heparina/metabolismo , Plantas Medicinales , Polisacáridos/metabolismo , Arginina , Sitios de Unión , Cofactor II de Heparina/química , Humanos , Lisina , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.