Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis.

BACKGROUND: The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity. METHODS: The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAE-cellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests. RESULTS: Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity. CONCLUSIONS: An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II. GENERAL SIGNIFICANCE: Old culture medium of A. platensis may represent an important source for the spirulan-like PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest.

Polysaccharides from the skin of the ray Raja radula. Partial characterization and anticoagulant activity.

INTRODUCTION: The polysaccharide fraction from the skin of the ray Raja radula was extracted, characterized and assayed for anticoagulant activity. MATERIALS AND METHODS: A whole polysaccharidic fraction was extracted from the skin of the ray Raja radula by papain digestion followed by cetylpyridinium chloride and ethanol precipitation and was subjected to gel chromatography and anion exchange chromatography, acetate cellulose electrophoresis and characterized by physicochemical procedures. APTT and anti Xa assays were performed to assess the anticoagulant activity of the polysaccharidic fractions in comparison with unfractionated heparin. RESULTS: Gel and anion-exchange chromatography revealed two negatively charged polysaccharidic populations different in both molecular weight and charge. Infrared spectra suggested the occurrence of uronic acids and acetylated hexosamines. The second polysaccharide was highly sulfated, with a sulfate content of approximately 29%. These data suggested that dermatan sulfate (DS) is the sulfate rich polysaccharide whereas hyaluronic acid (HA) is the polysaccharide devoid of sulfate groups. Molecular mass characterization indicated that their average molecular masses were 22 kDa and 85 kDa, respectively. The sulfated polysaccharide, i.e. presumably DS, accounted alone for the observed concentration-dependent anticoagulant activity which was, as measured by APTT, 2 to 3-fold lower than that of heparin. In addition, it had a significant anti-Xa activity. CONCLUSION: A major-sulfated polysaccharide, likely a dermatan sulfate, was extracted from the ray Raja radula skin. The results indicated that it exhibited a high anticoagulant activity and suggested that it was mediated by both heparin cofactor II and antithrombin.

Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray (Raja radula) skin dermatan sulfate.

INTRODUCTION: The kinetics of the thrombin inhibition by heparin cofactor II (HCII) and antithrombin (AT) have been studied as a function of the concentration of a dermatan sulfate (DS) from the skin of the ray Raja radula. MATERIALS AND METHODS: The initial concentrations of inhibitor (I), HCII or AT, and thrombin (E) were set at equimolecular levels (3.10(-9) M). Analysis of the experimental data obtained for DS concentrations ranging from 10(-8) to 10(-4) M was performed according to a previously described model in which DS binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin. RESULTS: The apparent rate constant of the thrombin inhibition, k(app), by either HCII or AT, increased in a concentration-dependent manner for DS concentrations up to 10(-5) M or 10(-6) M, respectively. At higher DS concentrations, k(app) remained unchanged for thrombin inhibition by HCII whereas a decrease in k(app) was observed for the thrombin-AT reaction. The dissociation constant of the polysaccharide-inhibitor complex, K(DSI), and the rate constant of the thrombin inhibition by this complex, k, were (7.81+/-0.75).10(-7) M and (2.84+/-0.42).10(9) M(-1).min(-1), whereas they were (4.93+/-0.31).10(-7) M and (2.47+/-0.28).10(8) M(-1).min(-1), when the inhibitor was either HCII or AT, respectively. CONCLUSION: DS from ray skin catalyzes the thrombin inhibition by HCII or AT primarily by forming a DS-inhibitor complex more reactive than the free inhibitor towards the protease. The affinity of DS for HCII was approximately 2-fold higher whereas the catalyzed reaction rate constant was approximately 20-fold higher when compared to AT.

Characterization of a novel dermatan sulfate with high antithrombin activity from ray skin (Raja radula).

INTRODUCTION: A novel dermatan sulfate (DS) from the skin of the ray Raja radula with high anticoagulant activity was identified and its monosaccharide composition and anticoagulant mode of action and potency were determined. MATERIALS AND METHODS: The DS isolated from the ray skin was identified by chondroitinase treatment and characterized by FT-IR and (1)H NMR spectroscopy. Its anticoagulant activity was checked by activated partial thromboplastin time (aPTT), thrombin time (TT), thrombin generation (TG), heparin cofactor II (HCII) and antithrombin (AT)-mediated inhibition of thrombin. The effects on platelet activation and aggregation were investigated using flow cytometry and aggregometry, respectively. RESULTS: Chemical backbone structures of DS from Raja radula were close to that of DS from porcine intestinal mucosa. However, (1)H NMR indicated that iduronic acid was the major hexuronic acid moiety in the ray skin DS and also suggested that the amount of 2-O-sulfonated iduronic acid was higher in comparison with mammalian DS along with the occurrence of 4-O-sulfonated N-acetylgalactosamine residues. The anticoagulant effect of the ray skin DS was mainly due to the potentiation of thrombin inhibition by HCII but also, although to a lesser extent, by AT and was higher than that of the DS standard. Moreover, it had no effect on platelet activation and aggregation induced by various agonists. CONCLUSION: Altogether, these results indicated that DS from raja radula skin is an anticoagulant drug of interest potentially useful in anticoagulant therapy.

Effect of oversulfated dermatan sulfate derivatives on platelet aggregation.

We have investigated the effect on human platelet aggregation of native dermatan sulfate (DS) and three oversulfated DS derivatives with different sulfur contents, and compared it with that of unfractionated heparin. An inhibitory effect on collagen-induced platelet aggregation was observed only with unfractionated heparin at high concentrations, whereas no inhibitory effect was observed when arachidonic acid was used. Heparin was the most potent inhibitor of the thrombin-induced platelet aggregation in platelet-rich plasma (PRP), whereas the oversulfated DS had a higher potency than the native DS. All these glycosaminoglycans (GAGs) also inhibited thrombin-induced aggregation of washed platelets in the presence of antithrombin (AT) or heparin cofactor II (HCII) but not in their absence. Heparin was by far the most potent inhibitor of washed platelet aggregation in the presence of AT, whereas the inhibitory effects of the DS (native or oversulfated) were lower but dependent on the sulfur content. In the presence of HCII, DSb, a slightly oversulfated DS, had the highest inhibitory effect, whereas heparin and DSd, the most oversulfated derivative, had lower potencies in this case. These data suggest that the inhibition of thrombin-induced platelet aggregation by the oversulfated DS derivatives is related to their ability to potentiate thrombin inactivation by AT or HCII. Hence, the oversulfated DS derivatives may not have an effect per se on the inhibition of platelet aggregation. They may constitute a new class of anticoagulants with enhanced anticoagulant effects in comparison with the native DS, but with only minor side-effects of bleeding in comparison with heparin.

In vitro and in vivo hemocompatibility evaluation of a new dermatan sulfate-modified PET patch for vascular repair surgery.

The development of new vascular devices requires to study the effects of materials on blood cells and on coagulation, both in vitro and in vivo. In this study, we have developed a new material by grafting dermatan sulfate (DS) from shark skin onto polyethylene terephthalate (PET). We have evaluated the haemocompatibility of PET-DS material in vitro by measuring thrombin generation, plasma recalcification time, hemolytic activity, and platelet adhesion and in vivo with a model of vascular patch in rat abdominal aorta. In vitro, our results have shown that PET-DS is a nonhemolytic material, able to inhibit thrombin generation and platelet adhesion. In vivo studies by Doppler echographic evaluation 20 days after implantation have shown that the PET-DS patch was integrated in the vessel wall and covered by a layer of cells. In conclusion, PET-DS has good haemocompatibility properties and could be a promising tool for vascular surgery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2001-2009, 2017.

Thrombin inhibition by antithrombin in the presence of oversulfated dermatan sulfates.

DSS1 and DSS2 are two oversulfated dermatan sulfate derivatives with sulfur contents of 7.8% and 11.5%, respectively. DSS1 and DSS2 both enhanced the rate at which antithrombin (AT) inactivates thrombin according to a concentration dependent manner. The analysis of the experimental data, using our previously described kinetic model [Biomaterials1997, 18, 203] (i) suggested that both DSS1 and DSS2 catalyzed the thrombin-AT reaction according to a mechanism in which the oversulfated derivative quickly formed with AT a complex, which was more reactive towards thrombin than the free inhibitor and (ii) allowed us to determine the dissociation constants of the polysaccharide-inhibitor complexes, which were (1.15 +/- 0.74) x 10(-7) and (7.17 +/- 0.65) x 10(-9) M, and the catalyzed reaction rate constants, which were (2.29 +/- 0.15) x 10(8) and (8.71 +/- 0.08) x 10(8) M(-1) min(-1), for DSS1 and DSS2, respectively. These data suggested that the oversulfation confers an affinity for AT to dermatan sulfate and that the higher the sulfur content the higher the affinity for AT. They also suggested that the reactivities of the polysaccharide-AT complexes formed towards the protease increased with the sulfur content.

Grafting of dermatan sulfate on polyethylene terephtalate to enhance biointegration.

The aim of the present study was to achieve the immobilization of dermatan sulfate (DS) on polyethylene terephthalate (PET) surfaces and to evaluate its biocompatibility. DS obtained from the skin of Scyliorhinus canicula shark was immobilized via carbodiimide on knitted PET fabrics, modified with carboxyl groups. PET-DS characterization was performed by SEM, ATR-FTIR and contact angle measurements. Biocompatibility was evaluated by investigating plasma protein adsorption and endothelial cell proliferation, as well as by subcutaneous implantations in rats. The results indicated that DS immobilization on PET was achieved at ~8 µg/cm². ATR-FTIR evidenced the presence of sulfate groups on the PET surface. In turn, contact angle measurements indicated an increase in the surface wettability. DS immobilization increased albumin adsorption on the PET surface, whereas it decreased that of fibrinogen. In vitro cell culture revealed that endothelial cell proliferation was also enhanced on PET-DS. Histological results after 15 days of subcutaneous implantation showed a better integration of PET-DS samples in comparison to those of nonmodified PET. In summary, DS was successfully grafted onto the surface of PET, providing it new physicochemical characteristics and biological properties for PET, thus enhancing its biointegration.

Anticoagulant activity of a dermatan sulfate from the skin of the shark Scyliorhinus canicula.

A dermatan sulfate isolated from the shark Scyliorhinus canicula skin by enzymatic digestion followed by purification with anion exchange chromatography was identified by chondroitinase and nitrous acid treatment and partially characterized by Fourier-transform infrared spectroscopy. Dermatan sulfate was the major glycosaminoglycan and represented 75% of the polysaccharide fraction in the sharkskin. This dermatan sulfate had a 38.6 kDa average molecular weight and 23% sulfate content. The anticoagulant action of this dermatan sulfate was checked by several coagulometric and colorimetric assays such as the activated partial thromboplastin time, thrombin time, thrombin generation and heparin cofactor II and antithrombin-mediated inhibition of thrombin and compared with that of porcine intestinal mucosa dermatan sulfate. The effects on platelet activation and aggregation were investigated using flow cytometry and aggregometry, respectively. The dermatan sulfate prolonged activated partial thromboplastin time and thrombin time, delayed and inhibited thrombin generation in a concentration-dependent manner. The specific anticoagulant activity of the sharkskin dermatan sulfate was 43 UI/mg. The anticoagulant effect of sharkskin dermatan sulfate was higher than that of the porcine dermatan sulfate and was due to the potentiation of thrombin inhibition by heparin cofactor II. Moreover, it had no effect on platelet aggregation and activation induced by various agonists and thereby constitutes a potentially useful drug of interest in anticoagulant therapy.

Highly sulfated dermatan sulfate from the skin of the ray Raja montagui: anticoagulant activity and mechanism of action.

The dermatan sulfate (DS) isolated from the ray skin Raja montagui was identified and characterized. Its average molecular weight (Mw) and sulfate content were 39 kDa and 25% w/w, respectively. This DS prolonged thrombin time and activated partial thromboplastin time and inhibited the thrombin generation in a concentration-dependent manner whereas it had no effect on the anti-Xa assay and on platelet function. Data from the anti-IIa assay allowed the assessment of the specific anticoagulant activity which was 40 units/mg. The kinetics of the thrombin inhibition by heparin cofactor II (HCII) has been studied as a function of DS concentration according to a kinetic model in which the polysaccharide binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin. This DS accelerated thrombin inhibition exclusively by HCII. The dissociation constant of the DS-HCII complex, K(DSHCII), and the rate constant of the thrombin inhibition by this complex, k, were (2.93+/-0.25)x10(-6)M and (2.2+/-0.35)x10(9)M(-1)min(-1), respectively. Our findings indicated that the major polysaccharide in the skin of the ray Raja montagui was a DS endowed with a high anticoagulant effect mediated by HCII and which may constitute an anticoagulant drug of interest in anticoagulant therapy.