Jacqueline Tapon-Bretaudière

Anticoagulant properties of a fucoidan fraction

Fucoïdans are a family of high molecular weight sulphated polysaccharides in the Mr range 8 x 10(5) -10(6), widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata, was undertaken to obtain a low molecular weight polysaccharide (Mr 20,000 +/- 5,000) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor IIa and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoïdan revealed a potent antithrombin activity: studied in an antithrombin III depleted plasma or in the presence of purified heparin cofactor II, the fucoïdan was as efficient as heparin and dermatan sulphate on heparin cofactor II potentiation, at the same concentration by weight. In whole plasma or in the presence of the purified inhibitor, an anti-factor IIa activity mediated by antithrombin III was detected (30 times less potent than for heparin, on a weight to weight basis). In contrast, no anti-factor Xa activity was detected in the presence of the degraded fucoïdan, under the same experimental conditions. These fucoïdans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.

Interactions of poly(lactic acid) and poly(lactic acid-co-ethylene oxide) nanoparticles with the plasma factors of the coagulation system

When surfactant-stabilized biodegradable poly(lactic acid) (PLA) particles are injected into rats, the rate of clearance from blood is fast. The rate can be strongly reduced by using particles made from diblock copolymers of PLA and poly(ethylene oxide) (PLA-PEO), resulting in an increased duration of contact with the components of the coagulation system. Thus, possible adverse effects such as activation of the coagulation cascade could occur. In this paper, the interactions of surfactant-stabilized PLA and PLA-PEO nanoparticle suspensions with the plasma factors of the coagulation system are presented. PLA suspensions stabilized by sodium cholate (PLA-Ch) interact with thrombin, factor V and calcium ions. Formation of complexes and aggregates is induced by addition of calcium ions to PLA-Ch suspensions in the presence or in the absence of plasma. On the contrary, PLA-PEO suspensions are remarkably inert towards the coagulation factors and calcium ions, even when cholate is present. Steric repulsion owing to the high surface density of PEO is sufficient to avoid strong interations with the proteins and formation of aggregates between particles.

Anticoagulant activity of dextran derivatives. Part II: Mechanism of thrombin inactivation.

The mechanism of anticoagulant activity of dextrans substituted with carboxylic and benzylamide sulphonate groups is studied by various coagulation tests. These derivatives exhibit a heparin-like antithrombic activity which requires the presence of antithrombin III; however they are less effective than heparin on a weight basis. They also exert a direct antithrombic activity by an antithrombic III independent pathway; but this action is negligible compared to the thrombin inhibition observed in the presence of antithrombin III. Dextran derivatives have been prepared with antithrombic properties similar to those of heparin.

Electron microscopy of the conformational changes of alpha 2-macroglobulin from human plasma.

High resolution electron microscopy reveals that fully active alpha 2‐macroglobulin (α2M) from fresh human plasma presents a very characteristic tetrameric structure. This native conformation of the α2M molecule is described here for the first time, along with its various orientations in negatively stained preparations. Although the native form is sensitive to inactivation, glutaraldehyde fixation is not necessary for its observation except when ammonium salts are used. The tetrameric structure of α2M undergoes a drastic conformational change when the protein is treated either with trypsin, thrombin or methylamine, as evidenced by the appearance of the typical)+(structure already described in the literature. The various aspects of this second conformation correspond to different orientations of the molecules in the stain film, and depend upon the nature of the support.

Influence of the oversulfation method and the degree of sulfation on the anticoagulant properties of dermatan sulfate derivatives

Six oversulfated dermatan sulfate (DS) derivatives, differing in their tissue origin (porcine skin, bovine and porcine intestinal mucosa), and the oversulfation method of preparation, have been tested for their anticoagulant properties. In the first method, the SO3-trimethylamine complex is added to a DS sodium salt dissolved in formamide while it is added to a DS-benzethonium salt dissolved in dimethyl formamide in the second method. The rate of sulfation of these compounds ranged from 7.8 to 11.5 percent of sulfur on a weight basis, whereas it is 6% and 12% for natural DS and for heparin respectively. The anticoagulant potency was assessed by determining the catalytic effect of each glycosaminoglycan on the inhibition of thrombin added to (i) plasma (ii) purified heparin cofactor II(HC II) or (iii) purified antithrombin III(AT III). The catalytic effect on Factor Xa inhibition in the presence of AT III has also been investigated. The increased sulfation is found to enhance the antithrombin activity of the native dermatan sulfate whatever the method used, while the Factor Xa inhibition by AT III could be catalysed only by the most sulfated derivative obtained by the second method. The two derivatives which were less oversulfated, by the first oversulfation method, exhibit equal or even higher catalytic effects on thrombin inhibition when compared to the four other derivatives. The use of the first oversulfation method provides slightly oversulfated derivatives which exhibit strong anticoagulant properties and may constitute effective antithrombotic drugs with no bleeding tendency, a side effect perhaps related to a high rate of sulfation.

Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin.

The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from 10(-9) to 10(-4) M. The initial concentrations of inhibitor (l) and thrombin (E) were set at equimolar levels (CI = CE = 10(-8) M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from 10(-9) to 10(-6) M and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902-2913] for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. KH,I, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 10(-7) M and 1.3 x 10(9) M-1 min-1, respectively, for AT, compared to a KH,I of 2.0 x 10(-6) M and k of 6.4 x 10(9) M-1 min-1 for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10(-6) M, the decrease in the reaction rate was in keeping with the formation of a heparin-thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of the free protease.

Antithrombic properties of insoluble modified polystyrene : Part I

Chlorosulfonated polystyrenes grafted with arginyl methyl ester have been synthetized and characterized by elemental analyses. When suspended in plasma or in fibrinogen solutions, these insoluble polymers cause an increase in the thrombin clotting time. Consequently the antithrombin activity observed is not dependent upon to the presence of antithrombin III. Binding between the resin and thrombin can be demonstrated. An approximative value of the affinity constant is calculated: 2.3 X 10(6) 1/M. At high ionic strength, thrombin can be desorbed and exhibits normal coagulation properties. The prolongation of the thrombin time therefore can be attributed to the thrombin-resin interaction.

Heparin-like functionalized polymer surfaces: discrimination between catalytic and adsorption processes during the course of thrombin inhibition.

Thrombus formation on blood-contacting artificial surfaces is a major problem. Antithrombogenic polymer surfaces have been obtained either by heparin binding, or by grafting sulphonate and/or amino acid sulphonamide groups on insoluble polystyrene. In addition to their capacity to adsorb thrombin, such surfaces were shown to be able to catalyse its inhibition by antithrombin III (AT), i.e. they are endowed with heparin-like activity. The results were mainly obtained by using clotting assays. In many cases, delineating adsorption and catalytic processes by such assays is not possible when evaluating anticoagulant polymer surfaces. To overcome this problem, the kinetics of thrombin adsorption and inhibitions by AT and heparin cofactor II (HC) in the presence of such surfaces have been measured by using an assay performed with a thrombin-specific chromogenic substrate. A simple kinetic model of thrombin consumption is proposed. The relevant calculations, carried out with the help of a computer program, lead to determination of relative second order rate constants of thrombin adsorption and inhibitions by AT and HC in the presence of the polymers. In addition to thrombin adsorption, polystyrene surfaces bearing only sulphonate groups catalyse inhibition by AT, whereas polystyrene surfaces bearing either aspartate, glycinate or isophthalate sulphonamide groups catalyse both inhibitions by AT and HC.

The stability of heparin-coated liposomes in plasma and their effect on its coagulation

Abstract The potential use of liposomes as drug carriers is still limited by their poor stability under storage conditions and in biological systems. To improve their stability and blood compatibility, positively, neutral and negatively charged large unilamellar vesicles were coated with heparin. As expected, addition of heparin to a positively charged phosphatidylcholine/cholesterol/stearylamine mixed micellar solution or preformed liposomes, resulted in rapid liposome aggregation. On the contrary, no aggregation was observed with heparin and neutral or negatively charged lipid components of mixed micelles or liposome bilayers. No heparin release from the heparin-coated liposomes was observed during prolonged incubation of the liposome carrier with buffer and human plasma. The results indicate that, compared with conventional liposomes, the stability of heparin-coated liposomes was significantly increased. This was even more effective when dicetylphosphate, a negatively charged lipid, was included in the liposome structure.