Characterization, chemical modifications and in vitro anticoagulant properties of an exopolysaccharide produced by Alteromonas infernus.

A new low-molecular-weight 'heparin-like' component was obtained from an exopolysaccharide produced by a mesophilic strain found in deep-sea hydrothermal vents. Data concerning the structure of the native high-molecular-weight exopolysaccharide (10(6) g/mol, 10% sulfate content) are reported for the first time. Two depolymerization processes were used to obtain low-molecular-weight (24-35x10(3) g/mol) oversulfated fractions (sulfate content 20 or 40%). Nuclear magnetic resonance studies indicated that after sulfation (40%), the low-molecular-weight fraction obtained by free radical depolymerization was less sulfated in the 6-O-position than the fraction depolymerized by acid hydrolysis. The free radical depolymerized product also had sulfated residues in the 4-O-position and disulfated ones in the 2,3-O-positions. Moreover, the compounds generated by the free radical process were more homogeneous with respect to molecular mass. Also for the first time, the anticoagulant activity of the low-molecular-weight exopolysaccharide fractions is reported. When the fractions obtained after sulfation and depolymerization were compared with heparins, anticoagulant activity was detected in oversulfated fractions, but not in native exopolysaccharide. The free radical depolymerized fraction inhibited thrombin generation in both contact-activated and thromboplastin-activated plasma, showing a prolonged lag phase only in the contact-activated assay. Affinity co-electrophoresis studies suggested that a single population of polysaccharide chains binds to antithrombin and that only a subpopulation strongly interacts with heparin cofactor II.

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.

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.

Respective roles of antithrombin III and alpha 2 macroglobulin in thrombin inactivation.

In order to investigate the mechanism of thrombin inactivation in the presence of both antithrombin III (AT III) and alpha 2-macroglobulin (alpha 2 M), thrombin and the inhibitors have been purified from human material and thrombin inactivation studied using purified reagents either alone or added to defibrinated plasma. Comparison of clotting and amidolytic activities of residual thrombin allowed to measure the amount of thrombin bound to alpha 2 M. In a purified reagent system as well as in plasma, part of exogenous thrombin is bound to alpha 2 M. The amount of bound thrombin is related to alpha 2 M concentration. Conversely, previous plasma alpha 2 M depletion by immunoabsorption increases the consumption of heparin-cofactor activity by exogenous thrombi. Thus AT III and alpha 2 M compete for thrombin inactivation. This finding could be of practical interest in clinical situations associating high plasma alpha 2 M levels and a decrease of AT III concentrations.

Modulation of vascular human endothelial and rat smooth muscle cell growth by a fucosylated chondroitin sulfate from echinoderm.

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 +/- 5 microg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 microg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 +/- 7.2%) and 27% (126.9 +/- 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 microg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 +/- 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 +/- 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form. +98% (198.2 +/- 25%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

Plasmatic antiproteinase activity enhancement by insoluble functionalized polystyrene surfaces.

Antithrombogenic functional polymer surfaces have been obtained by grafting heparin or by substituting insoluble polystyrene with sulphonate and/or amino acid sulphamide groups. Their heparin-like properties have been related to their catalytic effects on the antithrombin III - thrombin complex formation. Amongst these antithrombogenic surfaces, this study demonstrates that some insoluble amino acid sulphamide derivatives of polystyrene strongly potentiate heparin cofactor II, in addition to antithrombin III. In contrast, an insoluble polystyrene sulphonate and, to a lesser extent, an insoluble heparin copolymer, are better catalysts of antithrombin III. It is hypothesized that such different behaviours result from different conformations of the species adsorbed onto the surfaces. The conclusions support the possible use of such amino acid sulphamide groups to prepare antithrombogenic surfaces in contact with blood.

Inactivation of thrombin by a fucosylated chondroitin sulfate from echinoderm.

A polysaccharide extracted from the sea cucumber body wall has the same backbone structure as the mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. These branches confer high anticoagulant activity to the polysaccharide. Since the sea cucumber chondroitin sulfate has analogy in structure with mammalian glycosaminoglycans and sulfated fucans from brown algae, we compared its anticoagulant action with that of heparin and of a homopolymeric sulfated fucan with approximately the same level of sulfation as the sulfated fucose branches found in the sea cucumber polysaccharide. These various compounds differ not only in their anticoagulant potencies but also in the mechanisms of thrombin inhibition. Fucosylated chondroitin sulfate, like heparin, requires antithrombin or heparin cofactor II for thrombin inhibition. Sulfated fucans from brown algae have an antithrombin effect mediated by antithrombin and heparin cofactor II, plus a direct antithrombin effect more pronounced for some fractions. But even in the case of these two polysaccharides, we observed some differences. In contrast with heparin, total inhibition of thrombin in the presence of antithrombin is not achieved with fucosylated chondroitin sulfate, possibly reflecting a less specific interaction. Fucosylated chondroitin sulfate is able to inhibit thrombin generation after stimulation by both contact-activated and thromboplastin-activated systems. It delayed only the contact-induced thrombin generation, as expected for an anticoagulant without direct thrombin inhibition. Overall, the specific spatial array of the sulfated fucose branches in the fucosylated chondroitin sulfate not only confer high anticoagulant activity to the polysaccharide but also determine differences in the way it inhibits thrombin.

Thrombin binding properties of insoluble modified polystyrene: Part II.

Antithrombin III (AT III) inhibits thrombin via an arginine-serine interaction. Insoluble polystyrene resins grafted with arginyl methyl ester have been synthesized, and their interaction with thrombin tested. One of these resins was selected for its high affinity for thrombin. In this paper we report the characteristics of this thrombin resin interaction. Using this substituted polystyrene resin as a support for affinity chromatography, we have compared the binding of thrombin with that of other proteins (prothrombin, Factor IXa, trypsin and AT III). It was found that 0.7 mg of highly purified human thrombin (2,100 U/mg) was bound to 1 g of resin. This could only be eluted at high ionic strength (1.5 M) and the amidolytic and clotting activities of the eluted thrombin remained unchanged. The binding of thrombin to the resin involves the active site of the enzyme but also other residues since, when DIP thrombin was used, the inactive enzyme could be eluted at lower ionic strength (1.0 M). This resin seems to be specific for thrombin because it does not bind the other serine-proteases (trypsin or Factor IXa), prothrombin (the inactive precursor of thrombin) or AT III. The arginyl residues of the resin are important for the specificity of the interaction with Factor IIa since prolyl residues are totally ineffective. Chromatography performed on such a resin is a very efficient method of purifying thrombin, and may be very useful for the removal of thrombin as a contaminant of plasma protein fractions.

[Heparin inhibition of the antithrombin activity of alpha-2-macroglobulin]. / Inhibition par l'héparine de l'activité antithrombique de l'alpha-2-macroglobuline.

The interaction between thrombin and alpha-2-macroglobulin was studied on human purified materials, either in the presence or in the absence of heparin, by kinetic analysis of thrombin inhibition and polyacrylamide gel electrophoresis. In the absence of heparin, binding of thrombin to alpha-2-macroglobulin, shown by electrophoresis, leads to the loss of the coagulant property of the enzyme. In the presence of heparin the rate of inhibition of thrombin clotting activity by alpha-2-macroglobulin is strongly decreased. Heparin binds to thrombin, impairing the formation of thrombin-alpha-2-macroglobulin complex. These data show that heparin paradoxically protects thrombin from inhibition by alpha-2-macroglobulin whereas it increases the enzyme inhibition by antithrombin III. Such a phenomenon could be of practical interest for treatment of thrombosis in patients with high plasma level of alpha-2-macroglobulin and low level of antithrombin III, such as occurs in the nephrotic syndrome.

High-performance affinity chromatography of human thrombin on modified polystyrene resins.

Cross-linked polystyrenes modified with L-arginyl methyl ester mimic the binding site of antithrombin III and thrombin substrates. They can be used as stationary phases in high-performance affinity chromatography of thrombin. Under isocratic conditions, thrombin is strongly adsorbed on the resins when the sodium chloride concentration is lower than 0.5 M. The bound enzyme can be selectively desorbed when the salt concentration is raised to about 1.2 M. With a linear salt gradient, the specific elution of thrombin can be effected with a high recovery of its enzymatic activity. The decomposition products of thrombin, when treated with sodium dodecyl sulphate, are not retained by the stationary phase. The effects of the flow-rate and salt gradient slope on the adsorption and desorption of alpha-thrombin demonstrate the importance of kinetic parameters.

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

High resolution electron microscopy reveals that fully active alpha 2-macroglobulin (alpha2M) from fresh human plasma presents a very characteristic tetrameric structure. This native conformation of the alpha2M 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 alpha2M 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.

Immobilization of dermatan sulphate on a silica matrix and its possible use as an affinity chromatography support for heparin cofactor II purification.

Dermatan sulphate (DS) is a glycosaminoglycan which catalyses specifically thrombin inhibition by a plasmatic inhibitor, Heparin cofactor II (HCII). DS was insolubilized on a silica matrix to study its interaction with HCII. The immobilization of DS was performed with a good yield on a silica previously coated with polysaccharides in order to neutralize the negatively charged silanol groups. The value of the affinity constant of insolubilized DS for HCII, measured by the adsorption isotherm, is consistent with the value obtained for soluble DS. The DS bound to the silica matrix was also tested as a chromatographic support for the purification of HCII from human plasma; the optimum conditions for HCII adsorption and desorption were determined. The eluted HCII was obtained with a good yield (21%) and with no contamination by antithrombin III, the other main plasmatic inhibitor of thrombin.

Thrombin purification by one-step preparative affinity chromatography on modified polystyrenes.

Insoluble polystyrenes substituted with sulphonate and L-arginyl methyl ester have been synthesized. Using their specific affinity for thrombin, we developed a simple one-step chromatographic procedure for thrombin purification. As a control, insoluble polystyrenes substituted only with sulphonate groups were tested. The results obtained confirmed the importance of the arginyl residues grafted onto these polymers to obtain an affinity matrix useful for purifying thrombin with a high specific activity and a good recovery.

Affinity chromatography of thrombin on modified polystyrene resins.

Insoluble polystyrenes substituted with sulphonate and L-arginyl methyl ester (PAOM) present substituents mimicking the reactive binding site of antithrombin III. These materials have a specific affinity for thrombin. The binding of the enzyme is reversible and the eluted thrombin remains active. Consequently, these resins can be used as stationary phases in affinity liquid chromatography in order to purify thrombin with a high biological activity. The influence of different characteristics of such polymers (substitution ratio, average particle size, affinity constant, synthesis conditions) on the purification performance is studied. Human prothrombin complex concentrate is activated and applied onto the gel. A purified human thrombin of high specific activity is separated with a high recovery of biological activity of the enzyme.

Prothrombin Poissy: a new variant of human prothrombin.

A new congenital dysprothrombinaemia is described in a newborn baby girl who presented severe bleeding from the second day of life. Routine coagulation tests showed very prolonged prothrombin time and activated partial thromboplastin time with about 2% prothrombin activity in a one-stage assay. Staphylocoagulase and Echis carinatus venom prothrombin assays were respectively 35% and 25%. The prothrombin antigen level was 47% and its migration in crossed immuno-electrophoresis was abnormal. Family study revealed the presence of both normal and abnormal prothrombin in the plasma of three family members: the father, the mother and the brother. Thrombin generation in a system free from natural inhibitors showed that the abnormal prothrombin was slowly and incompletely activated. The propositus is thought to be homozygous for a 'lazy' dysprothrombin.

Recombinant hirudin (HBW 023): biological data of ten patients with severe venous thrombo-embolism.

This study reports on the biological data of ten patients with acute venous thrombo-embolism. They were treated for 5 days with continuous intravenous infusion of a fixed dose (0.05 mg/kg/hr) of a recombinant hirudin (r-H HBW 023 Behringwerke, Germany). The plasma level of r-H (HBW 023), assessed by an anti-factor IIa amidolytic activity, was stable after Day 2 and showed considerable individual variations. It correlated with APTT ratio, suggesting that this test is a reliable tool to monitor therapy. In contrast, thrombin time was constantly over 120 sec (control 15 sec) and consequently was not a useful parameter. Prothrombin time showed a slight, but significant, prolongation, which was correlated with the increase of APTT ratio. There was no bleeding time prolongation, platelet count, or ATIII level decrease. Levels of thrombin-antithrombin III complexes, and D-dimers, which were high in all patients on admission, decreased during the course of the treatment but remained abnormal on Day 5, showing an ongoing hemostasis and fibrinolysis activation: this is consistent with the delayed, but only slightly decreased thrombin generation evidenced by thrombin generation test performed on Day 3. These results suggest that thrombin inhibition by rH-hirudin at this dosage is only partial, which allows the generation of traces of thrombin needed for the feed-back thrombin production generated by factor V and VIII activation.