Marcel Jozefowicz

Anticoagulant effects of sulphonated polyurethanes.

Sulphonated polyurethanes have been shown to have excellent blood contacting properties. In this paper, similar polyurethanes which are water soluble have been investigated to determine their influence on thrombus formation. These polymers were shown to delay clotting times in the following ways: by direct complex formation between the polymer and thrombin; by interference with fibrin polymerization; and by complex interactions between polymer, thrombin, plasma antiproteases and fibrinogen in plasma.

Interactions of biospecific functional polymers with blood proteins and cells.

Biospecific functional polymers, i.e. synthetic or artificial polymers substituted with specific chemical functional groups carried by the macromolecular chain are designed to interact with living systems. These polymers are either insoluble or soluble, derived from polystyrene and dextran. Polymers substituted with aryl sulfonate and carboxyl groups specifically interact with antithrombin III and serine-proteases involved in the coagulation of blood. As a consequence, these polymers possess heparin-like activity and are therefore of low thrombogenicity when exposed to flowing blood. Other functional polymers have been prepared in order to interact with various components of the immune system. Soluble and insoluble functional polymers in contact with cells can affect both cell proliferation and metabolism. Some functional polymers have the ability to inhibit or to stimulate cell growth while others can alter cell function without a change in growth characteristics. The functional polymers described have possible applications as plasma expanders, non-thrombogenic catheters, non-complement activating surfaces and other applications in oncology, biotechnology and immunochemistry.

Heparin-like activity of insoluble sulphonated polystyrene resins. Part I: Influence of the surface density, nature and binding of substituted anionic groups.

It was previously demonstrated that copolystyrene (sulphonate-amino acid sulphamide) resins possessed an anticoagulant heparin-like activity in the presence of blood plasma. Taking into account the variable surfaces of swollen resins developed by these dry resins, it is now shown that the antithrombic activity of crosslinked sulphonated polystyrene is linearly dependent on the surface density of the sulphonate groups. This fact implies that the presence of such isolated groups is sufficient to obtain a catalytic site for increasing the rate of inactivation of thrombin by plasmatic proteins. It is also shown that replacing sulphonate groups either by directly backbone-bonded carboxylate groups or by methionine linked by amide bonds to polystyrene backbone is not sufficient to endow the resulting resins with a significant anticoagulant activity.

Mechanism of factor IXa inhibition by antithrombin in the presence of unfractionated and low molecular weight heparins and fucoidan

Heparin exerts its anticoagulant activity by catalysing the inhibition of coagulation proteases by antithrombin (AT). Its main target is thrombin but it also catalyses the inhibition of the other serine-proteases of the coagulation cascade, such as factor IXa (fIXa). The aim of this study was to compare the catalysis of inhibition of blood fIXa by antithrombin in the presence of several sulfated polysaccharides with anticoagulant activity, i.e. heparin, three widely used in therapeutics low molecular weight heparins (LMWH) and fucoidan. Plots of the second-order rate constants of the fIXa-antithrombin reaction vs. the concentration of added heparin and LMWH are bell-shaped and fit the kinetic model established for thrombin-antithrombin reaction by Jordan R., Beeler D., Rosenberg R. (1979) J. Biol. Chem., 254, 2902-2913. In the ascending branch, the catalyst (C) binds quickly to the inhibitor (I) to form a catalyst-inhibitor (CI) complex which is more reactive towards the enzyme (E) than the free inhibitor, leading to the formation of an inactive enzyme-inhibitor complex (EI) and the release of free catalyst, in a rate-limiting second step. After a maximum corresponding to an optimal catalyst concentration, the decrease in the reaction rate was in keeping with the formation of a catalyst-enzyme (CE) complex, whose inactivation by the CI complex was slower than that of the free enzyme. Maximum second-order rate constants for the inhibition of fIXa by AT were 105, 6.8, 12.24 and 22 microM-1 min-1 with heparin, Enoxaparin, Fraxiparin and Fragmin, respectively, leading to 3500-, 225-, 405- and 728-fold increases in the inhibition rate in the absence of polysaccharide, respectively. Fucoidan yielded 23-fold increase in the fIXa-antithrombin interaction rate. The kinetic profiles obtained with this polysaccharide exhibited ascending branch which correlated well with the kinetic model based on the formation of binary complexes (CI or CE). Fucoidan was covalently conjugated with a fluorescent probe (DTAF) and used in conjunction with fluorescence anisotropy to follow its binding to antithrombin, heparin cofactor II (HCII), thrombin and fIXa. The binding of fucoidan to these proteins occurred with low affinities when compared to heparin and LMWH. Fucoidan had higher affinity for the inhibitor HCII compared to antithrombin and enzymes. These data suggest that binding of heparins and fucoidan to the inhibitor (CI) is required for the polysaccharide-dependent enhancement in the rate of neutralization of the enzyme by the inhibitor.

Heparin-like activity of insoluble sulphonated polystyrene resins Part III: Binding of dicarboxylic amino acids

It has been demonstrated previously that polystyrene sulphonate possesses anticoagulant properties and that the binding of some amino acids could enhance the heparin-like properties of such resins. These properties depend on the surface density of the active groups, the nature and binding of the group and on the net change borne by the polymer. In this paper, we describe the preparation of copolystyrene (sulphonate-dicarboxylic amino acid sulphamide) resins. By measuring their antithrombotic-surface-activity, we demonstrate that the activity developed by each carboxyl group is at least roughly the same as the activity of one sulphonate group, except in the case of aspartic acid sulphamide resin for which a cooperative effect is shown. The anticoagulant properties of resins bearing phosphonate or monocarboxylic amino acid sulphamides are also examined.

Anticoagulant activity of amino acid modified polystyrene resins: influence of the carboxylic acid function

In previous papers, we have described the preparation and heparin-like properties of insoluble modified polystyrene resins. We now report results obtained with amino acid sulphamide resins that are virtually devoid of sulphonate groups and with resins bearing both sulphonate groups as well as amino acid sulphamides with spacers of various lengths. The absence of sulphonate groups does not affect the anticoagulant activity of the former type of resin. The biologic activity of the latter type of resin is dependent upon the length of the spacer between the amino and carboxylic acid functions. Maximal anticoagulant potency is attained with a spacer consisting of three methylene groups. Biologic activity is reduced with spacers that are less than or greater than this critical size.

Catalysis of the generation of thrombin-antithrombin complex by insoluble anticoagulant polystyrene derivatives

The inhibition of thrombin by antithrombin III is known to be accelerated by heparin through the formation of complexes between the muccopolysaccharide and both proteins. In the preceding papers, we reported that polystyrene derivatives absorb thrombin and its inhibitor with a higher affinity for the protease than for the antiprotease. These complexes are responsible for the catalysis of the generation of thrombin-antithrombin complex which was observed either with purified proteins or in plasma. The protease-antiprotease complex has an affinity for the polymer surface which is higher than that of antithrombin but lower than that of thrombin. Therefore, the thrombin-antithrombin complex generated on the insoluble material is desorbed by thrombin and a catalytic anticoagulant effect can be observed with these polymers.

Regulation by sulphonate groups of complement activation induced by hydroxymethyl groups on polystyrene surfaces

Reducing the complement-activating capacity of a polymer surface is important in improving its blood compatibility. Polystyrene surfaces bearing hydroxymethyl (CH2OH) groups activate the alternative pathway of complement. This activation depends strongly on the density of the groups. Polystyrene surfaces bearing sulphonate (SO3-) groups adsorb proteins, resulting in an apparent activation. Polystyrene surfaces bearing both types of groups in close proportions are not activators in human serum, due to the adsorption of a protein of the alternative pathway, which has a protecting effect, not found when a polymer surface bearing hydroxyl groups is mixed in serum with another polymer surface bearing SO3- groups. In the presence of purified proteins of alternative pathway, C3 convertase activity can be created on each of these surfaces by deposition of C3b, but their susceptibility to inactivation by regulatory proteins H and I depends on the types of chemical groups present on the surface and whether the surfaces were passivated or not before C3b deposition.

Affinity of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: I. In vitro adsorption studies

In previous papers, we described insoluble polystyrene derivatives which exhibit a heparin-like antithrombic activity in plasma. In order to ascertain the heparin-like mechanism of this activity we have studied the interactions of thrombin and antithrombin III with two polymers of this series: sulphonated polystyrene and sulphonate-glutamic acid sulphonamide polystyrene. The adsorption was measured using purified enzyme and enzyme inhibitor and polymer beads whose average diameter was about 25 micron. The maxima of adsorption approximately correspond to a monolayer of protein. The results are discussed with respect to the most common isotherms used in chemisorption and the affinities of the enzyme and its inhibitor for both materials are evaluated: kT congruent to 10(7) (M/I)-1, kAT congruent to 3.10(5) (M/I)-1.

Complement activation and adsorption of protein fragments by functionalized polymer surfaces in human serum

The interactions between blood and polymer surfaces used in extracorporeal circulations result in variable activations of the immune system of complement. Measuring concentrations of C3a or C5a in supernatant blood or serum after contact with the surface has been the most usual way of assessing this activation. Most polymer surfaces bearing various chemical groups were found to adsorb C3a and sometimes C5a. After taking into account adsorption, a good correlation was found between total C3a generated and CH50 units consumed by most of the polymer samples tested. Measuring only C3a remaining in the fluid phase should not be considered sufficient to conclude that a material surface is not an activator of complement.