C. Fougnot

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.

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.

Heparin-like tubings. I. Preparation, characterization and biological in vitro activity assessment.

In order to prepare tubular materials which could be used in blood-circulating medical devices, polystyrene was grafted by irradiation on to polyethylene tubings. A chemical surface treatment was used which resulted in the functionalization of the inner face of the tubing. This procedure is described and the chemical assessment of the constitution of the functionalized polymer has been completed. Tubing, the inner face of which is made of polyethylene-polystyrene copolymer in which polystyrene moieties were substituted with sulphonate and aspartic acid sulphamid groups, was tested for antithrombic properties in a circulating device under controlled transport conditions and by use of purified proteins.

Adsorption of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: II. Competition with the other plasma proteins.

In the preceding paper, we described results concerning the adsorption of purified thrombin and antithrombin III on two insoluble anticoagulant polystyrene derivatives. We now report similar results obtained in a plasma system. In each case, the purified protein was mixed with fresh platelet poor plasma in order to maintain the same concentrations of all the other plasma proteins. The thrombin molecule was modified by alkyl phosphorylation of the active serine site prior to mixing with plasma. The adsorption of antithrombin was found to be reduced 8 to 9 times when the protein solution was substituted by diluted plasma. In contrast, the thrombin adsorption only depends on the substituents bound on the polymeric chain. These results are supported by those of the study of the competition between purified antithrombin and albumin.

Heparin-like tubings: III. Kinetics and mechanism of thrombin, antithrombin III and thrombin-antithrombin complex adsorption under controlled-flow conditions

In previous papers, we described treated tubular materials which exhibit an heparin-like antithrombic activity under dynamic conditions. In order to ascertain the heparin-like mechanism of this activity, we have studied the interactions of thrombin, antithrombin III and thrombin-antithrombin III complex with the inner face of these treated tubings under controlled-flow conditions. Moreover, the kinetics of the adsorption of thrombin were studied at different flow rates to establish the rate-determining step.

Heparin-like tubings. II. Mechanism of the thrombin-antithrombin III reaction at the surface.

In a previous paper we described the surface treatment of tubings made of polystyrene grafted by irradiation onto polyethylene tubular materials. As a result, polystyrene moieties of their inner face were substituted by sulphonate and aspartic acid sulphamide groups. In order to study the mechanism of the thrombin-antithrombin III reaction occurring at the modified surface and to determine the kinetics of the reaction, step by step experiments were set up involving either the protease adsorbed at the surface reacting with the antiprotease circulating in the solution or the opposite. These procedures allowed us to demonstrate the heparin-like catalytic activity of the tubings.

Adsorption of plasma proteins onto anticoagulant polystyrene derivatives: a fluorescence study

Quenching of fluorescence was used to monitor adsorption of thrombin (T), antithrombin (AT) and their inactive complex (T-AT) onto three anticoagulant biomaterials made of polystyrene beads bearing the functional groups of heparin. An adsorption capacity of 0.12 mumol of T per mg of polymer allowed the formation of a monolayer of protein at the polymer surface. An affinity constant of 3 x 10(7) l.mol-1 between thrombin and polymer was estimated, whatever the polymer used. The affinity of T-AT was similar although weaker. Desorption of proteins from the polymeric interface by means of polycations (polybrene and polylysine) showed that the inactive complex T-AT is more quantitatively and easily released than thrombin.

Interactions of Some Plasmatic Proteins with Anticoagulant Polystyrene Resins: Mechanism of Catalytic Activity Towards the Thrombin— Antithrombin Reaction

Polymeric materials having antithrombogenic activity are very important and their development is expected in the field of artificial organs such as the artificial vessel or the devices for extracorporeal circulation. In previous papers1,2 we described that the binding of sulfonate and amino acid sulfamide groups onto cross- linked polystyrene endows these materials with antithrombic activity which requires the presence of a plasma cofactor, antithrombin mi These insoluble materials operate as catalysts of the inactivation of thrombin by its inhibitor as does soluble heparin2. The catalytic effect of this mucopolysaccharide was demonstrated to require the formation of complexes between heparin and either antithrombin III or thrombin or both4, 5, 6.