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.

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 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.

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.

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.

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.

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.

Interactions of human platelets with insoluble anticoagulant modified polystyrene resins.

The interactions of two insoluble anticoagulant polystyrene derivatives with human platelets were studied in an in vitro system similar to a chromatography column. Blood was pumped through the column and platelets were counted before and after passage through the column. Interaction of platelets with the beads led to retention of platelets in the column. The same experiment was performed with several donors. Different pretreatments were assayed and compared for both materials: platelet poor plasma, antithrombin III-depleted platelet poor plasma and an antithrombin III solution. Platelet retention depends on the polymer composition: the material containing glutamic acid sulphamide groups, which has a larger anticoagulant activity in plasma than the materials only substituted by sulphonate groups, is always less reactive towards platelets. The differences in the effects of pretreatment on both materials can be correlated with the variations of antithrombin adsorption on the synthetic surfaces.

Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces.

Heparin-like materials, characterized by a defined superficial density of functional groups which activate antithrombin III (AT III), when in contact with blood specifically inhibit thrombin as soon as it appears. This paper describes an isotopic method to estimate this density and to visualize the distribution of the affinity sites concerned, both directly with AT III labelled with 125Iodine and indirectly with an anti AT III monoclonal antibody labelled with 111Indium.

Interactions of anticoagulant insoluble modified polystyrene resins with plasmatic proteins.

In previous papers, we have described the preparation and heparin-like properties of insoluble modified polystyrene resins. We now report results about the interactions of several coagulation factors with some of these materials. Most of the non-activated factors are neither adsorbed nor modified, except factor V and prekallikrein. In contrast thrombin, antithrombin III and factor Xa adsorb on the surface of such insoluble polymers. Thrombin can be desorbed by addition of a polycationic compound. The inactivation of thrombin or factor Xa by antithrombin is catalysed by the presence in the mixture of these insoluble materials as it is with soluble heparin. The initial velocity of these reactions is second order for both types of catalysis-homogeneous or heterogeneous - as previously reported for soluble heparin. In the case of these insoluble materials, the inhibition of protease by antiprotease seems to be accelerated when complexes are formed between the polymer and the proteins.

ESCA studies on heparin-like materials used for extracorporeal shunts.

Amino-acids sulfamide substituted polystyrene surfaces exhibit an heparin-like activity. Similar treatments were achieved on small diameter tubings made of polystyrene grafted on polyethylene. Electron spectroscopy for Chemical Analysis (ESCA) appeared as the suitable method for a chemical characterization of the surface (approximately 50-100 A depth) to be in contact with circulating blood. These tubings were implanted as extracorporeal shunts on dogs. The variations of local concentrations of labelled platelets, red cells and fibrinogen were recorded in situ. Depending on the conditions of preparation and implantation, a slight adhesion of platelet was observed. By using such type of materials with an improved mechanical compliance, small diameter antithrombogenic tubings could be developed, provided the chlorosulfonation prior to amino-acid grafting is mild.