Hirudin-thrombin complex : an approach to prepare a blood compatible surface

The incorporation of pharmacologically active agents within biomaterials is a technique used to prepare haemocompatible surfaces. Presently, hirudin a well characterized antithrombotic drug and a potent inhibitor of thrombin, is gaining clinical acceptance. This study proposes the use of a hirudin-thrombin complex as a unique protein complex effective in creating a non thrombogenic surface and preventing the activation of the clotting pathway. The inhibition of thrombin clotting activity by hirudin was investigated. Hirudin was able to bind to the noncatalytic fibrinogen binding site on thrombin and thus block the thrombin catalyzed cleavage of fibrinogen to form fibrinopeptide A (FPA). The behavior of hirudin-thrombin complex, preadsorbed thrombin neutralized with hirudin, free thrombin, and hirudin were investigated onto the glass bead surface. The Hirudin-thrombin complex was adsorbed onto the surface by two methods: (i) by adsorbing the complex from solution and (ii) by addition of hirudin to beads preadsorbed with thrombin. The surface was then assayed for its ability to interact with fibrinogen and S-2238. Hirudin-thrombin complex adsorbed onto the glass beads by both methods did not stimulate the cleavage of FPA from fibrinogen, nor did it cleave S-2238. In other experiments, pure thrombin was also adsorbed onto the glass surface and its ability to cleave fibrinogen and hydrolyze S-2238 was investigated. Thrombin adsorbed on the surface retained its ability to cleave fibrinogen and S-2238. Hirudin adsorbed onto the glass surface did not retain its activity to bind to thrombin, and thus did not prevent activation of thrombin and its substrates. These results suggest that by physically adsorbing the hirudin-thrombin complex onto a surface, the thrombin component was neutralized in stimulating further pathways in the coagulation system. Thus, it may be possible to use this inert complex of hirudin and thrombin as a coating material to improve the haemocompatibility of blood contacting surfaces.