This study describes the protein interaction properties of hirudin-thrombin complex adsorbed onto a surface. Hirudin-thrombin complex, preadsorbed thrombin bound with hirudin, and thrombin were coated onto the surfaces of glass beads. The activity of the thrombin component adsorbed onto the surface, and hence the ability of hirudin to bind and inactivate thrombin, was determined by measuring the activity of thrombin to cleave fibrinogen to generate fibrin and to cleave a chromogenic substrate, 5-2238. Pure thrombin adsorbed on the surface (without hirudin) retained its activity to cleave fibrinogen and cleave S-2238. Hirudin-thrombin complex adsorbed on the surface did not stimulate the activation of fibrinogen, nor did it cleave S-2238.
Thrombin first adsorbed onto the surface was able to bind hirudin; this complex did not activate fibrinogen or the hydrolysis of S-2238. However, when hirudin was first adsorbed onto the surface followed by incubation with thrombin, the protein did not bind and neutralize thrombin, and therefore lost its biologic activity. Furthermore, all proteins and the hirudin-thrombin complex adsorbed onto the surface, as determined by protein binding assays. These results suggest that the hirudin-thrombin complex physically adsorbed onto the surface did not stimulate subsequent pathways in the coagulation system, mainly the activation of fibrinogen to fibrin.