SURFACE-ADSORPTION AND FIBRINOGEN INTERACTIONS WITH HIRUDIN-THROMBIN COMPLEX

This study describes the protein interaction properties of hirudin-thr ombin complex adsorbed onto a surface. Hirudin-thrombin complex, pread sorbed thrombin bound with hirudin, and thrombin were coated onto the surfaces of glass beads. The activity of the thrombin component adsorb ed onto the surface, and hence the ability of hirudin to bind and inac tivate thrombin, was determined by measuring the activity of thrombin to cleave fibrinogen to generate fibrin and to cleave a chromogenic su bstrate, S-2238. Pure thrombin adsorbed on the surface (without hirudi n) retained its activity to cleave fibrinogen and cleave S-2238. Hirud in-thrombin complex adsorbed on the surface did not stimulate the acti vation of fibrinogen, nor did it cleave S-2238. Thrombin first adsorbe d onto the surface was able to bind hirudin;this complex did not activ ate 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 i ts biologic activity. Furthermore, all proteins and the hirudin-thromb in complex adsorbed onto the surface, as determined by protein binding assays. These results suggest that the hirudin-thrombin complex physi cally adsorbed onto the surface did not stimulate subsequent pathways in the coagulation system, mainly the activation of fibrinogen to fibr in. (C) 1996 John Wiley & Sons, Inc.