HEPARIN SURFACE IMMOBILIZATION THROUGH HYDROPHILIC SPACERS - THROMBINAND ANTITHROMBIN-III BINDING-KINETICS

The immobilization of heparin onto polymeric surfaces using hydrophili c spacer groups has been effective in curtailing surface induced throm bus formation. In this study, the effect of hydrophilic spacers (PEO) on the binding kinetics of immobilized heparin with antithrombin Ill ( ATIII) and thrombin was investigated. Monodispersed, low molecular wei ght heparin was fractionated on an ATIII affinity column to isolate hi gh-ATIII affinity heparin. This high-ATIII affinity fraction was immob ilized onto a styrene/p-amino styrene random copolymer surface using h ydrophilic poly(ethylene oxide) (PEO) spacer groups. Styrene/p-amino s tyrene random copolymer was chosen as the model surface to provide qua ntitative and reproducible surface concentrations of available amine g roups, grafted PEO spacers, and immobilized heparin. The polymer subst rate was coated onto glass beads, tolylene diisocyanate modified PEO w as covalently coupled to the surface, followed by heparin immobilizati on. The bioactivity of immobilized heparin was 16.2%, relative to free heparin, and a 1:1 binding ratio between hepar-in and PEO was achieve d. The binding of ATIII and thrombin to control surfaces (no heparin), soluble heparin, heparin immobilized directly onto the surface, and h eparin immobilized via spacer groups, were compared. Soluble heparin b ound both thrombin and ATIII, while heparin immobilized directly onto the surface bound only thrombin. Spacer-immobilized heparin bound both ATIII and thrombin, although to a lesser extent than soluble heparin. Thus, the enhanced bioactivity of spacer-immobilized heparin, compare d to direct-immobilization, may be attributed to the retention of ATII I binding.