Heparin surface immobilization through hydrophilic spacers: Thrombin and antithrombin III binding kinetics

Abstract

The immobilization of heparin onto polymeric surfaces using hydrophilic spacer groups has been effective in curtailing surface induced thrombus formation. In this study, the effect of hydrophilic spacers (PEO) on the binding kinetics of immobilized heparin with antithrombin III (ATIII) and thrombin was investigated. Monodispersed, low molecular weight heparin was fractionated on an ATIII affinity column to isolate high-ATIII affinity heparin. This high-ATIII affinity fraction was immobilized onto a styrene/p-amino styrene random copolymer surface using hydrophilic poly(ethylene oxide) (PEO) spacer groups. Styrene/p-amino styrene random copolymer was chosen as the model surface to provide quantitative and reproducible surface concentrations of available amine groups, grafted PEO spacers, and immobilized heparin. The polymer substrate was coated onto glass beads, tolylene diisocyanate modified PEO was covalently coupled to the surface, followed by heparin immobilization. The bioactivity of immobilized heparin was 16.2%, relative to free heparin, and a 1:1 binding ratio between heparin and PEO was achieved. The binding of ATIII and thrombin to control surfaces (no heparin), soluble heparin, heparin immobilized directly onto the surface, and heparin immobilized via spacer groups, were compared. Soluble heparin bound 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, compared to direct-immobilization, may be attributed to the retention of ATIII binding.