Antithrombogenic Biomaterials: Surface Modification with an Antithrombin-Heparin Covalent Complex

Abstract

<p>Surface-induced thrombosis is a continuing issue in the development of biomaterials for blood contacting applications. Protein adsorption is a key factor in thrombosis since it occurs rapidly upon contact of a material with blood, initiating coagulation and other adverse reactions including platelet adhesion. The research presented in this thesis explores the use of a unique antithrombin-heparin covalent complex (ATH) for surface modification to provide antithrombogenicity. ATH was tethered to surfaces by various methods. Polyethylene oxide (PEO) was investigated as a linker-spacer molecule for surface attachment of ATH as well as for its antifouling properties.</p> <p>In the first phase of the work gold was used as a model substrate. ATH was attached by three different methods: direct attachment, attachment via a short chain linker, and attachment via PEO. Analogous heparin-modified surfaces were prepared for comparison. Surfaces were characterized using contact angle measurements, x-ray photoelectron spectroscopy (XPS), ellipsometry and quartz-crystal microbalance (QCM). The data suggested that the heparin moiety of ATH was directed away from the surface, in an orientation allowing ready interaction with blood components. The ATH-modified surfaces showed greater antithrombin binding than the heparin-modified surfaces as measured by radioactive labelling and Western blotting analysis. Antithrombin binding was found to occur predominantly through the active pentasaccharide sequence of the heparin moiety of ATH, demonstrating the potential of the ATH for catalytic anticoagulant function. From measurements of the ratio of total heparin to active heparin (anti-factor Xa assay), ATH-modified surfaces were shown to have greater bioactivity than heparin-modified surfaces. The adhesion of platelets to gold and modified gold surfaces was measured from flowing whole blood <em>in vitro</em> using a cone-and-plate device and was lower on all of the modified surfaces compared to bare gold. PEO-ATH surfaces were also shown to prolong plasma clotting times compared to control and heparinized surfaces.</p> <p>In subsequent work, surface modification methods were developed for polyurethane (PU) substrates. Isocyanate groups were introduced into the PU surface for attachment of PEO and ATH was attached to the “distal” end of the PEO. Surfaces using PEO of varying molecular weight and end group were investigated to determine conditions for maximum anticoagulant activity and minimum non-specific protein adsorption. Surfaces were characterized using contact angle measurements and XPS, and protein interactions were studied using radiolabelling. The optimum balance of bioactivity and protein resistance was found to occur with PEO of low to mid range MW (ie. MW 300-600). These PU-PEO-ATH surfaces showed low fibrinogen adsorption and high selectivity for antithrombin. Consistent with results using gold substrates, platelet adhesion remained low when ATH was attached to polyurethane surfaces grafted with PEO. A hetero-bifunctional amino-carboxy-PEO (PEO-COOH surface) was compared with a “conventional” homo-bifunctional dihydroxy-PEO (PEO-OH surface) with respect to their effectiveness as linkers for attachment of ATH. The PEO-COOH-ATH surface was shown to bind slightly greater amounts of antithrombin, indicating higher catalytic anticoagulant activity. Thrombin binding was measured to determine whether the surfaces could provide direct anticoagulant activity. The PEO-OH-ATH surface bound high amounts of thrombin, indicating potential for direct thrombin inhibition. It is hypothesized that the PEO properties (MW and functional end group) may have an effect on the orientation of ATH on the surface thus influencing its "preference" for catalytic vs. direct anticoagulant function.</p> <p>This thesis provides new information regarding the interactions of proteins and platelets with ATH immobilized on biomaterials. ATH-modified surfaces were superior to analogous heparin-modified surfaces with respect to antithrombin binding and catalytic anticoagulant ability. Immobilized ATH was also shown to bind thrombin, suggesting potential for direct anticoagulant activity. It can thus be seen as a unique surface modifier with dual functioning anticoagulant activity. The modification of polyurethane with ATH using PEO as a protein resistant linker-spacer, may provide a material of improved antithrombogenicity for the construction of blood contacting devices.</p>