Uwe Streller

Bestimmung der Blutverträglichkeit von Materialoberflächen mit immobilisiertem Thrombininhibitor

A major complication with biomaterials in blood contact is blood coagulation followed by thrombus formation and stenosis. The immobilization of bioactive substances to either inhibit blood coagulation proteins or platelets are keypoints in research efforts to improve the hemocompatibility of biomaterials. We report herein a strategy based on the surface immobilization of small synthetic amidine derivatives known as selective and potent inhibitors acting directly on thrombins active site. The immobilization of biomimetic synthetic coagulation inhibitors to the surface of biomaterials could achieve a directed prevention of blood coagulation. Starting with 4-aminobenzamidine we sythesized an inhibitor and could show through molecular modeling that several of the relevant aminoacids participate in the interaction of the thrombin inhibitor with the binding pocket. A covalent immobilization was realized using thin films of poly(octadecene-alt-maleic anhydride) (PO-MA) attached to a solid support. We prepared four different surfaces. The first was attained through hydrolysis of the anhydride moieties of the copolymer film and resulted in a negatively charged surface (M1). The second was a polyethylene glycol (PEG) modified surface (M2). These two served as reference samples. The immobilization of the thrombin inhibitor was accomplished by either direct modification of thin films of PO-MA (M3) or using a bifunctional PEG spacer, linked to PO-MA (M4). The in vitro hemocompatibility assays were conducted using in-house-built incubation chambers with an advantageous relation of testing surface to blood volume. The generated materials (M1 4) were incubated with freshly drawn human blood anticoagulated with heparin for 2 hours at 37 °C. After incubation, the blood was analyzed with respect to changes in cell numbers and activation markers for coagulation (TAT), complement activation (C5a) and thrombogenicity (PF4) using commercially available ELISA assays. The surface of the incubated materials was analyzed for adherent platelets and fibrin deposition by SEM. We could show that the highest activation for all the parameters was attained for the highly negatively charged surface M1. The PEG-surface showed considerably less activation probably due to the well known inhibition of protein adsorption. The immobilization of thrombin inhibitor further reduced the activation of coagulation and complement compared ORIGINAL ARBEITEN Claudia Sperling: Bestimmung der Blutverträglichkeit von Materialoberflächen mit immobilisiertem Thrombininhibitor Abb. 9: REM Aufnahme der Probenoberfläche nach der Inkubation mit humanem Vollblut. M 1: Poly(octadecen-alt-maleinsäureanhydrid) hydrolysiert, M 2: PO-MA + Jeffamin, M 3: PO-MA + TI, M 4: PO-MA + PEG + TI