The effect of immobilization of thrombin inhibitors onto self-assembled monolayers on the adsorption and activity of thrombin q Sido ´ nio C. Freitas a, b , Ma ´ rio A. Barbosa a, b , M. Cristina L. Martins a, * a INEB – Instituto de Engenharia Biome ´dica, Divisa ˜o de Biomateriais, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal b Universidade do Porto, Faculdade de Engenharia, Departamento de Engenharia Metalu ´rgica e Materiais, Porto, Portugal article info Article history: Received 11 November 2009 Accepted 15 January 2010 Available online 11 February 2010 Keywords: Thrombin Self-assembled monolayers Surface functionalization Peptide immobilization Protein adsorption abstract Thrombus formation is the major problem associated with biomaterials for blood contact medical devices. The immobilization of inhibitors to thrombin, a serine protease that plays a central role on the coagulation system, on the surface of biomaterials should be a good strategy to avoid blood clotting and increase their hemocompatibility. The aim of this work is the design of a nanostructured surface with capacity to adsorb and inactivate thrombin. The pentapeptide sequence D-Phenylalanine–Proline– Arginine–Proline–Glycine (fPRPG), that was described as a thrombin inhibitor, was immobilized onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs). Surface containing different amounts of fPRPG were prepared using different concentrations of N,N 0 -Carbonyldiimidazole (CDI) during immobilization. The efficiency of fPRPG immobilization was followed using ellipsometry, contact angle measurements, Infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS). Thrombin adsorption was quantified using radiolabelled thrombin and its activity in solution and after adsorption on the developed surfaces was assessed using a chromogenic assay. It was found that, although the immobilization of fPRPG on to EG4-SAMs does not increase its selectivity to thrombin, the activity of the adsorbed thrombin was inhibited in a peptide concentration dependent way. We concluded that SAMs with fPRPG immobilized in high amounts can be used as thrombin- inhibitor surfaces, which is a good step on the development of new surfaces for blood contact devices. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Blood contact medical devices (stents, catheters, vascular grafts) often induce thrombus formation and inflammation. These two processes are interlinked and involve the activation of the coagu- lation and the complement systems, and the activation and adhe- sion of platelets and leukocytes. These processes are usually initiated by the adsorption of plasma proteins which is dependent of the chemical and physical properties of the surface [1,2]. The key protein of coagulation system is thrombin which is produced after tissue or vessel injury or after the contact of blood with a foreign surface like a surface of a biomaterial. This serine protease cleaves fibrinogen into fibrin which is polymerized, crosslinked and stabilized into a blood clot by factor XIII, also activated by thrombin. In addition, thrombin stimulates its own production by the activation of factors V, VIII and XI and induces platelet activation which supports the blood clotting [1,3]. Thrombin have two basic anion-binding exosites (ABE I and ABE II). The ABE I, adjacent to the active site is known as the fibrinogen recognition exosite, and the ABE II is identified as the heparin- binding site [4]. Thrombin cleaves, almost exclusively, peptide bonds after arginine residues. Its active-site region comprise three distinct pockets: the specific pocket S1 contains an aspartic acid (Asp189) which determines its preference to substrates with a basic P1 residues like arginine; the S2 pocket accommodates relatively small and hydrophobic substrate residues as proline and the S3/4 pocket can harbour hydrophobic and aromatic residues as phenylalanine [4,5]. One strategy to increase the hemocompatibility of synthetic materials is the coating of the surface with thrombin inhibitors [6–8]. Heparin is the most widely used drug to prevent blood clotting, having similar properties with heparan sulfate of the endothelium. This compound has been immobilized on the surface of several materials in order to create anticoagulant surfaces [9]. Neverthe- less, heparin only inhibits thrombin indirectly, by the activation of antithrombin III (AT III), which could be a problem in some septic situations where the amount of AT III is very low [10]. In addition, q No benefit of any kind will be received either directly or indirectly by the authors. * Corresponding author. Tel.: þ351 22 6074982; fax: þ351 22 6094567. E-mail address: cmartins@ineb.up.pt (M.C.L. Martins). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.01.097 Biomaterials 31 (2010) 3772–3780