Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation q S.C. Freitas a,b , T.B. Cereija c , A.C. Figueiredo c , H. Osório d , P.J.B. Pereira c , M.A. Barbosa a,b,e , M.C.L. Martins a,e,⇑ a INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal b Universidade do Porto, Faculdade de Engenharia, Portugal c IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal d IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal e Universidade do Porto, Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal article info Article history: Received 8 February 2012 Received in revised form 5 July 2012 Accepted 16 July 2012 Available online 27 July 2012 Keywords: Hemocompatibility Coagulation Thrombin Surface functionalization Protein adsorption abstract Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assem- bled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after sur- face immobilization, boophilin was biotinylated after the formation of a boophilin–thrombin complex to minimize the biotinylation of the residues involved in thrombin–boophilin interaction. The extent of boo- philin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using 125 I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophi- lin-free surfaces, demonstrating boophilin’s potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Thrombus formation is still one of the major complications affecting blood-contacting medical devices [1]. Blood clotting in- duced by the surface of these devices is due to a complex series of highly interlinked events, starting with the adsorption of plasma proteins, followed by the activation of complement and coagulation systems and by the adhesion and activation of platelets and leuko- cytes [2]. Initial protein adsorption is dependent on the chemical and physical properties of the biomaterial surface [1]. One strategy to increase the hemocompatibility of these devices could be the surface immobilization of antithrombotic agents, such as thrombin inhibitors [3], since this enzyme plays a central role in the coagula- tion system. Thrombin is produced at the end of the coagulation cascade and is responsible for converting fibrinogen to fibrin. After polymerization, fibrin is crosslinked and stabilized into an insoluble gel by the thrombin-activated factor XIIIa. Thrombin is also able to stimulate platelet activation and to promote its own production through activation of factors V, VIII and XI [4]. In addition to these procoagulant activities, thrombin plays important roles in inflam- mation [5] and in the activation of complement factors [6]. The indirect thrombin inhibitor heparin has been used to coat the surface of several biomaterials [7,8]. However, heparin-coated surfaces have severe limitations. They only inhibit thrombin after binding of endogenous antithrombin (AT), which limits the thera- peutic use of heparin-coated surfaces in patients with low amounts of AT (e.g., in some septic situations) [9,10]. Other disadvantages include the inability of the heparin–AT complex to inhibit enzy- matically active and procoagulant fibrin-bound thrombin and the scavenging of heparin by platelet factor 4 released by activated platelets [11,12]. 1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.07.020 q No benefit of any kind will be received either directly or indirectly by the authors. ⇑ Corresponding author at: INEB – Instituto de Engenharia Biomédica, Univer- sidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. Tel.: +351 22 6074984; fax: +351 22 6094567. E-mail address: cmartins@ineb.up.pt (M.C.L. Martins). Acta Biomaterialia 8 (2012) 4101–4110 Contents lists available at SciVerse ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat