bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation

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-assembled monolayers (sam) via neutravidin. in order to maintain its proteinase inhibitory capacity after surface 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 boophilin 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 ¹²⁵i-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 boophilin-free surfaces, demonstrating boophilin’s potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.