title: Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation.
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-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.
authors: INEB-Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors: S C
authors: SC
authors: Freitas
authors: T B
authors: TB
authors: Cereija
authors: A C
authors: AC
authors: Figueiredo
authors: H
authors: H
authors: Osório
authors: P J B
authors: PJ
authors: Pereira
authors: M A
authors: MA
authors: Barbosa
authors: M C L
authors: MC
authors: Martins
mesh_terms: D000327:Adsorption / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001672:Biocompatible Materials / Q000494:pharmacology*; D057005:Bioengineering; D019921:Biotinylation / Q000187:drug effects; D001777:Blood Coagulation / Q000187:drug effects; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D055767:Immobilized Proteins / Q000378:metabolism; D008422:Materials Testing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013499:Surface Properties; D013917:Thrombin / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000991:Antithrombins; D001672:Biocompatible Materials; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D013917:Thrombin