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Covalent Linkage of Recombinant Hirudin to a Novel Ionic Poly(Carbonate) Urethane Polymer with Protein Binding Sites: Determination of Surface Antithrombin Activity
Author(s) -
Phaneuf Matthew D.,
Szycher Michael,
Berceli Scott A.,
Dempsey Donald J.,
Quist William C.,
LoGerfo Frank W.
Publication year - 1998
Publication title -
artificial organs
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.684
H-Index - 76
eISSN - 1525-1594
pISSN - 0160-564X
DOI - 10.1046/j.1525-1594.1998.05091.x
Subject(s) - hirudin , covalent bond , antithrombin , recombinant dna , chemistry , ionic bonding , polymer , polymer chemistry , biochemistry , heparin , thrombin , organic chemistry , biology , gene , platelet , immunology , ion
Surface thrombus formation on implantable biomaterials such as polyurethane is a major concern when utilizing these materials in the clinical setting. Thrombin, which is responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous surface modification strategies in an effort to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) onto a novel polyurethane polymer synthesized with carboxylic acid groups which served as protein attachment sites. The in vitro efficacy of thrombin inhibition by this novel biomaterial surface was then evaluated. Bovine serum albumin (BSA), which was selected as the basecoat protein, was reacted with sulfo‐SMCC in a 1:50 molar ratio. This BSA‐SMCC complex was then covalently linked to the carboxylated polyurethane (cPU) surface via the crosslinker EDC (cPU‐BSA‐SMCC). This cPU‐BSA‐SMCC surface was then reacted with Traut's‐ modified 125 I‐rHir, a procedure which created free sulfhydryl groups on rHir (cPU‐BSA‐SMCC‐S‐ 125 I‐rHir). Using these crosslinking procedures, the cPU‐BSA‐SMCC‐S‐ 125 I‐rHir segments bound 188 ± 40 ng/cm 2 (n =60) whereas the controls with nonspecifically bound 125 I‐rHir (Mitrathane + EDC + BSA + 125 I‐rHir‐SH and cPU‐BSA + 125 I‐rHir‐SH) bound 13 ± 8 ng/cm 2 and 4 ± 8 ng/cm 2 , respectively. Evaluation of these cPU‐BSA‐SMCC‐S‐ 125 I‐rHir segments for 131 I‐thrombin inhibition using a chromogenic assay for thrombin showed that a maximum of 2.64 NIHU thrombin was inhibited in contrast to the controls which inhibited 0.76 and 0.70 NIHU. Controls with nonspecifically bound 125 I‐rHir also had 0.31 and 0.76 NIHU 131 I‐thrombin adherent to their respective surfaces whereas the maximum 131 I‐thrombin binding to the cPU‐BSA‐SMCC‐S‐rHir segments was 1.51 NIHU. Exposure to 131 I‐thrombin did not result in any release of covalently bound 125 I‐rHir from the cPU‐BSA‐SMCC‐S‐ 125 I‐rHir segments. Thus, these results demonstrate that rHir can be covalently bound to this novel polyurethane surface and still maintain potent antithrombin activity.