Covalent Linkage of Recombinant Hirudin to a Novel Ionic Poly(Carbonate) Urethane Polymer with Protein Binding Sites: Determination of Surface Antithrombin Activity

Abstract

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 EDU (cPU-BSA-SMCC). This cPU-BSA-SMCC surface was then reacted with Traut's-modified 125I-rHir, a procedure which created free sulfhydryl groups on rHir (cPU-BSA-SMCC-S-125I-rHir). Using these crosslinking procedures, the cPU-BSA-SMCC-S-125I-rHir segments bound 188 +/- 40 ng/cm2 (n = 60) whereas the controls with non-specifically bound 125I-rHir (Mitrathane + EDC + BSA + 125I-rHir-SH and cPU-BSA + 125I-rHir-SH) bound 13 +/- 8 ng/cm2 and 4 +/- 8 ng/cm2, respectively. Evaluation of these cPU-BSA-SMCC-S-125I-rHir segments for 131I-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 bound 0.76 and 0.70 NIHU. Controls with nonspecifically bound 125I-rHir also had 0.31 and 0.76 NIHU 131I-thrombin adherent to their respective surfaces whereas the maximum 131I-thrombin binding to the cPU-BSA-SMCC-S-rHir segments was 1.51 NIHU. Exposure to 131I-thrombin did not result in any release of covalently bound 125I-rHir from the cPU-BSA-SMCC-S-125I-rHir segments. Thus, these results demonstrate that rHir can be covalently bound to this novel polyurethane surface and still maintain potent antithrombin activity.

Bioactive materials in the circulatory system

“…Active substances, such as the enzymes urokinase [25] and lumbrokinase [26], hirudin [27], human thrombomodulin [28], or inhibitors for activation and aggregation of platelets, such as prostacyclin [29], have been used to modify polymeric surfaces. The bioactive substances are either tightly bound to the surface or simply blended into the polymer system for controlled release.…”

Modified Prosthetic Vascular Conduits

“…This material was shown to retain antithrombin properties in vitro; however, no in vivo studies have been conducted to date. 26 Hirudin, in combination with iloprost, an inhibitor of platelet aggregation and promoter of vasodilation, has been used to modify ePTFE grafts. Heise et al 27 used these 2 drugs, along with PEG, as the delivery mechanism to coat 4-mm ePTFE grafts.…”

A biologically active VEGF construct in vitro: Implications for bioengineering‐improved prosthetic vascular grafts

“…17,18 Briefly, VEGF was radiolabeled with 200 ci 125 I using an iodo-bead reaction for tracing purposes. I-VEGF-SH complex was purified by gel filtration using a PD-10 column.…”