title: Covalent linkage of recombinant hirudin to poly(ethylene terephthalate) (Dacron): creation of a novel antithrombin surface.
abstract: Thrombus formation and intimal hyperplasia on the surface of implantable biomaterials such as poly(ethylene terepthalate) (Dacron) vascular grafts are major concerns when utilizing these materials in the clinical setting. Thrombin, a pivotal enzyme in the blood coagulation cascade primarily responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous strategies to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) to a modified Dacron surface and characterize the in vitro efficacy of thrombin inhibition by this novel biomaterial surface. Bovine serum albumin (BSA), which was selected as the "basecoat' protein, was reacted with various molar ratios of the cross-linker sulphosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulpho-SMCC; 1:5-1:50). These BSA-SMCC complexes were then covalently linked to sodium hydroxide-hydrolysed Dacron (HD) segments via the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Covalent linkage of these complexes to HD (HD-BSA-SMCC) was not affected by any of the sulpho-SMCC cross-linker ratios assayed. rHir, which was initially reacted with 2-iminothiolane hydrochloride (Traut's reagent) in order to create sulphydryl groups, was then covalently bound to these HD-BSA-SMCC surfaces (HD-BSA-SMCC-S-rHir). The 1:50 (BSA: sulpho-SMCC) HD-BSA-SMCC-S-rHir segments bound 22-fold more rHir (111 ng per mg Dacron) compared to control segments and also possessed the greatest thrombin inhibition of the segments evaluated using a chromogenic substrate assay for thrombin. Further characterization of the HD-BSA-SMCC-S-rHir segments demonstrated that maximum thrombin inhibition was 20.43 NIHU, 14.6-fold greater inhibition than control segments (1.4 NIHU). Thrombin inhibition results were confirmed by 125I-thrombin binding experiments, which demonstrated that the 1:50 HD-BSA-SMCC-S-rHir segments had significantly greater specific thrombin adhesion compared to control segments. Non-specific 125I-thrombin binding to and release from the 1:50 HD-BSA-SMCC-S-rHir segments was also significantly less than the control segments. Thus, these results demonstrate that rHir can be covalently bound to a clinically utilized biomaterial (Dacron) while still maintaining its ability to bind and inhibit thrombin.
authors.affiliation: Deaconess Hospital/Harvard Medical School, Vascular Surgery Research, Boston, MA 02215, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Phaneuf
authors.forename: S A
authors.initials: SA
authors.lastname: Berceli
authors.forename: M J
authors.initials: MJ
authors.lastname: Bide
authors.forename: W C
authors.initials: WC
authors.lastname: Quist
authors.forename: F W
authors.initials: FW
authors.lastname: LoGerfo
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001672:Biocompatible Materials / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008301:Maleimides; D011093:Polyethylene Terephthalates / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012709:Serum Albumin; D013499:Surface Properties; D013917:Thrombin / Q000378:metabolism
chemical_list: D000991:Antithrombins; D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D006629:Hirudins; D008301:Maleimides; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D012709:Serum Albumin; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D013917:Thrombin