Peptide Modified Gold Coated Polyurethane Surfaces as Thrombin Scavengers

Abstract

Gold, as a chemically inert metal, does not form a stable oxide, but has strong specific interactions with sulfur functions. It has been found that thiols or disulfides can chemisorb to gold under mild conditions (room temperature), and form densely packed monolayers on the gold surface due to the high density of binding sites (gold atoms). Thus, it is possible to form closely packed and stable monolayers of thiolates containing desirable bioactive moieties. Thiol-gold chemistry may therefore be considered as a potentially important tool in the surface modification of materials for biomedical applications. In order to develop surfaces with antithrombogenic properties, a number of thrombin inhibitors (heparin, hirudin and PPACK) have been bonded or immobilized to the material surfaces. In the present study, a series of short peptides, cys-pro-arg (CPR), cys-phe-pro-arg (CFPR) and cys-(D)-phe-pro-arg (C[D]FPR), analogues of PR and FPR respectively, were chosen as potential thrombin inhibitors to attach to gold-coated polyurethane surfaces via the cysteine residue. Their inhibitory activity against thrombin was verified by a chromogenic substrate assay. C(D)FPR, showed a relatively high level of inhibition activity. The surfaces were characterized by water contact angle, XPS, AFM, SEM, ellipsometry, and infrared reflection-absorption spectroscopy, and the adsorption of thrombin from buffer and modified plasma was investigated. It was found that the peptide modified gold surfaces adsorbed significantly more thrombin than the unmodified control surfaces. The C(D)FPR-modified gold surface showed the highest thrombin adsorption both from buffer and plasma. This results is in accord with previous studies showing that the D form of phenylalananine in the FPR peptide creates a favourable site geometry for binding to thrombin. The activity of thrombin adsorbed on these peptide modified gold surfaces was also investigated using a chromogenic substrate assay. Inhibition of adsorbed thrombin was demonstrated, and the C(D)FPR surface showed the strongest inhibitory activity. The presence of thrombin on the peptide surfaces following exposure to modified plasma was verified by elution of proteins and identification of thrombin in the eluate. Probing of the eluates with antibodies to 25 plasma proteins showed that the peptide surfaces are relatively non-adsorptive, suggesting they have some degree of selectivity for thrombin binding.