Marion P. Olivieri

Human serum albumin and fibrinogen interactions with an adsorbed RGD-containing peptide

The modification of polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) with an arginine-glycine-aspartic acid cell adhesion peptide, RGD peptide (PepTite Adhesive Coating; Telios Pharmaceuticals, San Diego, CA) has been previously investigated. Initial animal studies showed this RGD peptide to accelerate healing and assist in the formation of an endothelial cell lining of the lumenal side of PET and PTFE fabrics in a cardiovascular application. It is of interest to determine how this RGD peptide is able to influence cellular events through intervening layers of plasma proteins that spontaneously adsorb upon implantation. This study examined the interaction of predeposited RGD-containing peptide with human serum albumin (HSA) or fibrinogen that was characterized using multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy, ellipsometry, and contact angle analysis. It was determined that fibrinogen-containing films consistently exhibited more mass than films of the RGD peptide, HSA, or HSA adsorbed onto RGD peptide-containing films. MAIR-IR spectra of RGD peptide films before and after HSA adsorption were similar in absorption and intensity; however, ellipsometry indicated HSA introduction had created thicker, less dense films. Fibrinogen, on the other hand, when adsorbed onto RGD peptide films provided increased relative mass in a more compact arrangement. Contact angle analyses of each of the dried films showed their surface energies to remain high, but the polar components of RGD peptide films were reduced after either serum protein adsorption. These phenomena may be related to the minimal thrombus accumulation that was noted during the initial animal studies, that promoted subsequent healing.

Using Conformational Analysis to Identify Structurally Conserved Regions of MAP Peptides that Exhibit Cellular Attachment Ability

A natural bioadhesive obtained from Mytilus edulis , mussel adhesive protein, MAP or mefp-1, is frequently used for cellular attachment. MAP is approximately 114 kD, and generally composed of repeating decapeptide units, A-K-P-S-Y-Hyp-Hyp-T-DOPA-K, MAP-RD. Prior nuclear magnetic resonance (NMR) spectroscopy and molecular modeling of MAP-RD revealed an overall bent-helix. NMR spectroscopy and molecular modeling of a MAP fourteen residue peptide, P-S-Y-Hyp-Hyp-T-Y-K-A-K-P-S-Y-Hyp, MAP-14, are presented. Additionally, a molecular model built and minimized from MAP-RD and MAP-14 produced a twenty-six-residue MAP peptide, (MAP-26), that maintained regional structural consistency with both MAP-RD and MAP-14. Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy and ellipsometry of the MAP-14 as well as that of L-DOPA-containing MAP-14, (MAP-14(D)), showed uniform film formation near a monolayer in thickness was L-DOPA dependent. Significantly more undifferentiated leukocyte cells (MOLT-4) attached to and spread on MAP-14 (D) films (applied at 2 w g cm m 2 ) compared with intact MAP, MAP-RD or tissue culture treated polystyrene, indicating a cellular binding domain presence in the MAP-14 sequence. The culmination of biophysical data indicate the lysine-alanine-lysine (K-A-K) sequence as structurally conserved and responsible for the cellular attachment ability noted for MAP14(D) and ultimately MAP.

Structural and Biophysical Characterization of a Cyclic Bioadhesive with Cell Attachment Ability

Structural and cellular attachment analysis identified overall bent helical regions of adhesive peptides identified within mussel adhesive protein (MAP) capable of also attaching cells. DOPA (L-DOPA, 3,4-dihydroxyphenylalanine) is frequently identified and credited for the attachment ability of several marine proteins. Newly designed cyclic peptides (DOPA-G-G-C-G-K-A-K-G-C [cyc-DOPA] & Y-G-G-C-G-K-A-K-G-C [cyc-Y]) derived from structurally conserved regions of several MAP peptides were examined to assist in the understanding of both surface and cellular attachment. Solution-state proton nuclear magnetic resonance (NMR) spectroscopy coupled with molecular modeling and dynamics revealed minimal differences in the structures of the proposed cellular attachment domain within these two peptides. Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy, ellipsometry, and advancing contact angle analyses showed that formation of thin films by these peptides was L-DOPA- and pH-dependent. When compared with control surfaces, undifferentiated leukocyte cells (MOLT-4) significantly attached and spread onto films created from the cyc-DOPA. The culmination of these structural, biophysical, and cellular attachment techniques reveal a conformation of cyc-DOPA that is capable of both adsorbing to surfaces and then attaching cells that spread. This work supports the sequence K-A-K as the cellular attachment domain, especially when held in a reliable structural conformation.