Paper BI-MoA10
Platelets Adhere to Adsorbed Albumin through a Receptor-Mediated Process

Monday, November 9, 2009, 5:00 pm, Room K

Since albumin (Alb) lacks known sequences recognized by platelet receptors, it is not supposed to support platelet adhesion. However, studies have suggested that platelets may be able to adhere to adsorbed Alb (1, 2) with adhesion related to adsorption-induced Alb unfolding (1), although the mechanisms of this remain unclear. To address this issue, we conducted studies to definitively determine if platelets adhere to adsorbed Alb, whether adhesion is related to adsorbed Alb conformation,and if it occurs by a receptor-mediated process. Alb was adsorbed at 0.1, 1.0, and 10 mg/mL on various alkanethiol SAM surfaces to vary the degree of unfolding in the adsorbed Alb. The adsorption-induced conformational changes in Alb was quantified by CD spectropolarimetry (3). Platelet adhesion studies were carried out and the platelet response determined by LDH assay and SEM. A series of platelet adhesion inhibitors and protein modification agents were used to probe the mechanisms of platelet adhesion. Platelet adhesion to adsorbed Alb was negligible when adsorbed Alb retained most of its native structure (< 34% loss in alpha-helix), but began to linearly increase with the degree of adsorption-induced unfolding thereafter (r²=0.92). SDS-PAGE results showed that the platelet suspension was free of residual proteins and anti-Alb polyclonal antibodies completely inhibited platelet adhesion to adsorbed Alb, but had negligible effect on adsorbed Fg (used as a control); thus confirming that the platelets were adhering to adsorbed Alb and not some other residual protein in the system. Addition of an RGDS peptide to the platelet suspension strongly inhibited platelet adhesion to adsorbed Alb (~60% reduction on CH₃ SAM; p < 0.01), while the addition of RGES peptide had no inhibitory effect. Neutralization of arginine residues in the adsorbed Alb layer using 2,3-butanedione reduced platelet adhesion to a similar degree as exposing the platelets to the RGDS peptide in solution. These results indicate that the adhesion of nonactivated platelets to adsorbed Alb is primarily mediated by RGD-specific receptors and the degree to which the binding domains in adsorbed Alb are exposed and/or formed is directly proportional to the degree of adsorption-induced unfolding of the protein. Further studies will be carried out to identify the specific platelet receptors and Alb domains that mediate adhesion. These results indicate that we have much yet to learn about the mechanisms that influence platelet adhesion to adsorbed proteins.

1. Hylton et al, J. Biomed. Mater. Res. 73A, 349 (2005).

2. Rodrigues et al, Biomaterials 27, 5357 (2006).

3. Sivaraman et al, Langmuir 25, 3050 (2009).