Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.
Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDP peptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen.
Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St. Louis, Missouri, USA.
Vitaliy
V
Ovod
Evan A
EA
Scott
Megan M
MM
Flake
Stanley R
SR
Parker
Randall J
RJ
Bateman
Donald L
DL
Elbert
D000327:Adsorption; D019921:Biotinylation; D002460:Cell Line; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008422:Materials Testing; D011093:Polyethylene Terephthalates / Q000737:chemistry*; D013499:Surface Properties
D011093:Polyethylene Terephthalates; D005340:Fibrinogen; D008239:Lysine