title: Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: a mass spectrometric and isotope labeling study.
abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called "aspirin resistance". We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to "aspirin resistance" in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the α-chain: αK191, αK208, αK224, αK429, αK457, αK539, αK562, in the β-chain: βK233, and in the γ-chain: γK170 and γK273. Glycations were found at βK133 and γK75, alternatively γK85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [(14)C-acetyl]salicylic acid and [(14)C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 μM aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm, Sweden. jan.svensson@ki.se
authors.forename: Jan
authors.initials: J
authors.lastname: Svensson
authors.forename: Ann-Charlotte
authors.initials: AC
authors.lastname: Bergman
authors.forename: Ulf
authors.initials: U
authors.lastname: Adamson
authors.forename: Margareta
authors.initials: M
authors.lastname: Blombäck
authors.forename: Håkan
authors.initials: H
authors.lastname: Wallén
authors.forename: Gun
authors.initials: G
authors.lastname: Jörneskog
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001241:Aspirin / Q000737:chemistry*; D005340:Fibrinogen / Q000737:chemistry*; D005343:Fibrinolytic Agents / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry; D006031:Glycosylation; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
chemical_list: D005343:Fibrinolytic Agents; D005340:Fibrinogen; D005947:Glucose; D008239:Lysine; D001241:Aspirin