Evidence for S-nitrosothiol-dependent changes in fibrinogen that do not involve transnitrosation or thiolation.
S-nitrosoglutathione (GSNO, 50 microM) inhibited the initial rate of thrombin-catalyzed human and bovine fibrinogen polymerization by approximately 50% to 68% respectively. Inhibition was also observed with other structurally varied S-nitrosothiols (RSNOs) including sugar derivatives of S-nitroso-N-acetylpenicillamine (SNAP). The fact that the same concentration of GSNO had no effect on thrombin-dependent hydrolysis of tosylglycylprolylarginine-4-nitroanilide acetate suggested that this inhibition was due to GSNO-induced changes in fibrinogen structure. This result was confirmed by CD spectroscopy where GSNO or S-nitrosohomocysteine increased the alpha-helical content of fibrinogen by approximately 15% and 11%, respectively. S-carboxymethylamido derivatives of glutathione or homocysteine had no effect on the fibrinogen secondary structure. The GSNO-dependent secondary structural effects were reversed on gel filtration chromatography, suggesting that the effects were allosteric. Further evidence for fibrinogen-GSNO interactions was obtained from GSNO-dependent quenching of the intrinsic fibrinogen Trp fluorescence and the perturbation of the GSNO circular dichroic absorbance as a function of [fibrinogen]. The K(d)s of 3 to 10 microM for fibrinogen-GSNO interactions with a stoichiometry of 2:1 (GSNO:fibrinogen) were estimated from isothermal titration calorimetry and fluorescence quenching, respectively. These results suggest that RSNOs induce changes to fibrinogen structure by interacting at specific aromatic rich domains. Three such putative RSNO-binding domains have been identified in the unordered, aromatic residue-rich C-termini of the alpha-chains of fibrinogen.
Akhter
Shirin
S
Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada N9B 3P4.akhter@server.uwindsor.ca
Vignini
Arianna
A
Wen
Zhong
Z
English
Ann
A
Wang
Peng G
PG
Mutus
Bulent
B
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005340:Fibrinogen / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D017433:Protein Structure, Secondary / Q000187:drug effects; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D026403:S-Nitrosothiols / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D026403:S-Nitrosothiols; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D014364:Tryptophan; D005340:Fibrinogen; D013917:Thrombin; D019803:Glutathione Disulfide