Evidence for S -nitrosothiol-dependent changes in fibrinogen that do not involve transnitrosation or thiolation

Abstract

S-nitrosoglutathione (GSNO, 50 M) inhibited the initial rate of thrombin-catalyzed human and bovine fibrinogen polymerization by Ϸ50% 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 thrombindependent 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 ␣-helical content of fibrinogen by Ϸ15% 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 ds of 3 to 10 M 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 ␣-chains of fibrinogen.

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…Measurements of both the release of fibrinopeptide (by HPLC) and the generation of fibrin monomers (by electrophoresis) confirmed that oxidation-induced inhibition of clotting activity derived from an effect on fibrin monomer polymerisation, not from inhibition of thrombin activity [186]. S-nitrosothiols can induce changes in fibrinogen structure by interacting at specific domains rich in aromatic amino acids [187].…”

Influence of Glutathione and its Derivatives on Fibrin Polymerization

“…S-Nitrosoglutathione and other nitrosothiols have been previously reported to inhibit the initial rates of fibrin polymerization at low micromolar concentrations, although the exact mechanism remains unclear. 32 Herein, the dose-dependent effects of GSNO were evaluated for the entire process of fibrin polymerization to gain a deeper understanding of which phases of fibrin formation are affected by nitrosothiol species. Similar to results described above for GSH, inhibition of fibrin formation was observed in the presence of GSNO with a maximum inhibition occurring at 500 µM ( Figure 5).…”

S‐nitrosothiols as selective antithrombotic agents – possible mechanisms

“…Conversely, exposure of fibrinogen to the RSNO compound GSNO suppresses fibrin polymerization (i.e. an antithrombotic effect) through what appears to be an allosteric interaction separate from covalent modification of the fibrinogen molecule (Akhter et al, 2002;Geer et al, 2008). RSNOs may further oppose thrombosis by inhibiting the action of transglutaminase enzymes, including coagulation factor XIII (Catani et al, 1998;Lai et al, 2001).…”