Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: Structural and functional effects

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in th e pathogenesis of inflammation and atherothrombosis. This study investigate d the biochemical and functional effects of alpha-thrombin oxidation by MPO S. This system, in the presence of 100 mu M L-tyrosine, caused in the throm bin molecule loss of tryptophan and lysine residues and formation of dityro sine, chloramine and carbonyl groups. The same changes could be directly in duced by thrombin incubation with reagent HOCl, but not with H2O2 alone. Ex posure to either MPOS or HOCl caused major functional abnormalities in huma n cu-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being t he k(cat)/K-m value for Protein C hydrolysis roughly reduced 13-fold and th e affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed seve ral changes, arising from a perturbation of the S2-S3 specificity of the en zyme. Ox-thrombin was also characterized by a 5-fold decrease of the k(cat) /K-m value for both fibrinopeptide A and B release from fibrinogen, a 5.8-f old increase of the EC50 value for platelet activation and a 2-fold decreas e of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Per turbed interactions with Protein C and the heparin-ATIII complex were the m ost relevant functional abnormalities of ox-thrombin.