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 the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.
De Cristofaro
R
R
Hemostasis Research Center, Department of Internal Medicine, Catholic University School of Medicine, Rome, Italy. rdecristofaro@rm.unicatt.it
Landolfi
R
R
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000378:metabolism / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D006490:Hemostatics; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006868:Hydrolysis; D006997:Hypochlorous Acid / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D010455:Peptides / Q000737:chemistry; D009195:Peroxidase / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D011486:Protein C / Q000737:chemistry / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism
D002712:Chlorides; D006490:Hemostatics; D010455:Peptides; D011486:Protein C; D018180:Thrombomodulin; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D011732:Pyridoxal Phosphate; D006997:Hypochlorous Acid; D000990:Antithrombin III; D006493:Heparin; D006861:Hydrogen Peroxide; D009195:Peroxidase; D013917:Thrombin; D008239:Lysine