Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis

• Published in: Free radical biology & medicine Vol. 86; pp. 279 - 294
• Main Authors: Sokolov, Alexej V., Acquasaliente, Laura, Kostevich, Valeria A., Frasson, Roberta, Zakharova, Elena T., Pontarollo, Giulia, Vasilyev, Vadim B., De Filippis, Vincenzo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2015
• Subjects: Aged; Arthritis, Rheumatoid - enzymology; Case-Control Studies; Ceruloplasmin; Ceruloplasmin - chemistry; Ceruloplasmin - physiology; Female; Humans; Male; Middle Aged; Models, Molecular; Myeloperoxidase; Oxidants; Peroxidase - chemistry; Peroxidase - physiology; Protein Interaction Domains and Motifs; Protein–protein interaction; Proteolysis; Rheumatoid arthritis; Surface plasmon resonance; Thrombin; Thrombin - chemistry; Thrombin - physiology; PAR; Ceruloplasmin; TBS; PPACK; Thrombin; DLS; WB; CP; MPO; Protein–protein interaction; RA; SEC; S2238; SPR; Oxidants; PMSF; Proteolysis; Rheumatoid arthritis; ROS; Myeloperoxidase; HBS-EP; Surface plasmon resonance; ABTS
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.05.016

Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe2+ to Fe3+, decreasing the concentration of Fe2+ available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (Kd=130nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at 481Arg–Ser482 and 887Lys–Val888 bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (Kd=60–270nM), independent of proteolysis, and inhibits CP ferroxidase activity (KI=220±20nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen γ′-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, βT-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity. Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease. [Display omitted] •Ceruloplasmin (CP) oxidizes toxic Fe2+, inhibits leukocyte myeloperoxidase, and is cleaved by the coagulative protease thrombin exclusively at two sites•The nicked protein (CP*) lacks myeloperoxidase inhibitory function but retains the ferroxidase activity of the intact protein•Proteolytically degraded CP has been found in the synovial fluid of rheumatoid arthritis patients•Thrombin binds to CP and inhibits ferroxidase activity by a mechanism independent of proteolysis•Thrombin acts as a stimulator of oxidant species production by abrogating the myeloperoxidase inhibitory function of CP and inhibiting the CP ferroxidase function