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

Human ceruloplasmin (CF) is a multifunctional copper binding protein produced in the liver. CF oxidizes Fe2+ to Fe3+, decreasing the concentration of Fe2+ available for generating harmful oxidant species. CF is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (K-d=130 nM), 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 CF in vitro at (481)Arg-Ser(482) and (887)Lys-Val(888) 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 CF is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CF 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 CF (K-d=60-270 nM), independent of proteolysis, and inhibits CF ferroxidase activity (K-I=220 +/- 20 nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen gamma'-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, beta(T)-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CF, 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 CF via proteolysis and by competitively inhibiting CF 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. (C) 2015 Elsevier Inc. All rights reserved.