title: Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis.
abstract: Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe(2+) to Fe(3+), decreasing the concentration of Fe(2+) 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 (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 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.
authors.affiliation: Institute for Experimental Medicine, Pavlov str., 12, Saint Petersburg, 197376 Russia; State University of Saint Petersburg, University Embankment, 4-7, Saint Petersburg, 199034 Russia.
authors.forename: Alexej V
authors.initials: AV
authors.lastname: Sokolov
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, via Marzolo, 5, Padua, 35131 Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Acquasaliente
authors.affiliation: Institute for Experimental Medicine, Pavlov str., 12, Saint Petersburg, 197376 Russia.
authors.forename: Valeria A
authors.initials: VA
authors.lastname: Kostevich
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, via Marzolo, 5, Padua, 35131 Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Frasson
authors.affiliation: Institute for Experimental Medicine, Pavlov str., 12, Saint Petersburg, 197376 Russia.
authors.forename: Elena T
authors.initials: ET
authors.lastname: Zakharova
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, via Marzolo, 5, Padua, 35131 Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Pontarollo
authors.affiliation: Institute for Experimental Medicine, Pavlov str., 12, Saint Petersburg, 197376 Russia; State University of Saint Petersburg, University Embankment, 4-7, Saint Petersburg, 199034 Russia.
authors.forename: Vadim B
authors.initials: VB
authors.lastname: Vasilyev
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, via Marzolo, 5, Padua, 35131 Italy. Electronic address: vincenzo.defilippis@unipd.it.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Filippis
mesh_terms: D000368:Aged; D001172:Arthritis, Rheumatoid / Q000201:enzymology*; D016022:Case-Control Studies; D002570:Ceruloplasmin / Q000737:chemistry* / Q000502:physiology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D009195:Peroxidase / Q000737:chemistry / Q000502:physiology; D054730:Protein Interaction Domains and Motifs; D013917:Thrombin / Q000737:chemistry* / Q000502:physiology
chemical_list: D009195:Peroxidase; D002570:Ceruloplasmin; D013917:Thrombin
keywords: Ceruloplasmin; Myeloperoxidase; Oxidants; Protein–protein interaction; Proteolysis; Rheumatoid arthritis; Surface plasmon resonance; Thrombin