Thrombin-Cleaved Osteopontin Is Regulated by Thrombin-Activatable Carboxypeptidase B in Rheumatoid Arthritis.

The pro-inflammatory cytokine osteopontin (OPN) is present in rheumatoid arthritis (RA) synovial fluids (SF). OPN can be cleaved by thrombin exposing the cryptic C-terminal α4β1/α9β1 integrin-binding motif (SVVYGLR). We have previously shown that thrombin-activated proCPB (CPB) can remove the C-terminal arginine from the SVVYGLR motif abrogating its α4β1 binding. Using peptides based on the C-terminus of thrombin-cleaved OPN (OPN-R) and thrombin/CPB-cleaved OPN (OPN-L), rabbit anti-OPN-R and anti-OPN-L antibodies were generated. Specificity of these antibodies was demonstrated by western blot analysis. Specific ELISAs for OPN-R and OPN-L showed significant elevation of OPN-R and OPN-L levels in SF from patients with RA (n=26) as compared to osteoarthritis (OA, n=18) and psoriatic arthritis (PA, n=10). (OPN-R median values: 138.6 ng/mL (RA), 10.6 ng/mL (OA), and 2.2 ng/mL (PA) p <0.001; OPN-L median values 205.3 ng/mL (RA), 25.9 ng/mL (OA), and undetectable in PA p <0.003). Multiplex cytokine analysis showed that elevated OPN-R and OPN-L levels in RA were significantly correlated with multiple inflammatory cytokines including IL-6, IL-12p40 and FGF-2, while OPN-FL levels only exhibited a statistical correlation with IL-6. Synovial lining cells expressed functional TM that supported the activation of proCPB to CPB by thrombin. ProCPB activation by thrombin, which may occur either systemically or locally within the inflamed joint space, inactivates thrombin-cleaved OPN and C5a and represents a homeostatic response in modulating the inflammatory process in RA.