Differentially Cleaved Forms of Osteopontin by Thrombin and Carboxypeptidase B (TAFIa) Regulates Neutrophil Viability and Synoviocyte Binding in Rheumatoid Arthritis

Osteopontin (OPN) is a pro-inflammatory cytokine that plays a significant role in rheumatoid arthritis (RA). We have recently shown that OPN is a bona fide substrate for thrombin. OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal a4b1 and a9b1 integrin-binding motif (SVVYGLR). Procarboxypeptidase B (proCPB or thrombin-activatable fibrinolysis inhibitor TAFI), upon activation to CPB (TAFIa) by the thrombin/thrombomodulin complex on endothelial cell surface, removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell-binding. We have developed specific ELISAs for OPN-R and OPN-L. While intact OPN (OPN-FL) was significantly elevated in RA synovial fluid (SF) (n=26, median level 352.5 ng/mL), as compared to its normal plasma level, it was not significantly higher in osteoarthritis (OA, n=13, 157.9 ng/mL, .142) or psoriatic arthritis (PsA, n=10, 143.4 ng/mL, .074) SF samples. On the other hand, highly significant elevation of OPN-R and OPN-L levels was detected in the RA SF, as compared to OA and PsA (median values of OPN-R 138.6 ng/mL, 10.6 ng/mL and 2.2 ng/mL, p <0.003 and OPN-L 205.3 ng/mL, 25.9 ng/mL and undetectable, p <0.006 in RA, OA and PsA respectively). Significant disease heterogeneity was noted. The median value for the ratio of OPN-R and OPN-L to total OPN was approximately 0.5, indicating extensive enzymatic cleavage activity in a significant subset of patients. In RA SF, elevated OPN-R and OPN-L levels were significantly correlated with multiple inflammatory cytokines, including IL-6, IL-12p40, and FGF-2 while OPN-FL levels only correlated with IL-6. Fibroblast-like synoviocytes, in addition to producing OPN locally, released proCPB into SF and expressed functional cell surface thrombomodulin that supports the activation of proCPB to CPB by thrombin. Immunohistochemical studies demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the SF. OPN had an anti-apoptotic effect on neutrophils, which was retained by but diminished in OPN-R and OPN-L. Cultured synoviocytes expressed a4b1 integrins and adhesion of synoviocytes to immobilized OPN-R, but not OPN-L, was significantly enhanced compared to OPN-FL. Synoviocytes bound to immobilized SVVYGLR, but not SVVYGL, dose-dependently, consistent with binding via the a4b1 receptor on synoviocytes. Our results suggest that the sequential thrombin activation of OPN (OPN-R) and its subsequent inactivation by CPB (OPN-L) may represent an important local homeostatic mechanism that regulates neutrophil viability and synoviocyte adhesion and play an important role in the pathogenesis of RA in a subset of patients.