Objective Osteopontin (OPN) is a pro-inflammatory cytokine important in rheumatoid arthritis (RA). OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal α4β1 and α9β1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also termed thrombin-activatable fibrinolysis inhibitor (TAFI), removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell binding. We investigated the roles of OPN-R and OPN-L in: (i) synoviocyte adhesion, which contributes to formation of invasive pannus, and (ii) neutrophil survival, which affects inflammatory infiltrates, in RA. Methods and Results We developed ELISAs specific for OPN-R and OPN-L, and demonstrate elevations of OPN-R and OPN-L in RA, but not in osteoarthritis or psoriatic arthritis, synovial fluid samples. OPN-R and OPN-L levels correlated with multiple inflammatory cytokines including TNFα and IL-6. Immunohistochemical analyses demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an anti-apoptotic effect on neutrophils. OPN-R, but not OPN-L, augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to α4β1. Conclusion Thrombin activation of OPN (OPN-R) and its subsequent inactivation by thrombin-activatable CPB (OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA, by regulating neutrophil viability and reducing synoviocyte adhesion.

Background: Chemerin is a chemokine/adipokine whose activity depends on proteolytic processing.Results: Specific ELISAs demonstrate that in plasma the precursor is dominant, whereas in synovial fluid from arthritis patients and CSF from glioblastoma patients, chem158K dominates. Low levels of active chem157S were found. Conclusion: Chemerin proteolysis occurs during inflammation. Significance: This is the first report about levels of different chemerin isoforms in biological samples.

Background: Osteopontin (OPN) is highly expressed in glioblastoma (GBM) and possesses inflammatory activity modulated by proteolytic cleavage. Results: Cleaved OPN was increased in GBM and led to more adhesion of GBM cells. OPN conferred resistance to apoptosis in GBM cells. Conclusion: Increased osteopontin proteolysis increased GBM cell resistance to apoptosis. Significance: OPN cleavage links coagulation and inflammation providing a favorable niche for GBM development.

Objective-To determine whether procarboxypeptidase B (pCPB)Ϫ/Ϫ mice are susceptible to accelerated abdominal aortic aneurysm (AAA) development secondary to unregulated OPN-mediated mural inflammation in the absence of CPB inhibition. Methods and Results-Thrombin/thrombomodulin cleaves thrombin-activatable pCPB or thrombin-activatable fibrinolysis inhibitor, activating CPB, which inhibits the generation of plasmin and inactivates proinflammatory mediators (complement C5a and thrombin-cleaved osteopontin [OPN] Key Words: thrombosis Ⅲ abdominal aortic aneurysm Ⅲ carboxypeptidase Ⅲ osteopontin Ⅲ plasmin A bdominal aortic aneurysm (AAA) is a common and potentially lethal disease, found in approximately 6% to 8% of men older than 60 years. AAA rupture is the most serious consequence of the disease and accounts for approximately 15 000 deaths in the United States annually. 1,2 The pathogenesis of AAA disease is complex, involving chronic transmural inflammation, characterized by macrophage and lymphocyte infiltration and proteolytic degradation of medial elastin and interstitial collagens by matrix metalloproteinases (MMPs). Structural degradation of the aortic wall in combination with hemodynamic stress results in tissue failure and aneurysmal dilatation. 3 Osteopontin (OPN) is a multifunctional protein that is both a component of the extracellular matrix (ECM) in mineralized tissues and a circulating proinflammatory cytokine. 4 OPN is a prominent component of human atherosclerotic tissues and has recently been demonstrated to play a key role in AAA pathogenesis. 5 Compared with apolipoprotein E (ApoE) Ϫ/Ϫ mice, ApoE Ϫ/Ϫ OPN Ϫ/Ϫ double-knockout mice are protected from angiotensin II-induced accelerated atherosclerosis and AAA formation. 6 Structurally, OPN contains an RGD site (159 -161) that mediates binding to a number of integrins, including ␣v␤1, ␣v␤3, ␣v␤5, and ␣5␤1. Thrombin cleavage of OPN exposes a hitherto cryptic C-terminus ( 162 SVVYGLR 168 ) that mediates the binding of thrombincleaved OPN (OPN-R) to a new subset of integrins, consisting of ␣4␤1 and ␣9␤1. 7-9 OPN-R demonstrates enhanced cell binding and cell migration compared with intact OPN in vitro, 10,11 suggesting that thrombin cleavage of OPN may augment its proinflammatory properties.When thrombin binds to thrombomodulin on the endothelial cell surface, it undergoes a remarkable change in its substrate recognition, switching from procoagulant factors, such as fibrinogen, to protein C and functionally inhibiting progressive thrombosis. 12 In addition to inhibiting coagulation via the activation of protein C, the thrombinthrombomodulin complex also influences fibrinolysis by activating a second physiological substrate, the plasma carboxypeptidase, thrombin-activatable procarboxypeptidase B (pCPB), or thrombin-activatable fibrinolysis inhibitor. 13,14

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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.