Several components of the coagulation pathway are involved in inflammation (37
). For example, the procoagulant fibrin is proinflammatory by promoting the migration and activation of inflammatory cells and the production of proinflammatory cytokines (38
), whereas the anticoagulant protein C is antiinflammatory by promoting the cytoprotective pathways that inhibit apoptosis or cytokine productions (39
). CPB possesses both antifibrinolytic activity (and thus potentially proinflammatory activity) and antiinflammatory activity, and its role in inflammatory arthritis has not been characterized. In this study, we demonstrated that CPB protected against inflammatory arthritis in mice, exerting an antiinflammatory effect by inactivating the anaphylatoxin C5a. Our results with TA suggest that this effect is not mediated by the fibrin-plasmin pathway. Our findings in mice translated to human RA, in which carriage of the CPB2
1040T allele (encoding the long half-life Ile325 CPB) protected against erosive damage of RA joints. Furthermore, we found that the long half-life Ile325 CPB neutralized C5a activity more effectively than its short half-life counterpart, a difference that may underlie the protection conferred by the CPB2
1040T allele. Together, our findings suggest that CPB is a molecular link between inflammation and coagulation in RA. Activated during coagulation, CPB dampens inflammation in the synovial joints and ultimately reduces joint damage in RA.
The RA joint provides an environment suitable for activation of CPB. In RA synovial fluid, molecular markers of thrombin activation (thrombin-anti-thrombin III complexes and thrombin fragments) are significantly elevated (33
). In addition, TM levels are increased in RA synovial fluids, and TM is expressed in RA joint tissue, mainly in endothelial cells and synovial lining cells (40
). By forming the thrombin/TM complex, TM dramatically increases the rate of activation of both protein C and CPB approximately 1,000-fold (12
). Although the 2 proteins activated by the thrombin-TM complex are both antiinflammatory, they have opposing effects on coagulation: whereas protein C has anticoagulant activity, CPB has antifibrinolytic activity. Thus, coagulation and inflammation are interlinked processes that are tightly regulated in the RA joint.
Although CPB markedly inhibits fibrinolysis in vitro, CPB-deficient mice have no overt phenotype or coagulation abnormalities (20
). Moreover, restoration of antifibrinolytic activity by TA in CPB-deficient mice exacerbated inflammatory arthritis, which suggests that inhibition of fibrinolysis is not the main mechanism by which CPB protects against inflammatory arthritis. Indeed, our mouse data suggest that CPB’s primary role in inflammatory arthritis is an antiinflammatory one. Among CPB’s known proinflammatory substrates, OPN and bradykinin were not important in CAIA. C5a, in contrast, is known to be involved in autoimmune arthritis in humans and mice (42
). By cleaving C5a, CPB suppressed immune cell migration, a process important in RA pathogenesis.
Unlike in mice, there is no known case of CPB deficiency in humans. However, our human genomic data are consistent with our mouse phenotypic data showing that Cpb2
provides a protective effect on inflammatory arthritis. RA patients carrying the CPB2
allele encoding long half-life CPB (1040T) were less likely to progress to radiographically severe arthritis. We found that 1 copy of the allele was sufficient to provide protection, consistent with our results from mice heterozygously deficient in CPB. Interestingly, the CPB2
genotype was not associated with disease susceptibility, which suggests that the genetic factors that drive development of RA are distinct from those that drive progression of RA. CPB2
genotyping could potentially be performed in order to screen for RA patients who are at risk of progressing to a severe, erosive form of the disease or to predict the response of RA patients to therapeutic inhibitors targeting CPB substrates, such as anti-C5a therapy. Even though the CPB2
1040T allele had a protective effect on RA, it is also associated with an increased risk of certain thrombotic diseases (44
), which demonstrates the diverse role of CPB in inflammation and coagulation.
Although erosive changes were reduced in patients carrying the CPB2
1040T allele, we observed no differences in other disease activity indices, including DAS28, health assessment questionnaire score, tender joint count, and CRP, between CPB2
1040T allele carriers and 1040C homozygotes. CPB may have a direct role in bone and/or cartilage erosion in addition to its role in inflammation, given that there is evidence that C5a plays a direct role in bone and cartilage erosion by mediating osteoclast activation (48
) and by inducing MMP expression by RA FLSs (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:
). Alternatively, the differences in CPB half-life may not be sufficient to affect inflammatory markers at a single time point, but could potentially dampen chronic inflammatory responses over time and thereby reduce erosive progression.
Multiple genetic factors associated with RA have been discovered during the last decade (49
). Understanding their contribution to RA pathogenesis requires evaluation of their biological effects. However, for the vast majority of genetic variants associated with RA, there is no known functional effect of the variants. Here we demonstrated that long half-life CPB had greater C5a-neutralizing capability. Nevertheless, the molecular mechanisms by which the genetic variants of CPB affect RA may extend beyond regulation of inflammation mediated by C5a, because C5 inhibition using a neutralizing antibody provided only a trend toward modest benefit in human RA (50
), and C5a-receptor inhibition failed to provide benefit (51
). Thus, it remains possible that CPB’s effect in RA is mediated through multiple substrates.
Once activated, CPB has a short half-life of 5–10 minutes at 37°C (52
). No physiological inhibitors of CPB have been identified, and inactivation of CPB is achieved only through spontaneous structural destabilization of CPB after release of its activation peptide (53
). Therefore, the stability of CPB is an important determinant of its functional activity. In addition, CPB levels are higher in RA synovial fluids than in OA synovial fluids, possibly secondary to chronic inflammation and activation of the coagulation pathway as part of a compensatory antiinflammatory mechanism. Therefore, it is highly plausible that differences in the half-life of distinct CPB variants could influence disease progression in RA. CPB levels in plasma, however, did not differ between RA patients and healthy individuals, which suggests that CPB is produced locally in RA. We have previously demonstrated that cultured FLSs express CPB (15
), and showed in the present study that immune cells also produced CPB. Local production of CPB might be important in modulating local inflammatory processes.
Together, our findings suggest that CPB, a component of the coagulation system, plays a critical role in downregulating local inflammation in autoimmune arthritis. CPB exerted not only an antifibrinolytic effect, but also an antiinflammatory one. By cleaving C5a, CPB dampened inflammation in the synovial joints in a mouse model of autoimmune arthritis.