The links between inflammation and coagulation have been the subject of intense research. On the one hand, inflammation activates the coagulation cascade and is prothrombotic; on the other hand, coagulation can also initiate and perpetuate inflammation. The molecules that are implicated in this cross-talk include tissue factor (TF), fibrin, the TF-generated coagulation proteases activated factor X and thrombin, and the protease activated receptors (PARs). In rheumatoid arthritis, we and other workers have shown that joint inflammation is accompanied by massive activation of coagulation proteases 12, and fibrin deposition can perpetuate inflammation in a murine model of RA 3. Inhibition of thrombin activation and factor VII can also reduce synovial inflammation in these models 45.

TF is a glycoprotein that binds the serine protease activated factor VII (FVIIa) to initiate coagulation. Two major forms of TF are recognized; one cell bound, and the other in plasma or soluble form. Most of the known biological functions are attributed to the cell-bound form, but there are reports that soluble forms of TF may play a role in coagulation or hemostasis 6 and may be a link between tissue inflammation and thrombosis 7. Soluble tissue factor (sTF) by itself can induce inflammatory arthritis when injected into mouse joints 89.

The precise mechanisms linking TF-dependent coagulation activation to extravascular inflammation are not fully understood. Thrombin activation of PAR-1 and PAR-4 can lead to G-protein-mediated cellular activation, as well as to NF-κB-mediated expression of P-selectin, E-selectin, vascular cell adhesion molecule 1 and intracellular adhesion molecule 1 adhesion molecules that favor leukocyte migration and activation in the vascular lining 10. Fibrin, the final product of the coagulation cascade, can also be proinflammatory. Fibrin induces endothelial expression of adhesion molecules 11, and the fibrin degradation products are neutrophil chemotaxins 12. Fibrin deposition in human glomerulonephritis and arthritis is associated with more severe disease 1314; in animal models of glomerulonephritis, arthritis and nerve injury fibrin exacerbates inflammation and tissue damage 3151617.

To assess the inflammatory effects of TF and the mechanisms involved, we studied the effects of TF when injected extravascularly. We showed that sTF injected into the mouse footpad is a potent proinflammatory stimulus and is critically dependent on both platelet PAR-4 and fibrin. These findings provide a better understanding of the role of TF activation in inflammation, and suggest potential targets for interrupting this pathway in disease states.

Extravascular fibrin deposition is a hallmark of chronic inflammation and plays a role in perpetuating inflammation in rheumatoid arthritis, glomerulonephritis and experimental allergic encephalomyelitis 31625. TF-initiated coagulation accounts for fibrin formation and may also trigger inflammation through the action of downstream coagulation proteases such as thrombin and FVIIa on PARs. The relative roles of the different PARs in mediating inflammation are not well understood, however, and differing studies have implicated different PARs. We therefore chose to study the effects of sTF injected into the mouse footpad and the underlying mechanisms of its effects.

The injection of recombinant murine sTF into the mouse footpad results in an acute inflammation of extravascular tissues characterized by footpad swelling, histological signs of inflammation and fibrin deposition. This effect is mediated principally by the classical pathway of coagulation activation, through the formation of thrombin and fibrin, as inflammation was effectively blocked by administration of the thrombin inhibitor hirudin and an inhibitor of FVIIa. Depletion of fibrinogen by ancrod also attenuated inflammation in this model. These findings confirm that fibrin formation is an essential step in the link between coagulation and inflammation.

To determine whether PAR activation can also play a role in this model, we tested mice deficient for the individual PARs. To our surprise, only PAR4-deficient mice showed a phenotype, in that these mice were completely protected from sTF-induced inflammation. This contrasts with more chronic models that require an immune stimulus, such as glomerulonephritis and arthritis, in which PAR1 and PAR2 signaling seem to play a role 2627. Indeed, in antigen-induced arthritis, we found that TF/FVIIa activates PAR2 and subsequent arthritis – but in the same conditions, PAR4-deficient mice were indistinguishable from wildtype mice 28. The key role of PAR4 suggested to us that platelet activation may be critical for inflammation to develop following sTF injection, as PAR-4 is the main platelet protease receptor in mice. This was confirmed when we performed the same experiments on normal mice that were rendered thrombocytopenic by the administration of antiplatelet antibody. The fact that PAR-3 had no effect at all on inflammation suggests that PAR-4 is the main pathway by which proteases activate mouse platelets in vivo.

One could question the physiological relevance of sTF in inflammation. Circulating forms of TF have been measured in different disease states 17, although it is likely to be much less abundant than cell-bound TF. There is much debate regarding what soluble TF consists of and whether it is biologically active 29. A large proportion is probably in the form of microparticles but an alternatively spliced variant of natural TF has also been described 3031. Both forms have been reported to possess functional procoagulant activity 632, and increased levels of microparticular TF have been linked to vascular disease. The proinflammatory effects of the form of sTF we used in this study (containing amino acids 1 to 219) in vivo resembled those reported by Bokarewa and colleagues, who observed a chronic erosive arthritis after injection of a similar dose of human TF_1–219 into mouse knees 89. A difference is the time course, with an acute response in hours after injection in our hands, whereas in Bokarewa and colleagues' study, arthritis was observed from day 4 up to day 60 after injection.

Fibrin formation and deposition was significantly diminished in PAR-4-deficient mice and underlines the important role of fibrin in inflammation. These may be mediated by fibrin-induced macrophage secretion of proinflammatory cytokines such as IL-1β and TNFα 333435 and of the chemokines MIP-1α, MIP-1β and MIP-2 36. The beneficial effect of fibrin depletion in different models of inflammation and neurodegenerative diseases reinforces the important role of fibrin deposition in perpetuating inflammation.

Finally, it is important to point out that both fibrin formation and platelet activation were needed for inflammation to develop in this model, and the absence of either of the two events abrogated inflammation. When PAR4^-/- mice were injected with sTF, there was an indication that coagulation was activated, as TAT levels were comparable with injected wildtype animals (PAR-4^+/+ mice, 33.2 ± 13 ng/ml, n = 16; PAR 4^-/- mice, 42.7 ± 20.8 ng/ml, n = 13). PAR-4-deficient mice did not show any clinical or histological signs of inflammation, however, and fibrin scoring was significantly lower. These findings indicate that the development of inflammation following coagulation activation requires the participation of platelets, probably via PAR-4 signaling, as well as the formation of fibrin. Activation of one pathway alone is insufficient to trigger full-blown inflammation. The results also suggest that blockade of either of the two arms of this process can prevent the development of inflammation after coagulation activation. Direct thrombin inhibitors such as hirudin could act on both fibrin formation and PAR activation simultaneously, and therefore should reduce coagulation-induced inflammation. This concept has yet to be tested in inflammatory diseases in man, although it has been shown to be effective in murine arthritis and ischemia models. Furthermore, our results would suggest that inhibitors of platelet PAR activation may also have a beneficial role in inflammation.

Following subcutaneous injection of sTF in the mouse footpad, we observed both coagulation activation and acute inflammation. The inflammatory response required concomitant fibrin deposition and the participation of platelets. In this model, the platelet protease receptor PAR-4 plays a crucial role. In contrast, murine PAR-1, PAR-2 and PAR-3 did not have a major effect on inflammation. These results suggest that in chronic inflammatory diseases where fibrin deposition is significant, such as rheumatoid arthritis, inhibition of fibrin formation and platelet PAR can attenuate inflammation.

ELISA = enzyme-linked immunosorbent assay; FVIIa = activated factor VII; NF = nuclear factor; PAR = protease activated receptor; PEG = polyethylene glycol; sTF = soluble tissue factor; TAT = thrombin–antithrombin III; TF = tissue factor.

The authors declare that they have no competing interests.

NB and AS shared the design of the study and the writing of the manuscript. VC-P performed the experiments. JH provided the knockout mice used in the study. PS and NW generated the recombinant TF.