부산대학교 도서관

Thrombin is the main effector protease in the coagulation cascade, but it can also affect a wide array of cell types by signalling via protease activating receptors (PAR). The presence of these receptors on the surface of innate immune cells has been well reported but the full functional consequences of activation have yet to be defined. In a model of oxazolone-induced delayed type hypersensitivity (DTH) mice transgenically expressing hirudin on CD31 cells had normal CD4+ T-cell priming, but significantly reduced ear swelling, CD68+ infiltration & granuloma formation compared to wild type. This phenotype was bone marrow and PAR-1 dependent. Treatment of mice with a cell membrane localised thrombin inhibitor (PTL060) also delivered a protective phenotype. In vitro, stimulating BM-derived macrophages (BMM) with thrombin did not affect gross markers of macrophage polarisation but did lead to the formation of increased lipid rich microdomains. In conjunction with this, there was increased co-localisation of the LPS and IFNγ receptor within the lipid rafts. These thrombin stimulated cells were highly sensitive to low dose M1 stimuli, as evidenced by iNOS expression. The heightened sensitivity to M1 stimuli was shown to be dependent on cullin 3 mediated ABCA1 down regulation and alteration of lipid raft composition in response to thrombin. In vivo ABCA1 was also shown to be operational. Treatment of CD31-Hir-Tg mice with probucol (an inhibitor of ABCA1) prior to second exposure to oxazolone inhibited the transgenic phenotype. In the DTH model, mice expressing tissue factor pathway inhibitor on all CD31 cells had exaggerated CD68+ infiltration compared to wild-type. The hypothesised difference was due to the absence of a PAR-2 activation on macrophages in these mice. Consistent with this, treatment with a PAR-2 agonist was protective with respect to ear swelling and CD68+ infiltration. In vitro, PAR-2 signalling on BMM lead to an anti-inflammatory phenotype, reducing sensitivity to IFNγ by enhancing SOCS3 expression. The combination of inhibiting PAR-1 signalling while delivering a PAR-2 signal reduced ear swelling and CD68+ infiltration over and above PAR-1 antagonism or PAR-2 agonism alone. This was also seen using PTL0GC1 a cell membrane localising combination PAR-1 antagonist & PAR-2 agonist which has translatable potential. Finally, in a model of atherosclerosis, treatment with PTL0GC1 led to fewer recruited monocytes into the plaque and these CD68+ adopted a phenotype associated with plaque regression (ABCA1+, CCR7+, IL10+).