Fig. 5. Quantitative bioluminescence analyses of PAR-1 cleavage activities by APC and thrombin in living cells using GLase-PAR1 mutant cells.

a Bioluminescence imaging analysis of GLase-PAR1 cleaved by thrombin on the surface of living HEK293 cells. HEK293 cells were transiently transfected for 24 h with the expression vectors of GLase-PAR1 wild type, R41A-, or R46A- mutants. After incubation with thrombin (0.5 U/mL) for 10 s, luminescence signals of GLase-PAR1 were recorded for 5 min with an exposure time of 2 s. The luminescence images of GLase-PAR1 on the cell-surface (colored cyan) at 1, 2, 3, 4, and 5 min were shown. Scale bar was 50 µm. b Bioluminescence imaging analysis of GLase-PAR1 cleaved by thrombin or APC on the surface of living CHO-K1 cells. Cells were transiently transfected for 24 h with the expression vectors of both the GLase-PAR1 R41A/R46A double mutant and EPCR. After incubation with thrombin (0.05 U/mL) or APC (3 μg/mL) or control for 10 s, luminescence signals of GLase-PAR1 were recorded for 15 min with an exposure time of 2 s. The luminescence images of GLase-PAR1 on the cell-surface (colored cyan) at 0, 5, 10, and 15 min were shown. Scale bar was 20 µm