Fig. 6

Effects of PAR1 antagonism and agonism. a Atopaxar, a selective antagonist of PAR1 significantly reduced granzyme B total cumulative toxicity at 500 nM, 1 μM, and 10 μM pre-treatment doses after 5 days of co-incubation with granzyme B and IL-1β. The cumulative toxicity at the 1 μM dose of Atopaxar was not significantly different from toxicity of control media, which contained the same concentration of DMSO necessary to solubilize Atopaxar, nor was Atopaxar itself significantly more toxic than control media. *p < 0.05, ****p < 0.0001 relative to control media, ####p < 0.0001 relative to granzyme B+ IL-1β, repeated measures ANOVA with post hoc Tukey’s test, n = 8 per condition. Data are shown as the mean ± standard error of the mean from five separate experiments. b Thrombin (500 nM), an agonist of PAR1, causes mild neurotoxicity when added to neuronal cultures for 3 days. When granzyme B is added with thrombin, there is no additive toxic effect. *p < 0.05, ****p < 0.0001 relative to control media, one-way ANOVA with post hoc Tukey’s test, n = 10 per condition. Data are shown as the mean ± standard error of the mean from duplicate experiments. c The peptide activating sequence TFLLR (5 μM), an agonist of PAR1, reduced neuronal viability after 3 days to the same extent as 10 nM granzyme B. When granzyme B and TFLLR were combined, there was no additional increase in toxicity. RLLFT, the inverse peptide of the agonist sequence, was non-toxic by itself and did not increase toxicity when paired with granzyme B. ****p < 0.0001 relative to control media, one-way ANOVA with post hoc Tukey’s test, n = 10 per condition. Data are shown as the mean ± standard error of the mean from three separate experiments