Fig 2: Time-dependent activation of mitogenic pathways in the MC38 cell line after treatment with coagulation factors with or without EGFR inhibition. Western Blot analyses demonstrate the relative changes in the expression of phosphorylated p38, AKT and p44/42 MAPK after treatment with FXa (A–C), thrombin (D–F) or EGFR inhibition prior FXa treatment (G–I) for up to 16 h. Equal amounts of protein were loaded. Phosphorylated signals were normalized to endogenous levels of total p38, AKT or p44/42 MAPK, as well as the amount of total protein or β-Actin per lane. Shown is the fold induction relative to control. Mean ± SD of n independent experiments is presented for all data. n =3-6, One-way ANOVA, Dunnett posthoc test, p < 0.05 (*).

Fig 3: In vitro effects of FXa and PAR2 activation on proliferation and migration of murine CC cell line MC38 and involvement of EGFR. Proliferation. (A) Selective activation of PAR2 using AP2 induces significant proliferation, with the strongest stimulus being 10% FCS in the medium. Also, longterm stimulation with FXa enhances proliferation of the MC38 cell line. (B) Comparative analyses using BrdU assay exhibit equivalent increased proliferation rates after FXa stimulation of the cells. Migration. (C) Directed and (D) undirected cell migration of murine CC cells are significantly increased by FXa stimulation and selective PAR2 activation. Mean ± SD of n independent experiments is presented for all data. (A) n =7-11, (B) n =3-5, (C) n =5-7, (D) n =3, One-way ANOVA, Dunnett posthoc test, p < 0.05 (*). EGFR. (E) The involvement of EGFR was analyzed by pre-incubation of the cells with Erlotinib, an EGFR inhibitor, at varying concentrations and partially FXa stimulation. EGFR inhibition significantly decreased migration of the cell line used. Mean ± SD of n independent experiments is presented for all data. n =3-6, One-way ANOVA, Dunnett posthoc test, p < 0.05 (*). (F) The half-maximal inhibitory concentration (IC50) of the EGFR-inhibitor, Erlotinib, was determined as 161 nM utilizing a resazurin-/crystal violet assay, n =3-6, Dose-response curves and ED50 values were derived through non-linear regression modelling (log(inhibitor) vs. normalized response - Variable slope).

Fig 4: Characteristic in vivo values. Tail bleeding assay. (A) The direct FXa inhibitor Apixaban prolonged bleeding time significantly. Time was measured in mice that received 5 mg/kg Apixaban, 50 mg/kg Apixaban, or vehicle only (control) in both WT (white) and PAR2-KO (pink) mice. Mean ± SD of n independent experiments is presented for all data. n =5-6, Two-way ANOVA, p < 0.05 (*). Survival. (B) Kaplan-Meier estimates for treated (narrow line) and untreated (wide line) WT (black) and PAR2-KO (pink) mice. The survival duration of PAR2-KO animals was found to be significantly longer than that of WT animals. Apixaban did not demonstrate a substantial impact on this survival difference. Tumor growth in vivo. Untreated (C) and treated (D) WT (black) and PAR2-KO (pink) animals measured for maximum tumor extension. Apixaban demonstrates no significant effect.