Role of the PI3K-Akt-eNOS and PLCγ1-ERK signaling pathways on VE-cadherin cleavage induced by FXIa. (A) HUVECs were incubated with FXIa (30 nM). Cells were lysed and immunoblotted with antibodies for phosphorylated ERK1/2 T₂₀₂/Y₂₀₄, p38 MAPK T₁₈₀/Y₁₈₂. Akt S₄₇₃, eNOS S₁₁₇₇, PLCγ1 Y₇₈₃, Src Y₄₁₆, FAK Y₃₉₇, or tubulin (n = 3). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (B) HUVECs were incubated with FXIa, FXIa-PPACK, or FXI (30 nM) for 15 or 30 minutes. Cells were lysed and immunoblotted with antibodies for phosphorylated ERK1/2 T₂₀₂/Y₂₀₄ or Akt S₄₇₃. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Results are representative of 3 experiments. (C) HUVECs were incubated with vehicle (dimethyl sulfoxide [DMSO]) or FXIa for 6 hours in the absence or presence of the Src inhibitor, PP2 (10 μM); the protein kinase A (PKA) inhibitor, H-89 (10 μM); the eNOS inhibitor, L-NAME (5 μM); or the ERK1/2 inhibitor, LY3214996 (1 μM). Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3).