Figure 4.
FXIa–PAI-1 complex interaction with VLDLR induces VE-cadherin shedding. (A) HUVECs were incubated with FXIa (30 nM) for 6 hours in the absence or presence of the low-density lipoprotein receptor antagonist, RAP (50 ng/mL). Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. 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 (30 nM) in the absence or presence of PPACK (100 μM), GI254023X (5 μM), or RAP (50 ng/mL) for 6 hours. HUVECs cell surfaces were biotinylated, and cell lysates were precipitated with NeutrAvidin agarose beads. The precipitates were probed with an anti–N-terminal VE-cadherin or anti–PAI-1 antibodies. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (C) HUVECs were incubated with FXIa (30 nM) at 4°C for 2 hours. Cells were washed and incubated with PPACK (100 μM) for 30 minutes; lysed in the presence of PPACK; followed by immunoprecipitation with an anti-FXI LC and western blotting with an anti–PAI-1, anti-VLDLR, or anti–platelet EC adhesion molecule 1 (PECAM-1) antibodies. Results are representative of 3 experiments. (D) HUVECs were incubated with FXIa, FXI, or FXIa-PPACK (30 nM) at 4°C for 2 hours in the absence or presence of RAP (50 ng/mL). Cells were washed and incubated with PPACK (100 μM) for 30 minutes, lysed in the presence of PPACK followed by with an anti-FXI light chain immunoprecipitation and western blotting with an anti–PAI-1 antibody or anti-VLDLR antibodies. Results are representative of 3 experiments.

FXIa–PAI-1 complex interaction with VLDLR induces VE-cadherin shedding. (A) HUVECs were incubated with FXIa (30 nM) for 6 hours in the absence or presence of the low-density lipoprotein receptor antagonist, RAP (50 ng/mL). Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. 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 (30 nM) in the absence or presence of PPACK (100 μM), GI254023X (5 μM), or RAP (50 ng/mL) for 6 hours. HUVECs cell surfaces were biotinylated, and cell lysates were precipitated with NeutrAvidin agarose beads. The precipitates were probed with an anti–N-terminal VE-cadherin or anti–PAI-1 antibodies. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (C) HUVECs were incubated with FXIa (30 nM) at 4°C for 2 hours. Cells were washed and incubated with PPACK (100 μM) for 30 minutes; lysed in the presence of PPACK; followed by immunoprecipitation with an anti-FXI LC and western blotting with an anti–PAI-1, anti-VLDLR, or anti–platelet EC adhesion molecule 1 (PECAM-1) antibodies. Results are representative of 3 experiments. (D) HUVECs were incubated with FXIa, FXI, or FXIa-PPACK (30 nM) at 4°C for 2 hours in the absence or presence of RAP (50 ng/mL). Cells were washed and incubated with PPACK (100 μM) for 30 minutes, lysed in the presence of PPACK followed by with an anti-FXI light chain immunoprecipitation and western blotting with an anti–PAI-1 antibody or anti-VLDLR antibodies. Results are representative of 3 experiments.

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