PMC

A. Time course of PI3 kinase/Akt activation by FXa. HUVECs were incubated with 50 nM FXa for indicated time periods. Whole cell lysates were extracted and immunoblotted for phosphor-Akt. Actin was used as a loading control. The immunoblot is representative of 3 independent experiments. B. and C. Inhibition of PI3 Kinase had no effect on Xa-induced TF protein and activity. HUVECs were cultured and pretreated for 1h with or without 10 μM LY 294002 (an PI3K inhibitor). The cultures were then incubated for 6 hours with or without 50 nM FXa. TF protein was shown by Western blot and activity was assayed by a chromogenic assay as described in material and methods.

A. Factor Xa-induced TF expression in HUVECs in a concentration-dependent manner. HUVECs were incubated with increasing concentrations of Factor Xa for 6 hours. Lower panel: Time course of FXa–induced TF expression. HUVECs were incubated with 50 nM FXa for indicated time periods. Whole cell lysates were extracted and immunoblotted for TF. Actin was used as a loading control. The immunoblot is representative of 4 independent experiments.
B. FXa increased tissue factor activity on the surface of HUVECs in a concentration-dependent manner. HUVECs were cultured and incubated with or without increasing concentration of FXa for 6 hours. Tissue factor activity was assayed by a chromogenic assay as described in material and methods. TF activity was not affected by Hirudin pretreatment, but was inhibited by AT-III. C. TF protein level was not affected by hirudin, but was suppressed by antithrombin-III.

A. Time course of P44/42 MAPK ERK1/2 activation by FXa. HUVECs were incubated with or without 50 nM FXa for 0, 5, 10, 30, or 60 minutes. Whole lysates were prepared and immunoblotted for active ERK using phosphospecific antibody. B. TF protein expression was significantly attenuated by a selective inhibitor of MEK 1/2 (PD 98059). HUVECs were pre-incubated for 1 hour with or without 10 μM PD 98059. The cultures were then incubated for 6 hour with or without Factor Xa. Immunoblots are representative of 3 independent experiments. C. Inhibition of MEK 1/2 - ERK1/2 attenuated FXa-induced TF activity. HUVECs were cultured in a 24 well plates and pretreated for 1hour with or without 10 μM of the MEK 1/2 inhibitor PD 98059. The cultures were then incubated for 6 hours with or without Factor Xa. Tissue factor activity was assayed by a chromogenic assay as described in material and methods. * P<0.05, compared to FXa treatment.

A. Factor Xa stimulated mobilization of NF-κB (P65) to the nucleus. HUVECs were incubated with 50 nM FXa for the indicated times. Nuclear and cytosolic extracts were prepared and immunoblotted for IκBα and NF-κB P65. Immunoblots are representative of 4 independent experiments. B. Tissue factor expression was greatly attenuated by NF-κB inhibitor. HUVECs were preincubated for 1 hour with or without 10 μM Bay 11-7802, an IKK inhibitor. The cultures were then incubated for 6 hour with or without Factor Xa. Immunoblots are representative of 3 independent experiments. C. Inhibition of NF-kB attenuated Xa-induced TF activity. HUVECs were cultured in 24 well plate and pretreated for 1h without or with 10 μM Bay 11-7802 (an IKK inhibitor). The cultures were then incubated for 6 h without or with Factor Xa. Tissue factor activity was assayed by a chromogenic assay as described in material and methods. * P<0.05, compared to FXa treatment. D. The effect of inhibition of MEK1/2–P44/42 MAPK on NF- κB. HUVECs were incubated with 50 nM FXa in the presence or absence of PD 98059 for the indicated times. Nuclear and cytosolic extracts were prepared and immunoblotted for IκBα and NF-κB P65. Immunoblots are representative of 4 independent experiments.