Figure 4.

PI3K signaling is required for THB-mediated TFPI down-regulation. (A) HPMCs were transfected with control or PAR-1–targeting siRNA. Untransfected, control, and PAR-1 siRNA–transfected cells were treated with PBS and thrombin, and cell lysates were Western blotted for TFPI. Total Akt antigen was the loading control. (B) HPMCs were treated with THB for 0, 5, and 15 minutes in the presence and absence of PX-866 (1 µM). Cells were lysed, resolved on a SDS-PAGE, and probed for phosphorylated Akt. Akt antigen was used as a loading control. HPMCs were treated with THB in the presence of decreasing concentrations of PX-866 (C and D). (C) Isolated mRNA was transcribed into cDNA and probed for changes in TFPI mRNA. GAPDH mRNA was used as the normalization control. ^$P = 0.01; *P < 0.05 when compared with THB-treated HPMCs. Data represent n = 3/treatment. (D) Cell lysates and CM from THB-treated cells were probed for TFPI. β-actin antigen was used as the loading control. The figure is representative of three independent experiments. Graphed data represent n = 3/treatment. *P < 0.05 when compared with treatment with THB alone. (E) CM from PBS-, THB-, and THB/PX866-treated HPMCs were collected and assayed for TFPI activity in a cell-free factor X activation assay. The illustrated data represent n = 3/treatment. *P < 0.05.