Pretreatment with thrombin does not inhibit VEGF-induced exocytosis. (A) Thrombin pretreatment does not block VEGF activation of exocytosis. HAECs were transduced with adenovirus expressing dominant negative Akt or adenovirus expressing β-galactosidase (LacZ) at an MOI of 200 for 48 hours, then treated with 50 ng/mL VEGF for 2 hours. Cells were then washed and treated with thrombin 1 U/mL for 1 hour, and exocytosis was measured with an ELISA for VWF as above (n = 3 ± SD; **P < .01 versus VEGF + LacZ). (B) VEGF but not thrombin or A23187 increases Akt phosphorylation. HAECs were pretreated with 50 ng/mL VEGF, 1 U/mL thrombin, or 1 μM A23187 for 30 minutes, and total cell lysates were immunoblotted with antibody to phospho-Akt (top row) or total Akt (bottom row). (C) Thrombin does not activate phosphorylation of eNOS. HAECs were transduced with adenoviral vectors expressing constitutively active Akt [Akt (CA)], dominant-negative Akt [Akt (DN)], or β-galactosidase (LacZ) at an MOI of 200 for 48 hours. Cells were then treated with 1 U/mL thrombin for 30 minutes, and total cell lysates were immunoblotted with antibody to phospho-eNOS or eNOS. (D) VEGF but not thrombin pretreatment decreases VEGF-induced exocytosis. HAECs were pretreated with 1 mM L-NAME for 16 hours, washed, and then incubated with 50 ng/mL VEGF or 1 U/mL thrombin for 1 hour. Cells were washed and treated with VEGF 50 ng/mL for 1 hour, and the amount of VWF released from cells into the media was measured by an ELISA (n = 3 ± SD; **P < .01 for VEGF versus VEGF + L-NAME).