PLD activity is required for Stx B-induced VWF secretion. Confluent HUVECs were incubated with 0.2 μg/mL Stx1B (A) or Stx2B (B) for the indicated time, and PLD activity in cell lysates was assayed. Values are normalized to untreated cells (0 minutes) and shown as the mean ± SE for 3 experiments. (C) Confluent HUVECs in 24-well dishes were stimulated for 30 minutes with 0.2 μg/mL Stx1B or Stx2B. Secreted VWF propeptide was quantified by ELISA for each condition. Values shown are the mean ± SE from 3 replicates. Mean VWF propeptide concentrations were 6 ng/mL, 11 ng/mL, and 15 ng/mL for control, Stx1B-, and Stx2B-treated cells, respectively. (D) Confluent HUVECs in 24-well dishes were pretreated for 30 minutes with or without 25mM n-butanol or t-butanol, then stimulated for 30 minutes with 0.2 μg/mL Stx1B. Cell surface-associated VWF was quantified by ELISA in triplicate for each condition. Values shown are the mean ± SE from 4 independent experiments. (E) Confluent HUVECs were perfused at 2.5 dyn/cm² with 200 ng/mL Stx2B, with or without preincubation with n-butanol or t-butanol. VWF strings were counted in 10 fields, and values are shown as the mean ± SE, normalized to the Stx2B condition for each experiment. Experiments were conducted ≥ 3 times with similar results. For each panel, values for Stx1B or Stx2B are significantly different from control cells (P < .05). (D-E) Inhibition by n-butanol is significant (P < .01).