A cell-permeable peptide comprising the N-terminal CaMBD of RalGDS inhibits thrombin-induced WPB exocytosis. (A) The biotin-labeled peptides TAT-CaMBD, TAT (negative control), and TAT-Ral-c (positive control) were coupled to streptavidin-sepharose and used in a pull-down experiment from HUVEC lysate as described in “CaM pull-down assays.” The bottom panel shows total CaM levels to show equal expression. (B) RalA activation in HUVECs that were pretreated with TAT-CaMBD or TAT (negative control) upon thrombin stimulation was determined using a Ral-GTP–specific pulldown. HUVECs were preincubated for 30 minutes in SF medium in the presence or absence of 200 μg/mL TAT-CaMBD or TAT peptide. Subsequently, cells were stimulated with 1 U/mL thrombin (T₂) or SF-medium (−) for 2 minutes in the presence or absence of 200 μg/mL TAT-CaMBD or TAT peptide. The bottom panel shows total RalA as a loading control. (C) HUVECs were preincubated for 30 minutes with SF medium, 200 μg/mL TAT, or 200 μg/mL TAT-CaMBD. Subsequently, 1 U/mL thrombin, 10 mM epinephrine, and 100 mM IBMX or SF medium (unstimulated) was added for 15 minutes. WPBs were visualized by immunofluorescent staining of VWF. The number of remaining WPBs per cell upon stimulation in absence or presence of TAT or TAT-CaMBD was quantified using confocal microscopy. ***P < .001 by Student t test.