Impaired basal and thrombin-induced vWF secretion from WPBs in Gα12 depleted HUVECs. HUVECs were treated with a pool of 4 different oligonucleotides directed against Gα12 or a control siRNA. (A) Epifluorescent images of vWF immunostaining (green) in control siRNA- or Gα12 siRNA–treated HUVECs after incubation for 15 minutes with 25 nM thrombin or serum-free medium alone (unstimulated). Scale bars = 20 µm. (B) Intracellular vWF staining (corrected integrated density) in individual, randomly selected control siRNA and Gα12 siRNA–treated cells (mean ± SEM; 10-30 cells/group; *P < .05 vs basal signal in control cells). (C) Number of fluorescent WPBs per randomly selected control and Gα12 siRNA–treated cell (*P < .05 vs unstimulated control; n = 10-30 cells/group; red horizontal bars depict median values). (D) Confluent HUVECs were incubated with normal human platelets and exposed to uniform and constant shear for 5 minutes. After washing, platelets bound to HUVEC-anchored vWF strings were detected by anti-GpIb monoclonal antibody staining (red) and rabbit anti-human vWF polyclonal antibody staining (green). (E) Graph shows fluorescence intensity of individual platelets per vWF string per field (percentage of control siRNA treatment; *P < .05 vs control; n = 6).