Rab27a-GFP is recruited in a postsynthesis step, and Rab27a knockdown increases stimulus-induced exocytosis of WPBs and results in fewer peripheral WPBs. (A) HUVECs were transfected with Rab27a-GFP and a maximum intensity projection made of confocal images of immunofluorescence staining of VWF (red) and Rab27a-GFP. Boxed images represent magnified images of Rab27a-GFP (B,D) and VWF expression (C,E) in immature (B,C) and more mature WPBs (D,E). (F) HUVECs were treated in 2 consecutive transfection rounds with 3 different siRNA oligonucleotides or a control siRNA oligonucleotide. Cells were incubated with serum-free media for 30 minutes or release media with PMA (100 ng/mL), cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. Rab27a mRNA expression levels in knockdown compared with mock-treated cells as determined by quantitative PCR are above the relevant bars. (G,I) Mock and (H,J) Rab27a siRNA-treated cells were plated out subconfluently after one round of nucleofection and microinjected with siRNA and VWF-GFP. (G,H) Confocal images were acquired of VWF-GFP and rhodamine phalloidin (red). (I,J) A 40-μm circle was placed at the cell nucleus, and the percentage of VWF-positive structures (white) present inside and outside the circle was determined (“Quantification of WPB distribution” in “Methods”). (K) Fifteen mock and 12 knockdown cells were selected from each condition from 2 separate experiments. The percentage present inside the 40-μm circle was plotted. ***P < .001 by Student t test and χ² test. Bars represent 10 μm. (F,K) Error bars represent SD.