Formation of VWF strings in mock and Rab27a-depleted cells under flow. HUVECs were treated in 2 consecutive rounds with siRNA oligonucleotides directed against Rab27a or a control oligonucleotide, then seeded into μ-slides. HUVECs were stimulated with PMA and fixed under flow (“Flow analysis and quantification of VWF string length” in “Methods”). Fixed VWF strings were labeled with anti-VWF antibodies and imaged by confocal laser scanning microscopy. (A) Representative images of immunolabeled VWF strings released from mock (top panel) and Rab27a-depleted (bottom panels) cells. Arrow represents direction of flow. Bar represents 50 μm. (Inset graph) Number of strings per cell taking into account the number of cells per field of view. Data were collated from at least 5 fields of view taken from 3 separate experiments. Error bars represent SD. (B,C) The length of VWF strings released from either control or Rab27a-depleted cells was measured using LASAF software and binned into 10-μm divisions. Data were collated from at least 5 fields of view taken from 3 separate experiments. (B) Distribution of strings from Rab27a-depleted cells compared with mock-treated cells. Rab27a-depleted cells show a significant increase in shorter strings compared with mock control siRNA-treated cells. ***P < .001 by Mann-Whitney U test. (C) Distribution of strings more than and less than 100 μm.