Promyelocytic extracellular chromatin triggers PS exposure on HUVECs. On the third day of PBS or ATRA treatment, extracellular chromatin from NB4 cells was isolated. HUVECs were incubated with or without isolated extracellular chromatin (50-fold concentrated) for 24 hours. Cells were stained with CD31-Alexa Fluor 488 (A-B) and with Alexa 647–lactadherin and FITC–annexin V (C). (A) Representative confocal microscopy image showed morphology of HUVECs incubated with culture medium without extracellular chromatin. (B-C) Stimulation with ATRA-derived chromatin led to the retraction of cell margins and extension of filopods (arrows) and PS exposure on the filopods (arrows), which costained with lactadherin (red) and annexin V (green). The inset bar represents 5 μm in panels A-C. One of 6 independent experiments is shown. (D) Kinetics of PS reversal on ECs with different treatment. (E) For inhibition assays HUVECs were incubated with isolated ATRA-derived chromatin in the presence or absence of DNase I or APC. One of 6 independent experiments is shown. Each point represents mean ± SD. *P < .001 vs PBS-treated group in panel D; *P < .01, **P < .001 vs no inhibitor group and ^#P < .05 vs DNase-treated group in panel E.