Activation of the E-selectin promoter by PF4. Plasmids containing either the full-length (-840) E-selectin promoter or variants (B) driving expression of luciferase were transfected into HUVECs. (A) HUVECs were incubated in the absence or presence of PF4 (20 μg/mL) for 4 hours prior to measurement of luciferase activity. Data are expressed as fold increase in the presence of PF4 compared to buffer alone and are the mean ± SEM of 3 independent experiments performed in triplicate; n.s. indicates lack of statistical significance by 2-tailed t test (P > .05); asterisk, statistical significance compared to the full-length (840) promoter (P < .001); B, promoterless luciferase activity; Luc, luciferase coding sequence; E-selectin and SV40 indicate the promoters present. (B) Schematic diagram depicting promoter construct used in these experiments. The 5′ promoter region of the E-selectin gene is depicted (top). Consensus binding sites for AP-1 and NF-κB are shown as well as a palindromic site (PS) thought to be important for E-selectin expression. (C) Polymyxin B inhibition of LPS (100 ng/mL), but not PF4 (20 μg/mL), demonstrates that PF4 is not contaminated with LPS. P indicates polymyxin B treatment. Ability of platelet releasate to activate the E-selectin promoter is also shown (Plt). Data are mean ± SEM of 3 independent experiments performed in triplicate.