PF4 retention is inhibited by surfen and restricts MK development in vitro. (A) Representative maximum intensity projection images of isolated, mature (day 5) MKs adhered to fibrinogen-coated slips overnight and treated with surfen (5 μM) for 0, 1, 2, and 4 hours. Actin (red), DAPI/DNA (blue), and PF4 (green). The scale bar represents 10 μm. (B) Quantification of PF4 fluorescence intensity normalized to background fluorescence within each image. Data represent an average of 15 individual cells. (C) Western blots of PF4 in MK lysates at 0 and 4 hours of surfen (5 μM) treatment with tubulin as loading control. (D) Quantification of signal intensity from panel C. (E) Media PF4 concentrations during treatment of mature (day 5) MKs with surfen (5 μM) over 4 hours determined from media sample by ELISA; n = 4. (F) GMFI of CD41 and CD42b staining of MKs during differentiation in FM supplemented with 2.5 μM or 5 μM surfen on day 3. The arrow indicates treatment time point; n = 4. (G) The percentage of the total single-cell population collected residing with either the 2N, 4N, 8N, or 16N peak after propidium iodide staining; n = 5. Significance was determined by comparing each group with the corresponding WT with FM treatment. Significance: ns is indicated by P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. All error bars represent the SEM.