CD44 from NB4 and APL cells ligates with fibrinogen (Fbg). (A) Leukemia cells and neutrophils were mixed with Fbg (1 mg/mL) and thrombin (1 U/mL) for 2 hours. Dense fibrin networks formed among NB4 and APL cells (arrows). Almost no fibrin was located on the membrane of neutrophils. Scale bar, 10 µm. (B) Representative immunofluorescence images from direct staining of CD44 (green) and Fbg (red). Scale bar, 10 µm. (C) The MFI of Fbg in panel B. (D) The MFI of Fbg deposited on the membrane of NB4/APL cells was correlated with the MFI of CD44. (E) Competition assays. A nonlabeled Fbg was added to the mixture at different concentrations. The fluorescence of Fbg was detected by using flow cytometry. (F) APL/NB4 cells were incubated with Fbg, and images were obtained at the indicated time points. Fbg deposited on the surface of leukemic cells (arrowheads). Thrombin was added 60 minutes after leukemic cells were mixed with Fbg, and the mixture was treated as described in “Methods.” Leukemia cells were wrapped by fibrin networks (arrows). Scale bar, 10 µm. (G-H) The target sequence and the percent knockdown are shown as follows. The expression of CD44 was measured by western blotting, and the mean gray value was measured by using ImageJ. (I) NB4 cells/APL blasts with/without CD44 knockdown were cultured with Fbg for 1 hour. Fluorescence images showed decreased Fbg deposition on the CD44 knockdown cells. Scale bar, 20 µm. *P < .05, **P < .01. AU, arbitrary units; Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole.