Platelet-associated CD154 in ITP. (A) (top panel) CD154 was quantitated by ELISA in whole platelet lysates from healthy donors (control), ITP patients (group 1), and patients with nonimmune thrombocytopenia (group 2). Median value is indicated by □. Boxes indicate 25th and 75th percentiles; error bars, 10th and 90th percentiles. ○ indicates values outside these ranges. Statistical comparison (Mann-Whitney U test) shows a significant difference between group 1 and control (P < .001) or group 2 (P < .001) platelet CD154 levels. (Bottom panel) Surface expression of CD154 on activated platelets from an ITP patient compared with a healthy donor (control). Flow cytometry profile representative of 4 experiments. (B) CD154 detection by immunofluorescence in ITP megakaryocytes. Red fluorescence corresponds to GPIIb/IIIa. Green fluorescence corresponds to CD154. Yellow fluorescence corresponds to the superposition of red and green fluorescences. (i,iii) Control megakaryocytes. (ii, iv) ITP megakaryocytes. (i, ii) Double GPIIb/IIIa plus CD154 staining. (iii, iv) CD154 staining only. (C) Detection of CD154 mRNA by in situ hybridization in ITP megakaryocytes. Highlighted (arrows) are (i) a control megakaryocyte (arrowhead) and (ii) an ITP megakaryocyte (arrow).