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M. Brusson et al.
terms of percentage of Lu/BCAM-positive RBCs and asso- ciated mean fluorescence intensity (Figure 4C), confirming that HC increases both the percentage of Lu/BCAM-posi- tive RBCs and the expression of this protein within each positive RBC. This increase was reflected by higher amounts of Lu/BCAM during HC treatment detected by western blot (Figure 4D).
We have shown that Lu/BCAM long isoform was con- stitutively phosphorylated in PV RBCs18 and that inhibi-
tion of this phosphorylation was associated with less PV RBC adhesion to laminin.17 In order to determine whether the increased adhesion induced by HC was associated with Lu/BCAM activation, we tested the phosphorylation state of Lu/BCAM long isoform. Radiophospholabeling experiments confirmed that Lu/BCAM was phosphorylat- ed in PV RBCs and showed increased phosphorylation during HC treatment (Figure 4E, top panel). This increased phosphorylation was not only due to increased Lu/BCAM
AB
C
D Figure 4. Longitudinal analyses of red blood cell (RBC) adhesion and Lu/BCAM expression and activa- tion in 4 polycythemia vera (PV)
E
patients during
bamide (HC) treatment. All results were obtained with RBCs from 4 PV patients before (UT) and during (HC) HC treatment. (A) Typical images of RBCs adhering to laminin 521 at 3 dyn/cm2. (B) Mean number of RBCs/mm2. (C) Flow cytometry analysis of Lu/BCAM expression: (top) his- tograms (before: dotted; during: solid); (bottom) percentage of RBCs expressing Lu/BCAM and mean fluorescence intensity (MFI). (D) Western blot analysis of Lu/BCAM expression; the upper band corresponds to the long iso- form Lu and the lower one to the short isoform Lu(v13). (E) Lu/BCAM phosphorylation rate. The top (P) and bottom (T) panels show the phosphorylation and the total amount of the immunopuri- fied proteins, respectively. The phosphorylated fraction is deter- mined by the P/T ratio.
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haematologica | 2018; 103(6)