Dose-dependent effects of eltrombopag iron chelation on platelet formation

Abstract

Iron deficiency is associated with thrombocytosis in patients, although thrombocytopenia can occur in cases of severe iron deficiency anemia. Eltrombopag (EP), a thrombopoietic agent approved for immune thrombocytopenia, also acts as an iron chelator. Our study demonstrates that megakaryocytes (MKs) exhibit an increased requirement for iron as they mature and acquire the ability to form proplatelets and release platelets. Although low EP concentrations maintain MK functions, high EP concentrations disrupt iron homeostasis, reducing proplatelet formation. Mechanistically, EP-dependent iron chelation impairs MK cytoskeletal dynamics, induces higher extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, and reduces posttranslational glutathionylation of tubulin protein. Addition of exogenous iron or oxidized glutathione to high-dose EP-treated MKs counteracts the negative effect on iron status and ERK1/2 signaling, thereby rescuing proplatelet formation. Overall, these data reveal the complex role of iron status on MK cytoskeletal dynamics and platelet biogenesis and may explain the varied clinical manifestations of iron deficiency on platelet counts.

Graphical abstract

Figure 1.

Evaluation of iron homeostasis during the late phase of physiological thrombopoiesis. (A) Experimental strategy used to investigate the in vitro transition of MKs (10 days of culture, early-stage) into MKs bearing proplatelets (13 days of culture, late-stage). (B-C) Representative images of (B) MKs and (C) quantification of proplatelet formation (PPF) in early and late stages. The extent of PPF was calculated as the percentage of proplatelet-bearing MKs compared with total MKs. n = 3. Scale bar, 50 μm. Objective, 20×. (D-E) Evaluation of intracellular iron labile pool using the calcein-AM assay in 10-day and 13-day culture MKs. Representative dot plot of flow cytometric analysis (D) and mean fluorescence intensity (MFI) quantification (E). n = 4. (F) Flow cytometry analysis of intracellular iron levels in early- and late-stage MKs assessed with the BioTracker 575 red Fe²⁺ dye. (G-I) Changes in iron homeostasis were evaluated by assessing the protein levels of key iron regulators in MKs before and after proplatelet formation. Representative western blot analysis (G) and relative quantification of TfR1 (H) and FTL (I) expression. Glyceraldehyde-3-phosphate dehydrogenase was revealed to ensure equal protein loading. n = 3. (J-K) Mitochondrial superoxide anion production was assessed using MitoSOX red probe in early- and late-stage MKs through flow cytometric analysis. Representative dot plot (J) and relative quantification of MFI (K). n = 3. (L-M) BODIPY probe was used to measure lipid peroxidation during MK maturation (10-day and 13-day culture) by flow cytometry analysis. Representative dot plot showing the fluorescence level in treated cells (L) and relative quantification (M). n = 4. ∗P < .05; ∗∗∗P < .001. Data are shown as mean ± standard deviation (SD). The image was created with BioRender.com.

Figure 2.

EP’s iron-chelating properties affect proplatelet formation by altering cell signaling and cytoskeletal dynamics. (A-B) Early-stage MKs were incubated with EP (0.5, 1, or 10 μg/mL) or DFO (10 μM) for 3 days and the expression levels of TfR1 and FTL were evaluated by western blot (A) and quantified (B). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was revealed to ensure equal protein loading. n = 4. (C) Intracellular iron content was evaluated using the calcein-AM assay in early-stage MKs stimulated with increasing EP concentrations or DFO for 72 hours. Treated MKs were incubated with the calcein-AM probe and analyzed by flow cytometry. MFI was normalized on EP 0.5 μg/mL–treated cells. n = 3. (D) Mature MKs were incubated with calcein-AM for 10 minutes and, after washing, cells were treated with EP (0.5, 1, or 10 μg/mL) or DFO (10 μM) for 1 hour. Intracellular iron content was evaluated analyzing calcein-AM fluorescence by flow cytometry. MFI was normalized on EP 0.5 μg/mL–treated cells. n = 3. (E-F) Proplatelet formation was evaluated in immunofluorescence (E) upon β₁-tubulin staining (green signal). Nuclei were stained with a Hoechst-33258 solution (blue signal, 100 ng/mL in phosphate-buffered saline). Scale bar, 100 μm. Objective, 20×. n = 4. Relative quantification is provided in the graph (F). (G) Assessment of proplatelet formation in MKs treated for 3 days with EP 10 μg/mL or EP 10 μg/mL plus FAC 50 μM in the last 24 hours of culture. Western blot analysis of TfR1 and FTL levels are provided on the top of the graph. GAPDH was revealed to ensure equal protein loading. n = 3. (H-I) UT7 cells were stimulated with TPO (10 ng/mL), EP 0.5 or 10 μg/mL, or DFO (10 μM) for 3 hours and then seeded onto fibronectin-coated coverslips (25 μg/mL) for an additional 3 hours at 37°C. Cells were then stained with a β₁-tubulin antibody (green signal) and nuclei were counterstained with Hoechst-33285 solution (blue signal) (H). Scale bar, 100 μm. Objective, 20×. Bar graph (I) shows cell spreading fold increase normalized to TPO-treated cells. n = 5. (J-K) Western blot analysis (J) and relative quantification (K) of ERK1/2 and AKT phosphorylation levels in early-stage MKs stimulated with EP (0.5, 1, or 10 μg/mL) or DFO for 72 hours. n = 3. (L-M) Western blot analysis (L) and relative quantification (M) of ERK1/2 and AKT phosphorylation levels in MKs stimulated with EP 0.5 or 10 μg/mL and EP 10 μg/mL plus FAC 50 μM. n = 3. (N-O) MKs at day 13 of culture were stimulated for 30 minutes with EP (EP 0.5 or 10 μg/mL) or DFO (10 μM). β-tubulin was immunoprecipitated from cell lysates and levels of bound GSH to β₁-tubulin were evaluated by western blot. Input: β₁-tubulin in total cell lysates. GAPDH was detected from lysed MKs to ensure equal protein loading (N). Relative quantification of the amount of GSH bound to β₁-tubulin (O). n = 4. (P-Q) Early-stage MKs were stimulated for 3 days with EP (0.5 or 10 μg/mL) or EP 10 μg/mL plus GSSG (25 mM) added in the last 24 hours. Levels of glutathionylated β₁-tubulin were assessed upon β-tubulin immunoprecipitation and GSH western blot. Input: β₁-tubulin in total cell lysates. GAPDH was detected from lysed MKs to ensure equal protein loading (P). Representative western blot (P) and relative quantification are provided (Q). n = 4. (R-S) Early-stage MKs at day 10 of culture were stimulated for 3 days with EP 0.5 or 10 μg/mL, EP 10 μg/mL plus GSH (GSSG, 25 mM) in the last 24 hours. Representative images (R) and quantification of proplatelet formation in the different experimental conditions (S). Scale bar, 50 μm. Objective, 20×. n = 4. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data are shown as mean ± SD. p-AKT, phosphorylated protein kinase B; p-ERK, phosphorylated extracellular-signal regulated kinase; WB, Western blot.