Iron deficiency alters megakaryopoiesis and platelet phenotype independent of thrombopoietin

Abstract

Iron deficiency is a common cause of reactive thrombocytosis, however, the exact pathways have not been revealed. Here we aimed to study the mechanisms behind iron deficiency-induced thrombocytosis. Within few weeks, iron-depleted diet caused iron deficiency in young Sprague-Dawley rats, as reflected by a drop in hemoglobin, mean corpuscular volume, hepatic iron content and hepcidin mRNA in the liver. Thrombocytosis established in parallel. Moreover, platelets produced in iron deficient animals displayed a higher mean platelet volume and increased aggregation. Bone marrow studies revealed subtle alterations that are suggestive of expansion of megakaryocyte progenitors, an increase in megakaryocyte ploidy and accelerated megakaryocyte differentiation. Iron deficiency did not alter the production of hematopoietic growth factors such as thrombopoietin, interleukin 6 or interleukin 11. Megakaryocytic cell lines grown in iron-depleted conditions exhibited reduced proliferation but increased ploidy and cell size. Our data suggest that iron deficiency increases megakaryopoietic differentiation and alters platelet phenotype without changes in megakaryocyte growth factors, specifically TPO. Iron deficiency-induced thrombocytosis may have evolved to maintain or increase the coagulation capacity in conditions with chronic bleeding.

Figure 1

Induction of iron deficiency and anemia in rats. ID diet rapidly leads to drop in Hb (A), MCV (B) (n=4 per group) and hepatic iron content (C) (n=8 per group). Liver hepcidin mRNA was higher in control animals and only at detection limit in ID as measured by real time PCR (D; n=8 per group).

Figure 2

Iron deficiency induces thrombocytosis and enhances platelet aggregation. Development of ID was paralleled by a rise in platelet counts (A) and an increase in MPV (B; n=4 per group). Light transmission aggregometry demonstrated that, in ID, platelet aggregation was enhanced as reflected by the significantly higher area under the aggregometry curve (C; n=8 per group).

Figure 3

Morphometric analysis of megakaryocytes. BM histology samples were stained for CD61 to identify MEGs (A). Total MEG area (B), number of MEGs (C) and mean MEG area (D) were obtained by morphometrical analysis. No statistically significant differences between the groups could be observed for any of the parameters. N=8 per group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Iron deficiency inhibits proliferation but increases ploidy in megakaryocytic cell lines. HEL, Dami and CMK cells were grown in serum‐free medium either repleted with iron (control) or without iron‐ and transferrin (ID). ID led to a significant decrease in proliferation as assessed by MTT assays (A) but to an increase in polyploid cells (>4n), measured by flow cytometry (B (representative flow cytometry histograms of Hoechst‐stained HEL cells); C). In parallel, cell size increased (demonstrated for HEL cells, D). The results from 2 to 3 independent experiments are shown.