Erythropoietin Stimulates Thrombopoiesis in the Absence of C-Mpl Signaling

Thrombopoietin (TPO) is essential for normal megakaryopoiesis, and mice and humans lacking the TPO receptor c-Mpl have significantly impaired platelet production. However, in the c-Mpl-null mouse model platelet counts, while reduced to ~10% of normal, are not zero, suggesting that another cytokine is able to support some degree of residual thrombopoiesis. We and others have reported that elimination or severe reduction of stem cell factor, G-CSF, IL-3, IL-6 or IL-11 does not eliminate residual thrombopoiesis. Because megakaryocytes (MKs) and erythrocytes are derived from a common progenitor, we asked if erythropoietin (EPO) can stimulate thrombopoiesis in c-Mpl-null mice. We administered 90 u recombinant EPO or vehicle by subcutaneous injection every 3 days to c-Mpl-null or WT control mice and measured baseline and weekly platelet counts. In three independent experiments, at 2 weeks platelet counts in c-Mpl-null mice receiving EPO were significantly higher that at baseline (5–7 mice per group, average of mean platelet counts 425,000/mm3 vs. 285,000/mm3, p=0.0015). There was a trend towards higher platelet counts in WT mice receiving EPO but this did not reach statistical significance. No difference in platelet counts was observed in mice injected with vehicle. In one experiment c-Mpl-null or WT mice were injected with EPO for 4 weeks and the platelet response in the c-Mpl-null animals was sustained for the duration of the experiment. Western blotting showed that murine MKs express the EPO receptor. To determine if EPO stimulates MK production directly we stimulated WT murine MKs in vitro with either 6 u/ml EPO, 100 ng/ml rhTPO or both and monitored activation of ERK and STAT5 signaling by immunoblotting. Stimulation of MKs with EPO resulted in phosphorylation of ERK and STAT5 (15- and 14-fold above baseline, respectively), compared to TPO (97- and 75-fold above baseline). Stimulation with EPO and TPO together had an additive effect (phospho-ERK increased 121-fold and phospho-STAT5 increased 100-fold). To determine if EPO acts primarily on early or late MKs, we harvested bone marrow from c-Mpl-null mice after 2 weeks of treatment with EPO or vehicle and measured CFU-MK frequency and MK ploidy. Although there was a small increase in the frequency of CFU-MK in mice treated with EPO compared to vehicle, these differences were not significant (n=3, p=0.7), possibly due to the difficulty in assaying CFU-MK in vitro without TPO. In addition, EPO did not significantly enhance MK ploidy in c-Mpl-null mice, although MKs in the 32N and greater peaks were slightly more numerous. Therefore, we conclude that EPO can augment platelet production in the absence of c-Mpl signaling, although it is not yet clear if EPO primarily acts on early or late cells. Additional experiments are underway to determine if ablating EPO receptors in a TPO-null mouse model will eliminate residual thrombopoiesis. These findings may have clinical relevance for treating patients with congenital amegakaryocytic thrombocytopenia and other causes of thrombocytopenia in which c-Mpl signaling is impaired.