In Newborn Mice, Tpo Signaling Is Required for Normal Megakaryocyte Proliferation, Maturation, Platelet Formation, and Platelet Survival

Congenital amegakaryocytic thrombocytopenia (CAMT) is a disorder caused by c-mpl mutations that block or severely reduce thrombopoietin (Tpo) signaling, leading to amegakaryocytic thrombocytopenia and ultimately aplastic anemia. In neonates and infants, CAMT can have different clinical manifestations than in adults, and cases have been described of newborn infants who presented with severe thrombocytopenia but normal numbers of immature-appearing megakaryocytes (MKs) in the bone marrow. To elucidate the potential mechanisms underlying the developmental differences in the manifestations of Tpo deficiency, we evaluated MKs and platelets in newborn c-mpl^-/- mice on postnatal day 1 (P1). Since the liver is the main site of megakaryopoiesis at that developmental stage (Liu et al., Blood 2014), liver MKs in P1 mice were immunohistochemically stained for VWF, quantified, measured, and ultrastructurally evaluated using transmission electron microscopy (TEM), as previously described. Histological analysis showed that MKs in newborn c-mpl^-/- mice were present at a concentration of 30% and were significantly smaller compared to WT pups, a finding previously reported in adult mice. Unlike in adult c-mpl^-/- mice, however, which have ultrastructurally normal MKs, TEM analysis of neonatal c-mpl^-/- liver MKs revealed a significant shift toward immature cells and maturational abnormalities in 68% of MKs, including a disorganized demarcation system, reduced granules, and an abnormally wide peripheral zone. Ultrastructural analysis of platelets from c-mpl^-/- P5 mice also revealed a larger open canalicular system and more glycosomes compared to age-matched WT platelets. These findings were not associated with increased levels of baseline P-selectin expression, suggesting that they were not the result of activation during processing. Next, we serially evaluated platelet counts in WT and c-mpl^-/- mice during the transition from fetal/neonatal to adult megakaryopoiesis. At P2, c-mpl^-/- platelet counts were 170±54x10³/µl, representing 26% of those in P2 WT mice (648±137x10³/µl). Over the first two weeks of life, WT platelet counts doubled and reached nearly adult levels, as previously described. In contrast, c-mpl^-/- platelet counts failed to increase, and remained stably low at P14. Since we previously showed that the rise in platelet counts during this period was associated with a one-day prolongation of the neonatal platelet lifespan (Liu et al., Blood 2014), we evaluated platelet survival in c-mpl^-/- compared to WT mice, using in vivo biotinylation. These studies suggested that the platelet lifespan was significantly shorter in c-mpl^-/- compared to WT newborn (P2) mice. In contrast, we found no differences in platelet lifespan between c-mpl^-/- and WT adult mice. Taken together, our findings suggest that, in fetal and neonatal life, Tpo signaling is required for normal MK proliferation, maturation, platelet formation, and platelet survival. This is in contrast to adult life, when Tpo deficiency leads only to reduced MK and platelet counts. We hypothesize that these differences reflect the lack of Tpo-independent pathways of MK maturation in the environment of the fetal/neonatal liver, and have implications in the developmental stage-specific clinical manifestations of CAMT.