Thrombocytopenia is a common clinical problem among neonates, affecting 30% of all infants admitted to the neonatal intensive care unit, and up to 70% of those born prematurely. The neonatal predisposition to develop thrombocytopenia has been largely attributed to the increasingly recognized substantial differences between fetal/neonatal and adult megakaryocytes (MKs). However, little is known about overall platelet production rates and platelet survival during neonatal life. To address this question, we evaluated the sites of thrombopoiesis, platelet counts, reticulated platelet percentages (RP%), blood volume, and platelet survival in C57/B6 mice during the first 2 wks of life. During that period, the MK concentration decreased sharply in the liver (the main site of platelet production during fetal life), while it increased steadily in the BM, where it reached nearly adult levels by day 14. During this transition, the spleen helped support thrombopoiesis through a 6-fold increase in MK concentration (which peaked at day 10), prior to returning to baseline levels. Consistent with our recent findings in human MKs (Liu et al., Blood), murine neonatal MKs were small but cytoplasmically mature by immunofluorescence and electron microscopy.
In regard to platelet production, platelet counts in one day old pups were approximately 50% of adults, but then increased and reached nearly adult values by day 14. During the same time period, the body weight and total blood volume increased ∼5-fold. Combined, these changes led to a 10-fold increase in the total platelet number over the first 2 wks of life. The RP%, a measure of new platelet release, was ∼2-fold higher in 3- and 10-day old pups compared to adults, suggesting that increased platelet production contributes to the rapid increase in total platelet number during this period of development. To evaluate for potential developmental differences in platelet survival, we conducted in vivo biotinylation studies in newborn and adult mice. These showed that neonatal platelets have a significantly longer half life than adult platelets (90 vs. 50 hrs, respectively; p<0.05). To confirm this finding in humans, we compared the survival of platelets isolated from human cord blood (CB) vs. adult peripheral blood (PB) stored in vitro for 5 days, using the mitochondrial dye JC-1 to identify life platelets and Annexin V as a marker of apoptotic platelets. In agreement with the murine findings, CB platelets consistently exhibited significantly higher percentages of JC-1 positive platelets compared to PB platelets (63.9±7.5% vs. 23.9±15.9% on day 3, p<0.001) and significantly lower percentages of Annexin V positive platelets (37.1±17.3% vs. 68.3±18.5% on day 3, p<0.001).
Platelet survival is regulated by the concentrations of Bcl-2 family members, primarily by the anti-apoptotic protein Bcl-xL. We therefore evaluated the concentrations of Bcl-2 and Bcl-xL in CB- and PB-derived platelets by Western Blot. Interestingly, we found no differences in Bcl-xL expression, but Bcl-2 protein concentrations were significantly higher in neonatal compared to adult platelets (n=5). Bcl-2 expression has previously been shown to be regulated by GATA-1 (Tanaka et al., Blood), and we recently demonstrated that neonatal MKs have significantly higher GATA-1 levels than adult MKs (Liu et al., Blood). In order to determine whether Bcl-2 expression is regulated by GATA-1 in MKs, we quantified Bcl-2 protein levels in a GATA-1-deficient megakaryocytic cell line (G1ME cells) following transduction with GATA-1-containing retrovirus (MIGR1-GATA-1-GFP) or with empty control virus (MIGR1-empty-GFP). In these studies (n=3), Bcl-2 levels were significantly higher in GATA-1 expressing G1ME cells vs. in controls.
In conclusion, our findings suggest that increases in platelet production and platelet lifespan both contribute to meet the demands associated with rapid growth and increasing platelet counts during early post-natal life. The long survival of neonatal platelets is associated with high Bcl-2 levels, which result from the elevated GATA-1 concentrations found in neonatal MKs. A better understanding of the developmental differences in platelet production and survival is critical to gaining insight not only on the pathogenesis and potential therapies of neonatal thrombocytopenia, but also on the delayed platelet engraftment that frequently complicates CB transplants.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.