No abstract

Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis in vitro. We have now examined whether PF4 regulates megakaryopoiesis in vivo by studying PF4 knockout mice and transgenic mice that overexpress human (h) PF4. Steady-state platelet count and thrombocrit in these animals was inversely related to platelet PF4 content. Growth of megakaryocyte colonies was also inversely related to platelet PF4 content. Function-blocking anti-PF4 antibody reversed this inhibition of megakaryocyte colony growth, indicating the importance of local PF4 released from developing megakaryocytes. The effect of megakaryocyte damage and release of PF4 on 5-fluorouracil-induced marrow failure was then examined. Severity of thrombocytopenia and time to recovery of platelet counts were inversely related to initial PF4 content. Recovery was faster and more extensive, especially in PF4-overexpressing mice, after treatment with anti-PF4 blocking antibodies, suggesting a means to limit the duration of such a chemotherapy-induced thrombocytopenia, especially in individuals with high endogenous levels of PF4. We found that approximately 8% of 250 healthy adults have elevated (> 2 times average) platelet PF4 content. These individuals with high levels of platelet PF4 may be especially sensitive to developing thrombocytopenia after bone marrow injury and may benefit from approaches that block the effects of released PF4. IntroductionMegakaryopoiesis is a complex process that is still not fully understood. Early studies identified thrombopoietin (TPO) as the predominant cytokine responsible for regulating platelet counts. However, many other cytokines have been postulated to participate in regulating megakaryopoiesis by increasing TPO expression in the liver (eg,), enhancing megakaryocyte chemotaxis (eg, stromal-derived factor-1 [SDF-1]), 1 or directly stimulating megakaryocyte development (eg, IL-11). 2 A pathway by which megakaryopoiesis is auto-down-regulated has been suggested based on in vitro studies of platelet factor 4 (PF4) and later by studies of other chemokines that are also stored in ␣-granules, including the related CXC chemokines, neutrophil activating peptide-2 (NAP-2), and 3,4 and the more distantly related CC chemokines, RANTES (regulated upon activation, normal T-cell-expressed and secreted), 5 and MIP-1␣ (macrophage inflammatory peptide-1␣). 4,5 PF4 is a 7.8-kDa protein that is produced primarily in megakaryocytes and expressed in platelets as a tetramer, where it comprises a significant portion of the content of ␣-granules (2.5% on a molar basis). 6 The biological role of PF4 is not fully understood. Unlike other chemokines that have clearly defined chemokine receptors, PF4 appears to function by binding with high affinity to glycosaminoglycans on cell surfaces. 7-9 PF4 has been proposed to participate in many important biological process based primarily on in vitro studies, including roles in angiogenesis, 10 inflammation, 11 atherosclerosis, 12,13 thrombosis, [14][15][16][17] and megakaryopoiesis. 4,5,18 Studies...

Multiple observations support the existence of developmental differences in megakaryocytopoiesis. We have previously shown that neonatal megakaryocyte (MK) progenitors are hyperproliferative and give rise to MKs smaller and of lower ploidy than adult MKs. Based on these characteristics, neonatal MKs have been considered immature. The molecular mechanisms underlying these differences are unclear, but contribute to the pathogenesis of disorders of neonatal megakaryocytopoiesis. In the present study, we demonstrate that low-ploidy neonatal MKs, contrary to traditional belief, are more mature than adult lowploidy MKs. These mature MKs are generated at a 10-fold higher rate than adult MKs, and result from a developmental uncoupling of proliferation, polyploidization, and terminal differentiation. This pattern is associated with up-regulated thrombopoietin (TPO) signaling through mammalian target of rapamycin (mTOR) and elevated levels of full-length GATA-1 and its targets. Blocking of mTOR with rapamycin suppressed the maturation of neonatal MKs without affecting ploidy, in contrast to the synchronous inhibition of polyploidization and cytoplasmic maturation in adult MKs. We propose that these mechanisms allow fetuses/neonates to populate their rapidly expanding bone marrow and intravascular spaces while maintaining normal platelet counts, but also set the stage for disorders restricted to fetal/neonatal MK progenitors, including the Down syndrome-transient myeloproliferative disorder and the thrombocytopenia absent radius syndrome. IntroductionMegakaryocytopoiesis is the process by which hematopoietic stem cells undergo lineage commitment to become megakaryocyte (MK) progenitors, which proliferate and generate immature MKs. These immature MKs then undergo successive rounds of endomitosis that give rise to unique highly polyploid cells. The process of polyploidization is associated with the increasing production of proteins necessary for platelet formation and function, 1 including membrane receptors such as CD41/61 and CD42, and platelet granule components such as VWF, platelet factor 4, and P-selectin. Polyploidization is also accompanied by progressive ultrastructural changes, particularly the formation of a complex demarcation membrane system (DMS), which, together with an accumulation of ␣ granules, characterizes fully mature MKs. These events set the stage for the production of proplatelets and the release of platelets by mature MKs. 2 Over the last decades, a mounting body of evidence has supported the existence of substantial biologic differences between fetal/neonatal and adult MKs. Several in vitro studies have shown that MK progenitors from fetuses and neonates proliferate at a much higher rate than adult progenitors. [3][4][5] Neonatal MKs, however, are significantly smaller and of lower ploidy (and produce fewer platelets) than MKs from adults. [6][7][8] Based on these characteristics, MKs from fetuses and neonates have been considered to be immature compared with adult MKs. 9 Whereas the cellular and m...

ABSTRACT:We serially evaluated the effects of sepsis and/or necrotizing enterocolitis (NEC) on neonatal thrombopoiesis, using a panel of tests that included platelet counts, thrombopoietin concentrations (Tpo), circulating megakaryocyte progenitor concentrations (CMPs), and reticulated platelets (RPs). Variables analyzed included sepsis type, time after onset of sepsis, platelet counts, and gestational (GA) and postconceptional ages (PCA). Twenty neonates were enrolled. Ten had Gram-negative, six had Gram-positive, and four had presumed sepsis. Four neonates had NEC stage II or higher, and six developed thrombocytopenia. Overall, septic neonates had significantly elevated Tpo concentrations and circulating megakaryocyte progenitors. The highest Tpo levels were associated with Gramnegative or presumed sepsis. RP percentages were increased only in neonates with low platelet counts, while RP counts (RP% ϫ platelet count) were elevated in neonates with high platelet counts. Our findings suggest that septic neonates up-regulate Tpo production, leading to increased megakaryocytopoiesis and platelet release, although the degree of upregulation is moderate. The changes in RP% and RP count most likely reflect increased thrombopoiesis with variable degrees of platelet consumption. In addition, our findings suggest that different factors, likely including level of illness and/or specific platelet or bacterial products, can down-regulate the magnitude of the thrombopoietic response.

Background: Sick neonates frequently develop severe thrombocytopenia. Objective and Methods: In order to test the ability of fetal mice to increase their megakaryocyte size and ploidy in response to thrombocytopenia, we injected an antiplatelet antibody (MWReg30) into pregnant mice daily for 7 days, and into nonpregnant adult mice to serve as controls. After that time, platelet counts were obtained and megakaryocytes in the bone marrow, liver, and spleen were stained with anti-von Willebrand factor antibody, individually measured, and quantified. Results: Our study demonstrated that megakaryocytopoiesis in newborn mice shares many features of human fetal/neonatal megakaryocytopoiesis, including the small size of megakaryocytes. In response to thrombocytopenia, adult mice increased megakaryocyte volume and concentration, primarily in the spleen. Newborn mice, in contrast, increased the megakaryocyte concentration in the spleen, but exhibited no increase in megakaryocyte volume in any of the organs studied. In fact, the megakaryocyte mass was significantly lower in the bone marrow of thrombocytopenic neonates than in age-matched controls. Conclusions: We concluded that fetuses have a limited ability to increase their megakaryocyte mass in response to consumptive thrombocytopenia, compared to adult mice. These observations provide further evidence for the existence of biological differences between fetal/neonatal and adult megakaryocytopoiesis.