Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocytederived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P ؊ , LAMP-1 ؊ and expressed fulllength filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P ؊ and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet micropar-

Patients with a combined immunodeficiency characterized by normal numbers, but impaired function, of T and B cells had a homozygous p.Tyr20His mutation in transferrin receptor 1 (TfR1), encoded by TFRC. The mutation disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 surface expression. Iron citrate rescued the lymphocyte defects and transduction of wild type, but not mutant, TfR1 rescued impaired transferrin uptake in patient fibroblasts. TfrcY20H/Y20H mice recapitulated the patients’ immunologic defects. Despite the critical role of TfR1 in erythrocyte development and function, the patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares the patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

Objectives: Thrombocytopenia is common in neonatal intensive care units (NICU), with 18 to 35% of patients developing this problem before hospital discharge. It might be even more common among extremely low birth weight neonates (ELBW, p1000 g birth weight). However, little is known about thrombocytopenia in the ELBW population. We sought to determine the incidence, timing, causes, platelet transfusions given, and outcomes of thrombocytopenia among ELBW neonates. Results: Multiple platelet counts were obtained in all 284 (range, 4 to 441 platelet counts/patient). Of the 284, 208 (73%) had one or more platelet counts p150 000/ml. Most were detected during the first days of life; 80% were detected during the first week and only 20% were detected thereafter. Thrombcytopenia was more common among the smallest patients; 85% incidence among those p800 g, 60% among those 801 to 900 g, and 53% among those 901 to 1000 g. Platelet transfusions were given to 129 of the 208 thrombocytopenic neonates. More than 90% were given prophylactically (the patient was not bleeding). The mortality rate among those that received platelet transfusions was twice that of those that received no platelet transfusions (P<0.01). In 48% of cases, the cause of the thrombocytopenia went undiagnosed. The most common explanations were being small for gestational age or delivered to a hypertensive mother, DIC, bacterial infection, fungal infection, and necrotizing enterocolitis, respectively. Conclusions:We observed thrombocytopenia among ELBW neonates at a rate more than twice that reported among the general NICU population. Much remains to be discovered about the etiology and best treatments of thrombocytopenia among ELBW neonates.

Objective: Identifying a platelet count as abnormal (thrombocytopenia or thrombocytosis) can facilitate recognizing various disease states. However, the published reference ranges for platelet counts in neonates may be imprecise, as they were generated from relatively small sample sizes and compiled before modern platelet enumeration methods. Study design:We derived new neonatal reference ranges for platelet counts and mean platelet volume (MPV) measurements using electronic data accumulated during a recent 6-year period from a multihospital healthcare system.Result: Platelet counts were obtained between the first and the 90th day after birth, from 47 291 neonates delivered at 22 to 42 weeks gestation. The first platelet counts obtained in the first 3 days of life, increased over the range of 22 to 42 weeks gestation. In those born p32 weeks gestation, the lower reference range (5th percentile) was 104 200 ml À1 , but it was 123 100 ml À1 in late-preterm and -term neonates. Advancing postnatal age had a significant effect on platelet counts; during the first 9 weeks, the counts fit a sinusoidal pattern with two peaks; one at 2 to 3 weeks and a second at 6 to 7 weeks. The upper limit of expected counts (95th percentile) during these peaks were as high as 750 000 ml À1 . Conclusion:The figures herein describe reference ranges for platelet counts and MPV determinations of neonates at various gestational ages during their first 90 days. Expected values differ substantially from the 150 000 ml À1 to 450 000 ml À1 range previously used to define neonatal thrombocytopenia and thrombocytosis. The new definitions will render the diagnoses of neonatal thrombocytopenia and thrombocytosis less commonly than when the old definitions were used, because the new ranges are wider than 150 000 ml À1 to 450 000 ml À1 .

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...

BACKGROUND Recent studies have detected an association between red blood cell (RBC) transfusions and NEC. We hypothesized that RBC transfusions increase the risk of NEC in premature infants, and investigated whether the risk of ‘transfusion-associated’ NEC is higher in infants with lower hematocrits and advanced postnatal age. METHODS Retrospective comparison of NEC patients and controls born at <34 weeks gestation. RESULTS The frequency of RBC transfusions was similar in NEC patients (47/93, 51%) and controls (52/91, 58%). Late-onset NEC (> 4 weeks of age) was more frequently associated with a history of transfusion(s) than early-onset NEC (adjusted OR=6.7; 95% CI=1.5–31.2; p=0.02). Compared to non-transfused patients, RBC-transfused patients were born at an earlier gestational age, had greater intensive care needs (including at the time of onset of NEC), and longer hospital stay. A history of RBC transfusions within 48-hours prior to NEC onset was noted in 38% of patients, most of whom were extremely low birth weight (ELBW) infants. CONCLUSIONS In most patients, RBC transfusions were temporally unrelated to NEC and may be merely a marker of overall severity of illness. However, the relationship between RBC transfusions and NEC requires further evaluation in ELBW infants using a prospective cohort design.

Eltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log β=35). Clinically achievable concentrations (1 µM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators.

There is great variability in platelet transfusion practices among US and Canadian neonatologists, suggesting clinical equipoise in many clinical scenarios. Prospective randomized clinical trials to generate evidence-based neonatal platelet transfusion guidelines are needed.