Key Points• Dynein-dependent microtubule sliding drives proplatelet elongation under static and physiological shear stress conditions. • Proplatelet formation is a process that can be divided into repetitive phases: extension, pause, and retraction.Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy. Furthermore, we show that microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein under static and physiological shear stress by using fluorescence recovery after photobleaching in proplatelets with fluorescence-tagged b1-tubulin. A refined understanding of the specific mechanisms regulating platelet production will yield strategies to treat patients with thrombocythemia or thrombocytopenia. (Blood. 2015;125(5):860-868)

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Platelet transfusions are frequently given to neonates with platelet counts (PCs) below an arbitrary trigger, 1,2 but studies have shown a poor correlation between low PC and bleeding, 2,3 highlighting the need for better tests to identify infants at risk.Neonates have a unique primary hemostatic system, characterized by hypofunctional platelets counterbalanced by factors that enhance clotting (high hematocrit, mean corpuscular volume, and von Willebrand factor concentrations). 4 Thus, we hypothesized that a global test of primary hemostasis, the Platelet Function Analyzer-100 (PFA-100), an in vitro equivalent to the bleeding time, would be a better marker of moderate to severe bleeding among preterm neonates with thrombocytopenia than the PC. The PFA-100 (Dade Behring) measures the time it takes for blood to occlude an aperture (closure time [CT]) following stimulation with collagen and epinephrine (CT-Epi) or collagen and adenosine diphosphate (CT-ADP). Based on a previous finding that the CT-ADP was better correlated with the PC than the CT-Epi in neonates, 5 we focused on the CT-ADP.

A total of 17 patients (31.5%) received 21 PTX at a mean (SD) PC of 49 (21) × 10 3 /μL. They exhibited significant 1-day increases in PC (mean [SD] change, 31 [9]; P = .002) and decreases in CT-ADP (mean [SE] change, −51 [22]; P = .03) but no significant changes in BS. Fresh frozen plasma (9 transfusions given to 8 infants [14.8%]) or erythrocyte transfusions were not associated with CT-ADP or BS changes (Table ). DiscussionThe main finding of this study was an association between changes in BS and changes in CT-ADP (but not PC), suggesting that primary hemostasis and bleeding are dynamic and more interconnected than PC and bleeding in extremely preterm neonates with thrombocytopenia. Limitations of our study included the small number of infants with PCs less than 50 × 10 3 /μL because of platelet transfusion practices in our neonatal intensive care units and the relatively small number of the more clinically relevant bleeding grades (ie, 3-4). The limited number of CT-ADPs obtained in each infant was because of the relatively high blood volume required by the test (800 μL). We also recognize that all associations presented may be subject to residual or unmeasured confounding variables.Platelet transfusions given at the thresholds used in this study increased PCs but did not reduce BSs. This was consistent with the Platelet Transfusion Thresholds in Premature Neonates (PlaNeT-2) study, in which PTX for PCs less 50 × 10 3 /μL did not reduce bleeding. 6 Together, these findings suggest that when PCs are not extremely low, other factors could be contributors to bleeding in neonates with thrombocytopenia, although the PC threshold below which a PTX reduces bleeding is unknown. Overall, 8 infants in our cohort also received 9 fresh frozen plasma transfusions for clinical bleeding. However, these transfusions did not reduce CT-ADP or BS and did not affect their association. Implementing CT-ADP or other measures of primary hemostasis in clinical practice may lead to novel approaches to manage thrombocytopenia in preterm neonates.

AimThis study examined the relationship between hypothalamic-associated hormones and behavioural and eating disorders in children with low birthweight.MethodsWe included 100 children (mean age 9.7 years): 39 were born preterm at <32 gestational weeks, 28 were full-term, but small for gestational age, and 33 were full-term controls. Behavioural histories were analysed, together with fasting blood samples of leptin, insulin, insulin-like growth factor-1 (IGF-I), prolactin, glucagon and cortisol.ResultsPreterm children had lower prolactin (p = 0.01) and higher IGF-I than controls (p < 0.05, adjusted for confounders), despite being significantly shorter than the predicted target height (p < 0.001). More preterm children displayed behavioural disorders (38% versus 10%, p < 0.001) and eating disorders (26% versus 8%, p < 0.05) than full-term children. These disorders were associated with lower leptin (p < 0.01), insulin (p < 0.05) and IGF-I (p < 0.05), but correlations between these hormones and leptin were similar among the groups. Combined behavioural and eating disorders were only observed in preterm children, who were also the shortest in height.ConclusionBehavioural and eating disorders among preterm children were associated with low leptin, insulin and IGF-1. Low prolactin in all preterm children indicated an increased dopaminergic tonus, which might inhibit body weight incrementation. This raises speculation about IGF-I receptor insensitivity.

BackgroundPreterm infants commonly receive red blood cell (RBC), platelet and fresh frozen plasma (FFP) transfusions. The aim of this Neonatal Transfusion Network survey was to describe current transfusion practices in Europe and to compare our findings to three recent randomised controlled trials to understand how clinical practice relates to the trial data.MethodsFrom October to December 2020, we performed an online survey among 597 neonatal intensive care units (NICUs) caring for infants with a gestational age (GA) of <32 weeks in 18 European countries.ResultsResponses from 343 NICUs (response rate: 57%) are presented and showed substantial variation in clinical practice. For RBC transfusions, 70% of NICUs transfused at thresholds above the restrictive thresholds tested in the recent trials and 22% below the restrictive thresholds. For platelet transfusions, 57% of NICUs transfused at platelet count thresholds above 25×109/L in non-bleeding infants of GA of <28 weeks, while the 25×109/L threshold was associated with a lower risk of harm in a recent trial. FFP transfusions were administered for coagulopathy without active bleeding in 39% and for hypotension in 25% of NICUs. Transfusion volume, duration and rate varied by factors up to several folds between NICUs.ConclusionsTransfusion thresholds and aspects of administration vary widely across European NICUs. In general, transfusion thresholds used tend to be more liberal compared with data from recent trials supporting the use of more restrictive thresholds. Further research is needed to identify the barriers and enablers to incorporation of recent trial findings into neonatal transfusion practice.

Eltrombopag (ELT) is a thrombopoietic agent approved for immune thrombocytopenia and also a potent iron chelator. Here we found that ELT exhibited dose-dependent opposing effects on in vitro megakaryopoiesis: low concentrations (≤6 µM, ELT6) stimulated megakaryopoiesis, but high concentrations (30 µM, ELT30) suppressed megakaryocyte (MK) differentiation and proliferation. The suppressive effects of ELT30 were reproduced by other iron chelators, supporting iron chelation as a likely mechanism. During MK differentiation, committed MK progenitors (CD34+/CD41+ and CD34−/CD41+ cells) were significantly more sensitive than undifferentiated progenitors (CD34+/CD41− cells) to the suppressive effects of ELT30, which resulted from both decreased proliferation and increased apoptosis. The antiproliferative effects of ELT30 were reversed by increased iron in the culture, as were the proapoptotic effects when exposure to ELT30 was short. Because committed MK progenitors exhibited the highest proliferative rate and the highest sensitivity to iron chelation, we tested whether their iron status influenced their response to ELT during rapid cell expansion. In these studies, iron deficiency reduced the proliferation of CD41+ cells in response to all ELT concentrations. Severe iron deficiency also reduced the number of MKs generated in response to high thrombopoietin concentrations by ∼50%, compared with iron-replete cultures. Our findings support the hypothesis that although iron deficiency can stimulate certain cells and steps in megakaryopoiesis, it can also limit the proliferation of committed MK progenitors, with severity of iron deficiency and degree of thrombopoietic stimulation influencing the ultimate output. Further studies are needed to clarify how megakaryopoiesis, iron deficiency, and ELT stimulation are clinically interrelated.