Neonatal Platelet Research Articles

CDK8/19 inhibition triggers a switch from mitosis to endomitosis in cord blood megakaryocytes.

Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK-mediator complex, which regulates gene transcription in a cell-specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood-derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders.

Platelet antibody detection assays: a single-laboratory comparison of MAIPA, PIFT, and microsphere-based multiplex assays Pak-Lx

Background and ObjectivesThe identification of platelet antibodies is essential for diagnosing and managing conditions such as fetal and neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and immune platelet refractoriness. Monoclonal antibody immobilization of platelet antigens (MAIPA) is the standard method for detecting anti-human platelet antigen (HPA) antibodies, while the detection of anti-HLA antibodies once relied on the complement-dependent cytotoxicity method, however advanced technologies such as enzyme-linked immunosorbent assay and Luminex have significantly improved sensitivity and accuracy in identifying these antibodies. Flow cytometry-based techniques (platelet immunofluorescence test - PIFT) and Luminex platform-driven microsphere-based multiplex assays (Pak-Lx) are widely employed in platelet immunology laboratories owing to their remarkable flexibility and versatility. The present study compared the sensitivity, specificity, and concordance of these different serological techniques used in platelet antibody identification. Material and MethodsOne hundred serum samples from patients suspected of immune-mediated platelet disorders were examined. Initially, the samples underwent testing using the MAIPA method. Subsequently, the results were compared with three alternative methods: PIFT and microsphere-based multiplex assays for both HLA and HPA antibodies. ResultsPak-Lx demonstrated a 94 % agreement with MAIPA, while PIFT had 88 % agreement for HPA antibodies. For HLA antibody detection, Pak-Lx versus DLX had 75 % concordance, MAIPA versus DLX showed 77 %, and PIFT versus DLX displayed an 81 % concordance rate. Remarkably, there were no significant differences in concordance levels between Pak-Lx and PIFT compared to MAIPA and DLX for anti-HPA and HLA antibodies, respectively. ConclusionThis study found no significant differences in concordance among the tested assays for detecting anti-HPA and anti-HLA antibodies. These data suggest that no single method can detect all clinically important antibodies. Therefore, it is advisable that each laboratory develops customized protocols based on their expertise and employs complementary methods for comprehensive patient assessments.

Judicious Transfusion of Platelets Among Neonates: A Systematic Review.

In newborns, especially premature babies, there is a high association between thrombocytopenia and bleeding, particularly intraventricular hemorrhage, which may be due to immaturity. It was usual clinical practice that neonates should be transfused with higher platelet counts than older children or adults to reduce their risk of bleeding. However, after keen observations, we noticed that bleeding and mortality were more common in newborns who received more platelet transfusions. The mechanisms underlying the adverse effects of platelet transfusions in neonates may be due to higher antigenicity and immunological factors. We know that neonatal platelets are hyporeactive; this hyporeactivity is balanced by factors in the neonatal blood that promote coagulation, such as increased hematocrit, von Willebrand factor, and fibrinogen, which, on balance, leads to normal primary neonatal hemostasis. Platelets are very similar to adults in number, but functional capabilities were less, and for the reasons mentioned above, particularly bleeding time was short. Theologically, neonatal platelet lifespan was high to compensate for less production. We started this review because we observed that many babies were not having bleeding symptoms in some instances of severe thrombocytopenia. Many well-active babies are receiving unnecessary transfusions, as human blood is precious, and many young neonatologists are going on protocol-based excessive transfusions. This stimulated us to write a review.

Why might cord blood be a better source of platelets for transfusion to neonates?

Thrombocytopenia (defined as a platelet count <150×109/L) is a common condition in preterm neonates and may occur in 18-35% of all infants admitted to the Neonatal Intensive Care Unit (NICU). Neonatal platelet functionality in terms of reactivity is often described as reduced compared to adults, even in healthy, term neonates. However, this platelet "hyporeactivity" does not correspond to a global functional impairment of the normal delicately balanced neonatal hemostatic system. The extent to which neonatal thrombocytopenia and platelet hyporeactivity contribute to the bleeding risk in preterm neonates remains unknown. Prophylactic platelet transfusions are often administered to them to reduce the risk of bleeding. However, recent literature indicates that adopting a higher platelet transfusion threshold than a lower one results in significantly higher death rates or major bleeding and can be harmful. Although the mechanism by which this occurs is not entirely clear, a mismatch between adult transfused platelets and the neonatal hemostatic system, as well as volume overload, are speculated to be potentially involved. Therefore, future research should consider novel transfusion products that may be more suitable for premature neonates. Blood products derived from umbilical cord blood (UCB) are promising, as they might perfectly match neonatal blood features. Here, we discuss the current knowledge about UCB-derived products, focusing on UCB-derived platelet concentrates and their potential for future clinical application. We will discuss how they may overcome the potential risks of transfusing adult-derived platelets to premature infants while maintaining efficacy.

Is it possible to predict morbidities in neonates born to mothers with immune thrombocytopenic purpura?: A retrospective cross-sectional study.

Immune thrombocytopenic purpura (ITP) comprises ~1% to 4% of thrombocytopenia cases during pregnancy. Factors predicting neonatal thrombocytopenia and associated morbidities due to maternal ITP are unclear. The present study aimed to assess the neonatal outcomes of pregnant women with ITP. Fifty-five pregnant women with ITP and their babies, born between January/2013 and April/2021, were retrospectively reviewed. Maternal and neonatal thrombocytopenia cases other than ITP were excluded from the study. Physical examination, blood count, and cranial/abdominal ultrasonography findings of the newborns were recorded. Neonatal thrombocytopenia was defined as a platelet count < 150 × 109/L. Relationship between neonatal thrombocytopenia and maternal factors was investigated. Thrombocytopenia was detected in 17/55 babies (30.9%), and 8/17 (47.1%) had symptoms of bleeding, all but one being mild bleeding. There was a significant correlation between neonatal platelet counts of < 100 × 109/L and maternal splenectomy history. Incidence of moderate and severe thrombocytopenia was higher (statistically insignificant) in neonates of mothers with ITP. No significant correlation was determined between maternal and neonatal platelet counts. There was a weak insignificant correlation between platelet counts of neonates of mothers with or without thrombocytopenia. A significant correlation was found between the presence of splenectomy before delivery in the mother and a platelet count of < 100 × 109/L in the neonate. Moderate and severe thrombocytopenia was higher in neonates of mothers diagnosed with ITP before pregnancy and needed treatment during pregnancy and/or delivery, but the difference was insignificant. Close follow-up of babies born to mothers with ITP after birth is crucial since there is no significant prediction criterion for developing neonatal thrombocytopenia and associated morbidities.

Platelet proteomics emerges from the womb: mass spectrometry insights into neonatal platelet biology

Platelet proteomics emerges from the womb: mass spectrometry insights into neonatal platelet biology

Platelet transfusion practices among neonatologists in Saudi Arabia: A nationwide cross-sectional survey.

The etiologies of thrombocytopenia are highly diverse; however, early neonatal thrombocytopenia might be more common among extremely low-weight neonates. Therefore, in this study, we aimed to examine the current neonatal platelet (PLT) transfusion practices in Saudi Arabia. This is a cross-sectional online survey study that was conducted between October and December 2022. Convenience sampling was used to recruit the participants. In this study, we developed a questionnaire based on an extensive literature review to examine current neonatal PLT transfusion practices. A total of 81 neonatologists participated. The vast majority of them (85.2%) were practicing in a level 3 neonatal intensive care unit, with 60.0% of them reporting that they transfuse PLTs over 1 hour. Around 53% reported that they typically order 10 mL/kg per PLT transfusion. Up to 34.6% of the study participants reported that they use pooled whole-blood-derived PLT products in their practicing unit. Almost half (48.0%) of the study participants reported that they have written guidelines for PLT transfusion in their practicing unit, with 81.1% reporting that the PLT transfusion threshold was stated in the guidelines. Neonatal thrombocytopenia is typically treated with PLT transfusions. PLT transfusion criteria should be lowered in light of recent evidence suggesting that doing so may be counterproductive. However, there is some disagreement about whether a PLT count constitutes a medical emergency requiring a transfusion for a newborn baby. Furthermore, there is a great deal of variation in the administration of PLT infusions in Saudi Arabia because of the absence of clear protocols. Strict neonatal PLT transfusion standards and carefully planned clinical research are needed to address the risks and/or benefits of these diverse methods.

Neonatal Outcomes in Pregnant Women Complicated with Immune Thrombocytopenic Purpura

Background: Maternal antibodies that interact with both maternal and fetal platelets cause autoimmune thrombocytopenia. This happens in autoimmune diseases of the mother, such as immune thrombocytopenia purpura (ITP). Objective: The aim of the study was to evaluate the treatment and prognosis of newborns whose mothers had ITP. Methods: A multi center prospective study was carried out at the Duhok Obstetrics and Gynaecology Teaching Hospital and Kurdistan Private Hospital in Iraqi Kurdistan between November 2016 and November 2022. The requirements for inclusion were all expectant mothers with verified ITP diagnoses. Every newborn was monitored in the neonatal intensive care unit and had their thrombocytopenia assessed on their first day of life. Maternal age, parity, diagnosis time, history of previous splenectomy, platelet count at delivery and in the early stages of pregnancy, propensity for bleeding throughout pregnancy, treatment for ITP, and method of delivery based on obstetric circumstances were all noted. the baby's gender, gestational age at delivery, the neonatal platelet count at birth, haemorrhage, and thrombocytopenia therapy. Nominal variable statistics were expressed. Results: 25 neonates delivered by pregnant mothers with ITP during the trial period were all included in the analysis. The ITP patients' mothers were between 23 and 33 years old. There were 14 (56%) multigravida cases and 11 (44%) primigravida cases. Prior to three years of pregnancy, just one (4%) instance had a history of splenectomy. The mother's platelet count at delivery was less than 50 × 109/L in 7 (28%) cases. Thirteen (52%), utilised corticosteroids. Ten (40%) of the deliveries were caesarean sections. Two (8%) cases had gestational ages between 33 and 36.6 weeks, whereas 23 (92% of cases) had gestational ages between 37 and 38 weeks. In 12 (48%) cases, the birth weight ranged from 1200 to 2500 kg, whereas in the other cases, it was between 250 and 3300) kg in 13(52%) cases the new-born baby's gender was male. Twelve (48%) of the newborns had thrombocytopenia. Just three cases (about 25%) needed medical attention. Conclusion: Pregnancy counselling is necessary for a woman with a history of ITP in order to maximise her platelet count. While serious bleeding is uncommon in neonates with maternal ITP. Corticosteroids are an effective therapeutic option for both the mother and the new-born

Quantitative label-free mass spectrometry reveals content and signaling differences between neonatal and adult platelets

BackgroundRecent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. ObjectivesThis study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. MethodsWe isolated resting umbilical cord blood–derived platelets from healthy full-term neonates (n = 8) and resting blood platelets from healthy adults (n = 6) and compared protein and phosphoprotein contents using data-independent acquisition mass spectrometry. ResultsWe identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were significantly enriched in immunomodulatory proteins, including β2 microglobulin and CXCL12, whereas neonatal platelets were enriched in ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched in phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. ConclusionUsing label-free data-independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation between neonatal and adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Reduced Ca2+ mobilization in neonatal human platelets involves SARAF and pannexin-1.

Platelets from neonates have been shown to exhibit a reduced response to physiological agonists, such as thrombin; however, the mechanism behind these findings is poorly understood. Berna-Erro etal. now provide differences in SARAF and pannexin-1 expression and function between neonatal and maternal platelets that might shed some light on the underlying mechanism. Commentary on: Berna-Erro. SARAF overexpression impairs thrombin-induced Ca2+ homeostasis in neonatal platelets. Br J Haematol 2024;204:988-1004.

SARAF overexpression impairs thrombin-induced Ca2+ homeostasis in neonatal platelets.

Neonatal platelets present a reduced response to the platelet agonist, thrombin (Thr), thus resulting in a deficient Thr-induced aggregation. These alterations are more pronounced in premature newborns. Here, our aim was to uncover the causes underneath the impaired Ca2+ homeostasis described in neonatal platelets. Both Ca2+ mobilization and Ca2+ influx in response to Thr are decreased in neonatal platelets compared to maternal and control woman platelets. In neonatal platelets, we observed impaired Ca2+ mobilization in response to the PAR-1 agonist (SFLLRN) or by blocking SERCA3 function with tert-butylhydroquinone. Regarding SOCE, the STIM1 regulatory protein, SARAF, was found overexpressed in neonatal platelets, promoting an increase in STIM1/SARAF interaction even under resting conditions. Additionally, higher interaction between SARAF and PDCD61/ALG2 was also observed, reducing SARAF ubiquitination and prolonging its half-life. These results were reproduced by overexpressing SARAF in MEG01 and DAMI cells. Finally, we also observed that pannexin 1 permeability is enhanced in response to Thr in control woman and maternal platelets, but not in neonatal platelets, hence, leading to the deregulation of the Ca2+ entry found in neonatal platelets. Summarizing, we show that in neonatal platelets both Ca2+ accumulation in the intracellular stores and Thr-evoked Ca2+ entry through either capacitative channels or non-selective channels are altered in neonatal platelets, contributing to deregulated Ca2+ homeostasis in neonatal platelets and leading to the altered aggregation observed in these subjects.

Quantitative Label-Free Mass Spectrometry Identifies Molecular Content and Signaling Differences between Neonatal and Adult Platelets

Observational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by a limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Neonatal and adult platelets differ in reactivity and function, but we lack an understanding of underlying molecular differences. As proteins and kinase-driven phosphorylation signaling dictate platelet biology, we aimed to characterize altered protein and phosphoprotein content between neonatal and adult platelets to help explain differences in platelet reactivity and function. We isolated resting platelets from full term cord blood (n=9) and adult peripheral blood (n=7) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content. We identified 4745 high-confidence proteins in adult and neonatal platelets, including 331 differentially abundant proteins (≥1.5-fold change, p&amp;lt;0.05). Neonatal platelets had increased metabolic and ribosomal proteins, consistent with transcriptional data. Adult platelets were enriched for inflammatory proteins, including complement components, consistent with pro-inflammatory function. We also identified 2115 phospho-proteins (17852 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling that regulate platelet functions. Kinase enrichment analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities. Granule trafficking and degranulation proteins were among 1183 differentially abundant phosphoproteins in neonatal vs adult samples (p&amp;lt;0.05). For example, 31 different Reticulon 1 (RTN1) phosphopeptides were enriched in adult platelets (p&amp;lt;0.05). RTN1 regulates membrane trafficking and participates in SNARE-mediated exocytosis. Increased RTN1-directed membrane trafficking may promote increased activation and degranulation in adult vs neonatal platelets. Indeed, SNARE complex protein deficiency was previously suggested to mediate hyporeactive degranulation in neonatal platelets. We detected 2 phosphopeptides in adult samples that were absent from any neonatal sample. We reasoned that these could represent developmental stage-specific differences that exist only in adult platelets. One of the adult-specific phosphopeptides was in Rap1GAP2 (phospho-S588). Rap1GAP2 is a highly phosphorylated GTPase-activating protein that inhibits Rap1, a small guanine-nucleotide-binding protein that facilitates integrin activation in platelets. Rap1GAP2 binds synaptotagmin-like protein 1 (Slp1) to regulate dense granule secretion. These findings may link altered developmentally regulated Rap1GAP2 activities with platelet degranulation. A Platelet Cell Adhesion Molecule (PECAM) phosphopeptide (phospho-S726) was also found exclusively in adult platelets. PECAM facilitates inflammation and leukocyte transendothelial migration. PECAM phosphorylation in adult platelets may reflect an enhanced ability to participate in these processes. These findings reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, including pro-inflammatory activities and enhanced activation and/or degranulation propensity in adult platelets. RTN1-mediated SNARE complex function and Rap1 signaling activities represent novel and potentially modifiable targets to manipulate platelet activation and/or degranulation. Our results also help provide a biological rationale for the higher morbidity and mortality observed in preterm infants transfused with adult platelets, including the inadvertent introduction of serum complement and other pro-inflammatory molecules within transfusions of donated adult platelets.

Prednisone Plus Ivig Compared with Prednisone for Immune Thrombocytopenia during Pregnancy

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an isolated low platelet count and is the most common cause of thrombocytopenia during pregnancy, accounting for 1%-4% of thrombocytopenia in pregnant women. Women with ITP who have severe thrombocytopenia are at an increased risk for life-threatening obstetric complications. However, management of ITP during pregnancy can be challenging because treatment choices are limited. Corticosteroids or intravenous immunoglobulin (IVIg) are recommended as first-line treatments for pregnant women with ITP, but the response to initial treatment is approximately 39% and 38%, respectively, lower than that in nonpregnant patients with ITP. Therefore, in-depth studies investigating new solutions for the first-line treatment of ITP during pregnancy are urgently needed. Our previous retrospective study revealed an advantage of prednisone combined with IVIg compared to prednisone monotherapy for pregnant patients with ITP. Therefore, the aim of this prospective, randomized, open-label study was to further assess the efficacy and safety of prednisone plus IVIg compared to prednisone monotherapy in pregnant women with ITP. The study has been registered at clinicaltrials.gov. NCT05333744 Methods Eligible treatment-naive ITP patients during pregnancy with a platelet count &amp;lt; 30 × 10^9/L, accompanied by or without bleeding symptoms, were randomly assigned 1:1 to receive prednisone 20 mg per day for 4 weeks plus IVIg 400 mg/kg (total dose ≤20 g per day) for 5 days or prednisone 20 mg monotherapy per day for 4 weeks. Prednisone was gradually tapered to the maintenance dose of 5-10 mg per day until 6 weeks after delivery if there was a response to treatment; otherwise, prednisone was gradually tapered to withdrawal. The primary outcome was overall response (OR), defined as a platelet count ≥ 30 × 10^9/L, a doubling of the baseline platelet count and the absence of bleeding. Secondary endpoints included complete response (CR), time to response, platelet counts at delivery, platelet counts of newborns, and adverse events in parturients. CR was defined as platelet count ≥100×10^9/L measured on 2 occasions at least 7 days apart and the absence of bleeding. Results A total of 100 treatment-naive ITP patients during pregnancy were randomly assigned to either the prednisone plus IVIg (n=50) or prednisone monotherapy (n=50) groups. All the baseline characteristics were comparable between the two groups. The median ages of patients in the prednisone plus IVIg group and prednisone monotherapy group were 30.32 years (range, 22-43) and 30 years (range, 28-46), respectively. For patients in the combination and monotherapy groups, the mean estational ages at intervention start were 28.3 weeks and 30.9 weeks, respectively. All patients were followed up for more than 2 months. OR was observed in 23 (46.0%) patients in the combination group and 22 (44%) in the monotherapy group, and no significant difference was observed between the two groups (p=0.91). A significant difference was observed in the time to response between the prednisone plus IVIg group and prednisone monotherapy group (6.14 days vs. 9.84 days, p=0.001). However, the predelivery platelet transfusion of the prednisone plus IVIg group was greater than that of the prednisone monotherapy group. There were no significant differences in the delivery mode between the combined group and monotherapy group. No significant differences were found between the two groups in neonatal outcomes, particularly concerning the neonatal platelet counts. Conclusion Prednisone plus IVIg was an effective and safe treatment for ITP in pregnancy, and this therapy may be a new first-line treatment option for pregnant patients with ITP.

Risk of thrombocytopenia in neonates of thrombocytopenic mothers.

Maternal thrombocytopenia during pregnancy may occur due to several possible etiologies, with potential neonatal impact. The aim of the present study was to investigate whether there is a correlation between maternal and neonatal platelet count among women with thrombocytopenia during pregnancy. A cross-sectional retrospective study (2012-2019) was conducted at a tertiary medical center. Complete blood count was routinely measured in all patients on admission to the delivery ward. Thrombocytopenia was defined as a platelet count below 150 K/μL. Clinical and outcome parameters of thrombocytopenic mothers and their newborns were collected from the electronic files and analyzed by severity of maternal thrombocytopenia. Of 45 385 women with a documented platelet count at admission, 2841 (6.24%) had thrombocytopenia: 2623 (5.7%) mild (100-149 K/μL), 207 (0.45%) moderate (50-99 K/μL), and 11 (0.02%) severe (<50 K/μL). Eight newborns had thrombocytopenia; corresponding rates by severity of maternal thrombocytopenia were 0.11%, 1.43%, and 18.18% (P = 0.04). None of the thrombocytopenic neonates had an intraventricular hemorrhage or other bleeding complications. The correlation between maternal and neonatal platelet counts was weak (Pearson r = 0.038; P = 0.046). We suggest that although the chances of neonatal thrombocytopenia are higher with worsening maternal thrombocytopenia, actual occurrence is rare, and the correlation is poor. Therefore, maternal thrombocytopenia cannot be regarded as a significant risk factor for neonatal thrombocytopenia. Neonatal platelet count should be obtained when maternal thrombocytopenia is autoimmune or less than 100 K/μL.

Developmental Differences between Neonatal and Adult Platelets in Regard to Immune Protein Content and Regulation of Monocyte Inflammatory Responses

Preterm neonates frequently receive platelet transfusions (PTx), owing to their high incidence of thrombocytopenia and bleeding. However, a recent large randomized trial found a significantly higher incidence of death or major bleeding among more liberally transfused preterm neonates. The mechanisms underlying these deleterious effects are unknown, but it has been suggested that they are related to the developmental differences between adult and neonatal platelets, and to a potential “developmental mismatch” occurring when adult platelets (comparatively hyperreactive) are transfused into neonates. While most studies so far have focused on the hemostatic effects of PTx, it is now clear that platelets are key modulators of immune and inflammatory responses. We hypothesized that adult platelets would be more pro-inflammatory than neonatal platelets. To test this hypothesis, we first compared the proteome of resting cord blood (CB, n=9) and adult (n=7) platelets. Using data-independent acquisition mass spectrometry, we identified 4173 high-confidence proteins present in both adult and neonatal platelets. In a principal component analysis, neonatal and adult platelets clustered separately, and 359 proteins were present at significantly different relative abundances between the two groups (≥1.5-fold change, p&amp;lt;0.05). Pathway analysis of the 169 proteins significantly more abundant in the adult platelet proteome revealed enrichment for immune and inflammatory pathways, while neonatal platelets were enriched for pathways involved in metabolic processes. Next, we compared the effects of neonatal and adult platelets on the inflammatory response of human neonatal monocytes. Platelets and monocytes were isolated from healthy adult blood or full-term CB (n=6/group). Platelet P-selectin surface expression was determined by flow cytometry, and key platelet-derived immune regulators (RANTES, β-2 Microglobulin [β2M], and TGF-β) were quantified in the supernatant of resting and activated platelets by ELISA. After washing, neonatal and adult platelets had similarly low P-selectin surface expression levels. Following thrombin activation, P-selectin surface expression was significantly higher in adult than in neonatal platelets (MFI 4010±1645 vs. 1987±816, p=0.04). Thrombin activation increased RANTES and TGF-β release from adult and neonatal platelets to similar levels. In contrast, β2M levels were significantly higher in the supernatant from activated adult compared to neonatal platelets (37.9±10.2 vs 17.9±5.0 ng/mL, p=0.0004). Next, we co-cultured activated adult or neonatal platelets with neonatal monocytes and quantified the pro-inflammatory cytokines IL-8 and MCP-1 (CCL2) in the supernatants after 18 hours. The presence of activated platelets significantly increased IL-8 production from neonatal monocytes compared to monocytes alone, 2.9±1.2-fold when neonatal platelets were added and 4.2±1.8-fold with addition of adult platelets (p=0.01 and p=0.001, respectively). MCP-1 increased to similar levels with the addition of either neonatal or adult platelets. In a regression model, both P-selectin expression and platelet-released β2M levels were significantly associated with monocyte-produced IL-8 levels (p=0.001 and p=0.04, respectively). In summary, we found that proteins related to inflammatory and immune responses are more abundant in adult compared to neonatal platelets, and that activated adult platelets express more P-selectin and release more β2M than neonatal platelets, resulting in higher IL8 production by neonatal monocytes. These findings support the hypothesis that the pro-inflammatory effects of adult platelets might contribute to the increased morbidity and mortality associated with neonatal PTx.

OC 67.1 Characterizing the Differences in Adult and Neonatal Biophysical Platelet Function at the Single Cell Level

OC 67.1 Characterizing the Differences in Adult and Neonatal Biophysical Platelet Function at the Single Cell Level

Predictive effects of platelet-to-lymphocyte ratio on neonatal thrombocytopenia in primary immune thrombocytopenic mothers: a retrospective cohort study

BackgroundPrimary immune thrombocytopenia (ITP) can increase the risk of neonatal thrombocytopenia (NT). This study aimed to investigate the key factors for predicting the risk of NT.MethodsData were retrospectively collected from all pregnant women with ITP from 2015 to 2021. Newborns were divided into two groups according to the presence or absence of NT. The parameters between the two groups were then compared. Next, the correlation between maternal platelet-to-lymphocyte ratio (PLR) and neonatal platelet count was analyzed by logistic regression and generalized additive model. Additionally, the relationships among the platelet counts of siblings were also determined.ResultsA total of 147 maternal cases were included. NT was observed in 46 (31.72%) neonates. A history of previous children with NT appeared to have predictive value for NT (OR 16.484, 95% CI 2.212–122.858, P < 0.001), as the nadir gestational platelet (OR 0.958, 95% CI 0.93–0.988, P < 0.001). Correlation analysis of platelet count on postnatal day 1 and the nadir platelet count in 36 sibling neonates showed a positive correlation (r=0.684, P<0.001; r=0.900, P<0.05). PLR was divided into 3 groups via tertiles, and the incidence of NT was dramatically higher in the group with lower PLR during the second and third trimesters than in the other two groups (48.5% vs 33.3% vs 22%, P<0.05; 50% vs 21.3% vs 26.7%, P<0.001). Moreover, the risk of NT was markedly higher in the first trimester (PLR < 78.51; OR 0.975, 95% CI 0.951–0.999, P<0.05) and the second trimester (PLR < 20.41; OR, 0.899, 95% CI 0.820–0.985, P<0.05) compared to the third trimester.ConclusionOur findings suggest that a history of previous children with NT is a significant factor for predicting NT in subsequent pregnancies. PLR in the first, second and third trimesters can also be used as a reference to predict NT risk.

Fetal and neonatal alloimmune thrombocytopenia: No evidence of systemic inflammation as a modulator of disease severity. Couldplacental inflammation be key?

In fetal/neonatal alloimmune thrombocytopenia (FNAIT), maternal alloantibodies against paternal human platelet antigens (HPA) cross the placenta and lead to platelet destruction. The extent of thrombocytopenia varies among neonates, and inflammation may constitute an important trigger. A set of stable inflammatory markers was measured in serum samples from neonates with low platelet counts, of which n = 50 were diagnosed with FNAIT due to anti-HPA-1a antibodies and n = 50 were thrombocytopenic without detectable maternal HPA antibodies. Concentrations of C-reactive protein, soluble CD14, procalcitonin, and sFlt-1 did not differ between the two cohorts. There was no correlation between C-reactive protein or soluble CD14 and the platelet count, but a negative correlation between procalcitonin concentrations and the neonatal platelet count in both cohorts. sFlt-1 concentration and the platelet count were correlated in FNAIT cases exclusively. None of the inflammatory markers was statistically different between cases with and without intracranial haemorrhage. We were unable to identify systemic inflammation as a relevant factor for thrombocytopenia in FNAIT. The antiangiogenic enzyme sFlt-1, released by the placenta, did correlate with the platelet count in FNAIT cases. Our findings may give rise to the hypothesis that placental inflammation rather than systemic inflammation modulates disease severity in FNAIT.

Abstract 380: Adult But Not Neonatal Platelets Promotes Monocyte Trafficking In Neonatal Mice

Background: Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there is little clinical data to support this practice and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune related mRNA compared to adult platelets. In this study we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. Methods: Platelets and bone marrow isolated monocytes were co-cultured and monocyte phenotypes determined by RNA-seq and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet deficient thrombopoietin receptor mutant mice (TPOR -/- ) were transfused with adult or P7 platelets and monocyte phenotypes and trafficking were determined. Results: Adult and neonatal platelets had differential immune molecule expression, including Selp (P-selectin). Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6C hi ), but different trafficking phenotypes, as defined by CCR2 and CCR5 surface expression. Blocking P-selectin-PSGL-1 interaction, monocytes limited the adult platelet induced monocyte trafficking phenotype, as well as adult platelet induced monocyte migration in vitro . Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or P7 platelets; adult platelets increased monocyte CCR2 and CCR5 as well as monocyte chemokine migration, while P7 platelets did not. Conclusion: These data provide comparative insights into adult and neonatal platelet transfusion regulated monocyte functions.

EBNEO commentary: Platelet transfusions in neonates and brain development: The new frontier.

Preterm neonates have a high incidence of thrombocytopenia and intraventricular haemorrhage (IVH). Because of this, they are traditionally transfused at higher platelet count (PC) thresholds compared to older children and adults, under the premise that higher PCs decrease bleeding risk.1 Paradoxically, the largest randomised platelet transfusion threshold trial in preterm neonates (PlaNeT-2), found a higher incidence of death or major bleeding within 28 days of randomisation (primary outcome) and a higher incidence of bronchopulmonary dysplasia (BPD) among neonates randomised to a higher platelet transfusion threshold (<50 × 109/L), compared to those randomised to a lower threshold (<25 × 109/L).2 Neonates with a high baseline risk of bleeding and/or death benefited from the lower platelet transfusion threshold as much as low-risk neonates.3 In this manuscript, Moore and collaborators provide evidence that high platelet transfusion thresholds are associated with increased mortality and neurodevelopmental impairment (NDI) at 2 years of corrected age.4 The study had a high follow-up rate (92%), combining results from formal neurodevelopmental (ND) assessment tools (41% of participants), review of medical records, practitioners' notes and early intervention services. Participants were dichotomised as having a favourable or unfavourable outcome (death or severe CP, developmental delay or visual or hearing loss). The combination of different methods to assess ND outcomes is a limitation of the study and restricted the ability of investigators to evaluate subtle deficits or specific neurologic domains. Future trials should include standardised ND assessments at 2 years and be powered for this outcome. Nevertheless, the fact that only severe deficits were included made the findings even more concerning. Given the initial finding that neonates randomised to a higher platelet transfusion threshold had a higher incidence of BPD, the authors performed a post hoc analysis that found a higher need for respiratory support at 2 years among children randomised to the high threshold group. While BPD is common among preterm infants, oxygen dependence or need for respiratory support at 2 years of age is not. Thus, the higher incidence of this outcome among children randomised to the higher threshold suggests platelet transfusions given to high-risk preterm neonates might trigger or enhance pathways leading to severe lung disease. The mechanisms underlying increased morbidity and mortality associated with platelet transfusions are likely related to non-hemostatic effects of platelets. Platelets are critical regulators of immune and inflammatory responses, and it has been hypothesised that adult (transfused) platelets are more pro-inflammatory than neonatal platelets.5-8 In support of this hypothesis, pre-clinical studies suggest platelet transfusions trigger inflammation or dysregulated immune responses in neonates, in a context-dependent manner.9 Whether NDI results from the direct effects of transfused platelets on the developing brain or is mediated by higher rates of IVH and BPD observed in infants randomised to the high threshold group (both of which are associated with NDI), is unknown. Additional studies are needed to define the mechanisms underlying the deleterious and beneficial effects of platelet transfusions, which remain lifesaving in neonates with bleeding or severe thrombocytopenia. In the meantime, this study further supports the use of a restrictive, evidence-based approach to neonatal platelet transfusions. URL LINK: https://ebneo.org/ebneo-commentary-platelet-transfusions-in-neonates-and-brain-development-the-new-frontier/. The authors are supported by grants PO1 HL046925 (MSV), K99 HL156051 (PD) and P01HL152961 (CD). None.