Adult Platelets 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.

Changes in platelet transfusion thresholds utilized in the NICU since PlaNeT-2

Abstract Background Use of prophylactic platelet transfusions in the neonatal setting is a common practice due to concerns about bleeding despite evidence of a developmental mismatch with transfusion of adult platelets. The Platelets for Neonatal Transfusion-Study 2 (PlaNeT-2) published in January 2019 showed that using higher platelet thresholds (50 x 109/L) was associated with more adverse outcomes and no improvement in bleeding events compared to lower platelet thresholds (25 x 109/L). Following this trial, implementation of a lower threshold was provider dependent as there were no changes made to the neonatal intensive care unit (NICU) institutional policies. Our goal was to determine if there were any overall changes in individual clinical practice with thresholds used for platelet transfusions in our NICU and number of transfusions per patient. Methods Our center has a 52 bed level IV NICU that specializes in care for critical and/or preterm infants. We performed a retrospective review from 2017 to 2023 of patients admitted in the NICU who received platelet transfusion and had an available platelet count in the 12 hours prior to the transfusion. Results There were 272 platelet transfusions given to 107 unique NICU patients who had platelet counts available. There were 66 transfusions given to 32 patients pre-PlaNeT-2 (2017-2018) and 206 transfusions given to 75 patients post-PlaNeT-2 (2019-2023). The average platelet count pre-PLaNeT-2(2017 to 2018) was 43 x 109/L (SD±24 x 109/L) and post-PLaNeT-2 (2019 to 2023) was 36 x 109/L (SD±21 x 109/L, p=0.03). Prior to PLaNeT-2, 26% of platelet transfusions were for platelet counts ≤ 25 x 109/L and 35% were for platelet counts between 51 to 100x9/L. Post PLaNeT-2 the percentages changed to 35% and 21% respectively. The percentage of transfusions occurring for platelet counts between 26 to ≤50 x 109/L was 38% and 44%, respectively. The number of platelet transfusions per year did not decrease significantly after the PLaNeT-2 trial (average of 33/year versus 41/year, p=0.5). The number of transfusions per patient before and after PLaNeT-2 did not change (2.1 transfusions per patient versus 2.6 transfusions per patient, p=0.3). Conclusion Platelet thresholds utilized in our center decreased after the PLaNeT-2 trial with less transfusions occurring for platelet counts greater than 50 x 109/L. Although thresholds decreased, the number of transfusions per year and the number of transfusions per patient did not significantly change. Further studies are needed to understand changes in patient outcomes and platelet utilization in the NICU.

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

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.

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.

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.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Therapeutic potential of adult platelets and cord blood platelets in future clinical directions

Platelet rich plasma (PRP) is currently obtaining a natural concentration of autologous growth factors, and is used in different medical conditions. This review is aimed to establish several treatment options using adult PRP and scope of the therapeutic benefits of cord blood platelet rich plasma (CB-PRP) for future clinical directions. A literature search was done based on the clinical applications of PRP in the fields of dermatology, orthopaedic, dental, ophthalmology and gynaecology. The field keywords were searched in major indexing services for collecting the relevant articles from January 2010 to June 2023. PRP therapies are potential and efficient treatment options and established protocols for certain diseases but there is a need to establish CB-PRP opportunities of improving efficacy for promoting clinical application. Further research can focus on CB-PRP and it may be considered as immunologically compatible to use as allogenic. Keywords: platelet rich plasma; therapy; cord blood PRP; hair loss; poor ovarian reserve; osteoarthritis

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.

Transfusion of Adult, but Not Neonatal, Platelets Promotes Monocyte Trafficking in Neonatal Mice.

Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are 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 with 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. Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined. Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6Chi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on 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 postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not. These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.

The Usability of Platelet Mass Index Thresholds to Assess the Repeated Platelet Transfusion Requirements in Neonates.

To evaluate the usability of platelet mass index (PMI) thresholds to assess the repeated platelet transfusion requirements in neonates who have received transfusion within the previous six days. This is a retrospective cross-sectional study conducted with neonates who received prophylactic platelet transfusion. The PMI was calculated as platelet count (× 1000/mm3) × mean platelet volume (MPV) (fL). Platelet transfusions were divided into two groups as first (Group 1) and repeated transfusions (Group 2). The increment and percentage of increment in platelet counts, MPV and PMI after transfusion were compared between the two groups. The amounts of changes were calculated as: (Post-transfusion) - (Pre-transfusion values). The percentages of changes were calculated as: ([Post-transfusion - Pre-transfusion values]/Pre-tansfusion values) × 100. Eighty three platelet transfusions were analyzed in 28 neonates. The median gestational age and birth weight were 34.5 (26-37) weeks, and 2225 (752.5-2937.5) grams, respectively. There were 20 (24.1%) transfusions in Group 1, and 63 (75.9%) transfusions in Group 2. There were no differences in the amounts of changes in platelet counts, MPV and PMI between the groups (p > 0.05). When the percentages of changes were analyzed, it was found that the platelet counts and PMI in Group 1 increased to a greater extent compared to Group 2 (p = 0.026, p = 0.039, respectively), but no significant difference was found in MPV between the groups (p = 0.081). The lower percentage of change in PMI in Group 2 was associated with the lower percentage of change in platelet counts. Being transfused with adult platelets did not affect platelet volume of the neonates. Therefore, PMI thresholds can be used in neonates with a history of platelet transfusion.

Immunologic effects of red blood cell and platelet transfusions in neonates.

Premature neonates are frequently transfused red blood cells (RBCs) or platelets to raise hemoglobin or platelet counts. However, these transfusions may have unintended effects on the immune system. This review will summarize the newest discoveries on the immunologic effects of RBC and platelet transfusions in neonates, and their potential impact on neonatal outcomes. Neonatal RBC transfusions are associated with increases in plasma pro-inflammatory cytokines, but recent findings suggest sex-specific differential responses. At least one cytokine (monocyte chemoattractant protein-1) rises in females receiving RBC transfusions, but not in males. These inflammatory responses correlate with poorer neurodevelopmental outcomes in heavily transfused female infants, while preterm male infants seem to be more sensitive to severe anemia. Platelet transfusions in preterm neonates are associated with increased neonatal mortality and morbidity. The underlying mechanisms are unknown, but likely related to the immune/inflammatory effects of transfused platelets. Adult platelets are different from neonatal platelets, with the potential to be more pro-inflammatory. Early preclinical data suggest that platelet transfusions alter the neonatal systemic inflammatory response and enhance immune cell migration. RBC and platelet transfusions alter neonatal immune and inflammatory responses. Their pro-inflammatory effects might worsen neonatal disease or affect neurodevelopmental outcomes.

Neonatal Platelets: Lower G12/13 Expression Contributes to Reduced Secretion of Dense Granules.

Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor densities, reduced calcium mobilization, and functional impairments of G proteins. Reduced secretion of dense granules has been attributed to lower numbers of granules. Signaling studies with adult platelets have shown a regulating effect of the G12/13 signaling pathway on dense granule secretion via RhoA. We comparatively analyzed secretion profiles using flow cytometry and expression levels of Gq, Gi, and G12/13 using Western blot analysis in platelets from cord blood and adults. Furthermore, we evaluated Rho activation after in vitro platelet stimulation with thrombin using a pulldown assay. We observed a markedly reduced expression of the dense granule marker CD63 on neonatal platelets after thrombin stimulation. Gα12/13 expression was significantly decreased in neonatal platelets and correlated with lower Rho activation after thrombin stimulation. We conclude that lower expression of G12/13 in neonatal platelets results in attenuated activation of Rho and may contribute to reduced secretion of dense granules after exposure to thrombin.

Effects of Momordica charantia exosomes on platelet activation, adhesion, and aggregation.

The platelets play a crucial role in the progression of multiple medical conditions, such as stroke and tumor metastasis, where antiplatelet therapy may be a boon for treating these diseases. In this study, we have attempted to study the effects of extracted Momordica charantia exosomes (MCEs) on platelet activation, adhesion, and aggregation. Adult platelets isolated from healthy individuals were dose-dependently treated with MCEs (0.1, 40, and 200 μg/ml). We performed flow cytometry to detect the expression of platelet activation protein marker-activated GP IIb/IIIa (PAC-1) and P-selectin (CD62P). Platelet adhesion was analyzed through fluorescence labeling assays. The effect of MCEs on platelet-mediated cell migration of HCT116 cells was observed by transwell. Furthermore, the MCAO model of Sprague-Dawley rats was used to observe the effect of MCEs (200, 400, and 800 μg/kg) on platelet aggregation and maximum thrombotic agglutination in vivo . The results showed that 200 μg/ml MCEs exerted the most pronounced effect on platelet activation, adhesion, and aggregation. Experiments on animals showed that MCEs significantly inhibited platelet aggregation and attenuated the maximum thrombus agglutination. We concluded that MCEs inhibited platelet activation, adhesion, aggregation, and platelet-mediated migration of HCT116 cells, indicating the potential role MCEs may play in the treatment of stroke and tumor metastasis.

Platelet transfusions in a murine model of neonatal polymicrobial sepsis: Divergent effects on inflammation and mortality.

Platelet transfusions (PTxs) are often given to septic preterm neonates at high platelet count thresholds in an attempt to reduce bleeding risk. However, the largest randomized controlled trial (RCT) of neonatal transfusion thresholds found higher mortality and/or major bleeding in infants transfused at higher thresholds. Using a murine model, we investigated the effects of adult PTx on neonatal sepsis-induced mortality, systemic inflammation, and platelet consumption. Polymicrobial sepsis was induced via intraperitoneal injection of cecal slurry preparations (CS1, 2, 3) into P10 pups. Two hours after infection, pups were transfused with washed adult Green Flourescent Protein (GFP+) platelets or control. Weights, platelet counts, and GFP% were measured before 4 and 24 h post-infection. At 24 h, blood was collected for quantification of plasma cytokines. The CS batches varied in 24 h mortality (11%, 73%, and 30% in CS1, 2, and 3, respectively), due to differences in bacterial composition. PTx had differential effects on sepsis-induced mortality and systemic inflammatory cytokines, increasing both in mice infected with CS1 (low mortality) and decreasing both in mice infected with CS2 and 3. In a mathematical model of platelet kinetics, the consumption of transfused adult platelets was higher than that of endogenous neonatal platelets, regardless of CS batch. Our findings support the hypothesis that transfused adult platelets are consumed faster than endogenous neonatal platelets in sepsis and demonstrate that PTx can enhance or attenuate neonatal inflammation and mortality in a model of murine polymicrobial sepsis, depending on the composition of the inoculum and/or the severity of sepsis.

Management of Type II Diabetes Mellitus using Adult Autologous Adipose derived stem cells with Platelets Rich Plasma (PRP)

Type II diabetes mellitus (T2DM) is known as non-insulin dependent diabetes mellitus. Insulin impairment and insulin resistance are the major causes of type II diabetes mellitus. People with T2DM are more prone to macro-vascular and micro vascular impediments. Adult Autologous Adipose derived stem cells (ADSC) hold potential to develop into multilineages and they can secrete numerous growth factors that can regenerate the damaged neighboring cells. The chronic wound healing processes and diabetes-associated complications are reported to be treated by using stem cells by revascularization and by promoting the micro vascular remodeling. Objective: To evaluate the safety and efficacy of Adipose derived autologous stem cells and PRP in patients suffering from Type 2 Diabetes Mellitus (T2DM). Methods: We reported three cases aged 50, 69 and 72 years suffering from T2DM since 20, 10 and 20 years respectively. They were treated with adult autologous ADSC and Platelets Rich Plasma (PRP). Results: Over a follow up of twelve months significant control in glycemia as well as stable glycosylated hemoglobin with decrease in pain on Visual Analogous scale (VAS) score has been observed

Abstract 11291: Ectopic Olfactory Receptor Expression in Platelets as Novel Antithrombotic Targets

Introduction: Platelets are enriched in orphan G-protein-Coupled Receptors (GPCRs) that could potentially regulate platelet reactivity. A remote study in patients with hereditary anosmia reported dysfunctional platelet responses suggesting the presence of platelet olfactory receptor (ORs) that could regulate thrombosis. Hypothesis: Platelet ORs serve as an alternative pharmacological strategy to generate platelet cyclic adenosine monophosphate (cAMP) to prevent thrombosis. Methods: Ribonucleic Acid sequencing (mRNAseq) of human megakaryocytes (MKs) was conducted. Various ORs were validated in platelets by RT-qPCR and immunoblotting (IB). Reporter cell lines with stable integration of cloned ORs identified OR ligands and later validated in adult platelets. Fluorophores for OR second messengers (Fura2 for Ca 2+ and MQAE for Cl - ) and an ELISA for cAMP were used for adult platelets. Platelet reactivity in response to OR agonists was evaluated by FACS and aggregometry, and thrombosis by biomechanical platelet activation was evaluated using ex vivo microfluidics and flow cones to simulate disturbed flow (D-flow). OR agonist injection into mice and CRISPR-Cas9 OR knockdown was conducted, with platelet reactivity and thrombosis tested as proof-of-concept. Results: Only 15 of &gt;400 ORs in the genome were identified in MKs, and only 3 in all adult platelets (highest expression was OR2L13). A terpene family was identified as OR2L13 agonists, generating cAMP in the platelet, and structural modifications were made with IC 50 values in the low-high μM range for inhibiting agonist-induced platelet activation, ex vivo human blood thrombosis by microfluidics, and in vivo platelet activation in mice with prolonged tail bleeding. Deletion of OR2L13 (olfr168 -/- ) in mice augmented platelet reactivity with loss of OR agonist-mediated prolonged tail bleeding in olfr168 -/- . OR2L13 in human platelets is stored and trafficked by alpha granules following surface agonist stimulation and biomechanical activation. Conclusions: Like neurons, platelets have a fully functional OR signal transduction cascade which operates as a negative regulator of platelet reactivity and thrombosis in response to both chemical as well as biomechanical platelet activation.

Adult, but Not Neonatal, Platelet Transfusions Drive a Monocyte Trafficking Phenotype in Vitro and In Vivo

Although, thrombocytopenia can affect all age groups, neonates, especially pre-term, have an increased incidence of thrombocytopenia. Platelet transfusions may reduce the bleeding risk in neonates, but are also associated with adverse short and long-term immune and inflammatory outcomes. A randomized trial of platelet transfusions in neonates found that transfusion was associated with an increased risk of necrotizing enterocolitis, unilateral/bilateral retinopathy, and bronchopulmonary dysplasia. Past work from our research team found that neonatal platelets expressed lower levels of mRNA for many immune related molecules compared to adult platelets. We therefore sought to determine whether the transfusion of adult platelets to neonates resulted in developmental immune dysregulation, with a focus on platelet and monocyte interactions.To explore the interactions between monocytes and platelets, we isolated monocytes from adult mouse bone marrow and co-incubated monocytes with adult (>8 weeks old) or neonatal mouse platelets (7 days old mice) and determined inflammatory and trafficking monocyte phenotypes by flow cytometry and qRT-PCR. Monocytes treated with adult platelets had an increased inflammatory (Ly6C hi) and trafficking phenotype (CCR2 hi), while monocytes treated with neonatal platelets adopted an inflammatory, but not trafficking phenotype. As expected, adult platelets increased the expression of monocyte inflammatory (Nos2, Cxcl1, Ccl2) and trafficking (Ccr2) mRNA, while neonatal platelets also increased inflammatory mRNA expression, but did not increase Ccr2 expression. Adult platelets express more Selp (P-selectin) than neonatal platelets and P-selectin is a major mediator of platelet and monocyte interactions. We confirmed that adult platelets expressed more P-selectin protein compared to neonatal platelets, and found that blocking P-selectin decreased adult platelet induced CCR2 expression to levels similar to monocytes treated with neonatal platelets. Using a transwell chamber we assessed adult and neonatal platelet effects on monocyte migration towards the CCR2 ligand CCL2. Monocytes were treated with adult platelets had significantly greater monocyte migration compared to monocytes co-incubated with neonatal platelets.To model platelet transfusions in the setting of thrombocytopenia, we used 14d old thrombopoietin receptor knockout mice (TPOR -/-) that have low platelet counts, and infused adult or neonatal platelets. We observed a significant increase in inflammatory and trafficking monocytes in mice transfused with adult platelets compared to those transfused with neonatal platelets. Using an in vivo model of monocyte chemotaxis, mice were treated with CCL2 intraperitoneal after platelet transfusion. Adult platelet transfusions, but not neonatal, increased monocyte peritoneal trafficking to CCL2.These data provide comparative insights as to how adult and neonatal platelet transfusions regulate monocyte functions. Adult platelet transfusions to neonates are associated with an inflammatory and trafficking monocyte phenotype that is platelet P-selectin dependent and may have a major impact on neonatal platelet transfusion complications. DisclosuresPalis: Rubius Therapeutics: Consultancy.

Platelet Transfusions Can Increase or Decrease the Inflammatory Response and Mortality in a Murine Model of Neonatal Polymicrobial Sepsis

Thrombocytopenia affects 18-35% of all neonates in the Neonatal Intensive Care Unit and ~70% of those born extremely prematurely, with sepsis being a frequent cause. Platelet transfusions (PTx) are frequently given to septic preterm neonates at higher platelet count (PC) thresholds than those used in adults, in an attempt to reduce their bleeding risk. However, in the largest randomized trial of neonatal PTx thresholds, infants transfused at a higher PC threshold had a significantly higher mortality and/or major bleeding compared to infants transfused at a lower threshold. We hypothesized that the deleterious effects of PTx would be related to a potential “developmental mismatch” resulting from adult platelets being transfused into a neonate. Among other developmental differences, adult platelets (human and murine) exhibit significantly higher surface P-selectin expression following activation than neonatal platelets. P-selectin is essential for the interaction of platelets with immune cells. Thus, we hypothesized that adult platelets transfused into septic neonates would be consumed faster than endogenous neonatal platelets (due to higher potential for immune interaction), and would increase inflammation and mortality. To test these hypotheses, we used a published murine model of neonatal sepsis, consisting of injecting cecal slurry (CS) into C57BL/6 pups. CS batches were prepared by isolating the cecal content of adult C57BL/6 mice, which was weighted, aliquoted and frozen until use. Three different CS batches were prepared and injected IP into post-natal day 10 pups at a dose of 1.1 (CS1) or 1.0 mg/g (CS2 and 3). Two hours after infection, pups were transfused with washed platelets from adult GFP mice (5x10 7 platelets/g) or Tyrode's buffer (control). Weights, PCs and GFP platelet % were measured before, 4h and 24h post-infection. Blood was collected via terminal bleed at 24h, and plasma separated for quantification of 31 cytokines by multiplex. Despite identical preparation, CS batches varied greatly in their 24h mortality (11% vs 73% vs. 30% for CS1, 2 and 3, respectively). Moreover, PTx had different effects on the mortality of pups infected with different CS batches, increasing the 24h mortality of pups infected with CS1 (30% in transfused vs 11% in non-transfused, RR 2.70, 95% CI 1.02-7.15) but decreasing the mortality of pups infected with CS2 (46% vs. 73%) or CS3 (9% vs. 30%), with a combined RR of 0.52; 95% CI 0.30-0.91. Bacterial counts differed between CS batches, but did not correlate with mortality. Comparison of the microbiome composition using deep sequencing revealed an increased presence of pathogenic bacterial species (Legionella, Sutterella, and Helicobacter species) in CS2 and 3 compared to CS1, and a relative abundance of beneficial bacterial (Actinobacteria and Proteobacteria) in CS1. Different CS batches also elicited different cytokine responses, with significant differences noted in G-CSF, IL-1α, IL-1β, IL-3, IL-7, IL-12p70, and IL-15 levels (p<0.05). For all of these cytokines, except G-CSF, levels were lower in mice infected with CS1 compared to CS2 or 3. Next, we investigated the effects of PTx on the plasma cytokine profile of mice infected with CS1 or CS2/3 (combined), compared to their infected, non-transfused littermates. For nearly all cytokines, PTx increased the response after infection with CS1, but decreased it after infection with CS2/3, with a significant difference in mean global cytokine effect (p<0.0001). For individual cytokines, however, these differences only reached statistical significance for LIX (CXCL5, p=0.04) and approached significance for IL15 and IL17 (p=0.06). Finally, we developed a mathematical model to compare the consumption of endogenous neonatal platelets (GFP-) to that of transfused adult platelets (GFP+) in pups infected with CS1 vs. CS2. In both, the calculated percent consumption was higher for adult platelets than for neonatal platelets (54.8% vs. 32.6% for CS1 and 56.5% vs. 40.4% for CS2). In conclusion, our findings support the hypothesis that adult transfused platelets are consumed faster than endogenous platelets in early neonatal sepsis, and demonstrate that platelet transfusions can either enhance or attenuate the neonatal inflammatory response and the mortality in a model of murine polymicrobial sepsis, depending on the bacterial composition of the inoculum and/or the severity of the sepsis. DisclosuresStowell: Grifols: Speakers Bureau; Argenx: Speakers Bureau; Alexion: Consultancy.