Background Developmental ontogeny of neonatal thrombopoiesis retains characteristics that are distinct from adults although molecular mechanisms remain unestablished. Methods We applied multiparameter quantitative platelet responses with integrated ribosome profiling/transcriptomic studies to better define gene/pathway perturbations regulating the neonatal‐to‐adult transition. A bioinformatics pipeline was developed to identify stable, neonatal‐restricted platelet biomarkers for clinical application. Results Cord blood (CB) platelets retained the capacity for linear agonist–receptor coupling linked to phosphatidylserine (PS) exposure and α‐granule release, although a restricted block in cross‐agonist activation pathways was evident. Functional immaturity of synergistic signaling pathways was due to younger ontogenetic age and singular underdevelopment of the protein secretory gene network, with reciprocal expansion of developmental pathways (E2F, G2M checkpoint, c‐Myc) important for megakaryocytopoiesis. Genetic perturbations regulating vesicle transport and fusion (TOM1L1, VAMP3, SNAP23, and DNM1L) and PS exposure and procoagulant activity (CLCN3) were the most significant, providing a molecular explanation for globally attenuated responses. Integrated transcriptomic and ribosomal footprints identified highly abundant (ribosome‐protected) DEFA3 (encoding human defensin neutrophil peptide 3) and HBG1 as stable biomarkers of neonatal thrombopoiesis. Studies comparing CB‐ or adult‐derived megakaryocytopoiesis confirmed inducible and abundant DEFA3 antigenic expression in CB megakaryocytes, ~3.5‐fold greater than in leukocytes (the most abundant source in humans). An initial feasibility cohort of at‐risk pregnancies manifested by maternal/fetal hemorrhage (chimerism) were applied for detection and validation of platelet HBG1 and DEFA3 as neonatal thrombopoiesis markers, most consistent for HBG1, which displayed gestational age‐dependent expression. Conclusions These studies establish an ontogenetically divergent stage of neonatal thrombopoiesis, and provide initial feasibility studies to track disordered fetal‐to‐adult megakaryocytopoiesis in vivo.

Inflammatory stimuli have divergent effects on peripheral platelet counts, although the mechanisms of thrombocytopenic and thrombocytotic responses remain poorly understood. A candidate gene approach targeting 326 polymorphic genes enriched in thrombopoietic and cytokine signaling pathways was applied to identify single nucleotide variants (SNVs) implicated in enhanced platelet responses in cohorts with reactive (RT) or essential (myeloproliferative neoplasm [MPN]) thrombocytosis (ET). Cytokine profiles incorporating a 15-member subset, pathway topology, and functional interactive networks were distinct between ET and RT, consistent with distinct regulatory pathways of exaggerated thrombopoiesis. Genetic studies using aggregate (ET + RT) or ET-restricted cohorts identified associations with 2 IFNA16 (interferon-α16) SNVs, and the ET associations were validated in a second independent cohort (p-value 0.0002). Odds Ratio (OR) of the combined ET cohort (N = 105) was 4.92, restricted to the JAK2V617F-negative subset (OR 5.01). ET sub-stratification analysis by variant IFNA16 demonstrated statistically significant increase in interferon-α16 levels (p = 0.002) among 16 quantifiable cytokines. Recombinantly-expressed variant IFN-α16 encompassing 3 linked non-synonymous SNVs (E65 H95 P133) retained comparable antiviral (AV) and pSTAT signaling profiles as native IFN-α16 (V65 D95 A133) or IFN-α2, although both native and variant IFN-α16 demonstrated stage-restricted differences (compared to IFN-α2) of interferon-regulated genes in CD34+-stimulated megakaryocytes. These data implicate IFNA16 (IFN-α16 gene product) as a putative susceptibility locus (driver) within the broader disrupted cytokine network evident in MPNs, and provide a framework for dissecting functional interactive networks regulating stress or MPN thrombopoiesis.