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

Background: Recent 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. Objectives: This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. Methods: We isolated resting umbilical cord blood -derived platelets from healthy fullterm neonates ( n = 8) and resting blood platelets from healthy adults ( n = 6) and compared protein and phosphoprotein contents using data -independent acquisition mass spectrometry. Results: We identi fied 4770 platelet proteins with high con fidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were signi ficantly enriched in immunomodulatory proteins, including beta 2 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 traf ficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. Conclusion: Using label -free data -independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identi fied 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.