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

BACKGROUND AND OBJECTIVE: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. 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 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. RESULTS:We identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets clustered separately by principal component analysis. Adult platelets were significantly enriched for immunomodulatory proteins, including β2 microglobulin and CXCL12, whereas neonatal platelets were enriched for ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched for phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched for phosphorylated proteins involved in insulin growth factor signaling. CONCLUSIONS:Using label-free data independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation compared with 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.