Oral Abstract
Cell Biology/Immunology and Biochemistry (Basic and Preclinical Research) - Platelets
Christopher Thom, MD, PhD
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania
Disclosure information not submitted.
Thrombocytopenic infants are often given empiric transfusions of donated adult platelets to prevent bleeding. But, paradoxically, randomized controlled trials indicate liberal transfusion practices increase neonatal bleeding, and increase the incidence of neurocognitive impairment, respiratory complications, and death in preterm infants.
Platelets rapidly activate and degranulate to form blood clots. Neonatal platelets are less reactive to agonists than adult platelets. Adult platelet transfusions may thus harm neonatal physiology, but molecular mechanisms are not well understood. We used proteomics to define molecular changes in neonatal vs adult platelets. We aim to create safer neonatal transfusion practices and products.
Study
Design/Methods: We analyzed proteomic and phospho-proteomic data from resting platelets from umbilical cord blood platelets from healthy full-term neonates and adult peripheral blood. We confirmed developmental differences in key kinases and targets by western blot.
Results/Findings:
From 4770 platelet proteins, we quantified changes in >18,000 phosphorylation sites in 2160 proteins. Platelets from both neonates and adults had abundant phospho-proteins were enriched for actin and membrane proteins (e.g., GTPase enzymes) that facilitate platelet activation and granule secretion, linking the platelet phospho-proteome to these functions.
Neonatal and adult platelets contained 1209 differentially abundant phospho-proteins and clustered separately by principal component analysis. The 471 phospho-proteins enriched in adult platelets reflected increased kinase activity. Kinase enrichment analysis identified increased activities of protein kinase C (PRKC), as well as downstream PRKA pathways, both of which impact platelet activation and degranulation. By western blot, we confirmed that adult platelets have 3-fold more PRKC activity and an increase in total and phospho-PRKCD/PKCδ abundance vs neonatal platelets (Figure).
Reticulon 1 (RTN1) is a PRKC substrate enriched in adult platelets that could contribute to enhanced platelet degranulation. RTN1 is part of the SNARE complex, which mediates calcium release and platelet granule secretion. RTN1 contains 5 PRKC recognition motifs and was among the most enriched proteins (7.1-fold, p< 1x10-8) and phospho-proteins ( >200-fold, p< 1x10-4) in adult platelets, which we confirmed by western blot (Figure). Other SNARE complex proteins showed minimal changes.
Conclusions:
Developmental changes in platelet PRKC and/or RTN1 activity could alter activation and degranulation in adult vs neonatal platelets. Targeted modulation of this pathway could help develop platelet transfusion products with biological activities appropriate for neonatal physiology.