145 - Phosphoproteomics identifies molecular signaling differences between neonatal and adult platelets

Saturday, April 29, 2023

3:30 PM – 6:00 PM ET

Poster Number: 145
Publication Number: 145.239

Christopher Thom, Children's Hospital of Philadelphia, Philadelphia, PA, United States; Patricia Davenport, Boston Children's Hospital, Boston, MA, United States; Hossein fazelinia, CHOP, Philadelphia, PA, United States; Hua Ding, Children's Hospital of Philadelphia, Philadelphia, PA, United States; Jennifer Roof, CHOP, Philadelphia, PA, United States; Lynn A. Spruce, CHOP, Philadelphia, PA, United States; Harry Ischiropoulos, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Martha Sola-Visner, Boston Children's Hospital, Boston, MA, United States

Presenting Author(s)

Christopher Thom, MD, PhD (he/him/his)

Assistant Professor of Pediatrics
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Background: Observational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Proteins and kinase-driven protein phosphorylation pathways dictate platelet biology. Neonatal platelet proteome and phospho-proteome data are scarce.

Objective: We aimed to define altered protein and phosphoprotein content in neonatal vs adult platelets to help explain differences in platelet reactivity and function.

Design/Methods: We isolated resting platelets from full term neonatal cord blood and adult peripheral blood (n=9 per condition) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content.

Results:

We identified 4173 high-confidence proteins in adult and neonatal platelets, including 359 proteins related to metabolic, immune, and inflammatory pathways that differed between groups (≥1.5-fold change, p< 0.05).

We identified 3062 phosphorylated proteins (17575 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling, which directly regulate platelet activity and function. Kinase Enrichment Analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities.

Proteins that regulate granule trafficking and degranulation were among 1041 phosphoproteins (4017 phosphopeptides) with differential abundance in neonatal vs adult samples (p< 0.05). For example, 31 unique Reticulon 1 (RTN1) phosphopeptides were more abundant in adult platelets (p< 0.05). RTN1 regulates membrane trafficking in many cell types. Increased RTN1-directed membrane trafficking may underlie increased activation and degranulation propensity in adult platelets. Conversely, phosphorylated Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) was more abundant in resting neonatal platelets (6.8±2.3-fold increase vs adult platelets, p=4.5x10^-7). MARCKS phosphorylation regulates dense granule trafficking, and may promote enhanced serotonin-containing dense granule release observed in neonatal platelets.

Conclusion(s):

Our findings identify novel molecular targets that modulate platelet signaling and reactivity. These results also reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, and may provide biological rationale for clinical complications in preterm infants transfused with adult platelets.