Distinct sialylation profiles of neonatal and adult platelets revealed by lectin flow cytometry

Abstract Neonatal platelets differ from adult platelets in their activation thresholds, in vivo half-life, and hemostatic function. We and others have hypothesized that these differences in function may relate to underlying differences in platelet glycan repertoires. Sialic acids are negatively charged terminal sugars that cap platelet surface glycans and may regulate platelet clearance from the circulation. These residues can be linked via different glycosidic bonds, primarily α2,3 or α2,6 linkages. Specific plant-derived lectins can distinguish between these configurations, such as Sambucus nigra agglutinin (SNA) which binds preferentially to α2,6-linked sialic acid, and Maackia amurensis lectin II (MAL II), which binds α2,3-linked sialic acid. Additional lectins such as Erythrina cristagalli lectin (ECL) and peanut agglutinin (PNA) detect underlying β-galactose and Galβ1-3GalNAc residues, respectively, which become exposed upon desialylation. Building on recently published guidelines by the International Society on Thrombosis and Haemostasis Subcommittee on Platelet Physiology (Kauskot et al., 2025), we applied lectin spectral flow cytometry and lectin blotting to compare glycan profiles between neonatal and adult platelets. Umbilical-cord blood was obtained from healthy full-term neonates delivered by elective cesarean section; adult blood was collected from healthy volunteers not on antiplatelet agents. All procedures were conducted under IRB-approved protocols. Platelet-rich plasma (PRP) was either left untreated or treated with α2-3,6,8,9 neuraminidase A (New England Biolabs), which enzymatically remove surface sialic acid residues regardless of their glycosidic linkage. Platelets were labeled with antibodies against glycoprotein Ibα and P-selectin. After fixation and washing, platelets were stained with lectins (SNA–Cy5, MAL II–biotin, ECL–FITC, and PNA–Alexa Fluor 594) and analyzed using spectral flow cytometry. Paired neonatal and adult samples were processed in parallel. For lectin blotting, platelets were pelleted from PRP, washed, lysed, and equal amounts of protein were separated by SDS-PAGE. Western blots were incubated with fluorophore-conjugated lectins, and binding was visualized via direct fluorescence detection. Lectin flow cytometry enabled sensitive, single-cell detection of glycan motifs on platelets. Neonatal platelets exhibited significantly higher α2,6-linked sialic acid (SNA binding) compared to adult platelets (mean fold increase: 1.7 ± 0.4-fold; p= 0.001; n = 10). Platelet surface α2,3-linked sialic acid (MAL II binding) was similar between groups (1.1 ± 0.5-fold; p = 0.7). Both SNA and MAL II signals were markedly reduced following neuraminidase treatment, confirming the silaic-acid specific binding of these lectins. In the platelets that were not treated with neuraminidase, ECL and PNA binding were minimal and showed no difference between groups. Upon desialylation, both lectins showed increased signal intensity, consistent with unmasking of underlying N- and O-linked β-galactose motifs. Neonatal platelets showed greater increases in ECL (2.3 ± 0.9-fold; p = 0.001) and PNA (1.6 ± 0.9-fold; p = 0.05) binding, suggesting a larger reservoir of cleavable sialic acid. Lectin blot analysis confirmed elevated baseline SNA signal in neonatal platelet lysates, while MAL II staining showed no clear difference between groups. Baseline ECL and PNA reactivity remained low, but both increased following neuraminidase exposure, with neonatal samples exhibiting stronger signal than adult counterparts. In conclusion, neonatal platelets exhibit a distinct glycomic signature characterized by 1) more abundant α2,6-sialic acid and 2) enhanced exposure of terminal galactose residues following desialylation. To our knowledge, this study represents the first comparative analysis of platelet glycan architecture between neonatal and adult platelets using standardized lectin-based flow cytometry. These data provide mechanistic insight into developmental platelet biology and may inform age-specific transfusion strategies and neonatal thrombocytopenia management. These glycan differences may drive altered platelet reactivity and survival in the neonatal circulation. Future studies should define their impact on thrombosis risk, inflammatory responses, and the design of optimized platelet transfusion products for neonates.