High Dimensional Immunophenotyping of Neonatal and Adult Platelets: Differential Reactivity to Agonists, Maximal Marker Expression, and Abundance of Defined Platelet Subsets

Neonates admitted to neonatal intensive care units are often thrombocytopenic and receive platelet transfusions to prevent or treat bleeding. However, a large recent randomized trial found higher mortality and morbidity among preterm neonates receiving more platelet transfusions. The mechanisms underlying these findings are unknown but are thought to be related to functional differences between the transfused (adult) and recipient’s (neonatal) platelets. We recently demonstrated distinct procoagulant, proinflammatory, and proaggregatory platelet subpopulations in adults by 16-color spectral flow cytometry and hypothesized that neonatal and adult platelets would exhibit differential abundance of these subpopulations. The goal of this study was to characterize the platelet subpopulations present in cord vs adult blood in response to agonist stimulation.

All studies were IRB approved. Cord blood (CB) was collected from healthy full-term infants born by elective cesarean section. Adult blood was from healthy volunteers free of antiplatelet medications. Anticoagulated CB and adult blood +/- platelet agonist (varied concentrations of adenosine diphosphate (ADP), thrombin receptor activator peptide (TRAP), cross-linked collagen related peptide (CRP), or rhodocytin) was stained with a panel of 15 platelet surface markers (PAC-1, CD61, CD42a, CD29, CD31, CD32, CD62P, CD63, CD107a, CD154, annexin V, TLR9, TLT-1, CD36 and GPVI) and analyzed as described (Spurgeon & Frelinger, Cytometry A 2023). Platelet subsets were identified using FAUST.

CB (n=13, 7 male) and adult blood (n=13, 4 male) did not differ significantly with respect to platelet count (206 ± 6.4 vs 256 ± 48, CB vs. adult), immature platelet fraction (3.0 ± 1.3 vs. 2.2 ± 1.4), or mean platelet volume (9.5 ± 0.7 vs. 8.8 ± 0.7). At baseline, of the 15 measured platelet surface markers, only CD42a (GPIX) and CD32 (FcgRIIa) differed, even after correction for platelet size, on CB vs adult platelets (each was 1.7-fold higher on CB platelets). Consistent with prior reports, CB platelets showed significantly lower platelet surface PAC-1 and CD62P with low TRAP. In addition, TLR9 was reduced in CB vs adult platelets with CRP and CRP+TRAP. However, other activation markers (TLT-1, CD63, CD107a and CD154) did not significantly differ between CB and adult platelets activated with other agonists. This led us to investigate lower concentrations of ADP, TRAP and CRP using dose-response curves.

Increasing concentrations of ADP, TRAP, and CRP led to dose-dependent increases in 7 activation markers (PAC-1, CD62P, CD63, CD107a, CD154, TLT-1 and TLR9) in CB and adult platelets. The EC₅₀s for TRAP- and CRP-induced increase of these markers were significantly higher for CB vs adult platelets (TRAP 3-fold, range 2.7-3.2; CRP 9.2-fold, range 5.3-12.6) while EC₅₀s for ADP were similar in CB vs adult platelets. These results suggest that the developmental differences in platelet activation are highly agonist- and signaling pathway-dependent, with the GPVI-mediated pathway being the most hyporeactive in neonatal compared to adult platelets. In contrast to the higher TRAP and CRP EC₅₀s in CB vs adults for all markers, the maximal expression levels for 5 of the 7 surface markers (CD63, CD107a, CD154, TLT-1 and TLR9) were similar in CB vs adult platelets, while maximal PAC-1 and CD62P were significantly lower in CB vs adult platelets across all agonists.

High-dimensional (FAUST) analysis identified 12 platelet subsets based on the expression patterns of 6 markers (annexin V, PAC-1, CD62P, CD107a, CD63 and TLT-1). These platelet subsets were functionally categorized as resting, proinflammatory, proaggregatory, procoagulant, and combinations thereof. In baseline and agonist-stimulated samples, significant developmental differences in the abundance of subsets were observed. Notably, proinflammatory (FAUST07: Annexin V- PAC-1- CD62P+ CD107a+ CD63+ TLT-1+) platelets were more abundant in CB than adult blood across agonists, while proaggregatory/proinflammatory platelets (FAUST10: Annexin V- PAC-1+ CD62P+ CD107a+ CD63+ TLT-1+) were more abundant in adults.

In sum, high-parameter spectral flow cytometry provided new insights into neonatal platelet function and identified developmental differences in the abundance of platelet subpopulations. How these differences impact the neonatal responses to platelet transfusions remains to be investigated.