Ontogeny of peripheral blood platelet GPIIb/IIIa activation and granule release in preterm and term neonates

Introduction Erythrocytes undergo an overall well characterized switch from fetal to postnatal circulation, which is reflected by stage-specific expression of hemoglobin chains. Postnatal alterations in thrombopoiesis remain poorly understood. Since prematurity is the major risk factor for postnatal bleeding and recent studies have shown that transfusion of (adult) platelet concentrates might increase mortality, detailed understanding of the ontogeny of platelet phenotype and function is crucial.

Method We recruited 66 individuals, stratified into 6 cohorts: (I) neonates <32 weeks (wk) gestational age (GA); (II) neonates 32–37 wk GA; (III) term neonates, (IV) infants 28 wk-2 a; (V) 2–12 a; (VI) >13 a. Neonates were assessed at up to three time points t1: 0–2 d, t2: 3–7 d, and t3: 8–14 d. Platelets were investigated flow cytometrically and subpopulations analyzed using t-SNE and automated clustering algorithms. Integrin GPIIb/IIIa activity was assessed by monitoring spreading on fibrinogen-coated cover slips ([Fig. 1]).

Fig. 1 Continuous evolution of platelet subpopulations; Platelets in whole blood were stimulated with TRAP-6 [5 µM]. Platelet subpopulations show a contiuous shift from a CD63-/PAC-1- or CD63+/PAC-1- pattern towards a CD63+/PAC-1+ predominant pattern in adolescents. (A-C) Representative curve displayed in A. Figure legends displayed in B.

Results Platelets were immunophenotyped in response to selected agonists and compared to the resting condition by a complex multi-color panel. The surface expression of the surface receptors CD9, CD29, CD41a, CD49d/e/f, CD61, GPVI, CLEC-2, and TLR-4 was overall unaltered in respect to a marginally reduced platelet size in preterms. Platelet subpopulations were identified by FlowSom-based clustering. In response to TRAP-6, platelets among all neonatal cohorts (I-III) were found to be in the PAC-1high (Pop3, Pop4) or PAC-1intermediate(Pop2) cluster, in contrast to 51% (Pop3,4) or 15% (Pop2) in adolescents. This finding was further corroborated when neonatal platelets were seeded on fibrinogen-coated surfaces. These platelets showed a massive defect in spreading, implying an impaired outside-in activation defect (80% neonatal platelets in spreading stage 1+2 vs. adolescents: 80% in stage 3+4, p<0.05). In contrast, agonist-induced granule release indicated by CD62P or CD63 surface neo-exposition was unaltered or just slightly reduced in neonates suggesting that granule release in preterm/neonatal platelet is uncoupled of integrin activation. Coupling of agonist-triggered granule release and GPIIb/IIIa activation evolved continuously with age throughout all platelet subpopulations (Fig. 1). In neonates, we found increased platelet-leukocyte aggregates (PLA), especially with neutrophils or monocytes, but not with T- or B-cells. While blockade of PSGL-1 or GPIIb/IIIa reduced the number of PLAs in adults, antagonism of PSGL-1, but not GPIIb/IIIa reduced PLA formation in neonates, suggesting that GPIIb/IIIa function matures until adolescence.

Conclusion Our study shows that platelet function does not just switch from fetal to mature thrombopoiesis, but undergoes a continuous development until adolescence. The separate responsiveness of granule release and integrin activation might reflect specific platelet functions during the fetal and neonatal period compared to the adult platelet.

Conflict of Interest

No conflicts of interest to declare.

Publication History

Article published online:
20 February 2023