Gradually Increased Gpiib/Iiia Responsiveness from Preterm and Term Neonatal Infants to Adults Reveals a Complex Phenotypic Switch during Platelet Ontogenesis - Data from the Platelets in Neonatal Infants Study (PLINIUS)

Whereas erythropoiesis undergoes well described changes after birth due to a rapid switch from fetal to postnatal circulation and oxygen saturation, alterations in thrombopoiesis around birth remain incompletely understood. Platelets of neonatal infants are generally considered as hyporeactive upon various agonists, when compared to adult platelets, assumingly to maintain a balanced hemostatic state within a rather prothrombotic environment. Since prematurity is a major risk factor for intracranial hemorrhage and sepsis, this patient group is at high risk for severe complications by thrombocytopenia or platelet transfusions. However, there is a lack of detailed knowledge about how platelet activation pathways change around birth. The Platelets in Neonatal Infants Study (PLINIUS) aimed to comprehensively explore platelet function and activation by analyzing platelet receptor expression, core activation pathways and differential receptor modification upon a variety of agonists in a large cohort of preterm and term neonatal infants on a single cell level.

We expect to find ontogenetic differences by correlating the results with the infants' gestational age (GA) and with platelets from children and adults. The study was approved by the local ethics committee. Our cohort of 44 neonatal infants was stratified into three groups (<32 weeks GAM; 32-37 weeks; term neonates) and compared to 22 individuals in three control groups (<2 years; 2-12 years; >13 years). Blood was drawn at up to three time points after birth (0-2, 3-7, and 8-14 days). Resting and activated platelets were assessed by whole-blood multi-color flow cytometry and expression data for each subgroup was analyzed by FlowJo. t-SNE-based dimension reduction analyses were performed using FlowSOM. Expression levels of surface receptors for fibrinogen (CD41/CD61), von Willebrand factor (CD42a/CD42b) and subunits of integrin receptors for collagen (CD49b), fibronectin (CD49e), laminin (CD49f) and their shared b1 subunit (CD29) increased slightly age-dependent, but this corresponded with an increased forward scatter, indicating an overall unaltered receptor density on the platelet surface. Activation markers P-selectin (CD62P) and CD63 surface exposure upon stimulation with TRAP-6 in neonates was only slightly reduced when compared to the control subgroups. Unexpectedly, integrin GPIIb/IIIa activation (PAC1 binding) upon TRAP-6, ADP or U46619 was virtually blunted in all preterm and term neonates. The simultaneous measurement of all three markers allowed the identification of platelet subpopulations. The control subgroups revealed that most platelets developed a CD63⁺/PAC1⁺ phenotype, whereas preterm and term neonates remained predominantly in a CD63⁺/PAC1^- phenotype. This was reflected in tSNE analyses that resulted in 8 distinct platelet subpopulations. Stimulation with ADP or U46619 resulted in a comparable reactivity pattern with a large fraction of CD63^-/PAC1⁺ platelets, a subgroup that was virtually absent from all neonate groups. Of note, our consecutive measurements confirmed the initial reactivity pattern, excluding that maternal or birth-related effects could be a major confounder. Most strikingly, the transition occurred gradually from early preterms to adults, implying that the changes underlying thrombopoiesis and platelet function around birth are not the consequence of one single change. We found that the fraction of platelet-leukocyte aggregates (PLA) was significantly increased in preterm and term neonates of 40% without clinical signs of infection before reaching a median of 20% in adults. Blockade of PSGL-1 resulted in a 50% decrease of PLA formation, whereas eptifibatide did not have any effect.

In this comprehensive trial assessing the ontogenesis of platelet phenotypes and functions from extremely preterm neonates to adolescents in a longitudinal design. Our study provides evidence that activation induced alpha- and delta-granule release is uncoupled from GPIIb/IIIa activation in neonates despite an overall unaltered receptor density on the platelet surface. Changes in platelet responsiveness occurs gradually, which is not reflected by altered expression patterns in obvious platelet receptors. We propose an immune modulatory role of neonatal platelets beyond hemostasis, since the PSGL-1/CD62P axis but not GPIIb/IIIa mediates PLA formation in neonates.

No relevant conflicts of interest to declare.