Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia

Abstract

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

“…Although exosomes are released by many cell types, those contained in the plasma are mainly, but not only, platelet-derived, as determined by the expression of the CD41 and CD63 markers [ 25 , 32 ]. Since (i) these nanosized particles are considered to be minimaps of their cells of origin [ 33 ]; (ii) the formation and secretion of exosomes is regulated by Rab and SNARE proteins [ 34 , 35 ]; and (iii) variations in platelet transcriptome [ 36 ], signaling pathways [ 37 ] and SNARE proteins [ 15 ] have been recently found during development, in the present study, we examined whether the morphology and/or the content of plasma exosomes from neonates was different from those of adults.…”

“…These findings supported and expanded our recent observation that, in platelets, some SNARE proteins are developmentally regulated [ 15 , 36 ]. We also observed positive regulation of the expression of major proteins related to integrin αIIbβ3 signaling (RAP1A; integrins αIIb and β3; TLN1), suggesting that they may contribute to balance the defective activation of integrin αIIbβ3 in platelets of preterm and full-term newborns [ 37 ].…”

Functional significance of the platelet immune receptors GPVI and CLEC-2

“…There is no evidence to suggest that GPVI plays a role in development, although it is noteworthy that neonatal platelets are hyporesponsive to GPVI and CLEC-2 for reasons that are not yet known (154). In contrast, the podoplanin/CLEC-2 axis plays a critical role in several stages of embryonic development.…”

Morphometric image analysis of placental C-type lectin domain family 2, member D (CLEC2D) immuno-expression in HIV associated pre-eclampsia

“…In normal pregnancy, the villous stroma of the placenta contains a strong expression of podoplanin [37]. Platelets may be exposed to higher levels of placental podoplanin during angiogenesis, and suppression of CLEC2D may decrease platelet activation [38]. Hence, the diminished presence of CLEC2D in our findings, may also contribute to thrombocytopenia and the anti-angiogenic properties associated with PE.…”