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.
Highlights
Platelets are anuclear haematopoietic cells that play an essential role in haemostasis and its pathological counterpart thrombosis
ITAM-Receptor Signalling in Neonatal Platelets Hardy et al 1011 were from Santa Cruz (Heidelberg, Germany). α-PLCγ2Y1217 and α-Syk-525/526 were from Cell Signaling Technology (Leiden, The Netherlands). α-βActin was from SigmaAldrich and α-glycoprotein VI (GPVI) was from Abcam (Cambridge, UK)
The antibodies were as follows: The mouse C-type lectin-like receptor 2 (CLEC-2) monoclonal antibody 17D9 and α-IgG2bÃFITC were purchased from Bio-Rad (Hemel Hempstead, UK). α-PSelectinÃPE was from Novus Biologicals (Abingdon, UK). αCD41ÃAPC and α-CD41ÃPE were from eBioscience (Hatfield, UK) and BD Pharmigen (Oxford, UK), respectively. αCD41ÃFITC, α-CD42bÃFITC, α-CD49bÃFITC, α-GPVIÃFITC and α-IgGÃFITC were from Emfret (Eibelstadt, Germany). αGPIbα and immunoglobulin G (IgG) control used for immune depletion were from Emfret. α-CLEC-2ÃFITC was from AbD Serotec (Kidlington, UK)
Summary
Platelets are anuclear haematopoietic cells that play an essential role in haemostasis and its pathological counterpart thrombosis. Platelets play critical roles in other physiological and pathological processes including inflammation,[1] infection,[2] vascular integrity,[3] development[4] and cancer metastasis.[5] Currently, we have a rudimentary understanding of the role of platelets in many of these functions and how these processes vary during development and throughout adulthood. Thrombopoiesis takes place in multiple sites during development, beginning in the yolk sac before moving to the liver and to the bone marrow and spleen.[6,7,8] This means that, throughout development, circulating platelets are derived from more than one haematopoietic site. It is presently unclear to what extent this influences platelet function
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