Significant hypo-responsiveness to GPVI and CLEC-2 agonists in pre-term and full term neonatal platelets and following immune thrombocytopenia

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 GPVI and integrin a2b1, or to defects in downstream signalling events by comparison to platelet activation by CLEC-2. GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) g2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosinebased- 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 aIIbb3, accounted for at transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin aIIbb3 activation, P-Selectin expression, and Syk and PLCg2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to two weeks of age, as determined by measurement of integrin aIIbb3 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 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.