Developmental Stage–Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice

Abstract

Congenital amegakaryocytic thrombocytopenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near-absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL−/− mice. Liver MKs in c-MPL−/− neonates were reduced in number and size compared to WT age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL−/− MKs. Platelet counts were lower in c-MPL−/− compared to WT mice at birth and did not increase over the first two weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL−/− newborn mice (P2) compared to age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL−/− newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ platelets transfused into thrombocytopenic c-MPL−/− P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL−/− mice exhibited reduced P-selectin surface expression following stimulation compared to adult platelets of either genotype, and platelets from c-MPL−/− neonates exhibited reduced GPIIb/IIIa activation in response to thrombin compared to age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

CAMT-MPL: congenital amegakaryocytic thrombocytopenia caused by MPL mutations - heterogeneity of a monogenic disorder - a comprehensive analysis of 56 patients

“…Furthermore, both life-span and thrombin dependent activation of platelet GPIIb/IIIa are markedly reduced in neonatal Mpl -/- mice compared to adult Mpl -/- mice. 37 Our results indicate a possible functional impairment of platelets also in human fetuses and newborns with MPL defect which is in contrast to the assumption of a normal function of Mpl -/- platelets. 39 …”

Development of gates to measure the immature platelet fraction in C57BL/6J mice using the Sysmex XN-V series multispecies hematology analyzer

“…The IPF and IPC values obtained with the new gate on the XN-1000V analyzer closely matched our previously published values for WT and c-MPL −/− adult and newborn mice measured using the XT-2000iV, 9,17 and provided different profiles depending on the mechanism of the thrombocytopenia. As in our prior study, the IPF was similar in WT and c-MPL −/− P10 and adult mice, but the IPC was strikingly lower in the transgenic mice.…”

“…As in our prior study, the IPF was similar in WT and c-MPL −/− P10 and adult mice, but the IPC was strikingly lower in the transgenic mice. 9…”

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

“…3 ). 33 We speculate that a moderate but significantly reduced level of integrin αIIbβ3 in platelets from pre-term and full-term neonates contributes to the mild impairment in αIIbβ3 activation (i.e. fibrinogen binding) by all agonists.…”