Regulation of Platelet Lifespan in the Presence and Absence of TPO Signaling

It is well established that thrombopoietin (TPO), acting via its receptor Mpl, is the major regulator of platelet biogenesis. It has become increasingly clear that the primary mechanism by which TPO signaling stimulates thrombopoiesis is via Mpl-expressing hematopoietic progenitors. Mpl receptors on megakaryocytes and platelets act to control the amount of TPO available to hematopoietic stem and progenitor cell populations. TPO could potentially reduce platelet and/or megakaryocyte apoptosis and therefore increase the platelet count. However, the effect of TPO receptor signaling on platelet survival is unresolved. Increased TPO signaling after administration of TPO mimetic drugs in immune thrombocytopenia patients was suggested to increase platelet survival, while reduced platelet lifespan observed in thrombocytopenic Mpl deficient mice, was attributed to extrinsic causes, such as compromised vascular integrity, as well as unknown intrinsic factors. In the current study we investigated platelet survival in mouse models in the presence or absence of TPO signaling. We found that the survival of host and transfused wild-type (WT) platelets were reduced in thrombocytopenic Mpl deficient mice. The use of Mpl -/- c-Myb Plt4/Plt4 mice (platelet count of 4662 ± 851 x10 3 /µl) allowed us to determine platelet in vivo lifespan in the absence of thrombocytopenia. c-Myb Plt4/Plt4 mice display thrombocytosis (3936 ± 618 x10 3 /µl) independent of TPO signaling, driven by a point mutation in c-Myb (Carpinelli et al., PNAS 2004). In the absence of thrombocytopenia, Mpl deficiency did not negatively affect platelet lifespan in the host, nor diminish platelet survival in WT recipient animals post transfusion. The intrinsic apoptosis pathway regulates the survival of platelets, where the pro-survival protein Bcl-x L restrains the essential death mediators Bak and Bax. Accordingly, in mice loss of Bak and Bax almost doubles platelet lifespan and overexpression of pro-survival Bcl-2 extends platelet survival. In order to study the effect of increased TPO stimulation on platelet lifespan, mice with abnormally high level of circulating TPO, Tpo Tg mice (de Graaf et al., PNAS 2010), were crossed to Bcl-x Plt20/Plt20 mice harboring an intrinsic reduction in platelet lifespan due to a mutation in pro-survival Bcl-x L (Mason et al., Cell 2007). Chronic elevated TPO stimulation normalized platelet counts in Bcl-x Plt20/Plt20 mice, due to an expansion of megakaryocyte number in bone marrow and spleen. Nevertheless, platelet lifespan was not extended in Tpo Tg Bcl-x Plt20/Plt20 versus Bcl-x Plt20/Plt20 mice or in Tpo Tg versus WT mice. Similarly, platelets derived from Tpo Tg mice survived normally in WT recipient mice. Furthermore, the expression level of pro-survival Bcl-x L and Bcl-2 proteins in purified platelets from Tpo Tg and WT mice were not significantly different, indicating that TPO mediated effects on platelet survival were negligible. We conclude that TPO and its receptor Mpl are dispensable for platelet survival in adult mice. Disclosures No relevant conflicts of interest to declare.