SUMMARY The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8−/−Mlkl−/− and Fadd−/−Mlkl−/− mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8−/−Mlkl−/− and Fadd−/−Mlkl−/− mice compared to Casp8−/−Ripk3−/− or Fadd−/−Ripk3−/− mice, respectively. These results demonstrate that MLKL is an essential effector of necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity and excess cytokine production that occur when FADD or caspase-8 mediated apoptosis is abrogated.

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/ progenitor cells but lack expression on megakaryocytes and platelets (Mpl PF4cre/PF4cre). Mpl PF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.bone marrow | essential thrombocythemia T hrombopoietin (TPO) is the principal hematopoietic cytokine that regulates platelet production at steady state and is required for rapid responses to platelet loss. TPO acts by binding to a specific cell surface receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), leading to receptor dimerization, activation of intracellular signal transduction pathways, and responses of target cells. Mice lacking TPO or Mpl are severely thrombocytopenic and deficient in megakaryocytes and their progenitor cells, a phenotype consistent with a role for TPO in maintaining appropriate megakaryocyte numbers in vivo. In addition to its role in megakaryopoiesis, TPO is also an indispensible regulator of hematopoietic stem cells (HSC), essential for maintenance of quiescence and self-renewal (1).TPO is produced primarily in the liver (2) and upon binding to the Mpl receptor on target cells, is internalized and degraded. The prevailing model posits that circulating TPO concentration is inversely proportional to the "Mpl mass" contributed by the total number of megakaryocytes and platelets. In normal individuals, this model describes an effective feedback system to regulate TPO-driven megakaryocyte and platelet production according to need. The reciprocal relationship between platelet number and circulating TPO level is clearly evident in bone marrow transplant patients (1), and the key role of the TPO receptor is illustrated by the elevated circulating TPO in Mpl −/− mice (3) and the modest elevation of platelet counts in transgenic mice expressing low levels of Mpl (4, 5). However, the relationship between circulating TPO concentration and peripheral platelet counts is not always conserved in pathological states of thrombocytosis and thrombocyto...

The lifespan of platelets in circulation is brief, close to 10 days in humans and 5 days in mice. Bone marrow residing megakaryocytes produce around 100 billion platelets per day. In a healthy individual, the majority of platelets are not consumed by hemostatic processes, but rather their lifespan is controlled by programmed cell death, a canonical intrinsic apoptosis program. In the last decade, insights from genetically manipulated mouse models and pharmacological developments have helped to define the components of the intrinsic, or mitochondrial, apoptosis pathway that controls platelet lifespan. This review focuses on the molecular regulation of apoptosis in platelet survival, reviews thrombocytopenic conditions linked to enhanced platelet death, examines implications of chemotherapy-induced thrombocytopenia through apoptosis-inducing drugs in cancer therapy as well as discusses ex vivo aging of platelets.

Key Points Inactivation of Suz12 results in a rapid and marked exhaustion of the HSC pool. Lymphoid development is completely dependent on PRC2, but numerous myeloid lineages develop in the absence of PRC2.

Mature megakaryocytes depend on the function of Bcl-x L , a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x L does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl IntroductionMegakaryocytes are large polyploid cells responsible for the production of blood platelets. They develop primarily in the BM and spleen. On reaching maturity, megakaryocytes extend long pseudopodial projections called proplatelets into the circulation, and it is from these structures that platelets are released. [1][2][3] Once born, platelets have a brief lifespan in the circulation: 10 days in humans, 5 days in mice. 4,5 In recent years, it has become apparent that this finite existence is governed by the interplay between members of the Bcl-2 protein family, the critical regulators of the "intrinsic" or "mitochondrial" apoptosis pathway. 6 Platelets depend on the prosurvival protein Bcl-x L to maintain their viability. 7 Mutations in murine Bcl-x L cause dose-dependent cell-intrinsic reductions in platelet lifespan. 7-9 Pharmacologic blockade of Bcl-x L with the BH3 mimetic drugs ABT-737 10 or ABT-263 11 triggers platelet death 7,12-15 and thrombocytopenia in mice, 7,16 dogs, 15 and humans. 17,18 The function of Bcl-x L is to restrain the prodeath proteins Bak and Bax. When activated, Bak and Bax induce mitochondrial outer membrane permeabilization (MOMP), resulting in platelet apoptosis, which, at least in vitro, is characterized by cytochrome c release, caspase activation, and phosphatidylserine exposure. [12][13][14][15] At steady state in vivo, aged platelets that have escaped hemostatic consumption undergo Bak-mediated apoptosis and clearance from the circulation. Genetic deletion of Bak and Bax almost doubles platelet lifespan, 9 rescues the thrombocytopenia caused by loss of Bcl-x L , 9 and renders platelets refractory to the effects of ABT-737. 13 We recently demonstrated that Bcl-x L is also essential for mature megakaryocytes to proceed safely through the process of platelet shedding. 9 In vitro, loss of Bcl-x L triggers Bak-and Bax-mediated mitochondrial damage, caspase activation, and failure of proplatelet formation. In vivo, mature, shedding megakaryocytes lacking Bcl-x L exhibit severe dysmorphology and produce grossly abnormal platelets that survive only hours in the circulation. Bcl-x L is, however, dispensable for the growth and development of megakaryocytes, suggesting that other Bcl-2 family prosurvival proteins are necessary to promote survival during this process. Of the 5 prosurvival members of the Bcl-2 family, 6 3 are known to be expressed in megakaryocytes: Bcl-2, Bcl-x L , and Mcl-1. 9 Given its critical role in hematopoietic progenitors and lymphocy...