• PEAR1 is a negative regulator of megakaryocyte proliferation in vitro and thrombocyte formation in vivo.• PEAR1 regulates the PI3K/ PTEN pathway.Platelet endothelial aggregation receptor-1 (PEAR1) participates in platelet aggregation via sustaining aIIbb3 activation. To investigate the role of PEAR1 in platelet formation, we monitored and manipulated PEAR1 expression in vitro in differentiating human CD34 1 hematopoietic stem cells and in vivo in zebrafish embryos. PEAR1 expression rose during CD34 1 cell differentiation up to megakaryocyte (MK) maturation. Two different lentiviral short hairpin knockdowns of PEAR1 did not affect erythropoiesis in CD34 1 cells, but increased colony-forming unit MK cell numbers twofold vs control in clonogenic assays, without substantially modifying MK maturation. The PEAR1 knockdown resulted in a twofold reduction of the phosphatase and TENsin homolog (PTEN) phosphatase expression and modulated gene expression of several phosphatidylinositol 3-kinase (PI3K)-Akt and Notch pathway genes. In zebrafish, Pear1 expression increased progressively during the first 3 days of embryo development. Both ATG and splice-blocking PEAR1 morpholinos enhanced thrombopoiesis, without affecting erythropoiesis. Western blots of 3-day-old Pear1 knockdown zebrafish revealed elevated Akt phosphorylation, coupled to transcriptional downregulation of the PTEN isoform Ptena. Neutralization by morpholinos of Ptena, but not of Ptenb, phenocopied the Pear1 zebrafish knockdown and triggered enhanced Akt phosphorylation and thrombocyte formation. In summary, this is the first demonstration that PEAR1 influences the PI3K/PTEN pathway, a critical determinant of Akt phosphorylation, itself controlling megakaryopoiesis and thrombopoiesis. (Blood. 2013;121(26):5208-5217)

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Niemann-Pick type C is a lysosomal storage disease associated with mutations in NPC1 or NPC2, resulting in an accumulation of cholesterol in the endosomal-lysosomal system. Niemann-Pick type C has a clinical spectrum that ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative disease combined with remarkably, in some cases, hematological defects such as thrombocytopenia, anemia and petechial rash. A role of NPC1 in hematopoiesis was never shown. Here, we describe platelet function abnormalities in three unrelated patients with a proven genetic and biochemical NPC1 defect. Their platelets have reduced aggregations, P-selectin expression and ATP secretions that are compatible with the observed abnormal alpha and reduced dense granules as studied by electron microscopy and CD63 staining after platelet spreading. Their blood counts were normal. NPC1 expression was shown in platelets and megakaryocytes (MKs). In vitro differentiated MKs from NPC1 patients exhibit hyperproliferation of immature MKs with different CD63(+) granules and abnormal cellular accumulation of cholesterol as shown by filipin stainings. The role of NPC1 in megakaryopoiesis was further studied using zebrafish with GFP-labeled thrombocytes or DsRed-labeled erythrocytes. NPC1 depletion in zebrafish resulted in increased cell death in the brain and abnormal cellular accumulation of filipin. NPC1-depleted embryos presented with thrombocytopenia and mild anemia as studied by flow cytometry and real-time QPCR for specific blood cell markers. In conclusion, this is the first report, showing a role of NPC1 in platelet function and formation but further studies are needed to define how cholesterol storage interferes with these processes.

Summary. Regulators of G protein signaling (RGS) are intracellular signaling regulators that bind activated G protein a subunits (Ga) and increase their intrinsic GTPase activity via their common RGS homology domain. In addition to their GTPase accelerating activity (GAP), RGS proteins also contain other domains that regulate their receptor selectivity, their interaction with other proteins such as adenylyl cyclase or their subcellular localization via interaction with scaffold proteins such as tubulin, 14-3-3 or spinophilin. There are at least 37 different RGS family members in humans and numerous physiological functions have been assigned to these proteins, which have rather a tissue-specific expression pattern. The role of some RGS proteins was shown to be important for hematopoiesis. More recent studies also focused on their expression in platelets, and for R4 RGS subfamily members RGS2, RGS16 and RGS18, it could be demonstrated that they regulate megakaryopoiesis and/or platelet function. These functional studies mostly comprised in vitro experiments and in vivo studies using small animal models. Their role in human pathology related to platelet dysfunction remains still largely unknown, except for a case report with a RGS2 gain of function mutation. In addition to an introduction on RGS signaling and different effectors with a special focus on the R4 subfamily members, we here will give an overview of the studies related to the role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function.

RGS18 was originally identified as a R4 subfamily member of regulators of G-protein signaling (RGS) with specific expression in hematopoietic progenitors, myeloerythroid cells, and megakaryocytes, though its physiological role in hematopoiesis remained unknown. Here, we show that lentiviral RGS18 overexpression during differentiation of mouse Sca1(+) hematopoietic stem cells induced a 50% increase of megakaryocyte proliferation. RGS18 depletion in zebrafish results in thrombocytopenia, as 66 to 88% of the embryos lack thrombocytes after injection of an ATG or splice-blocking morpholino, respectively. These embryos have no defects in early hematopoiesis, erythropoiesis, or leukocyte number and migration. In addition, all RGS18 depleted embryos have curly tails and an almost absent response to acoustic stimuli. In situ hybridization in zebrafish, Xenopus, and mouse embryos shows RGS18 expression in thrombocytes and/or hematological tissues but also in brain and otic vesicles. RGS18 interferes with development of cilia in hair cells of the inner ear and neuromast cells. On the basis of literature evidence that RGS-R4 members interact with the G-protein-modulated Wnt/calcium pathway, Wnt5b- but not Wnt5a-depleted embryos phenocopy all RGS18 knockdown effects. In summary, our study is the first to show that RGS18 regulates megakaryopoiesis but also reveals its unexpected role in ciliogenesis, at least in lower vertebrates, via interference with Wnt signaling.