Key Points• Nbeal2 2/2 mice are a model of human GPS, characterized by macrothrombocytopenia and a-granule-deficient platelets.• NBEAL2 is required for normal platelet function and megakaryocyte development.Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and a-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2 2/2 mouse. As in GPS, Nbeal2 2/2 mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet a-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet a-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2 2/2 platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of a-granule biogenesis. Impaired Nbeal2 2/2 platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2 2/2 bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that a-granule secretion plays a significant role in platelet function, and they also indicate that abnormal a-granule formation in Nbeal2 2/2 mice has deleterious effects on megakaryocyte survival, development, and platelet production. (Blood. 2013;122(19):3349-3358)

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal/neonatal platelets. It is the most common cause of severe thrombocytopenia in neonates, but the frequency of FNIT-related miscarriage is unknown, and the mechanism(s) underlying fetal mortality have not been explored. Furthermore, although platelet αIIbβ3 integrin and GPIbα are the major antibody targets in immune thrombocytopenia, the reported incidence of anti-GPIbα-mediated FNIT is rare. Here, we developed mouse models of FNIT mediated by antibodies specific for GPIbα and β3 integrin and compared their pathogenesis. We found, unexpectedly, that miscarriage occurred in the majority of pregnancies in our model of anti-GPIbα-mediated FNIT, which was far more frequent than in anti-β3-mediated FNIT. Dams with anti-GPIbα antibodies exhibited extensive fibrin deposition and apoptosis/necrosis in their placentas, which severely impaired placental function. Furthermore, anti-GPIbα (but not anti-β3) antiserum activated platelets and enhanced fibrin formation in vitro and thrombus formation in vivo. Importantly, treatment with either intravenous IgG or a monoclonal antibody specific for the neonatal Fc receptor efficiently prevented anti-GPIbα-mediated FNIT. Thus, the maternal immune response to fetal GPIbα causes what we believe to be a previously unidentified, nonclassical FNIT (i.e., spontaneous miscarriage but not neonatal bleeding) in mice. These results suggest that a similar pathology may have masked the severity and frequency of human anti-GPIbα-mediated FNIT, but also point to possible therapeutic interventions.

Key Points Mice deficient in HRG have normal hemostasis, but demonstrate accelerated thrombosis via the contact system. HRG abrogates nucleic acid–driven coagulation and serves as a novel modulator of the contact system in vivo.

PurposeNon-small cell lung cancer (NSCLC) is the first leading cause of cancer-related death globally. Long noncoding RNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) was involved in the progression of multiple cancers by sponging target miRNA. We aimed to explore the pathological mechanism of KCNQ1OT1 in NSCLC progression.MethodsThe expression of KCNQ1OT1, miR-204-5p and autophagy-related gene 3 (ATG3) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay were conducted for the detection of cell proliferation and apoptosis, respectively. Western blot assay was performed to examine the protein levels of B-cell lymphoma-2 (BCL-2), BCL2-Associated X (Bax), cleaved caspase-3, cleaved caspase-9 and LC3Ⅱ/LC3Ⅰ and P62. The interaction between miR-204-5p and KCNQ1OT1 or ATG3 was validated by dual-luciferase reporter system and RNA immunoprecipitation (RIP) assay. Murine xenograft assay was conducted to explore the function of KCNQ1OT1 in vivo. Immunohistochemistry (IHC) staining assay was used for the analysis of ki67-positive cell percentage.ResultsThe expression of KCNQ1OT1 and ATG3 was up-regulated whereas miR-204-5p was down-regulated in NSCLC tumors and cells. MiR-204-5p was inversely correlated with KCNQ1OT1 or ATG3. In addition, KCNQ1OT1 knockdown facilitated apoptosis, inhibited autophagy and proliferation of NSCLC cells in vitro and blocked tumor growth in vivo. However, the miR-204-5p inhibitor reversed the effects. More importantly, ATG3 was a target gene of miR-204-5p and ATG3 overexpression restored the effect of miR-204-5p on NSCLC cell progression.ConclusionKCNQ1OT1 promotes cell proliferation and autophagy and inhibits cell apoptosis via regulating miR-204-5p/ATG3 axis, providing a promising target for NSCLC therapy.