Abnormal megakaryocyte development and platelet function in Nbeal2 mice Running title: Mouse model of gray platelet syndrome

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NBEAL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2 mouse. As in GPS, Nbeal2 mice exhibit splenomegaly, macrothrombocytopenia and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1 and platelet factor 4. The platelet α-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 platelets suggests that Nbeal2 acts independently of Vps33B/Vps16B at a later stage of α-granule biogenesis. Impaired Nbeal2 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 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 α-granule secretion plays a significant role in platelet function and they also indicate that abnormal α-granule formation in Nbeal2 mice has deleterious effects on megakaryocyte survival, development and platelet production. For personal use only. on July 10, 2017. by guest www.bloodjournal.org From