Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Platelet α-granules modulate the inflammatory response under systemic lipopolysaccharide injection in mice.

BACKGROUND: Beyond their role in hemostasis and thrombosis, platelets are also important mediators of inflammation by the release of hundreds of factors stored in their α-granules. Mutations in Nbeal2 cause gray platelet syndrome (GPS), characterized by the lack of platelet α-granules. This study aims to evaluate the immunological (proinflammatory) effects of platelet α-granules. STUDY DESIGN AND METHODS: We performed an experiment using Nbeal2-/- mice, the mouse model of GPS. Systemic inflammation was induced by intravenous injection of lipopolysaccharide (LPS). Inflammatory response was assessed by quantification of inflammatory soluble factors and platelet biological response modifiers. RESULTS: The lack of Nbeal2 (in Nbeal2 -/- mice, compared with controls) significantly reduced the recruitment of circulating neutrophils and monocytes. Moreover, after LPS injection, there was a significant increase in neutrophil and monocyte counts in control animals, compared with Nbeal2 -/- mice. The control of inflammation, evaluated by the production of anti-inflammatory cytokines, appeared to be greater in Nbeal2-/- mice compared with controls. Conversely, the production of certain inflammatory-soluble mediators known to characterize normal platelet secretion, such as soluble CD40 ligand (sCD40L), was decreased under experimental inflammation in Nbeal2 -/- mice. CONCLUSIONS: These results show that α-granules play a direct role in platelet-mediated inflammation balance, confirming the need to further investigate platelet-associated inflammatory pathophysiology and inflammatory adverse events related to blood transfusion.

Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice.

During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.

The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome.

BACKGROUND: Deficiencies in granule-bound substances in platelets cause congenital bleeding disorders known as storage pool deficiencies. For disorders such as gray platelet syndrome (GPS), in which thrombocytopenia, enlarged platelets and a paucity of α-granules are observed, only the clinical and histologic states have been defined. OBJECTIVES: In order to understand the molecular defect in GPS, the α-granule fraction protein composition from a normal individual was compared with that of a GPS patient by mass spectrometry (MS). METHODS: Platelet organelles were separated by sucrose gradient ultracentrifugation. Proteins from sedimented fractions were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis, reduced, alkylated, and digested with trypsin. Peptides were analyzed by liquid chromatography-tandem MS. Mascot was used for peptide/protein identification and to determine peptide false-positive rates. MassSieve was used to generate and compare parsimonious lists of proteins. RESULTS: As compared with control, the normalized peptide hits (NPHs) from soluble, biosynthetic α-granule proteins were markedly decreased or undetected in GPS platelets, whereas the NPHs from soluble, endocytosed α-granule proteins were only moderately affected. The NPHs from membrane-bound α-granule proteins were similar in normal platelets and GPS platelets, although P-selectin and Glut3 were slightly decreased, consistent with immunoelectron microscopy findings in resting platelets. We also identified proteins not previously known to be decreased in GPS, including latent transforming growth factor-ß-binding protein 1(LTBP1), a component of the transforming growth factor-ß (TGF-ß) complex. CONCLUSIONS: Our results support the existence of 'ghost granules' in GPS, point to the basic defect in GPS as failure to incorporate endogenously synthesized megakaryocytic proteins into α-granules, and identify specific new proteins as α-granule inhabitants.