The Nbeal2(-/-) mouse as a model for the gray platelet syndrome

Abstract

The gray platelet syndrome (GPS) is a rare, autosomal-recessive platelet disorder characterized by thrombocytopenia, large platelets lacking α-granules, and variable bleeding. GPS has been linked to mutations in the neurobeachin-like 2 gene (NBEAL2). We have recently characterized Nbeal2-deficient mice and shown that the absence of Nbeal2 results in defective protein sorting in megakaryocytes (MKs) and impaired α-granule biogenesis, a finding also seen for human MKs. In the mice, the lack of α-granules results in impaired aggregation, defective platelet adhesion to collagen under flow and reduced pro-coagulant activity; findings that translate into defective hemostasis and thrombosis in vivo indicating that α-granule secretion is critical for platelet plug stability. Furthermore, we revealed a role of α-granule proteins in ischemic stroke and wound healing. Thus, Nbeal2-deficient mice recapitulate the hallmarks of human GPS without showing its phenotypic heterogeneity and are a promising model to investigate the (patho-)physiological relevancy of α-granules.

Keywords: NBEAL2; gray platelet syndrome; platelet; α-granule.

Figure 1. Platelet ultrastructure in Nbeal2^−/− mice (A) and human GPS (B). Representative transmission electron microscopy (TEM) images of resting wild-type (+/+) and Nbeal2^−/− (−/−) platelets and those of a control human subject as well as a characterized GPS patient^, with a homozygous L388P mutation. Platelets deficient in NBEAL2 both show lack of α-granules (#) and an increased number of vacuoles (arrows) while platelet size was increased and dense granule (*) content was unaltered. Bars = 0.5 µm.

Figure 2. Confocal microscopy of MKs cultured in vitro from CD34⁺ cells from the peripheral blood of a GPS patient with a homozygous L388P mutation in NBEAL2, MK suspensions were incubated at day 14 of culture on polylysine-coated slides. Cells were fixed and permeabilized. Integrin αIIbβ3 and VWF were localized by a murine antibody (AP2) and VWF by a polyclonal antibody with bound IgG visualized using species-specific FITC (green) and Alexa-Fluor568-conjugated IgG. All technical details were as previously described. In the upper panel, a round small MK shows abundant labeling for VWF; in the middle panel a MK shows VWF staining along the proplatelet extension, the VWF labeling is decreased compared to control and is absent from the proplatelet tip; the lower panel shows a very mature MK with a long proplatelet string with typical swellings along its length while the VWF labeling is minimal. This suggests that VWF is not maintained in the MK. Please note that the scale of this panel is reduced to allow the proplatelet to be fully seen.