Defective Production, Turnover, and Secretion of the Platelet α-Granule Protein P-Selectin In Mice with Disrupted FOG1-NuRD Interaction

Abstract 547

We reported recently that point mutations in the GATA-1 co-factor FOG-1 that disrupt binding to the NuRD chromatin remodeling complex display defects in the erythroid and megakaryocyte (MK) lineages. Homozygous FOG-1 mutant (ki/ki) mice display a Gray Platelet Syndrome (GPS)-like macrothrombocytopenia. Specifically, ki/ki platelets exhibit a paucity of α-granules and increased numbers of lysosomal-like vacuoles, reminiscent of the platelet morphology observed in human patients with GPS due to GATA-1 mutations. To understand the molecular basis for the phenotype in ki/ki platelets, we compared mRNA and protein expression of α-granule proteins in wildtype (wt) and ki/ki MK. P-selectin mRNA in ki/ki MKs was 3–5 fold reduced when compared to controls, while platelet factor 4 (Pf4), platelet basic protein (PBP) and von Willebrand factor (Vwf), were only mildly reduced. Strikingly, P-selectin protein levels were more severely reduced in ki/ki MK and platelets being nearly undetectable by Western blot and immunohistochemistry. In contrast PF4, PBP, and vWF protein levels were normal. P-selectin loss was MK-specific as it was expressed at normal levels in endothelial cells. The dramatic decrease in P-selectin protein in ki/ki MK relative to the more moderate reduction in mRNA was due to a >5-fold decrease in protein half-life, as measured by metabolic pulse-chase and immunoprecipitation experiments. This suggests that P-selectin in ki/ki MKs is largely mistargeted to lysosomes. Nevertheless, immunofluorescence microscopy and volume de-convolution analyses of ki/ki platelets showed that the remaining P-selectin resided in vesicular compartments that were distinct from Lamp1 (a lysosomal marker)-containing granules, indicating that a fraction of P-selectin remains within α-like granules. Interestingly, the reduction in P-selectin levels in ki/ki platelets was accompanied by a defect in agonist-stimulated degranulation. While stimulation of wt platelets with AYP, thrombin or phrobol myristate acetate induced α-granule, dense granule and lysosome degranulation, ki/ki platelets failed to do so or undergo platelet aggregation. In summary, the GPS-like phenotype in FOG-1/NuRD interaction-defective MKs involves reduced P-selectin mRNA production, accelerated turnover of P-selectin, abnormal granule formation, and defective degranulation in response to diverse agonists. These findings may partially explain patients having platelet α-granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation with GPS. Future work will determine whether similar defects occur in human GPS.