PB0774 - Development and Characterization of Conditional Nbeal2 KO Mouse Strains for studies of Thrombo-Inflammation

Background: Deleterious mutations in the human NBEAL2 gene cause the rare bleeding disorder gray platelet syndrome, characterized by the absence of platelet alpha granules, macrothrombocytopenia and bleeding diathesis. A mouse Nbeal2 global KO strain recapitulates the human phenotype. In previous studies using this mouse strain, it has been shown that Nbeal2 and platelet alpha granules are critical in several models of thrombosis, inflammation and sepsis, however a global absence of Nbeal2 in this strain has also been linked with leukocyte and endothelial cell anomalies, making it difficult to determine if the causative and protective affects are due directly due to Nbeal2 deficiency or indirectly due to a loss of platelet alpha granules. Currently adoptive transfer is used to assess specific contributions of platelet and leukocytes in thrombo-inflammation however these studies can be restrictive depending on the type of thrombo-inflammatory model being assessed

Aims: Develop and characterize 3 conditional strains where Nbeal2 is absent specifically from platelets, endothelial cells and neutrophils.

Methods: Nbeal2LoxP mice were created using CRISPR/Cas9 technologies by inserting BamH1+loxP sites flanking the Nbeal2 gene. We crossed these mice with Cre transgenic mice to generate megakaryocyte/platelet specific; endothelial specific or neutrophil specific deletion of Nbeal2 (Figure 1).
Whole blood or platelet and neutrophils were isolated for phenotypic and functional studies.

Results: This study reports, for the first time, the development and characterization of mice deficient in Nbeal2 specifically in the megakaryocyte/ platelet; neutrophil and endothelial cell compartments. Consistent with previous studies in global KO strains we show Nbeal2PLT KO mice have decreased platelet counts (a) and decreased CD41/61 (b) and P selectin (c) activation compared to WT (Figure 2).

Conclusion(s): These new Nbeal2 mouse strains represent new animal models for studying the direct and indirect affects of Nbeal2 and alpha granules in thrombo-inflammatory models.