Deletion of Bcl-X in the Megakaryocyte Compartment Results in Profound Thrombocytopenia Caused by Impaired Platelet Production and Survival.

Abstract 1495

Poster Board I-518

Recent studies have suggested a role for the intrinsic apoptosis pathway in megakaryocytic differentiation and platelet shedding. The intrinsic pathway is regulated by the Bcl-2 family of pro- and anti- cell death proteins. We recently demonstrated that platelet life span is controlled by an intrinsic cell death pathway, whereby the anti-apoptotic protein Bcl-x_L constrains the pro-apoptotic activity of Bak to maintain platelet survival. As Bcl-x_L is expressed in megakaryocytes, we investigated whether this protein is required for megakaryocyte survival, differentiation and/or platelet shedding. We specifically deleted the Bcl-x gene in the megakaryocyte lineage by crossing mice carrying a floxed allele of Bcl-x with mice carrying a platelet factor 4-regulated Cre transgene. Bcl-x^fl/flCre⁺ mice were profoundly thrombocytopenic (26 ± 5 × 10³/μl, n=14) compared with Bcl-x^fl/flCre⁻ animals (1157 ± 202 × 10³/μl, n=13). Platelet life span in these mice was reduced to only 5 hours, as compared to 5 days in wild type littermates. This result confirmed that Bcl-x_L is absolutely required for platelet survival. To determine whether Bcl-x deletion has an impact on platelet production, we analyzed the megakaryocyte compartment in Bcl-x^fl/flCre⁺ and Bcl-x^fl/flCre⁻ mice. We observed that the number of megakaryocyte progenitors, and number of megakaryocytes in the bone marrow were increased in Bcl-x^fl/flCre⁺ mice (23 ± 9 megakaryocyte progenitors vs 11 ± 5, and 51 ± 9 megakaryocytes vs 12 ± 1). This result suggested that Bcl-x_L is not required for the survival of megakaryocytes or their progenitors. To determine whether Bcl-x_L is required for the last step of megakaryocyte differentiation, i.e. platelet shedding, we cultured fetal liver cells with thrombopoietin. Large megakaryocytes were isolated after 3 days of differentiation on discontinuous bovine serum albumin gradient. They were cultured for 3 more days in the same media and the percentage of megakaryocytes displaying proplatelets was determined each day. Interestingly, Bcl-x^fl/flCre⁺ megakaryocytes died much more quickly than Bcl-x^fl/flCre⁻ megakaryocytes, and almost none of those that survived were able to form proplatelets. Our study indicates that Bcl-x_L is not only essential for platelet survival, but it is also required for the survival of mature megakaryocytes at the stage of platelet shedding.