Abstract
Abstract 389
The survival of megakaryocytes and platelets is regulated by the intrinsic apoptosis pathway. Both cell types express Bak and Bax, the essential mediators of intrinsic apoptosis, which must be kept in check for cellular viability to be maintained. In platelets, Bak and Bax are restrained by the pro-survival protein Bcl-xL. Mutations that reduce the pro-survival activity of Bcl-xL cause dose-dependent cell-intrinsic reductions in circulating platelet life span in mice. Accordingly, pharmacological blockade of Bcl-xL with the BH3 mimetic drugs ABT-737 or ABT-263 (Navitoclax) triggers platelet death and thrombocytopenia in mice, dogs and humans. In mice, loss of Bak and Bax almost doubles platelet life span, and renders platelets refractory to the effects of ABT-737. In megakaryocytes, we and others have recently demonstrated that Bcl-xLand its pro-survival relative Mcl-1 are essential for restraint of the intrinsic apoptosis pathway. Their loss triggers Bak/Bax-mediated death. Conversely, ablation of Bak and Bax can protect megakaryocytes from acute apoptotic insults, such as treatment with carboplatin. Combined with the fact that platelet production is normal in the absence of Bak and Bax, these studies have brought into question the long-standing theory that megakaryocytes deliberately undergo apoptosis in order to shed platelets. However, whilst it is clear that the intrinsic apoptosis pathway is not required for thrombopoiesis, the role of the extrinsic pathway—the other major route to apoptotic cell death—has not been established.
In the current study we examined the functionality of, and physiological requirement for, the extrinsic apoptosis pathway in megakaryocytes and platelets. The extrinsic pathway is triggered when members of the tumor necrosis factor (TNF) superfamily such as Fas ligand (FasL) bind to cell surface death receptors (e.g. Fas). This induces receptor multimerization, recruitment of death domain adaptor proteins (e.g. FADD) and subsequent activation of Caspase-8, which is the essential mediator of extrinsic pathway-mediated cell death. We found that both megakaryocytes and platelets express critical components of the pathway, including FADD, Caspase-8 and Bid. Megakaryocytes, but not platelets, also expressed the death receptor Fas. Mature fetal liver-derived megakaryocytes treated with soluble FasL exhibited activation of Caspase-8 and the effector Caspases-3/7. This was accompanied by mitochondrial damage and a failure of pro-platelet formation.
To establish the requirement for the extrinsic pathway in megakaryocyte development and platelet production, we conditionally deleted Caspase-8 from the megakaryocyte lineage. Platelet counts and platelet life span in Casp8Pf4Δ/Pf4Δ mice were indistinguishable from those of wild-type littermates. Megakaryocyte numbers, morphology, ploidy and in vitro pro-platelet formation capacity were also normal. Caspase-8-deficient megakaryocytes were resistant to FasL treatment. Casp8Pf4Δ/Pf4Δ animals responded to experimentally-induced thrombocytopenia in a manner similar to wild-type. Collectively, these data indicate that the extrinsic apoptosis pathway is dispensable for the generation and survival of platelets. To examine any potential redundancy between the extrinsic and intrinsic apoptosis pathways, we generated Bak−/−BaxPf4Δ/Pf4ΔCasp8Pf4Δ/Pf4Δ triple knockout mice. The ability of these animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers and morphology were normal. Thus, platelet shedding by megakaryocytes does not require the intrinsic or extrinsic apoptosis pathways. Together with recent work demonstrating that the apoptotic initiator caspase, Caspase-9, is also dispensable for platelet production, we conclude that platelet biogenesis is not an apoptotic process.
Disclosures:
No relevant conflicts of interest to declare.
Pharmacological targeting of c-FLIPL and Bcl-2 family members promotes apoptosis in CD95L-resistant cells
