Megakaryocytes Possess a Functional Intrinsic Apoptosis Pathway That Must Be Restrained In Order to Survive and Produce Platelets

Abstract 550

It is widely held that megakaryocytes undergo a specialized form of apoptosis in order to shed platelets. Conversely, it is also believed that a range of insults including chemotherapeutic agents, autoantibodies and viruses, cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. However, the apoptotic pathways that megakaryocytes possess, and the role they play in survival and platelet production are ill-defined. We recently demonstrated that platelets contain a classical intrinsic mitochondrial apoptosis pathway that regulates their life span in vivo. The key components of this pathway are the Bcl-2 family pro-survival protein Bcl-x_L, and pro-death Bak and Bax. Deletion of Bak and Bax—the gatekeepers of the intrinsic pathway—blocks platelet apoptosis in response to genetic mutation or pharmacological insult, and significantly extends circulating platelet life span.

To elucidate the role of the intrinsic apoptosis pathway in megakaryocytes, we generated both hematopoietic- and megakaryocyte lineage-specific deletions of Bak and Bax in mice. Surprisingly, we found that the ability of Bak^−/−Bax^−/− animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers, morphology and ploidy were normal. Bak^−/−Bax^−/− megakaryocytes cultured in vitro showed no impairment of pro-platelet formation. Thus, classical intrinsic apoptosis is not required by megakaryocytes for the process of platelet shedding. Given that in platelets, Bak and Bax must be kept in check to maintain survival, we reasoned that the same might be true of megakaryocytes. If so, then it would be expected that one or more members of the Bcl-2 family of pro-survival proteins restrain Bak and Bax. Since Bcl-x_L fulfills this role in platelets, we generated mice lacking Bcl-x_L in the megakaryocyte lineage. Platelet counts in Bcl-x^{Pf4CΔ/Pf4CΔ} animals were approximately 2% of those observed in Bcl-x^fl/fl littermates. Platelet life span was reduced to 5 hours, versus 5 days in controls, underscoring the requirement for Bcl-x_L in mediating platelet survival. In addition, reticulated platelet analyses combined with mathematical modeling suggested that Bcl-x^{Pf4CΔ/Pf4CΔ} mice had an underlying platelet production defect. Further examination revealed that megakaryocyte numbers were significantly increased in both the bone marrow and spleen of Bcl-x^{Pf4CΔ/Pf4CΔ} animals relative to Bcl-x^fl/fl controls. Megakaryocyte progenitor numbers were doubled, and serum TPO levels were dramatically reduced, indicating a megakaryocyte compartment under considerable thrombopoietic stress.

In vitro cultures confirmed that Bcl-x^{Pf4CΔ/Pf4CΔ} megakaryocytes were able to develop and mature. Strikingly, however, at the point of pro-platelet formation, they underwent an abortive attempt to generate extensions and died. Death was accompanied by a dramatic increase in apoptotic effector caspase activity. This suggested that, like platelets, megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained in order to survive, and that Bcl-x_L is the factor that does so during pro-platelet formation and platelet shedding. To establish whether Bak and Bax can mediate megakaryocyte death, we examined the effect on mature wild type megakaryocytes of three pharmacological agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and a BH3 mimetic that inhibits Bcl-x_L, Bcl-2 and Bcl-w. All three triggered mitochondrial damage, caspase activation and cell death. Remarkably, genetic deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and the BH3 mimetic, but not staurosporine. Our results demonstrate that megakaryocytes can undergo classical Bak- and Bax-mediated apoptotic death. They do not activate the intrinsic pathway to facilitate platelet shedding, rather, the opposite is true: they must restrain it in order to survive and generate platelets. These findings offer a potential mechanism for the death of megakaryocytes in response to insults such as cancer chemotherapy. They also suggest that additional megakaryocyte cell death pathways remain to be elucidated.