Actin and Myosin-II Inhibition Increase Megakaryocyte Proplatelet Formation Through a Caspase Dependent Mechanism

Introduction Maximizing proplatelet formation from cultured megakaryocytes will be a critical step in ex-vivo production of platelets for transfusion. However, mechanisms triggering proplatelet formation are not completely identified. Apoptosis factors are suggested to play an important role in initiating the process of platelet release. However, their role is controversial. Megakaryocytes might employ the apoptotic machinery to facilitate platelet production through the intrinsic pathway and caspase 3/9 activation, and pro-survival factors, such as Bcl-xL, are necessary to enable megakaryocyte survival during proplatelet formation. However, removing the pro-apoptotic intrinsic pathway factors Bak and Bax did not limit proplatelet formation. To shed some light on this controversy we performed experiments in which we increased megakaryocyte proplatelet formation by inhibiting two vital cytoskeletal proteins, actin and myosin-II. We found that such an inhibition of actin or myosin-II late in megakaryocytopoiesis correlated with activation of the apoptosis pathways. We analyzed the effect of actin or myosin-II inhibition on the extrinsic and intrinsic apoptosis pathways and whether the increase in proplatelet formation due to actin or myosin inhibition was dependent on apoptosis. Methods Human cord blood derived CD34+ cells were isolated and cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 11 days. Megakaryocytes were isolated on day 8 and replated in culture medium containing only TPO. The day 8 megakaryocytes were treated with an actin inhibitor (AI, Latrunculin-A) 10µM or myosin-II inhibitor (MI, Blebbistatin) 20µM. On day 11 of culture, cell ploidy was analyzed by flow cytometry using propidium Iodide. Proplatelets were counted using inverted light microscopy and their structure characterized by fluorescence microscopy using a β-1 tubulin-specific antibody. Western blots were performed for Bcl-xL, Bak, and Bax. Mitochondrial outer membrane permeabilization (MOMP) and phosphatidylserine (PS) externalization (Annexin-V binding) were measured using flow cytometry. Caspases 3 and 7 were analyzed using a luminescence assay (CaspaseGlo3/7). In some experiments apoptosis was inhibited on day 8 with a pan-caspase inhibitor ZVAD-fmk (20µM). All results were compared to untreated control megakaryocytes. Results Treatment with either actin or myosin-II inhibitor increased the level of polyploidization in megakaryocytes (both p=0.03). There was no additive effect in final ploidy with combination of reagents. Proplatelet extension by megakaryocytes treated with AI or MI was higher than untreated control cells (p=0.02, p=0.05) and proplatelet structure was normal as assessed by fluorescence microscopy. Bcl-xL, Bak and Bax protein levels were not different from controls after AI or MI treatment. MOMP and PS externalization were increased in both AI (p=0.04) and MI (p=0.04) treated megakaryocytes compared to untreated controls. Caspase 3/7 activities were also increased in treated megakaryocytes. The addition of a pan-caspase inhibitor prevented the release of proplatelets in both AI and MI treated megakaryocytes. Discussion Inhibition of actin or myosin-II led to increased levels of polyploidization and proplatelet formation in cultured megakaryocytes. This increased proplatelet formation was simultaneous with increased final apoptosis activation. Apoptosis analysis showed no difference in protein levels of the intrinsic pathway pro-apoptotic (Bak and Bax) or pro-survival (Bcl-xL) factors. However, both flow cytometry and caspase 3/7 assays showed that the final apoptosis process was activated in cells treated with AI or MI. Thus the apoptosis activation was mediated through a pathway independent of the intrinsic pathway factors Bcl-xL, Bak and Bax. Proplatelet formation in response to AI or MI was inhibited by the pan-caspase inhibitor, suggesting that the effects of AI and MI on proplatelet formation are caspase dependent. These results suggest that inhibition of actin or myosin-II late in megakaryocytopoiesis increases proplatelet formation through activation of apoptosis. Our findings suggest that proplatelet formation is dependent on the activation of apoptosis but may not be mediated through the intrinsic pathway. Determining the pathway leading to final apoptosis and proplatelet formation deserves further investigation. Disclosures: No relevant conflicts of interest to declare.