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Megakaryocyte Mitochondrial Dynamics and Oxidative Metabolism Are Essential to Initiate Platelet Biogenesis

1Université de Paris, Innovative Therapies in Haemostasis, INSERM 1140, Paris, France, 2AP-HP, Necker Hospital, Clinical Biochemistry Department, Paris, France, 3Université de Paris, Institut Cochin, INSERM 1016, CNRS UMR 8104, Paris, France, 4Université Paris-Sud, Université Paris Saclay, 4HITh, INSERM UMR_S 1176, Kremlin Bicêtre, France, 5Université de Paris, PTICM, INSERM US 025, CNRS UMS 3612, Paris, France

Abstract Number: PB1606

Meeting: ISTH 2020 Congress

Theme: Platelets and Megakaryocytes » Megakaryocytes and Thrombopoiesis

Background: Increased platelet production is known to occur upon oxygen concentration rise. In several cell types a relation between reactive oxygen species (ROS) and mitochondrial dynamics exists but nothing is known about thrombopoiesis initiation.

Aims: We addressed the role of ROS and mitochondrial (mt) dynamics in the initiation of thrombopoiesis from human mature MK.

Methods: Primary human cord blood CD34+ cells were differentiated for 12-13 days to generate mature MK. These cells were treated for 5hrs with various pro- and anti-oxydants : N-acetylcysteine (NAC), MitoTEMPO, nicotinamide (NAM),deacetylation-null SIRT3 (DN-SIRT3) mutant. ROS levels were quantified in CD42b+ MK using DHE or MitoSOX probes and flow cytometry; MK morphology and mitochondrial dynamics were monitored using phase or immunofluorescent microscopy and MitoSOX, MitoTracker or Drp1 probes.

Results: The anti-oxidant NAC decreased by 1.6-fold, whereas the pro-oxidant NAM enhanced by 1.5-fold platelet biogenesis from MK. Quenching mtROS by MitoTEMPO decreased by 2-fold the % proplatelet-forming MK, whereas enhancing mtROS through DN-SIRT3, increased them by 2-fold. We delineated three stages of thrombopoiesis from mature MK according to roundness index and identified an intermediate stage with small cell deformations assessing thrombopoiesis onset. Analysis of ROS levels showed that thrombopoiesis onset correlated with 1.7-fold increase of mtROS levels and a 2-fold decrease in mitochondrial area in intermediate MK. A higher mitochondrial recruitment of fission activator Drp1 was also identified before detectable thrombopoiesis initiation. Blocking mitochondrial fission by Drp1 self-assembly inhibition through Mdivi, inhibited thrombopoiesis and lowered mtROS, whereas quenching mtROS inhibited mitochondrial recruitment of active Drp1 in round MK.

Conclusions: These observations indicate a role of mtROS and mitochondrial dynamics in platelet biogenesis onset. They provide evidence for a reciprocal relationship between mtROS and mitochondrial fission in MK that contributes to initiate platelet biogenesis.

To cite this abstract in AMA style:

Poirault-Chassac S, Nivet-antoine V, Houvert A, Kauskot A, Lauret E, Lai-Kuen R, Dusanter-Fourt I, Baruch D. Megakaryocyte Mitochondrial Dynamics and Oxidative Metabolism Are Essential to Initiate Platelet Biogenesis [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/megakaryocyte-mitochondrial-dynamics-and-oxidative-metabolism-are-essential-to-initiate-platelet-biogenesis/. Accessed January 28, 2022.

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