Abstract Number: PB1596
Meeting: ISTH 2020 Congress
Background: Necroptosis is a newly discovered pathway of programmed necrosis, with RIPK1 being a critical regulator of the pathway. RIPK1 promotes the sequential activation of two downstream targets, RIPK3 and MLKL. Previously, it has been shown that RIPK3 plays a role in regulating thrombosis and haemostasis (Zhang et al., 2017 PNAS), however characterisation of necroptosis signalling and the functional significance in megakaryocytes has not been explored.
Aims: To determine whether necroptosis signalling influences the megakaryocytic lineage.
Methods: Genetic mouse models included WT and MLKL KO counterparts. Western blot was employed on cell lysates from megakaryocytes and platelets to assess the expression of necroptosis signalling proteins. Cell viability and morphology was assessed by the cell titre glow assay and time lapse imaging of WT and MLKL KO megakaryocytes via confocal microscopy. Agonists induced platelet activation was measured via flow cytometry and bleeding times after 3-mm tail amputation were assessed.
Results: Bone marrow derived WT megakaryocytes expressed RIPK1, RIPK3, and MLKL proteins. Phospho MLKL was induced in response to activation of necroptosis (TSI -TNF-a/Smac mimetic/the caspase-8 inhibitor, Idun). WT megakaryocytes treated with TSI were sensitized to necroptosis, which could be rescued by Necrostatin-1, or genetic deletion of MLKL. Time lapse imaging demonstrated that the necroptotic process could be visualized as early as 1 h post TSI treatment, while TNF/SMAC induced apoptosis was apparent at 4h post treatment. Furthermore, MLKL KO mice exhibited increased re-bleeding indicating unstable thrombus formation. MLKL KO platelets showed reduced degranulation and integrin activation in response to thrombin, suggesting impaired platelet signalling.
Conclusions: For the first time, we demonstrate that bone marrow derived megakaryocytes harbour a functional necroptosis signalling cascade, triggered via TNF receptor signalling. MLKL KO mice have unstable thrombus formation and impaired platelet activation, demonstrating that the necroptosis pathway can influence function of the megakaryocytic lineage.
To cite this abstract in AMA style:Moujalled D, Gangatirkar P, Corbin J, Kauppi M, Lalaoui N, Hildebrand J, Silke J, Alexander W, Josefsson E. Megakaryocytes Possess a Functional Necroptosis Pathway [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/megakaryocytes-possess-a-functional-necroptosis-pathway/. Accessed November 30, 2021.
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