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Necroptosis executioner MLKL plays pivotal roles in agonist-induced platelet prothrombotic responses and lytic cell death in a temporal order

Cell Death & Differentiation volume 30, pages 1886–1899 (2023)Cite this article

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Abstract

Necroptosis is a form of programmed cell death executed by receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL). Platelets are circulating cells that play central roles in haemostasis and pathological thrombosis. In this study we demonstrate seminal contribution of MLKL in transformation of agonist-stimulated platelets to active haemostatic units progressing eventually to necrotic death on a temporal scale, thus attributing a yet unrecognized fundamental role to MLKL in platelet biology. Physiological agonists like thrombin instigated phosphorylation and subsequent oligomerization of MLKL in platelets in a RIPK3-independent but phosphoinositide 3-kinase (PI3K)/AKT-dependent manner. Inhibition of MLKL significantly curbed agonist-induced haemostatic responses in platelets that included platelet aggregation, integrin activation, granule secretion, procoagulant surface generation, rise in intracellular calcium, shedding of extracellular vesicles, platelet-leukocyte interactions and thrombus formation under arterial shear. MLKL inhibition, too, prompted impairment in mitochondrial oxidative phosphorylation and aerobic glycolysis in stimulated platelets, accompanied with disruption in mitochondrial transmembrane potential, augmented proton leak and drop in both mitochondrial calcium as well as ROS. These findings underscore the key role of MLKL in sustaining OXPHOS and aerobic glycolysis that underlie energy-intensive platelet activation responses. Prolonged exposure to thrombin provoked oligomerization and translocation of MLKL to plasma membranes forming focal clusters that led to progressive membrane permeabilization and decline in platelet viability, which was prevented by inhibitors of PI3K/MLKL. In summary, MLKL plays vital role in transitioning of stimulated platelets from relatively quiescent cells to functionally/metabolically active prothrombotic units and their ensuing progression to necroptotic death.

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Fig. 1: Thrombin induced phosphorylation of MLKL in PI3K/AKT-dependent manner.
Fig. 2: MLKL has a potential role in regulating thrombin-induced platelet functions.
Fig. 3: MLKL drives transformation of agonist-stimulated platelets to procoagulant phenotype.
Fig. 4: MLKL prompts formation of platelet thrombi on immobilized collagen under high shear.
Fig. 5: MLKL contributes to sustenance of well-coupled functional mitochondria in thrombin-stimulated platelets.
Fig. 6: Role of MLKL in eliciting membrane permeabilization upon sustained stimulation of platelets.
Fig. 7: Dimerization of MLKL in thrombin-stimulated platelets.
Fig. 8: Trafficking of MLKL to surface membrane of thrombin-stimulated platelets to accumulate as focal clusters.
Fig. 9: Scheme depicting the contribution of MLKL in agonist-induced platelet activation and necroptosis on a temporal scale.

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Data availability

All relevant data that support the findings of this study are within the paper. Raw data incorporated in Microsoft Excel and/or GraphPad Prism files are available on request.

Change history

  • 21 June 2023

    In this article the Mohammad Ekhlak’s ORCID has been corrected.

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Funding

This research was supported by a J.C. Bose National Fellowship and grants received by DD from Indian Council of Medical Research (ICMR) under Center for Advanced Research (CAR), and Science and Engineering Research Board (SERB), Government of India. ME and VS are recipients of CSIR-SRF and UGC-SRF, respectively.

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Authors and Affiliations

  1. Center for Advanced Research on Platelet Signaling and Thrombosis Biology, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India

    Mohammad Ekhlak, Paresh P. Kulkarni, Vipin Singh, Susheel N. Chaurasia & Debabrata Dash

  2. National Institute of Biomedical Genomics, Kalyani, India

    Saroj Kant Mohapatra

  3. Department of Neurology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India

    Rameshwar Nath Chaurasia

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  1. Mohammad Ekhlak
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DD designed and supervised the entire work; ME, PPK, VS, SNC, SKM and RNC provided samples and performed various experiments; DD, ME, and PPK analyzed results and wrote the manuscript.

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Correspondence to Debabrata Dash.

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Ekhlak, M., Kulkarni, P.P., Singh, V. et al. Necroptosis executioner MLKL plays pivotal roles in agonist-induced platelet prothrombotic responses and lytic cell death in a temporal order. Cell Death Differ 30, 1886–1899 (2023). https://doi.org/10.1038/s41418-023-01181-6

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