PB0789 - Temporal dynamics of MLKL: pivotal roles in agonist-induced platelet prothrombotic responses and lytic cell death.”

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Vipin Singh

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

Co-Author(s)

DD

Debabrata Dash

Professor
Center for Advanced Research on Platelet Signaling and Thrombosis Biology, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India
Varanasi, Uttar Pradesh, India
ME

Mohammad Ekhlak

Ph.D. Scholar
Center for Advanced Research on Platelet Signaling and Thrombosis Biology, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India
VARANASI, Uttar Pradesh, India
Susheel N. Chaurasia, MSc, PhD

Scientist-C
Center for Advanced Research on Platelet Signaling and Thrombosis Biology, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India
Varanasi, Uttar Pradesh, India
PK

Paresh P. Kulkarni P. Kulkarni

Associate Professor
Center for Advanced Research on Platelet Signaling and Thrombosis Biology, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India
Varanasi, Uttar Pradesh, India
RN

Rameshwar Nath Chaurasia

Professor
Department of Neurology, Banaras Hindu University Institute of Medical Sciences: Varanasi, Uttar Pradesh, IN
Varanasi, Uttar Pradesh, India
SK

Saroj Kant Mohapatra

Associate Professor (Infectious Diseases Division)
National Institute of Biomedical Genomics, Kalyani, India.
Varanasi, Uttar Pradesh, India

Background: Necroptosis, a regulated form of cell death, relies significantly on the presence of receptor-interacting serine/threonine protein kinase (RIPK) 1, 3, and mixed lineage kinase domain-like (MLKL). A study using mice with a megakaryocyte-specific MLKL knockout identified functional necroptosis signaling in these platelet precursors. The mice displayed extended tail re-bleeding times and impaired agonist-induced platelet activation. Additionally, RIPK3 exhibited an MLKL-independent role in thrombosis pathology. Despite these insights, the regulatory mechanisms and functions of MLKL in human platelets remain largely unexplored.

Aims: The aim of the study is to explore necroptosis signaling in human platelets and investigate the crucial role of MLKL in the transformation of agonist-challenged platelets into prothrombotic phenotypes.

Methods: We investigated the impact of MLKL inhibitors, on the responses of platelets to agonist stimulation using various techniques such as Western blotting, flow cytometry, nanosight, and shear-based thrombosis studies. Statistical significance in mean differences was assessed using repeated measures ANOVA and Dunnett's multiple comparison test.

Results: Physiological agonist thrombin induces phosphorylation and oligomerization of MLKL in platelets in a RIPK3-independent but PI3K/AKT-dependent manner. Inhibition of MLKL significantly reduces agonist-induced haemostatic responses in platelets, including platelet aggregation, integrin activation, granule secretion, rise in intracellular calcium, shedding of extracellular vesicles, platelet-leukocyte interactions, and thrombus formation under arterial shear. MLKL inhibition also impairs mitochondrial oxidative phosphorylation and aerobic glycolysis in stimulated platelets, accompanied by disruption in mitochondrial transmembrane potential, augmented proton leak, and drop in mitochondrial calcium and ROS levels.

Conclusion(s): MLKL, abundantly expressed in platelets, guides agonist-stimulated platelets toward active hemostatic units and necroptotic death. It plays a crucial role in platelet responses. Prolonged exposure to thrombin triggers MLKL-mediated necroptosis, a process that can be effectively thwarted by inhibitors targeting PI3K/MLKL, irrespective of RIPK3 dependence. Investigating MLKL in platelets enhances understanding of cell death processes, offering therapeutic potential in cardiovascular and hematologic diseases.