Background: Notch signaling dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets remains elusive.
Methods: Transcripts/peptides of Notch1 and Delta-like ligand (DLL)-4 were detected in platelets isolated from human blood by RT-qPCR, Western analysis and flow cytometry. Platelet aggregation, granule secretion and platelet-leucocyte interaction were analyzed by lumi-aggregometry and flow cytometry. Platelet-derived extracellular vesicles were documented with Nanoparticle Tracking Analyzer. Platelet thrombus on immobilized collagen was quantified using microfluidics platform. Intracellular calcium was monitored by fluorescence spectrophotometry. Whole blood coagulation was studied by thromboelastography. Ferric chloride-induced mouse mesenteric arteriolar thrombosis was imaged by intravital microscopy.
Results: We demonstrate expression of Notch1, its ligand DLL-4 and their respective transcripts in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated by thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to 'activated' phenotype through cleavage of Notch receptor and release of its intracellular domain (NICD), which was averted by inhibition of γ-secretase and phosphatidylinositol-3-kinase (PI3K). Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin.
Conclusions: Our study presents compelling evidence in support of non-canonical juxtacrine Notch signaling within platelet aggregates that synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, Notch pathway can be a potential anti-platelet/anti-thrombotic therapeutic target.
Funding: Research was supported by grants received by DD from JC Bose Fellowship (JCB/2017/000029), ICMR (71/4/2018-BMS/CAR), DBT (BT/PR-20645/BRB/10/1541/2016) and SERB (EMR/2015/000583). SNC, ME and VS are recipients of ICMR-Scientist-C, CSIR-SRF and UGC-SRF support, respectively. Funders had no role in design, analysis and reporting of study.
All the data are available in the main text or the supporting information. Source Data file have been provided for each figure included either in the manuscript or supplemental data.
- Debabrata Dash
- Debabrata Dash
- Debabrata Dash
- Debabrata Dash
- Susheel N Chaurasia
- Mohammad Ekhlak
- Vipin Singh
- Debabrata Dash
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: The animal study was ethically approved by the Central Animal Ethical Committee of Institute of Medical Sciences, Banaras Hindu University (Approval No. Dean/2017/CAEC/83). All efforts were made to minimize the number of animals used, and their suffering.
Human subjects: Blood samples were drawn from healthy adult human participants after obtaining written informed consent, strictly as per recommendations and as approved by the Institutional Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University (Approval No. Dean/2015-16/EC/76).
- Robert Baiocchi, The Ohio State University, United States
© 2022, Chaurasia et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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