xygen-compromised environments, such as high altitude, are associated with platelet hyperactivity. Platelets confined within the relatively impervious core of an aggregate/thrombus have restricted access to oxygen, yet they continue to perform energy-intensive procoagulant activities that sustain the thrombus. Studying platelet signaling under hypoxia is, therefore, critical to our understanding of the mechanistic basis of thrombus stability. We report here that hypoxia-inducible factor (HIF)-2a is translated from pre-existing mRNA and stabilized against proteolytic degradation in enucleate platelets exposed to hypoxia. Hypoxic stress, too, stimulates platelets to synthesize plasminogen-activator inhibitor-1 (PAI-1) and shed extracellular vesicles, both of which potentially contribute to the prothrombotic phenotype associated with hypoxia. Stabilization of HIF-a by administering hypoxia-mimetics to mice accelerates thrombus formation in mesenteric arterioles. In agreement, platelets from patients with chronic obstructive pulmonary disease and high altitude residents exhibiting thrombogenic attributes have abundant expression of HIF-2a and PAI-1. Thus, targeting platelet hypoxia signaling could be an effective antithrombotic strategy.

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.

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Background: Notch signaling is an evolutionarily conserved pathway that dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets that are generated as cytoplasmic buds from megakaryocytes still remains elusive. Methods: Platelets were isolated from human blood by differential centrifugation under informed consent. Expression of transcripts as well as peptides of Notch1 and DLL-4 in platelets was studied by employing RT-qPCR, Western analysis and flow cytometry. Platelet activation responses that include aggregation, secretion of granule contents and platelet-leucocyte interaction were analyzed by Born's aggregometry, flow cytometry, Western analysis and lumi-aggregometry. Shedding of extracellular vesicles from platelets was documented with Nanoparticle Tracking Analyzer. Platelet adhesion and thrombus growth on immobilized matrix was quantified by employing microfluidics platform. Intracellular free calcium in Fura-2-loaded platelets was monitored from ratiometric fluorescence spectrophotometry. Coagulation parameters in whole blood were studied by thromboelastography. Ferric chloride-induced mesenteric arteriolar thrombosis in murine model was imaged by intravital microscopy. Results: Here we demonstrate significant expression of Notch1 and its ligand, the Delta-like ligand (DLL)-4, as well as their respective transcripts, in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated when cells were challenged with physiological agonists like thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to 'activated' phenotype, associated with cleavage of Notch receptor and generation of its intracellular domain (NICD). DLL-4-mediated pro-thrombotic attributes were averted by pharmacological inhibition of γ-secretase and phosphatidylinositol 3-kinase. Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice, suggestive of synergism between thrombin- and DLL-4-mediated pathways. 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 Notch signaling that propagates in juxtacrine manner within platelet aggregates and synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, targeting Notch signaling can be investigated as a potential anti-platelet/anti-thrombotic therapeutic approach. Funding: This research was supported by J. C. Bose National Fellowship (JCB/2017/000029) and grants received by D. Dash from the Indian Council of Medical Research (ICMR) under CAR (71/4/2018-BMS/CAR), Department of Biotechnology (DBT) (BT/PR-20645/BRB/10/1541/2016) and Science and Engineering Research Board (SERB) (EMR/2015/000583), Government of India. S.N. Chaurasia is a recipient of financial assistance from the ICMR. M. Ekhlak is a recipient of CSIR-SRF and V. Singh is a recipient of UGC-SRF. D. Dash acknowledges assistance from the Humboldt Foundation, Germany. Funders have no role in the design, analysis and reporting of the study.