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Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of β‐oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin‐stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P‐selectin externalization and integrin αIIbβ3 activation. Both etomoxir and trimetazidine impeded agonist‐induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose‐ and incubation time‐ dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen‐epinephrine‐induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre‐administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride‐induced mesenteric thrombosis. In conclusion, β‐oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy‐intensive agonist‐induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

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.

Sonic hedgehog (Shh) is a morphogen in vertebrate embryos that is also associated with organ homeostasis in adults. We report here that human platelets, though enucleate, synthesize Shh from pre-existing mRNAs upon agonist stimulation, and mobilize it for surface expression and release on extracellular vesicles, thus alluding to its putative role in platelet activation. Shh, in turn, induced a wave of non-canonical signaling in platelets leading to activation of small GTPase RhoA and phosphorylation of myosin light chain (MLC) in AMP-activated protein kinase (AMPK)-dependent manner. Remarkably, agonist-induced thrombogenic responses in platelets, which include platelet aggregation, granule secretion and spreading on immobilized fibrinogen, were significantly attenuated by inhibition of Hedgehog signaling, thus implicating inputs from Shh in potentiation of agonist-mediated platelet activation. In consistence, inhibition of Shh pathway significantly impaired arterial thrombosis in mice. Taken together, above observations strongly support a feed-forward loop of platelet stimulation triggered locally by Shh, similar to ADP and thromboxane A2, that contributes significantly to stability of occlusive arterial thrombus and that can be investigated as potential therapeutic target in thrombotic disorders.

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.

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