Notch signaling functions in noncanonical juxtacrine manner in platelets to amplify thrombogenicity

Abstract

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.

Platelet function suggests cardioembolic aetiology in cryptogenic stroke

“…Fresh blood (1 ml) was taken in citrated kaolin vials and mixed gently. CaCl 2 (20 µl) was added to 340 µl sample to initiate coagulation cascade 34 . The mixture was placed in disposable TEG cups.…”

Ayurvedic preparations of Raudra Rasa inhibit agonist‐mediated platelet activation and restrict thrombogenicity without affecting cell viability

“…Binding of antibody was determined using enhanced chemiluminescence detection kit (Millipore). p‐AKT‐ and p‐GSK‐3β‐stained membranes were stripped and reprobed with anti‐AKT and anti‐GSK‐3β antibodies, as described [5]. Images were acquired on the UVP BioSpectrum 800 Imaging System and quantified using VisionWorks LS software (UVP, Upland, CA, USA).…”

“…Dean/2019/EC/1683). Platelets were isolated from fresh peripheral venous human blood by differential centrifugation, as described previously [5]. Cells were counted using automated cell counter (Multisizer 4, Beckman Coulter, Mumbai, India) and leukocyte contamination in platelet isolate was found to be less than 0.015%.…”

“…After incubation, cells were suspended in sheath fluid and subjected to flow cytometry (FACSCalibur, BD Biosciences). Data were analyzed by CELLQUEST PRO Software (Gurugram, India), as described previously [5].…”

“…dichloroacetate), and Shh and Notch signaling pathways (e.g. vismodegib and DAPT, respectively), that are widely tried as potent anticancer agents, have also been shown to possess strong anti-platelet/anti-thrombotic attributes [4,5]. Previously, it has been shown that Diabet, which is a polyherbal formulation, has potent anti-platelet activity [6].…”