To target glutamate signaling as an anti-platelets therapeutic strategy.

Platelets have considerable presence of glutamate in their dense granules coexistent with thrombogenic mediators like ADP, ATP, serotonin and Ca²⁺, all of whom are exocytosed upon platelet activation. Secreted ADP, ATP, and serotonin bind to respective receptors on platelet membrane and establish positive feedback loops to potentiate platelet stimulation. As platelets express glutamate receptors and serum glutamate has been reported to rises locally upto 400 µM following platelet activation, it is pertinent to ask whether glutamate can, too, behave in autocrine/paracrine manner to potentiate platelets activation.

Platelets were isolated from fresh human blood by differential centrifugation. An informed consent was taken from healthy volunteers, and the methods were carried with the guiding principles endorsed by the ºÃÉ«ÏÈÉú. The study methodologies confirmed to the standards set by the Declaration of Helsinki. Cytosolic calcium was measured using fura2/AM, released extracellular vesicles (EVs) was characterized by nanoparticle tracking analysis, immunoblotting was performed to study expression of protein using specific antibodies, Rho-GTP pull down assay was carried to evaluate charged-RhoA protein, MitoTracker Red was used to measure mitochondrial membrane potential, ROS was determined by H₂DCF-DA dye and mitochondrial respiration was measured using Oxygraph.

Glutamate induced synthesis of thrombogenic peptides, plasminogen activator inhibitor-1 and hypoxia-inducible factor-2α in platelets. It stimulated cytosolic calcium entry, up regulated RhoA-ROCK-myosin light chain/MLC phosphatase axis, and elicited shedding of EVs from platelets. Glutamate also raised mitochondrial transmembrane potential associated with generation of reactive oxygen species and activation of AMP-activated protein kinase (AMPK) in platelets.Inhibition was found in above results with CNQX, an AMPA glutamate receptor inhibitor.