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in plugging of the defect in vasculature. This response is termed ‘hemostasis’. A similar but more exaggerated response from platelets upon rupture of an atherosclerotic plaque leads to arterial thrombosis with potentially fatal consequences, such as myocardial infarction and ischemic stroke.3
Platelet responses to agonist stimulation, including adhesion, shape change, integrin activation, aggregation, exocytosis of granule contents and clot retraction are all
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energy intensive processes. Nevertheless, platelets
accomplish these responses while trapped within the rel- atively impervious boundaries of a thrombus, with restricted access to nutrients and oxygen. We hypothe- sized that it is imperative for activated platelets within a thrombotic milieu to adapt their energy metabolism to these challenges in order to sustain and hold the thrombus together. We employed a high-resolution bioenergetics screen to establish aerobic glycolysis and the consequent flux through the pentose phosphate pathway (PPP) as the metabolic signature of agonist-stimulated platelets. Using small-molecule modulators – dichloroacetate (DCA), diarylsulfonamide (DASA-58) (DASA), and dehy- droepiandrosterone sulphate (DHEA) – which target these pathways, we demonstrate that rewiring of metabolism is essential for activation of human platelets ex vivo as well as thrombus formation in a murine model in vivo. Considering that a substantial body of information on the safety and pharmacokinetics of DCA and DHEA in humans is already available,8-11 one can expect probable future translation of these drugs into clinical use as anti- platelet agents.
Methods
Ethical approval
The animal experiments were approved by the Central Animal Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University. All efforts were made to minimize the number of animals used, and their suffering. Peripheral venous blood sam- ples were collected from healthy participants after obtaining writ- ten informed consent, strictly in accordance with the recommen- dations and as approved by the ethical committee of the Institute of Medical Sciences, Banaras Hindu University.
Platelet preparation and mitochondrial respirometry
Platelets were isolated from fresh human blood by differential centrifugation, as already described.12 Mitochondrial respiration was measured using a high-resolution respirometer (Oxygraph- 2k, Oroboros Instruments) as previously described.12 Details are provided in the Online Supplement.
Measurements of glucose, lactate and NAPDH
Rates of glucose uptake and lactate secretion in thrombin (0.5 U/mL)-stimulated platelets were determined using an YSI 2900D Multiparameter Bioanalytical System (YSI Life Sciences), which uses immobilized enzyme electrodes and electrochemistry-based biosensing.13
The ratio of NADPH to total NADP(H) in washed human platelets was determined using a NADP/NADPH assay kit (Sigma). For this, NADP was decomposed at 60° C for 30 min fol- lowed by estimation of the amount of NADPH. To estimate total NADP(H), extract was directly processed for color development following the instructions of the kit’s manufacturer and absorbance was recorded at 450 nm.
Platelet aggregation and granule secretion
Platelets suspended in buffer B or in platelet-rich plasma were stirred (1200 rpm) at 37°C in an optical lumi-aggregometer (Chrono-log model 700-2) for 1 min. Platelet aggregation induced with thrombin or collagen was recorded as percent change in light transmittance where 100% reflects transmittance through a blank (buffer or platelet-poor plasma).12
haematologica | 2019; 104(4)
Aerobic glycolysis fuels platelet activation
Release of adenine nucleotides from platelet dense granules was measured with the Chronolume reagent (stock concentration, 0.2 μM luciferase/luciferin). Luminescence generated was monitored using the lumi-aggregometer in parallel with the platelet aggrega- tionmeasurement.12
Secretion from platelet α-granules was evaluated by quantifying surface expression of P-selectin as described previously.12
Details are provided in the Online Supplement.
Platelet surface integrin activation and GLUT3 expression
Platelet surface integrin activation was analyzed as described previously.12
In order to measure GLUT3 surface expression, platelets were fixed with an equal volume of 4% paraformaldehyde, washed and blocked with 2% bovine serum albumin for 40 min before being incubated with a GLUT3-specific antibody for 2 h, and then washed and stained with Alexa Fluor 488-conjugated goat anti- mouse antibody for 1 h. Cells were analyzed by flow cytometry as described above.
Further details are provided in the Online Supplement.
Measurement of intracellular reactive oxygen species
The intracellular levels of reactive oxygen species (ROS) were determined using a redox-sensitive cell-permeable dye, H2DCF- DA as described elsewhere.14 Details are available in the Online Supplement.
Immunoblotting
Immunoblotting experiments were performed as described pre- viously.12 Details are given in the Online Supplement.
Studies of coagulant activity
Externalization of phosphatidylserine, a measure of surface pro- coagulant activity, in stimulated platelets was assessed from annexin V binding, as described previously.15
Animal experiments
Intravital microscopy of ferric chloride-induced thrombosis of mice mesenteric arterioles was performed as described elsewhere,16 with some modifications.
Tail bleeding time experiments were carried out as described previously,17 with minor modifications.
Pulmonary embolism was induced by collagen-epinephrine in 12- to 20-week old Swiss albino mice of either sex as described previously.12
The methods are described in detail in the Online Supplement. Results
Platelets exhibit a higher rate of aerobic glycolysis upon agonist stimulation
We measured oxygen flux in platelets (suspended in Tyrode modified buffer under stirring) using a Clark amperometric electrode at high resolution (sampling at 2 s intervals). This system in a cuvette format closely resem- bles the platelet aggregometry of Born. When we exposed
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