Figure 4.

Hypoxia-mimetics induced increases in HIF-2α expression, shedding of extracellular vesicles and a rise in intracellular Ca²⁺ in human platelets. (A) Western blot showing the expression of HIF-2α in platelets treated with either dimethyloxalylglycine (DMOG, 1 mM) or deferoxamine (DFO, 1 mM) for 15 min at 37°C under normoxia. (B) Corresponding densitometric analysis of HIF-2α normalized to β-actin (n=4). (C) Platelets were exposed to DMOG (1 mM) or DFO (1 mM) for 15 min at 37°C in normoxic conditions. Platelet-derived extracellular vesicles (PEV) were isolated and analyzed with a Nanoparticle Tracking Analyzer (n=3). (D) Fura-2-loaded platelets were pretreated for 5 min with either calcium (1 mM) or EGTA (1 mM) and then incubated with DMOG (1 mM) or DFO (1 mM) for 15 min at 37°C under normoxic conditions. Intracellular Ca²⁺ was measured. RP, resting platelets. (E) Corresponding bar chart showing the intracellular calcium levels (n=4). (F) Fura-2-loaded platelets were pretreated with DMOG or DFO before the addition of MnCl₂ (2 mM) after 260 sec and fluorescence was recorded (n=3). Data are represented as the mean ± standard error of mean of at least three different experiments. *P<0.05; **P<0.01; ****P<0.00005; #P<0.05; ##P<0.01, analyzed by the Student t test.