Energy production in platelets during clot retraction. (A) Clot retraction in the presence of glycolysis and oxphos inhibitors. PRP was adjusted to a platelet concentration of 1 × 108/mL with PBS and then preincubated for 20 minutes at RT with vehicle as control or the glycolysis inhibitor 2-deoxy-D-glucose (2-DG, 30 mM, supplemented with 2 mM pyruvate) or a combination of mitochondrial inhibitors, 4 μM oligomycin, 5 μM rotenone, 15 μM antimycin (ORA) as well as with both the glycolysis and oxphos inhibitors (2-DG + pyruvate/ORA). Clot formation was then initiated by addition of thrombin to a final concentration of 2.5 U/mL and clots were kept at RT. Shown are triplicate samples, and photos were taken at different time points during clot retraction as indicated. The retraction assay was repeated twice with the same samples. In the upper panels, clot formation was induced successively from left to right (ie, beginning with control conditions) and in the bottom panels, from right to left (ie, beginning with 2-DG+pyruvate/ORA conditions). This allows to compensate for the delay in the onset of clot induction. Images are representative of 4 independent experiments using platelets from 4 different donors. (B) The extent of clot retraction was quantified as percent of the initial clot volume using the images shown in panel A. Results are expressed as means ± standard deviations. (C) Quantification of lactate release during clot retraction. The concentration of lactate was determined in the serum extruded from triplicates of control clots from 4 different donors (D1-D4) after 30 minutes of retraction and compared with parallel triplicate samples of resting platelets (“Methods”). Data comparison was done using paired 2-tailed t test.
