The number of mitochondria in platelets within a clot is similar to that in platelets activated by spreading on a glass surface. (A) Illustration of a clot retraction assay around 2 holders instead of the classical clot retraction assay used in Figures 5 and 7. The strong clot adhesion to both holders allows clot retraction but prevents clot collapse even after prolonged incubation times, thus allowing to visualize individual platelets within the retracted clot as shown in panels B and C. (B) PRP was adjusted to a platelet concentration of 1 × 10⁸/mL with PBS and then preincubated for 30 minutes at RT with mitoTracker (red). Clot formation was then initiated by the addition of thrombin to a final concentration of 2.5 U/mL, and clots were incubated for 15 minutes at 37°C and then fixed, embedded in gelatin, and flash frozen. Transversal clot slices of 14 μm thickness were processed for expansion and then stained using NHS-ester (cyan) to detect the entire platelet dimensions. Shown are maximal intensity projections of confocal image stacks of 3 representative examples, scale bar 10 μm, corresponding to 2.5 μm after correction for expansion. (C) Same conditions as in panel A, but retraction was allowed to proceed for 60 instead of 15 minutes at 37°C. (D) Comparison of the number of mitochondria/platelet in resting and spread platelets (as shown in Figure 1G), with the number of mitochondria per platelet in blood clots after 15 minutes (n = 23; 2 different donors) and 60 minutes (n = 29; 2 different donors) of retraction. Data comparison was performed using 1-way analysis of variance.