α-Actinin-1 deficiency results in impaired mitochondrial bioenergetics. (A) Mitochondrial respiration in platelets from Actn1^f/f or PF4-Actn1^−/− mice stimulated with medium (Med) or thrombin (Thr; 0.1 U/mL) was evaluated via Seahorse tracings (n = 5 independent experiments per group). The oxygen consumption rate (OCR) was measured with sequential injections of Med or thrombin (0.1 U/mL), oligomycin (Oligo), trifluoromethoxy carbonyl cyanide phenylhydrazone, or rotenone/antimycin A (Rot/AA). The following critical parameters of mitochondrial function were calculated: (B) basal respiration, (C) maximal respiration, and (D) ATP-linked respiration. Representative OCR (E) and extracellular acidification rate (ECAR) (F) data for Actn1^f/f or PF4-Actn1^−/− platelets treated with Med or Thr (0.1 U/mL) according to the real-time ATP rate assay protocol (n = 5 independent experiments per group). Metabolic flux analysis showing the quantification of (G) mitochondrial ATP (mitoATP) production, (H) GP ATP (glycoATP) production, and (I) total ATP production. The results are presented as the means ± SDs. (J) Representative TEM images from 3 independent experiments of mitochondria in Actn1^f/f or PF4-Actn1^−/− MKs. (K) The mitochondrial membrane potential (MMP) in platelets was analyzed via flow cytometry. The data are presented as bar graphs (n = 11 mice per group). (L) Representative traces of changes in mitochondrial calcium content. Mitochondrial calcium mobilization in thrombin-stimulated (0.025, 0.5, and 0.1 U/mL) and collagen-stimulated (2, 4, and 8 μg/mL) platelets was examined by flow cytometry (n = 8 mice per group). (M) The levels of mitochondrial ROS in the presence or absence of thrombin (0.025, 0.5, and 0.1 U/mL) or collagen (2, 4, and 8 μg/mL) were evaluated (n = 8 mice per group). The data are presented as the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. MFI, mean fluorescence intensity; ns, not significant.