Adenosine diphosphate (adp)

Human PRP (450 μl, 250 × 10⁹ platelets per litre) was incubated for 2 min at 37 °C in a two-channel aggregometer (Chrono-log corporation) before platelets were stimulated with ADP (5 μM, Sigma-Aldrich). ADP was preincubated with PPX (0–10 μg ml⁻¹) or apyrase (0.1 U ml⁻¹, Sigma-Aldrich) for 30 min at 37 °C. Change in light transmission during constant stirring of the samples was recorded for 6 min and normalized to light transmission of PPP.

Purified TcPINK1 at 1 μM was combined with 100 μM ¹³C-ATP (Sigma-Aldrich), 100 μM compound of interest, 500 μM MgSO₄, 5% D₂O (Sigma-Aldrich), and 5% DMSO in in 300 mM NaCl, 50 mM Tris–HCl pH 8, 2 mM DTT. For the ADP control, knowing that the natural abundance of ¹³C is 1.1%, 1 μM ¹⁵N-TcPINK1 was combined with 10 mM ADP (Sigma-Aldrich) in the same buffer to re-create a ¹³C signal equivalent to 100 μM ¹³C-ADP. Standard 2D ¹H-¹³C sensitivity-enhanced HSQC NMR spectra were acquired at 298 K every 5 min for 1.5 h on a 600 MHz Bruker Avance spectrometer equipped with a triple resonance (¹⁵N/¹³C/¹H) cryoprobe. The spectra were acquired with a carrier frequency of 600.3328216 MHz (4.7 ppm) for F2 (¹H) and 150.9697038 MHz (110 ppm) for F1 (¹³C); a sweep width of 13.0136 ppm for F2 (¹H) and 36 ppm for F1 (¹³C); 42 increments and 2 scans. Spectra were processed using TopSpin 4.0.6 (Bruker).

Standard laboratory reagents, except ADP, were obtained from Sigma (St. Louis, MO, USA). ADP was purchased from Merck (Darmstadt, Germany). BCECF/AM and Amplex UltraRed were obtained from TermoFisher Scientific (Waltham, MA, USA).

Oxygen consumption was measured polarographically using a respirometry system (System S 200A; Strathkelvin Instruments, Glasgow, Scotland). Respiration experiments using pyruvate or succinate at pH 7.15 and without Ca²⁺ were initially conducted to determine the viability of mitochondria for the rest of the experiments. Respiration was initiated by adding 10 mM complex I substrate Na⁺ pyruvate or the complex II substrate Na⁺ succinate. State 3 respiration was measured after adding 250 μM ADP (Sigma), and state 4 respiration was measured after complete phosphorylation of the added ADP. The respiratory control index (RCI) was calculated as the ratio of the rate of state 3 to state 4 respiration. Only mitochondria with an RCI of 10 or above with pyruvate or an RCI of 3 or above with succinate were used in the experiments. To assess the effects of pH and extra-matrix (e) [Ca²⁺]_e on O₂ consumption (respiration), we added either H₂O (control) or CaCl₂ for a final concentration of 150 μM to the mitochondrial suspension at three pHs (7.15, 6.9 or 6.5) before adding substrates.

All laboratory chemicals, except for ADP, were purchased from Sigma (St. Louis, MO, USA). ADP was purchased from Merck (Darmstadt, Germany). Experiments were carried out in a standard assay medium containing (in mM): 125 KCl, 20 Hepes, 2 K₂HPO₄, 1 MgCl₂, 0.1 EGTA, pH 7.0 (KOH)), supplemented with 0.025% fatty-acid-free bovine serum albumin (BSA).