Adenosine diphosphate (adp)

Oxygen consumption was measured polarographically in water-jacketed respiration chambers maintained at 37 °C (Oxygraph, Hanstech Instruments, King’s Lynn, UK) as previously detailed [56 (link)]. Following daily calibration with Buffer Z [50 mM K-MES (Sigma M0895), 30 mM KCl (Sigma P4504), 10 mM K₂HPO₄ (Fisher, Hampton, NH, USA, P290), 1 mM EGTA (Sigma E4378), 5 mM MgCl₂-6H₂O (Sigma M2670), 0.005 mM Glutamate (Sigma G8415), 0.002 mM Malate (Sigma M6773), and 0.05% BSA (Sigma A6003), pH to 7.1 at 4 °C] and Na₂SO₄, 10 µL of isolated SS or IMF mitochondria was independently resuspended in 965 µL of buffer Z, containing 20 mM creatine (Sigma C0708) warmed to 37 °C within the oxygraph chamber. Mitochondria were allowed to equilibrate before the addition of 10 µL Malate (272.21 mM; Sigma M7397) and 10 µL pyruvate (500 mM; Sigma P5280) followed by the addition of 5 µL of ADP (48.09 mM; MP Biomedicals, Irvine, CA, USA, 150259) to determine state 3 and state 4 respiration. Respiratory control ratio (RCR) was designated as state 3 respiration divided by state 4 respiration. Values were normalized post hoc to protein content by the Bradford method.

One hour before flow experiments, platelets in whole blood were stained with anti-CD41/61 PE (BioLegend #359806; 1 µg/mL whole blood). To activate platelets and induce expression of P-selectin concurrently with the platelet staining step, 20 µM adenosine diphosphate (ADP, MP Biomedical) was added to aliquots of whole blood 1 h before flow experiments. HUVEC on glass coverslips were manually scored using a scalpel to expose the underlying extracellular matrix³⁸ . The coverslip was then attached to a parallel plate flow chamber (PPFC, Glycotech) fitted with a silicone gasket (2 cm × 0.25 cm × 127 µm in height). Immediately before blood was perfused over the coverslip, particles were added to whole blood at the specified final concentration in number of particles per mL of whole blood. A syringe was attached to the chamber outlet, and a syringe pump was utilized to control whole blood over the coverslip, with the direction of blood flow perpendicular to the scalpel scores. The wall shear rate was held constant at 1000 s^−1,30. After 5 min of laminar blood flow, PBS buffer with calcium and magnesium and 1% BSA (pH 7.4) was perfused over the coverslip for 2 min to flush the chamber. After rinsing, a digital camera was utilized to take 10 images of endothelium-bound leukocytes and 10 fluorescent images of bound platelets utilizing a TRITC filter.

Firefly luciferase aggregates were formed by incubating luciferase (50 µM) in LRB (pH=7.4) plus 8M urea at 30°C for 30 min. The luciferase was then rapidly diluted 100-fold into LRB, snap frozen, and stored at −80°C until use. Hsp104 and Skd3 variants (1 µM monomer, unless otherwise indicated) were incubated with 50 nM aggregated firefly luciferase in the presence or absence of Hsc70 and Hdj2 (0.167 µM each) in LRB plus 5 mM ATP plus an ATP regeneration system (ARS; 1 mM creatine phosphate and 0.25 µM creatine kinase) at 37°C for 90 min (unless otherwise indicated). The nucleotide-dependence of Skd3 disaggregation activity was tested in the presence of ATP (Sigma), AMP-PNP (Roche), ATPγS (Roche), or ADP (MP Biomedicals) for 30 min at 37°C without ARS. Recovered luminescence was monitored in Nunc 96 Well Optical plates using a Tecan Infinite M1000 plate reader (DeSantis et al., 2012 (link)).