Carnitine palmitoyltransferase-1 (CPT1) and PDH activities.

CPT1 and PDH activities were determined in isolated platelets spectrophotometrically based on ref. 53 and utilizing a PDH activity kit (Pyruvate Dehydrogenase Enzyme Activity Microplate Assay Kit, catalog ab109902, Abcam), respectively. CPT1 activity of isolated platelets was determined by spectrophotometrically monitoring the generation of CoA-SH from 100 μM palmitoyl-CoA in the presence of 5 mM L-carnitine and 200 μM 5,5’-dinitro-bis-(2-nitrobenzoic acid) (DTNB) at an absorbance of 412 nm. PDH activity in platelets was quantified using a microplate assay kit according to manufacturer’s instructions (Abcam). Platelet proteins were solubilized by addition of detergent (1/10 volume) and then added to anti-PDH antibody–coated wells of a microplate to facilitate immunocapture of active PDH. PDH activity was then determined spectrophotometrically by monitoring the reduction of NAD+ to NADH, coupled to the reduction of a reporter dye at absorbance 450 nm.