Protamine sulfate induces mitochondrial hyperpolarization and a subsequent increase in reactive oxygen species production

Abstract

Protamine sulfate (PS) is widely used in heart surgery as an antidote for heparin, albeit its pharmacological effects are not fully understood and applications are often accompanied by unwanted side effects. Here we show the effect of PS on mitochondrial bioenergetics profile resulting in mitochondrial reactive oxygen species (ROS) production. Polarographic measurements were performed in parallel to membrane potential and ROS measurements by FACS analyzer using tetramethylrhodamine ethyl ester and MitoSOX fluorescent dyes, respectively. PS inhibited intact rat heart mitochondrial respiration (stimulated by ADP) to 76% (P, 0.001) from the baseline of 51.6 6 6.9 to 12.4 6 2.3 nmol O2×min21×ml21. The same effect was found when respiration was inhibited by antimycin A (101.0 6 8.9 vs. 38.0 6 9.9 nmol O2 ×min21×ml21, P, 0.001) and later stimulated by substrates of cytochrome oxidase (CytOx) i.e., ascorbate and tetramethyl phenylene diamine, suggesting that PS exerted its effect through inhibition of CytOx activity. Furthermore, the inhibition of mitochondrial respiration by PS was concentration dependent and accompanied by hyperpolarization of the mitochondrial membrane potential (Dcm), i.e., 18% increase at 50 mg/ml and an additional 3.3% increase at 250 mg/ml PS compared with control. This effect was associated with a strong consequent increase in the production of ROS, i.e., 85% and 88.6% compared with control respectively. We propose that this excessive increase in ROS concentrations results in mitochondrial dysfunction and thus might relate to the “protamine reaction,” contributing to the development of various cardiovascular adverse effects.