Objective: Protamine sulfate is widely used albeit its pharmacological effects are not fully
understood and applications are often accompanied by unwanted side effects. Here,
we show the effect of protamine sulfate (PS) on mitochondrial bioenergetics profile,
and the resulting mitochondrial reactive oxygen species (ROS) production.
Methods: Polarographic measurements were performed in parallel to membrane potential and ROS
measurements by FACS analyzer using tetramethylrhodamine ethyl ester (TMRE) and MitoSOX
fluorescent dyes, respectively.
Results: Protamine sulfate inhibited intact rat heart mitochondrial respiration stimulated
by ADP to 76% (p < 0.001) from the baseline of 51.6 ± 6.9 to 12.4 ± 2.3 nmol O2 min−1·mL−1. Same effect was found when respiration was priorly inhibited by antimycin A (101.0 ± 8.9
vs. 38.0 ± 9.9 nmol O2 min−1·mL−1, p < 0.001) and stimulated by the addition of substrates, ascorbate and tetramethyl
phenylene diamine (TMPD) for cytochrome oxidase (CytOx), suggesting that PS exerted
its effect through inhibition of CytOx activity. Furthermore, the inhibition of mitochondrial
respiration by PS occurred in a concentration-dependent manner and accompanied by
hyperpolarization of the mitochondrial membrane potential (Δψm), that is, 17.4% and further 4.8% increase to control, respectively. This effect
was associated with a strong consequent increase in the production of reactive oxygen
species, that is, 86.4 and 89.5% compared with control, respectively.
Conclusion: We propose, the 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 findings appearing after administration
of the drug.
