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 O 2 ×min 21 ×ml 21 . The same effect was found when respiration was inhibited by antimycin A (101.0 6 8.9 vs. 38.0 6 9.9 nmol O 2 ×min 21 ×ml 21 , 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 (Dc m ), 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.

Stress-mediated generation of deleterious ROS in healthy individuals - role of cytochrome c oxidase

“…Transient high ΔΨ m values have also been described by synthetic cannabinoids in human proximal tubule cells [80], by statins (lovastatin and simvastatin), which increased the ΔΨ m in HepG2 and Huh7 human hepatocarcinoma cells and HCC4006 human lung adenocarcinoma cells [81], and by honokiol, which induced in bladder cancer cells and increase of ΔΨ m and ROS formation, and at high doses apoptotic cell death [82]. Hyperpolarization of ΔΨ m was furthermore shown with protamine sulfate [83] and graphene oxide (a marker for air pollution) [84].…”

H1 and H2 antihistamines pretreatment for attenuation of protamine reactions after cardiopulmonary bypass: a randomized-controlled study

“…In a small study among 22 patients after CPB, protamine neutralization of heparin resulted in gradual drops in systemic vascular resistance and blood pressure eventually decreased once cardiac output was over-compensated (2). Lastly, a recent in vitro study using isolated mitochondria from rat hearts demonstrated that protamine caused an excessive increase in mitochondrial reactive oxygen species (ROS), resulting in mitochondrial dysfunction (37). The authors proposed that excessive formation of ROS might be the pathogenesis of the cardiovascular (both systemic hypotension and pulmonary hypertension) side effects of protamine.…”

Effect of Lidocaine Pre-Treatment on Protamine-Induced Pulmonary Vascular Reaction During the Repair of Congenital Heart Disease

“… 11 This can directly cause smooth muscle contraction, and the accumulation of platelets and leucocytes activation in the lungs, respectively, 12 , 13 and subsequently induce a large number of releases of proteolytic enzymes that could directly cause lung injury and the subsequent increase in reactive oxygen species production, which contributes to the development of lipid peroxidation. 14 , 15 As a result, pulmonary vasoconstriction and potential lung injury would likely be mediated through complement-mediated C 5a -induced TXA 2 generation and might thereby result in the development of various cardiovascular adverse effects, such as pulmonary artery pressure elevation, right ventricular/right atrial pressure elevation, and systemic hypotension. Therefore, the regimen of treatment is to reduce pulmonary artery pressure and inhibit inflammatory responses, in order to reduce the generation of TXA 2 , which may be useful.…”