Diazeniumdiolation of protamine sulfate reverses mitogenic effects on smooth muscle cells and fibroblasts

• Published in: Free radical biology & medicine 2015-05, Vol.82, p.13-21
• Main Authors: van Lith, Robert, Yang, Jian, Ameer, Guillermo A.
• Format: Article
• Language: English
• Subjects: Anticoagulants - chemistry; Anticoagulants - pharmacology; Antioxidants - pharmacology; Aorta - cytology; Azo Compounds - chemical synthesis; Azo Compounds - chemistry; Blood Coagulation - drug effects; Cell Line; Cell Proliferation - drug effects; Cell Survival - drug effects; Diazeniumdiolate; Fibroblasts - metabolism; Free radicals; Human Umbilical Vein Endothelial Cells; Humans; Muscle, Smooth, Vascular - metabolism; Myocytes, Smooth Muscle - metabolism; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Donors - chemistry; Oxidative Stress; Peptide; Prodrugs - chemical synthesis; Prodrugs - chemistry; Protamine sulfate; Protamines - chemistry
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.01.022

After vascular interventions, endothelial cells are typically injured or lacking, resulting in decreased NO synthesis to maintain vascular health. Moreover, inflammation as a result of the tissue injury and/or the presence of an implanted foreign polymer such as a vascular graft causes excessive generation of reactive oxygen species (ROS) (e.g., superoxide), which can react with NO. The combination of the above creates a general decline in NO bioavailability, as well as oxidative stress due to less available NO to scavenge ROS. Localized NO delivery is an attractive solution to alleviate these issues; however, NO donors typically exhibit unpredictable NO payload release when using nitrosothiols or the risk of nitrosamine formation for synthetic diazeniumdiolates. The objective of this study was therefore to synthesize an NO donor from a biological peptide that could revert to its native form upon NO release. To this effect, protamine sulfate (PS), an FDA-approved peptide with reported vasodilator and anticoagulant properties, was diazeniumdiolated to form PS/NO. PS/NO showed diazeniumdiolate-characteristic UV peaks and NO release in physiological solutions and was capable of scavenging radicals to decrease oxidative stress. Furthermore, PS/NO selectively inhibits the proliferation of smooth muscle cells and adventitial fibroblasts, thereby reversing reported mitogenic properties of PS. Endothelial cell growth, on the other hand, was promoted by PS/NO. Finally, PS retained its anticoagulant properties upon diazeniumdiolation at clinically relevant concentrations. In conclusion, we have synthesized an NO prodrug from a biological peptide, PS/NO, that selectively inhibits proliferation of smooth muscle cells and fibroblasts, retains anticoagulant properties, and reverts back to its native PS form upon NO payload release. •We converted a biological peptide, protamine sulfate, into a diazeniumdiolate NO donor, PS/NO.•PS/NO reverts back to native protamine sulfate upon NO release, minimizing toxicity risk.•Diazeniumdiolation of protamine sulfate reverses its mitogenic effects on vascular cells.•Diazeniumdiolated protamine sulfate can scavenge radicals and function as antioxidant.