Purpose: Metabolic stress, characterized by hyperglycemia and dyslipidemia, constitutes a major risk factor for cardiovascular disease. This pathological condition triggers platelet hyperactivation through oxidative stress mechanisms, promoting life-threatening atherothrombotic complications. Platelet activation under metabolic stress is primarily mediated by excessive reactive oxygen species (ROS) accumulation. This study investigated the therapeutic potential of novel thioredoxin mimetic peptides (TMP) as antioxidant agents for attenuating metabolic stress-induced platelet dysfunction and preventing cardiovascular thrombotic complications.
Methods: Human platelets were isolated from healthy donors using standard density gradient centrifugation techniques. Platelets were exposed to oxidized low-density lipoprotein (oxLDL) alone or combined with hyperglycemic conditions representing metabolic stress. Intracellular oxidative stress levels were quantified using CellROX Green reagent and comprehensive flow cytometry analysis. Platelet activation status was assessed through quantitative surface expression analysis of specific activation markers including CD62P (P-selectin), CD148 (DEP-1), and CD31 using FACS. NADPH oxidase inhibition was achieved using diphenyleneiodonium chloride and VAS2870 inhibitors. Thioredoxin reductase function was inhibited using Auranofin to evaluate endogenous antioxidant system capacity. TMP were employed to restore cellular redox homeostasis and evaluate therapeutic efficacy. Functional platelet-endothelium interactions were studied by static adhesion assays on TNF-α-activated human umbilical vein endothelial cells.
Results: Metabolic stress conditions induced significant platelet hyperactivation compared to hyperlipidemic conditions alone, evidenced by robust upregulation of CD62P, CD148, and CD31 surface expression levels. Metabolic stress substantially enhanced intracellular ROS accumulation in platelets. Unexpectedly, pharmacological inhibition of NADPH oxidases using DPI and VAS2870 failed to reduce metabolic stress-induced ROS generation or platelet activation, strongly suggesting involvement of alternative oxidative pathways. Thioredoxin reductase inhibition with Auranofin markedly potentiated both ROS accumulation and platelet activation responses, confirming the critical protective role of the endogenous thioredoxin system. Treatment with thioredoxin mimetic peptides (TMP) completely reversed Auranofin-induced oxidative stress and platelet hyperactivation. Most importantly, TMP treatment significantly attenuated metabolic stress-induced ROS accumulation and platelet activation markers. Additionally, TMP substantially reduced metabolic stress-induced platelet adhesion to activated endothelial cells, indicating protective effects on platelet-endothelial interactions.
Conclusions: This study establishes thioredoxin as a crucial regulator of platelet activation under metabolic stress conditions. TMP effectively reverse the key pathological steps in atherothrombosis development, including platelet hyperactivation and enhanced endothelial adhesion. These findings identify TMP as a promising therapeutic strategy for preventing thrombotic complications and subsequent cardiovascular disease in metabolic syndrome patients.
