Tissue Factor Decryption By Oxidative Stress-Induced Lipid Peroxidation: Potential Mechanisms
Authors: Ansari Shabbir , Pendurthi Usha R , Rao L. Vijaya Mohan 
Published: October 2019

Abstract
Cellular lipid peroxidation is known to contribute to the initiation and propagation of atherothrombosis. Recently, we showed that 4-hydroxynonenal (HNE), one of the most abundant reactive aldehydes generated from the oxidation of ω-6 fatty acids, enhanced tissue factor (TF) activity on monocytic cells by externalizing phosphatidylserine (PS) in p38 MAPK activation-dependent manner. However, at present, the link between HNE-induced oxidative stress and p38 MAPK activation and the relation of p38 MAPK activation to PS externalization is not fully known. In the present study, we investigated the role of mitochondrial electron transport chain and reactive oxygen species (ROS) generation in HNE-mediated TF decryption. In addition, we also investigated the thioredoxin reductase-thioredoxin-ASK-1 axis in regulating p38 MAPK activation and PS externalization in decrypting TF. To elucidate potential mechanisms of HNE-induced TF decryption, we first determined the role of specific mitochondrial electron transport chain complexes in regulating TF activity. Since THP-1 cells used in the study had a measurable basal TF activity, they were not further treated with LPS or other agonists to induce TF synthesis. The electron transport chain in these cells was disrupted by specific inhibitors and cell surface TF activity was measured by factor X activation assay. Inhibition of complex I and complex IV by rotenone and sodium azide, respectively, enhanced the procoagulant activity of basal level TF. However, the inhibition of complex I and IV had no significant effect on the HNE-mediated increase in TF activity. Interestingly, inhibition of ATP synthase/complex V by oligomycin significantly inhibited the HNE-mediated enhanced TF activity, indicating that HNE-mediated TF decryption may involve the generation of ATP. In agreement with earlier published studies in monocytes/macrophages, stimulation of THP-1 cells with ATP increased cell surface TF activity. However, at present, it is yet to be shown that HNE treatment actually increased the production of ATP and that this ATP is responsible for the HNE-mediated TF decryption. It is also possible that HNE, either through a generation of ROS in mitochondria or directly, can affect the activity of thioredoxin either by intracellular signaling or by directly forming an adduct with it. Therefore, we next investigated the effect of HNE on the activity of thioredoxin reductase, the enzyme known to regulate thioredoxin activity in the cell. Our data showed that HNE treatment inhibited the activity of thioredoxin reductase in a concentration-dependent manner, 40 µM of HNE inhibiting 50% of the activity and a complete inhibition at 80µM of HNE. To further determine the downstream signaling cascade involved in the PS externalization and TF decryption on exposure to HNE, we analyzed the effect of HNE on the activation of MKK3 and MKK6, the protein kinases known to activate p38 MAPK and the downstream signaling activator of thioredoxin/thioredoxin reductase pathway. HNE treatment increased the phosphorylation of MKK3 and MKK6 in a time-dependent manner. In summary, our data suggest that HNE may mediate TF decryption via modulation of thioredoxin/thioredoxin reductase system, which results in activation of MKK3/MKK6, which in turn activates p38 MAPK that is responsible for PS externalization. The study highlights the potential role of oxidative stress in regulating TF activity in thrombotic disorders and provides a mechanistic link between disorders associated with cellular oxidative stress and thrombosis.

Disclosures
No relevant conflicts of interest to declare.