4-Hydroxy-2-Nonenal (HNE), a Product of Lipid Peroxidation, Induces Tissue Factor Decryption By Two Independent Mechanisms

Oxidative stress plays a major role in the pathogenesis of various diseases, including diseases associated with coagulopathy, such as atherosclerosis, sepsis, and diabetes. We showed recently that 4-hydroxy-2-nonenal (HNE), the most abundant and stable unsaturated aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids, increases the procoagulant activity of cell surface tissue factor (TF) in monocytic cells (Vatsyayan et al., 2013 Arterioscler Thromb Vasc Biol 33:1601-1611). These studies revealed that HNE generates ROS and decrypts TF through p38 MAPK activation-dependent externalization of phosphatidylserine (PS) on the outer cell surface. However, at present, the link between HNE-induced ROS generation and p38 MAPK activation in the externalization of PS and TF activation is unclear. The present study is carried out to elucidate potential mechanisms involved in HNE-induced TF decryption. The colocalization of fluorescence staining signals for ROS generation and mitochondrial (correlation coefficient 0.96 ± 0.005) indicated that mitochondria are the major source of HNE-induced ROS generation in THP-1 cells. Next, to delineate the role of mitochondrial electron transport chain in HNE-induced ROS generation and TF decryption, THP-1 cells were pretreated with the specific inhibitors of the electron transport chain complexes I to V. The inhibitors of complex III and complex V, i.e., antimycin and oligomycin, respectively, inhibited the HNE-induced ROS generation suggesting that these complexes of the mitochondria are altered under HNE-induced oxidative stress to generate ROS. Pretreatment of the cells with antimycin and oligomycin significantly, but not fully, attenuated the HNE-induced TF decryption. Antimycin and oligomycin also inhibited the HNE-induced prothrombinase activity, indicating that HNE-induced ROS generation contributes to PS exposure that is crucial for TF decryption. However, inhibition of ROS generation by these inhibitors did not block the HNE-induced p38 MAPK activation. Since thioredoxin (Trx) and thioredoxin reductase (TrxR) were shown to control TF activity and HNE can inhibit the activities of Trx and TrxR, directly or through the generation of ROS, we next investigated the role of Trx-TrxR in the HNE-induced TF decryption. HNE inhibited TrxR activity in a dose- and time- dependent manner. Pretreatment of THP-1 cells with antioxidant N-acetylcysteine (NAC)prevented the HNE-induced inhibition of TrxR. Antimycin and oligomycin, the inhibitors of mitochondrial respiratory chain complexes that prevented HNE-induced ROS generation, failed to block the HNE-induced TrxR inhibition. To investigate whether HNE-induced inhibition of Trx or TrxR is responsible for HNE-induced TF decryption, we determined whether inhibition of the activity of Trx or TrxR by pharmacological inhibitors would also activate TF. Treatment of THP-1 cells with either curcumin (TrxR inhibitor) or PX-12 (Trx inhibitor) markedly increased TF activity. These inhibitors also induced p38 MAPK activation and increased the exposure of PS onto the cell surface. The reduced form of Trx binds ASK1 and oxidation/inactivation of Trx disrupts the complex, enabling ASK1 activation that could lead to activation of p38 MAPK activation. Therefore, we investigated whether HNE induces the activation of ASK1 under our experimental conditions and the role of ASK1 in the HNE-induced p38 MAPK activation-dependent TF decryption. We found no evidence for the activation of ASK1 in THP-1 cells following HNE treatment. Furthermore, the specific inhibitors of ASK1 failed to block the HNE-induced TF decryption. These data indicate that HNE inhibition of Trx-TrxR leads to p38 MAPK activation-dependent TF decryption, independent of ASK1 activation. In additional experiments, we found that blockade of thiol groups by phenylarsine oxide (PAO) attenuated HNE-induced PS exposure and TF decryption. In summary, our present data suggest that HNE induces TF decryption by two separate pathways - one is ROS-dependent but independent of p38 MAPK activation and the second is via Trx-TrxR- and p38 MAPK activation-dependent. Both the mechanisms result in the exposure of PS at the cell surface that contributes to TF decryption. Blockade of thiol groups by PAO appears to inhibit HNE-induced TF decryption by blocking the upstream signaling molecules that regulate HNE-induced PS exposure.