Involvement of NADPH oxidase in the shedding of tissue factor-bearing vesicles from human monocytic cells exposed to ice-cold temperature

Abstract

Recent rise in whole blood usage for traumatic hemorrhagic shock prompts reevaluation of leukocyte's impact on hemostatic function during cold storage. We investigated whether tissue factor (TF) production in human monocytic cells (THP-1) is influenced by cold storage or rewarming, employing mechanisms similar to apoptosis. We also explored the role of superoxide anion (·O₂⁻) generated from NADPH oxidase (NOX) in TF production. Methods: THP-1 cells incubated at 4°C for up to 24 h with/without test reagents were rewarmed at 37°C, and subject to flow cytometry. Cells were washed by centrifugation before the rewarming as required. TF in the supernatant of cells was also measured. Results: TF and the proportion of apoptotic cells increased during cold storage for up to 24 h. TF increased at 1–2 h after cell lavage following the cold storage, indicating functional shedding of TF-bearing vesicles, not by leakage through the cell membrane due to apoptotic cell damage. TF-bearing vesicles shed from THP-1 cells were distinct from apoptotic vesicles. SOD and catalase inhibited TF production during cold storage, but only SOD suppressed TF production after cell lavage. Western blot analysis confirmed the recruitment of p47^phox and p67^phox into the cell membrane during cold storage, indicating involvement of ·O₂⁻ from NOX in TF-bearing vesicle shedding. Conclusions: We found that extracellular addition of SOD successfully suppressed TF release from THP-1 cells exposed to cold, suggesting that ·O₂⁻ derived from NOX was involved in the TF release from human monocytic cells during both cold storage and rewarming.