Rui Yamaguchi1, Arisa Sakamoto2, Reona Yamaguchi3, Misa Haraguchi2, Shinji Narahara2, Hiroyuki Sugiuchi2, Takahiko Katoh4, Yasuo Yamaguchi5. 1. Department of Public Health, Faculty of Life Sciences, Kumamoto University School of Medicine, Kumamoto, Japan; Graduate School of Medical Science, Kumamoto Health Science University, Kumamoto, Japan. 2. Graduate School of Medical Science, Kumamoto Health Science University, Kumamoto, Japan. 3. Department of Neuroscience, Graduate School of Medicine and Faculty of Medicine, Kyoto University, Kyoto, Japan. 4. Department of Public Health, Faculty of Life Sciences, Kumamoto University School of Medicine, Kumamoto, Japan. 5. Graduate School of Medical Science, Kumamoto Health Science University, Kumamoto, Japan. Electronic address: yamaguti@kumamoto-hsu.ac.jp.
Abstract
BACKGROUND: Plasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. MATERIALS AND METHODS: The effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. RESULTS: Exposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-β1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. CONCLUSIONS: Di-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-β1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.
BACKGROUND: Plasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. MATERIALS AND METHODS: The effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. RESULTS: Exposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-β1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. CONCLUSIONS:Di-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-β1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.