Fig. 3: Cholesterol ester determines the ability of neutrophil-derived migrasomes to adsorb coagulation factors.
From: Neutrophil-derived migrasomes are an essential part of the coagulation system
a, Plot showing the differential abundance of lipids in plasma membranes of neutrophils (neu-membrane), erythrocytes, platelets and neutrophil-derived migrasomes. Total lipids were extracted from plasma membranes of neutrophils, erythrocytes, platelets and neutrophil-derived migrasomes and subjected to quantitative lipidomic analysis. The plot shows the ratio of each lipid to total lipids. n = 3 repeats. The data are presented as means ± s.e.m. b, Heat map of the distribution of lipids in plasma membranes of neutrophils, erythrocytes, platelets and neutrophil-derived migrasomes. c, Plot showing the differential abundance of cholesterol in plasma membranes of neutrophils, erythrocytes, platelets and neutrophil-derived migrasomes. n = 3 repeats. The data are presented as means ± s.e.m. d, Dragonfly confocal images of liposomes labelled with PE–Rhod (purple). Lipo-Mig contained PC, PE, PS, SM and ChE in similar ratios to neutrophil-derived migrasomes. Lipo-PLT was the same as Lipo-Mig except the ChE was replaced with cholesterol to mimic platelets. Lipo-Ctrl only contained PC, PE, PS and SM. Scale bars, 10 μm. e, Liposomes were incubated with s-plasma (37 °C for 1 h), then centrifuged down for western blot analysis using antibodies against coagulation factors. Numbers of liposomes (PE–Rhod+ vesicles) were counted by flow cytometry and the same number of each type of liposome was incubated with s-plasma. f, Dragonfly confocal images of liposomes labelled with PE–Rhod (purple). Lipo-Mem contained PC, PE, PS and SM in similar ratios to the plasma membranes of neutrophils. ChE was added to the composition of Lipo-Mem to make Lipo-MemC. Scale bars, 10 μm. g, Liposomes were incubated with s-plasma (37 °C for 1 h), then centrifuged down for western blot analysis using antibodies against coagulation factors. The numbers of liposomes (PE–Rhod+ vesicles) were counted by flow cytometry and the same number of liposomes from each sample was incubated with s-plasma. The western blot grey values were quantified using ImageJ. Source numerical data and unprocessed blots are available in Source Data Fig. 3. Cer, ceramide; DG, diglyceride; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TG, triglyceride.
