Figure 5.
Figure 5. 12/15-LO–mediated oxidation of membrane phospholipids initiates eosinophil-mediated thrombin formation. (A) Representative LC/MS/MS analysis of different 12-HETE-PE oxidation species in lipid extracts of in vitro–generated mouse eosinophils. Cps, counts per second. (B) Quantitative RT-PCR analysis of 12/15-LO mRNA (Alox15) and 12-LO mRNA (Alox12) in mouse monocytes (monos; CD11b+CD115+), neutrophils (PMN; CD11b+Ly6G+), eosinophils (eos; side scatterhiCD11b+Siglec-F+), and platelets (plts) after FACS. Expression was normalized to Atcb expression. rel., relative. (C) Western blot of 12/15-LO protein (74 kD) expression in WT and Alox15−/− mouse eosinophils. (D) Quantitative RT-PCR analysis of Alox5 and Alox15 mRNA (top) and Western blot analysis of 15-LO protein (bottom) expression in human neutrophils (huPMN) and human eosinophils (huEos) isolated by Ficoll density gradient centrifugation and magnetic cell separation. mRNA expression was normalized to Actb. (E) Flow cytometry analysis of 12/15-LO in eosinophils (side scatterhiSiglecF+) isolated from peripheral blood of WT and Alox15−/− mice. (F) Flow cytometry analysis of the side scatter (SSC)hi12/15-LO+ population in blood from WT, Alox15−/−, and ΔdblGATA1 mice. (G) LC/MS/MS-based quantification of different esterified 12- and 15-HETE-PE, nonesterified 5-, 8-, 11-, 12-, and 15-HETE species, and prostaglandins D2 and E2 (PGD2 and PGE2) in WT (blue) and Alox15−/− (red) mouse eosinophils. Levels are presented as nanograms per 106 cells. (H, left) Calibrated thrombin generation curve of mouse WT eosinophils, eosinophils treated with baicalein (Bai), and Alox15−/− eosinophils. (Right) Bar graphs show endogenous thrombin potential (ETP; nM*min) and peak of thrombin generation (peak; nM). (I) Plasmatic clotting time experiments with WT mouse eosinophils (EOS), mouse eosinophils treated with the 12/15-LO inhibitor baicalein, and Alox15−/− mouse eosinophils. Bar graphs display calculated clotting index. The black bar shows plasma with ADP alone. (J, left) Calibrated thrombin generation assay with lysates generated from human eosinophils in the presence of PRP reagent (see Materials and methods). (Right) Bar graphs show endogenous thrombin potential (nM*min) and peak of thrombin generation (nM). (K, left) Calibrated thrombin generation assays with WT and Alox15−/− mouse eosinophils in the presence of phosphatidylcholine/PS liposomes carrying oxidized 12-HETE-PE or nonoxidized PE (SAPE) species, as indicated. (Right) Bar graphs show endogenous thrombin potential (nM*min) and peak of thrombin generation (nM). Data are representative of at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test.

12/15-LO–mediated oxidation of membrane phospholipids initiates eosinophil-mediated thrombin formation. (A) Representative LC/MS/MS analysis of different 12-HETE-PE oxidation species in lipid extracts of in vitro–generated mouse eosinophils. Cps, counts per second. (B) Quantitative RT-PCR analysis of 12/15-LO mRNA (Alox15) and 12-LO mRNA (Alox12) in mouse monocytes (monos; CD11b+CD115+), neutrophils (PMN; CD11b+Ly6G+), eosinophils (eos; side scatterhiCD11b+Siglec-F+), and platelets (plts) after FACS. Expression was normalized to Atcb expression. rel., relative. (C) Western blot of 12/15-LO protein (74 kD) expression in WT and Alox15−/− mouse eosinophils. (D) Quantitative RT-PCR analysis of Alox5 and Alox15 mRNA (top) and Western blot analysis of 15-LO protein (bottom) expression in human neutrophils (huPMN) and human eosinophils (huEos) isolated by Ficoll density gradient centrifugation and magnetic cell separation. mRNA expression was normalized to Actb. (E) Flow cytometry analysis of 12/15-LO in eosinophils (side scatterhiSiglecF+) isolated from peripheral blood of WT and Alox15−/− mice. (F) Flow cytometry analysis of the side scatter (SSC)hi12/15-LO+ population in blood from WT, Alox15−/−, and ΔdblGATA1 mice. (G) LC/MS/MS-based quantification of different esterified 12- and 15-HETE-PE, nonesterified 5-, 8-, 11-, 12-, and 15-HETE species, and prostaglandins D2 and E2 (PGD2 and PGE2) in WT (blue) and Alox15−/− (red) mouse eosinophils. Levels are presented as nanograms per 106 cells. (H, left) Calibrated thrombin generation curve of mouse WT eosinophils, eosinophils treated with baicalein (Bai), and Alox15−/− eosinophils. (Right) Bar graphs show endogenous thrombin potential (ETP; nM*min) and peak of thrombin generation (peak; nM). (I) Plasmatic clotting time experiments with WT mouse eosinophils (EOS), mouse eosinophils treated with the 12/15-LO inhibitor baicalein, and Alox15−/− mouse eosinophils. Bar graphs display calculated clotting index. The black bar shows plasma with ADP alone. (J, left) Calibrated thrombin generation assay with lysates generated from human eosinophils in the presence of PRP reagent (see Materials and methods). (Right) Bar graphs show endogenous thrombin potential (nM*min) and peak of thrombin generation (nM). (K, left) Calibrated thrombin generation assays with WT and Alox15−/− mouse eosinophils in the presence of phosphatidylcholine/PS liposomes carrying oxidized 12-HETE-PE or nonoxidized PE (SAPE) species, as indicated. (Right) Bar graphs show endogenous thrombin potential (nM*min) and peak of thrombin generation (nM). Data are representative of at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test.

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