The mitochondrion is a substrate for the bactericidal sPLA2-IIA. (A) Quantification of sPLA2-IIA in human platelets by time-resolved immunofluorescence (n = 3; data are mean ± SEM). (B) sPLA2-IIA immunoblotting of mitochondria isolated with anti-TOM22 microbeads reveals binding of human recombinant sPLA2-IIA to mitochondria (supplemental Figure 3). (C) Mitochondria were incubated in (left) the absence or (right) presence of Alexa488-conjugated sPLA2-IIA and analyzed by hs-FCM. The significant shift in the fluorescent population size (right) indicates that sPLA2-IIA binds mitochondria. (D-E) Catalytic activity of human recombinant sPLA2-IIA (or PBS as vehicle) toward mitochondria. Mitochondrial membrane phospholipid hydrolysis by sPLA2-IIA yields (D) lysophospholipids and (E) fatty acids as quantified by mass spectrometry. (F) sPLA2-IIA affects mitochondrial structural integrity. Scanning electronic micrographs of mitochondria incubated in the (left) absence or (right) presence of human recombinant sPLA2-IIA. (G) Mitochondria (magenta) release mtDNA (blue) on incubation with recombinant sPLA2-IIA (upper panels). Extracellular mtDNA accumulation (arrow) is apparent in the presence of sPLA2-IIA. Differential interference contrast images are shown for reference (lower panels). (H) mtDNA extrusion is amplified in presence of human recombinant sPLA2-IIA (0.2 μg/mL, 30 minutes at 37°C), as quantified by Sytox Green nucleic acid stain assay (n = 6; data are mean ± SEM, *P < .05, Student t test).
