Profiling the eicosanoid networks that underlie the anti‐ and pro‐thrombotic effects of aspirin

Figures

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  FIGURE 1. Characterization of platelet COX-1 deletion. A, Control and platelet-COX-1-ko platelet lysates were subjected to SDS-PAGE and probed for COX-1 and GAPDH as indicated. B, Fixed and permeabilized platelets from control and platelet-COX-1-ko mice were stained for tubulin (green) and COX-1 (magenta) and imaged with Airyscan laser scanning confocal microscopy. Bars represent 2 μm. C, The buffy coat was prepared from global-COX-1-ko, control, and platelet-COX-1-ko mouse blood with the mixed platelet and leukocyte populations being isolated prior to staining for tubulin (green), DAPI (blue), and COX-1 (magenta) and imaging. Bars represent 10 μm. D, Aortas from global-COX-1-ko, control, and platelet-COX-1-ko mice were fixed, permeabilized, and stained for COX-1 (magenta) and the endothelial marker CD31 (green). Aortas were then dissected into rings that were cut open to visualize their luminal surface by confocal microscopy. Bars represent 50 μm. E,F, Kidney cortex (Kc), kidney medulla (Km), lungs (L), and platelet lysates were subjected to SDS-PAGE and probed for COX-1 presence. The images are representative of at least four mice per group

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  FIGURE 2. Tandem mass spectrometry analysis of lipid mediators produced in whole blood and IPA predictions of platelet activation and thrombosis. Plasma samples were prepared from mouse blood following incubation in vitro with vehicle or A23187 (50 μM) in stirring conditions (1000 rpm, 5 minutes) and subjected to tandem mass spectrometry analysis. A, Relative levels of mediators displayed in a heat map using a Z-score ranging from −4.12 (blue) to 4.12 (red). The individual products are represented on the bottom horizontal bar and their clustering on the top, while on the left the metabolites are grouped according to stimulated and unstimulated conditions and to the mouse strain. Hierarchical clustering analysis identified three different clusters of eicosanoids whose levels are significantly increased upon stimulation of whole blood with A23187 (50 μM): the cluster including TxB2, PGF2α, 11-HETE, and 15-HETE is indicated in the red box; the cluster including PGE2 and PGD2 is indicated in the blue box; and the cluster comprising 12-HETE, 5-HETE, and LTB4 in the green box. B, The absolute plasma levels of these eicosanoids as measured by tandem mass spectrometry. Results are from n = 4…

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  FIGURE 3. Tandem mass spectrometry analysis of eicosanoids produced in vivo and IPA predictions for platelet reactivity and thrombosis. A, Mass spectrometry measurements of COX-derived eicosanoids induced by systemic injection of AA (2.8 mg/kg, i.v.). Data are means ± SEM, n = 4–6 mice per group. *P < .05, **P < .01, ***P < .001, and ****P < .0001 (one-way ANOVA followed by Tukey’s post hoc test). B, The relative levels of eicosanoids are displayed in a heat map using a Z-score ranging from −4.20 (blue) to 4.20 (red). The individual products are identified on the bottom horizontal bar and their clustering on the top, while on the left the clustering of the mice depending on their eicosanoid signatures is shown. C, IPA was used to predict changes in processes linked to platelet reactivity and thrombosis based on the determination of in vivo eicosanoid synthesis. The bar charts show the most significant changes in platelet-COX-1-ko mice, aspirin-treated control mice, and global-COX-1-ko mice. Functions are listed from most significant to least. The x-axis displays the −(log) significance and the vertical dotted line denotes the cutoff for significance (P value of .05). Blue, red, and grey…

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  FIGURE 4. In vivo thrombosis. A, In vivo thrombus formation was studied in the carotid artery in response to exposure to 5% FeCl3 for 3 minutes. The time to occlusion was quantified in control mice and platelet-COX-1-ko mice under control conditions or following treatment with aspirin (10 mg/kg, i.v.). The data are means ± SEM, n = 6–8 mice per group. *P < .05 (one-way ANOVA followed by Tukey’s post hoc test). B, Traces of blood flow rate in carotid arteries during thrombosis model in (blue line) control mice and (red line) platelet-COX-1-ko mice

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  FIGURE 5. Model comparing the effects of aspirin in platelets vs the vasculature. A, Aspirin’s action in platelets, that is mimicked in p-COX-1-ko mice, results in the inhibition of COX-1-dependent pro-thrombotic eicosanoids, including TxA2, PGE2, and PGF2α, and in a beneficial antithrombotic effect. B, In the vasculature, aspirin blocks the activity of COX-1 and COX-2 and the synthesis of antithrombotic and vasorelaxant mediators, including PGI2 and PGD2. This effect favors thrombosis thus countering and limiting the beneficial effects of aspirin on the cardiovascular system mediated through inhibition of COX-1 in platelets. This figure was produced using Servier Medical Art (http://www.servier.com)