Fig 2: 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

Fig 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 indicate diseases or functions that are predicted as decreased, increased, or affected, respectively.

Fig 4: Increased mPGES1 protein abundance is dependent upon activation of NCC. Western blot (WB) analysis of PGE2 synthesis enzymes in tissue of kidney cortex. (A) mPGES1, total Cox2 (both bands), and Cox1 (B) quantification. Statistical significance evaluated by two-way ANOVA (see text), followed by Tukey’s post hoc tests (*p < 0.05).

Fig 5: TWG selectively inhibits 5-LOX. (A) Effect of 1 and 10 µg/mL TWG on the residual activities of isolated ovine COX-1, human recombinant COX-2 and 5-LOX, mPGES-1 in microsomes from A549 cells, and TXAS in homogenates of human platelets. Data are expressed as percentage of vehicle (=100%) and given as means ± S.E.M., n = 3 (for COX-1/2, 5-LOX) or n = 4 (for mPGES1, TXAs). Indomethacin (IND) 10 µM, MK886 10 µM, and zileuton 3 µM were used as positive controls. * p < 0.05, ** p < 0.01, *** p < 0.001, TWG vs. control group, one-way ANOVA and Dunnett’s multiple comparisons test. (B,C) Concentration response curves of TWG for inhibition of (B) isolated human recombinant 5-LOX incubated with 20 µM AA, and (C) 5-LO in human intact PMNL stimulated with 2.5 µM A23187 plus 20 µM AA. Data are means + S.E.M., n = 3.