Page 301 - Haematologica March 2020

CD36 activates platelet PDE3A
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Figure 6. High-fat diet fed mice correlate with increased plasma levels of oxidized phospholipids to reduced prostacyclin (PGI2) sensitivity via CD36. (A) Representative dot blots of mouse plasma probed with anti-oxidized phospholipids (HRP-conjugated-E06) and total-IgG (HRP-conjugated anti-mouse). (B) Whole blood from normal chow and high-fat fed animals were treated with PGI2 (100nM) for 1 minute (min). Blood was fixed, permeabilized and incubated with anti- pVASPser157 followed by secondary fluorescent-conjugate (Alexa 647) and analyzed by flow-cytometry. Representative heat map of fold increase in phosphoVASPser157. Quantification is presented as fold-change of median fluorescence intensity above basal (n=4, *P<0.05, Mann-Whitney U Test). (C) Whole blood from normal chow and high-fat diet fed animals were treated with PGI2 (100 nM) for 1 min followed by CRP (10 mg/mL) for 5 min. Blood was fixed and JON/A positive cells were analyzed by flow cytometry. (Left) Representative histograms. (Right) Data presented as percentage inhibition of JON/A binding, mean±standard error of mean (SEM) (n=4 *P<0.05, Mann-Whitney U Test). (D) Whole blood incubated alone or with PGI2 (20 nM) for 1 min was perfused at arterial shear 1000s-1 for 2 min over a collagen matrix (50 mg/mL). Images of adherent platelets were taken by fluorescence microscopy. (Top) surface coverage (%) presented as a function of time. (Bottom left) Representative images of arterial flow experiments, (Bottom right) Data presented as inhibition of surface coverage (%), mean±SEM (n=5; *P<0.05, Mann-Whitney U Test).
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