Page 298 - Haematologica March 2020

M. Berger et al.
Figure 3. Oxidized low-density lipoproteins (oxLDL) and oxPCCD36 alter prostacyclin (PGI2) inhibitory signaling via CD36. (A) Washed human platelets (5x108/mL) incubated with apyrase, indomethacin and EGTA were incubated with FA6-152 or IgG (1 mg/mL) for 20 minutes (min). Platelets were then incubated alone or with control native LDL (nLDL) or oxLDL (50 mg/mL) for 2 min and subsequently stimulated by PGI2 (50nM) for 1 min. Treated platelets were lysed in Laemmli buffer, sep- arated by SDS-PAGE and immunoblotted with anti-phosphoVASPser157 or anti-β tubulin. (Top) Representative blot of three independent experiments. (Bottom) Densitometry of pVASPser157 fold-change above basal mean±standard error of mean (SEM) (n=3 *P<0.05, Mann-Whitney U Test). (B) As in (A) except that platelets were probed for pVASPser239. (C) Platelets were treated as in (A) alone or with oxPCCD36 or PAPC (25 mM) for 2 min followed by PGI2 (50 nM) for 1 min. (Top) Representative blot of three independent experiments and (Bottom) Densitometry of pVASPser157 fold-change above basal, mean±SEM (n=3 *P<0.05, Mann- Whitney U Test). (D) Washed wild-type murine platelets (2.5x108/mL) were treated alone or with oxPCCD36 or PAPC (10 mM) for 2 min followed by a 1 min incubation with PGI2 (20 nM). Thrombin (0.05 U/mL)-stimulated aggregation was then measured under constant stirring (1000 rpm) at 37°C for 4 min. (Top) Representative aggregation traces and (Bottom) data presented as percentage aggregation, mean±SEM (n=3, P<0.05, Mann-Whitney U Test). (E) As described in (D) for CD36-/- platelets. Ns: not significant. (F) Washed murine WT or CD36-/- platelets (2x108/ml) were treated alone or with nLDL or oxLDL (50 mg/mL) for 2 min followed by a 1-min PGI2 (50 nM) incubation. Platelets were lysed, and intracellular cAMP concentrations were measured by enzyme immunoassay. Intracellular cAMP levels are presented as mean±SEM (n=3, *P<0.05, Mann-Whitney U Test). (G) As in (A) except that murine (Left) WT and (Right) CD36-/- platelets were used. Representative blot of three independent experiments. pVASPser157 is presented as beta-tubulin corrected fold-change above basal±SEM (n=3 *P<0.05, Mann-Whitney U Test).
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haematologica | 2020; 105(3)