Page 299 - Haematologica March 2020

CD36 activates platelet PDE3A
A
BCD
EFG
H
Figure 4. Oxidized low-density lipoproteins (oxLDL) and oxPCCD36 induce sustained phosphodiesterase 3A (PDE3A) activity in a CD36-dependent manner. (A). Washed human platelets (5x108/mL) incubated with apyrase, indomethacin and EGTA were (Left) incubated with nLDL (50 mg/mL) or oxLDL (10-100 mg/mL) for 2 minutes (min) or (Right) incubated with control native LDL (nLDL) (50 mg/mL) or oxLDL (50 mg/mL) for up to 60 min. Platelets were lysed, PDE3A was immunopre- cipitated and enzyme activity was measured. Data is expressed as % activity above basal activity and presented as mean±standard error of mean (SEM) (P<0.05; n=3, Kruskal-Wallis Test ). (B) Washed human platelets (5x108/mL) were incubated with oxLDL (50 mg/mL) for up to 60 min followed by PGI2 (50 nM) for 1 min. Platelets were lysed, and intracellular cyclic adenosine monophosphate (cAMP) levels were determined by enzyme immunoassay. Data are presented as mean±SEM (*P<0.05, n=3, Kruskal-Wallis Test). (C) Washed human platelets (5x108/mlL) were incubated with oxPCCD36 or PAPC (25 mM) for 2 min, and activity of immuno- precipitated PDE3A measured. Data are expressed as % activity above basal activity and presented as mean±SEM (P<0.05; n=3 Mann-Whitney U Test). (D) As in (C), except that PDE3A activity was measured in wild-type (WT) and CD36-/- washed platelets (*P<0.05; n=3 Mann-Whitney U Test). (E) As in (A) except that human platelets (5x108/mL) incubated with apyrase, indomethacin and EGTA were then treated with oxPCCD36 or PAPC (25 mM) for 2 min in the presence of absence of dasatinib (10 mM) or vehicle control (DMSO). PDE3A was immunoprecipitated, and enzyme activity was measured. Data are expressed as % activity above basal activity and presented as mean±SEM (P<0.05; n=4 Mann-Whitney U Test). (F) As in (A) except that platelets were treated with R406 (1 mM) or vehicle control (DMSO). (G) As in (A), except that platelets were treated with PKC inhibitors (RO31 10μM, BIM1 10μM), PMA (100nM), BAPTA (20 mM) or vehicle control (DMSO). (n=3, *P<0.05 compared to basal, Kruskal-Wallis Test ). (H) Washed human platelets (5x108/mL) treated with apyrase, indomethacin and EGTA were incubated with or oxLDL (50 mg/mL; 0-60 min) or thrombin (0.1 U/mL; 0-2min). Treated platelets were lysed in Laemmli buffer, separated by SDS-PAGE and immunoblotted with anti- phosphoPDE3Aserr312, phosphoPDE3Aser428 or anti-β tubulin. (Left) Representative blot of three independent experiments. (Right) Densitometry for phosphoPDE3Aser428 is presented as fold-change above basal, mean±SEM (n=3, *P<0.05, Kruskal-Wallis Test).
haematologica | 2020; 105(3)
813