Page 295 - Haematologica March 2020

CD36 activates platelet PDE3A
their ability to promote platelet hyperactivity. In vitro experimentation demonstrates that these modified lipoproteins can cause direct activation of platelets and also potentiate platelet activation induced by physiologi- cal agonists such as thrombin, ADP and epinephrine.7–10 However, the potential pathophysiological importance of these observations for thrombosis in vivo remain unclear.
The scavenger receptor CD36 has emerged as a poten- tial conduit for transducing plasma lipid stress into platelet hyperactivity and thrombosis, through the recognition of lipoprotein associated molecular patterns (LAMPs). CD36, alone or potentially in combination with Toll-Like Receptor (TLR)2 and TLR6 drive a complex series of intra- cellular signaling events that are associated with platelet activation.11–15 Upon ligation of CD36, Src family kinases constitutively associated with the receptor, drive the acti- vation of Syk, Vav-1, PLCγ2, ERK5 and JNK that are asso- ciated with platelet activation.13,16–18 More recently, data have emerged to suggest that the signaling events pro- mote the generation of reactive oxygen species (ROS).14,16,17 ROS in turn activate ERK to drive thrombosis directly by platelet hyperactivity and caspase-dependent procoagu- lant activity.18,19 Moreover, we found that ROS diminish sensitivity to the nitric oxide (NO)-stimulated cGMP-PKG inhibitory signaling cascade to reduce the threshold for platelet activation.17 These data suggest that the transla- tion of atherogenic lipid stress by platelet CD36 is func- tionally linked to both stimulation of activatory signaling pathways and to an as of yet ill-defined modulation of inhibitory pathways.
PGI2 is the most potent endogenous regulator of platelet function with both genetic and pharmacological modula- tion of the pathway linked to accelerated thrombosis in vivo.20 PGI2 activates a cyclic adenosine monophosphate (cAMP) signaling pathway that leads to subsequent activa- tion of protein kinase A (PKA) in platelets and results in the phosphorylation of numerous proteins,21 linked to the inhibition of Ca2+ mobilization, dense granule secretion, spreading, integrin αIIbβ3 activation and aggregation in vitro.20 To ensure optimal platelet function, cAMP levels are tightly regulated by the hydrolyzing enzymes phosphodi- esterase (PDE)2A and 3A. The pharmacological inhibition or genetic ablation of PDE3A in murine and human platelets reduces thrombotic potential.22,23 Thus, factors that alter platelet inhibition by influencing cAMP synthe- sis or hydrolysis may be critical modulators of atherothrombosis and potentially lead to a pro-thrombot- ic phenotype. Given the established link between oxi- dized lipid stress and excessive platelet activation, the aim of this study was to determine if oxidatively modified lipoproteins could promote platelet hyperactivity through modulation of the PGI2 /cAMP signaling pathway.
Methods
Reagents
Phospho-PKA Substrate (RRXS*/T*) Rabbit mAb and phospho- VASP-Ser157/239 antibodies were from Cell Signaling Technology (Danvers, USA). PDE3A antibodies were from the MRC Unit (Dundee University, Dundee, UK). Anti-β-Tubulin antibody was from Millipore (Nottingham, UK). BD Phosflow Lyse/Fix Buffer was from BD Biosciences (Oxford, UK). OxPC-E06 mAb was from Avanti Polar Lipids (Alabaster, USA). FITC-labeled Rat Anti- Mouse P-selectin (CD62P) and PE-labeled JON/A antibodies were
from Emfret Analytics (Würzburg, Germany). Alexa-Fluor 647 Goat anti-Rabbit IgG, Alexa-Fluor 488 Succinimidyl Ester and Pacific Blue Succinimidyl Ester were from ThermoFisher Scientific (Waltham, USA). PAR-1 peptide was from Anaspec (Fremont, USA). Anti-CD36 Antibody (FA6-152) was from Abcam (Cambridge, UK). Phosphodiesterase Activity Assay Kit was from Enzo Life Sciences (Exeter, UK). cAMP Biotrack EIA was from GE Healthcare (Buckinghamshire, UK). Horm Collagen was from Nycomed (Munich, Germany). PGI2 and Cholesterol Assay Kit were from Cayman Chemical (Cambridge, UK). Vena8 Endothelial+ biochips were from Cellix (Hertfordshire, UK). All other reagents were from Sigma-Aldrich (Dorset, UK).
Experimental animals
CD36-/- mice were provided by Prof. Maria Febbraio (University of Alberta, Canada). C57BL/6 were from Charles River (Kent, UK). For high-fat diet studies, male mice were fed a 45% Western diet (Special Diet Services) for 12–16 weeks. Sex/age-matched litter- mates were fed a normal chow for the duration of the experiments and used as controls. For all remaining experiments, male C57BL/6 and CD36-/- were used at eight weeks of age.
Isolation and oxidation of plasma low density lipoproteins
Low-density lipoproteins (density 1.019–1.063 g/mL) were pre- pared from fresh human plasma by sequential density ultracen- trifugation and oxidised with CuSO4 (10 mmol/L).14 Separate preparations of LDL were used to repeat the individual experi- ments.
Platelet aggregation, flow assays, flow cytometric analysis, intravital microscopy, immunoprecipitation, immunoblotting, phosphodiesterase enzyme activity assay and cyclic adenosine monophosphate measure- ment
Detailed methods are presented in the Online Supplementary Methods.
Statistical analysis
Experimental data was analyzed by Graphpad Prism 6 (La Jolla, CA, USA). Data are presented as means±standard error of the mean (SEM) of at least three different experiments. Differences between groups were calculated using Mann-Whitney U Test or Kruskal-Wallis Test for non-parametric testing and statistical sig- nificance accepted at P≤0.05.
All studies were approved by the Hull York Medical school Ethics committee and University of Leeds Research Ethics com- mittee.
Results
Oxidized low density lipoproteins cause platelet hyposensitivity to prostacyclin
Treatment of human washed platelets with PGI2 (20 nM) for one minute (min), at a time point that induces maximal cAMP-mediated signaling24 reduced thrombin (0.05 U/mL)-induced aggregation from 89.0±4.1 to 9.4±4.4% (P<0.01) (Figure 1A). Next, platelets were treat- ed with oxLDL or control native LDL (nLDL) (50 mg/mL) for 2 min prior to the addition of PGI2 (20 nM) and throm- bin (0.05 U/mL). The presence of oxLDL caused a partial recovery in thrombin-stimulated platelet aggregation to 50.0±9.3 (P<0.015 vs. control), without stimulating aggre- gation directly (Figure 1A). In contrast, PGI2-mediated
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