Page 303 - Haematologica March 2020

CD36 activates platelet PDE3A
4G). These three inhibitors blocked PDE3A activity induced by oxLDL, suggesting a PKC-dependent pathway (P<0.05). Platelet PDE3A activation is associated with phosphorylation of key serine residues.31 We examined two of the most characterised sites, ser312 (PKA and PKC sensitive) and ser428 (PKC sensitive). OxLDL led to a time- dependent phosphorylation of ser428 which peaked at 2 min and was still evident at 10 min, but had no effect on phosphoPDE3Aser312. In comparison, thrombin (0.1 U/mL) induced significantly less phosphorylation at ser428, which peaked at 1 min before returning to basal at 2 min (Figure 4H) but also induced phosphorylation of ser312. These data confirm that oxLDL and its associated oxidized phos- pholipids require the sequential ligation and activation of CD36, Src, Syk and PKC to activate PDE3A.
Dyslipidemia is associated with platelet prostacyclin hyposensitivity in mice
To demonstrate the functional importance of our obser- vations on dyslipidemia-driven thrombosis, we examined platelet sensitivity to PGI2 in mice fed a Western diet (45%). cAMP signaling, integrin αIIbβ3 activation and thrombosis were assessed in whole blood ex vivo, which allowed us to evaluate platelet function and thrombotic potential while avoiding any confounding effects of altered PGI2 synthesis in vivo associated with dyslipidemia.32 Western diet significantly raised choles- terol levels (Online Supplementary Figure S8) and the pres- ence of oxidized phospholipids in the plasma (Figure 5A). We then investigated the effect of dyslipidemia on cAMP signaling using multiplexed phosphoflow cytometry to allow the examination of signaling in the physiological conditions of whole blood.24 Platelets from high-fat fed WT animals produced significantly less phosphoVASP- ser157 when challenged with PGI2 (100 nM) than normal chow WT mice (4.9±0.4 fold vs. 6.2±0.2-fold increase over basal; P<0.05) (Figure 5B). The deletion of CD36 protected cAMP signaling in dyslipidemic blood, with phosphoVASP-ser157 remaining at control levels (Figure 5B). In parallel experiments, blood was stimulated with CRP- XL (10 mg/mL) in the presence and absence of PGI2 (100 nM), and αIIbβ3 activation was measured. In normal chow WT blood, PGI2 caused 75.7±3.9% inhibition of integrin activation (P<0.05 compared to absence of PGI2), which was blunted in high-fat fed WT blood (43.1%±7.6 inhibition, P<0.05 compared to normal chow) (Figure 5C, left). Conversely, in CD36-/- blood, PGI2 induced inhibition of integrin activation was not significantly different in nor- mal chow and high-fat fed conditions (66.5±8% and 79.3±6.5%, respectively) (Figure 5C, 1st and 2nd left).
When we assessed ex vivo thrombosis under flow, nor- mal chow WT blood formed small thrombi on immobi- lized collagen in a time dependent manner, which was abolished by PGI2 (20 nM) (Figure 5Di). High-fat fed WT blood showed an accelerated thrombotic response with increased surface area (11±3.6% compared to 16.2±4.3% at 2 mins). In addition, dyslipidemia caused significant hyposensitivity to PGI2, with the prostanoid causing 31.7±10.7% inhibition compared 61.6±5.6% with normal chow (P<0.05; 2 min) (Figure 5ii and iii). In contrast, accel- erated platelet deposition on collagen and platelet hyposensitivity to PGI2 was not detected in CD36-/- high- fat fed blood (Figure 5D). We repeated the experiments with 8-CPT-6-Phe-cAMP. If PDE3A activation was respon- sible for the increased thrombotic potential associated
with reduced sensitivity to PGI2, then CPT-6-Phe-cAMP- mediated inhibition of thrombosis would be unaffected by dyslipidemia. The cAMP analog caused a similar degree of inhibition of thrombosis in all genotypes but critically remained unaffected in the context of dyslipi- demia (WT-normal chow, 65.6±11.2%; WT-Western diet, 62.3±7.7%; CD36-/--normal chow, 70.4±2.0%; CD36-/-- Western diet, 62.3±5% inhibition) (Online Supplementary Figure S9). Here we show a physiological role for platelet CD36 in dyslipidemia, where it drives a phenotype of platelet hyperactivity by blocking cAMP-mediated inhibi- tion.
Oxidized low density lipoprotein potentiation of thrombosis in vivo is prevented by inhibition of phosphodiesterase 3A
To examine the role of oxLDL in thrombosis in vivo we used intravital microscopy following ferric chloride– induced carotid artery injury. Tail-vein injections of oxLDL (2.5 mg/kg-1 bodyweight)33,34 into WT mice accelerated post-injury thrombotic response at all time points com- pared to control PBS injection (Figures 6A-C and Online Supplementary Videos S1 and S2). Next animals were pre- treated with milrinone to explore in principle whether PDE3A inhibition might diminish prothrombotic effects of oxLDL. Consistent with previous studies, modulation of PDE3A activity reduced murine thrombosis (Figure 6A- C).23 Importantly, the presence of milrinone (10 mmol/L) significantly reduced the ability of oxLDL to promote thrombosis at all time points post injury (P<0.05) (Online Supplementary Video S3) suggesting the heightened throm- botic response in the presence of oxLDL could be related, at least in part, to changes in PDE3A activity.
Discussion
The presence of oxidized lipid epitopes, including oxLDL, is thought to promote platelet hyperactivity in subjects with obesity, CAD and stroke.5,6,35 It has been established that oxidative modifications are a hallmark of dyslipidemia and that they stimulate platelet activation directly through a number of distinct receptors.6,8,13 Interestingly, platelets from patients with CAD and dys- lipidemia show a reduced sensitivity to the inhibitory effects of PGI2. These observations, coupled to pharmaco- logical trials indicating that suppression of endothelial PGI2 synthesis increased rate of atherothrombotic events,36–39 suggest that platelet sensitivity to PGI2 could play an undefined role in the development of atherothrombotic events. Hence, the aim of this study was to investigate whether oxLDL may promote unwant- ed platelet activation through the modulation of platelet sensitivity to PGI2. Using a combination of pharmacologi- cal and genetic approaches, we show that oxidative lipid stress modulates platelet cAMP signaling leading to increased platelet activation. Our key findings include: (i) oxLDL and oxidized phospholipids decrease platelet sen- sitivity to PGI2, which is coupled to reduced platelet accu- mulation of cAMP and PKA mediated signaling; (ii) PGI2 hyposensitivity likely occurs via sustained activation of the cAMP hydrolysing enzyme PDE3A in response to oxLDL; (iii) the activation of PDE3A by oxLDL requires ligation of CD36; and (iv) that dyslipidemia induces platelet hyposensitivity to PGI2 in a CD36-dependent manner.
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