Page 304 - Haematologica March 2020

M. Berger et al.
In the first instance, we used three increasingly physio- logical systems to show that exposure of platelets to oxLDL opposes the inhibitory effects of PGI2. In contrast, oxLDL failed to affect platelet inhibition by 8-CPT-6-Phe- cAMP, a PDE-resistant cAMP analog, demonstrating firstly that the PKA signaling was intact and secondly that the effects of the oxidized lipoprotein may regulate cAMP availability. Exploration of the underlying mechanisms demonstrated that OxLDL prevented the accumulation of cAMP in response to both PGI2 and forskolin. Forskolin increases cAMP in a receptor-independent manner, there- by providing evidence that oxLDL did not affect the inter- action of PGI2 with the IP receptor or target adenylyl cyclases. This is consistent with previous studies demon- strating that reduced platelet sensitivity to PGI2 in patients with hypercholesterolemia was independent of any changes in cAMP synthesis by adenylyl cyclase.36 It was therefore possible that oxLDL could either prevent the synthesis of cAMP or accelerate its breakdown. We fur- ther found that oxLDL failed to modulate cAMP concen- trations in the presence of the PDE3 inhibitor milrinone, but not the PDE2 inhibitor EHNA, suggesting that cAMP hydrolysis by PDE3A was the potential mediator of PGI2 hyposensitivity. A role for PDE3A was confirmed using immunoprecipitation experiments showing that both oxLDL and oxPCCD36 accelerated the hydrolytic activity of PDE3A in both human and murine platelets through lig- ation of CD36. The activation of PDE3A downstream of CD36 required the activation of Src family kinases, Syk and PKC. This provides further evidence that CD36-SFK- Syk represents a multiprotein complex that transduces extracellular oxidative lipid stress to the intracellular sig- naling machinery of the platelet. Interestingly, hemostatic agonists such as thrombin and collagen also activate PDE3A through a PKC-dependent mechanism.31 These agonists are proposed to cause a rapid attenuation of cAMP signaling at sites of vascular injury to promote platelet-mediated hemostasis. However, in contrast to the rapid and short-lived activation of PDE3A activity by thrombin and collagen, oxLDL induced a sustained PDE3A response for up to 60 min (longest time tested). This was linked to a different activatory phosphorylation pattern of PDE3A by oxLDL and could suggest a distinct mechanism of activation induced by short-lived hemosta- tic agonists from that of oxLDL. Given the sustained acti- vation of platelet PDE3A in the presence of oxidative lipid stress, it is attractive to speculate that PDE3A may be par- tially activated in dyslipidemic disease states and thereby reduce the threshold for platelet activation by diminution of cAMP. Indeed, gain of function mutations of PDE3 are associated with stroke, underlining its role in vascular pathology.40 This concept is also supported by observa- tions that inhibition of PDE3A by cilostazol can have ben- eficial anti-thrombotic effects in high-risk groups charac- terized a prothrombotic phenotype.41–43
The pathophysiological consequences of platelet hyposensitivity to PGI2 and the potential importance of CD36 was explored in a murine model of high-fat feed- ing-induced dyslipidemia. Interestingly, we found that
even mild dyslipidemia was characterized by the presence of oxidized lipid epitopes in the plasma, which was unaf- fected by the absence of CD36. Whole blood phospho- flow cytometry was used to measure platelet phosphoVASP, as a marker of cAMP signaling, without the need for cell isolation. This demonstrated that mild dys- lipidemia was accompanied by reduced cAMP signaling. The functional importance of this blunted cAMP signaling response manifested as diminished platelet sensitivity to the inhibitory effects of PGI2 on integrin activation meas- ured by flow cytometry and ex vivo thrombosis. The assessment of thrombosis ex vivo was important to demonstrate that hyposensitivity of platelets to PGI2 was a primary platelet defect rather than a response to a dys- functional endothelium, where altered PGI2 production has been observed in models of dyslipidemia.32 To support our hypothesis that hyposensitivity to PGI2 was linked to PDE3A activity, we showed that cAMP signaling in dys- lipidemia was normal if a PDE-resistant cAMP analog (8- CPT-6-Phe-cAMP) was used, again confirming that PKA signaling downstream of cAMP was functional. Critically, genetic ablation of CD36 protected animals from the loss of PGI2 sensitivity and restored PKA signaling. Infusions of oxLDL into wild-type mice caused a robust potentia- tion of thrombosis by ferric chloride. However, mice were protected from the prothrombotic effects of oxLDL in vivo when PDE3A was pharmacologically inhibited. Using this approach, milrinone did not target only platelets and could therefore have an effect on other PDE3A expressing cells. However, the data are proof of principle that the pro- thrombotic effects of oxLDL in vivo, at least in part, may be prevented by therapeutic strategies based on enhancing or preserving cAMP signaling events in platelets. This ele- ment of the work requires further studies focussing on strategies for the specific targeting of PDE3A, and poten- tially PDE2, in platelets.
Together, our ex vivo and in vitro data suggest a previous- ly unrecognized mechanism contributing to platelet hyperactivity, where the ligation of CD36 by oxidized lipids modulates cAMP signaling by activating PDE3A leading to PGI2 hyposensitivity. These data may constitute a link to the observed PGI2 hyposensitivity in dyslipidemic high-risk populations and indicate a novel therapeutic strategy to target atherothrombotic risk in certain patient groups. Remarkably, current antiplatelet therapy exclu- sively targets platelet activatory pathways including cyclo-oxygenases (Aspirin), P2Y12 (Thienopyridines, non- Thienopyridines) or αIIbβ3 (Tirofiban; Eptifibatide) while platelet inhibitory pathways remain untargeted. Therefore, high-risk populations might remain at increased atherothrombotic risk despite optimal available pharmacological therapy, and impaired platelet inhibition might contribute to the residual cardiovascular risk.
Acknowledgments
The authors would like to thank the British Heart Foundation (PG/13/90/20578, PG/12/49/29441 and RG/16/5/32250) and the Rotations-Program of the Medical faculty of RWTH Aachen University for funding this study.
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