CD36 Activation Enhances Hydrogen Peroxide Generation in Human Platelets

Cardiovascular disease remains the leading cause of death in the United States and thrombosis following atherosclerotic plaque rupture represents a significant portion of this mortality. Our lab and others have previously shown that oxidized lipids in low density lipoprotein (LDL) particles (oxLDL) serve as a "danger" signal that is recognized by CD36, a scavenger receptor of the innate immune system, found on platelets, and other vascular cells, that promotes platelet hyper-reactivity and thrombogenesis. Recently published data from our lab suggests that oxLDL-CD36 signaling in platelets involves generation of reactive oxygen species (ROS) that are important signaling pathway regulators and contribute to the sustained activation of the redox-sensitive MAPK, ERK5. This pathway is an important contributor to platelet activation and thrombosis. It is not well understood how CD36-dependent ROS generation maintains platelets under hyperlipidemic conditions in a "hypersensitive" state. Our hypothesis is that oxLDL acting through CD36 stimulates specific hydroperoxide species to modulate platelet signaling pathways. Using reverse-phase HPLC to detect 7-hydroxycoumarin (COH), the stable fluorescent product of the reaction between hydroperoxides and coumarin boronic acid (CBA), we selectively measured intracellular hydroperoxide generation in washed human platelets. Platelets showed a dose-dependent increase in COH accumulation of 70 ± 20%, 81 ± 13%, and 84 ± 16% after stimulation with oxLDL 25 μg/mL, 50 μg/mL, or 100 μg/mL, respectively (p=0.0115, p<0.01, p<0.01). Equivalent doses of native LDL produced no significant increase in COH accumulation. Platelets also showed a time-dependent increase in COH with maximal accumulation after 60 minutes of 50 μg/mL oxLDL stimulation (p<0.001). Previously published functional aggregometry data suggested that PEG-catalase, an enzyme that promotes hydrogen peroxide (H₂O₂) breakdown, prevents oxLDL-stimulated platelet activation and aggregation. Here we showed that pre-treatment of platelets with PEG-catalase blunted oxLDL-stimulated COH accumulation by 42 ± 8.6% (p=0.0106). In addition to H₂O₂, CBA reacts with peroxynitrite, but inhibition of platelet nitric oxide synthase with L-N^G-Nitroarginine methyl ester (L-NAME) did not alter COH generation after oxLDL treatment when compared to oxLDL alone. Since CD36 signaling activates platelets through "non-classical" pathways, we examined how classic platelet agonists affected oxLDL-stimulated hydroperoxide generation. Treatment with collagen-related peptide (CRP) showed no significant increase in COH accumulation, but co-stimulation with oxLDL and CRP showed a 185 ± 26.4% increase in COH accumulation, as compared to oxLDL alone (p<0.0001), suggesting a synergistic effect between the oxLDL-CD36 and collagen-GPVI signaling pathways. This effect was not observed after thrombin or ADP treatment. Further studies are necessary to elucidate how oxLDL-stimulated hydroperoxide generation affects platelet signaling. Taken together our data suggests that oxLDL specifically generates H₂O₂ in platelets to promote a hyper-sensitive state and collagen-GPVI signaling may enhance this effect promoting thrombosis in pro-atherogenic conditions. Since mice and humans lacking CD36 do not demonstrate a bleeding diathesis and form normal thrombi, we reason that targeting CD36-mediated redox mechanisms has clinical potential to reduce the risk of atherothrombosis without compromising normal physiological hemostasis responses, unlike current anti-platelet agents in clinical use.