Protein Cysteine Sulfenylation By CD36-Dependent Reactive Oxygen Species Signaling Promotes Platelet Activation

Arterial thrombosis in the setting of dyslipidemia produces clinically significant events, including myocardial infarction and stroke. Oxidized lipids in circulating lipoproteins (oxLDL) are a risk factor for atherothrombosis in dyslipidemia and are recognized by platelet scavenger receptor CD36. OxLDL binding to CD36 promotes platelet activation and thrombosis by generating intracellular reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Downstream signaling events initiated by ROS in this setting are largely unknown. We hypothesize that H₂O₂ generated by CD36 signaling promotes oxidative cysteine modification of cellular regulators of arterial thrombosis. Platelets isolated from healthy human donors were stimulated with oxLDL and H₂O₂ levels were measured by HPLC fluorescence quantification of 7-hydroxycoumarin generated by H₂O₂-specific oxidation of the coumarin boronic acid probe. OxLDL induced time- and concentration-dependent H₂O₂ formation (up to 80 ±13% greater than unstimulated platelets), while control "native" LDL showed negligible H₂O₂ formation. Pre-treatment of platelets with a CD36-blocking antibody or with PEG-catalase, an enzyme that degrades H₂O₂, normalized H₂O₂ formation by oxLDL to levels observed in unstimulated platelets. To mimic pathophysiologic conditions platelets were sensitized with oxLDL before stimulating with classic activators: adenosine diphosphate (ADP) and collagen-related peptide (CRP). Sensitization synergistically increased H₂O₂ formation through collagen receptors, but not ADP receptors. Since H₂O₂ can induce transient protein cysteine sulfenylation as a posttranslational oxidative modification, we assayed platelet cysteine sulfenylation using an alkyne-containing benzothiazine-based probe, BTD. BTD was loaded into platelets prior to exposure to oxLDL and detected by click-chemistry with biotin-PEG-azide followed by detection by western blot with streptavidin. OxLDL, but not LDL, induced two-fold increase in sulfenylation in the platelet proteome within 15 minutes. Src family kinases (SFK) are known to be recruited to and activated by CD36 in a ligand-dependent manner and SFK were also previously shown to be sulfenylated by H₂O₂ at Cys185 and Cys277, maintaining the kinase in an activated state. To determine if SFK cysteines are oxidatively modified by CD36 signaling, lysates from BTD-loaded, oxLDL-stimulated platelets were biotinylated as above, immunoprecipitated with anti-Src antibody, and then analyzed by immunoblot to detect BTD incorporation. SFK were sulfenylated in a time-dependent manner and this was blocked by a CD36 blocking monoclonal antibody or by treatment with PEG-catalase. We then showed by immunoblot that SFK cysteine sulfenylation by CD36/H₂O₂ was associated with phosphorylation at Y416, a signature activation motif in the kinase domain. OxLDL induced Src Y416 phosphorylation was prevented by PEG-catalase. Platelet aggregometry was used to determine the functional impact of protein sulfenylation and revealed that oxLDL-induced platelet aggregation was inhibited in a concentration-dependent manner (IC₅₀ 2.02 mM) by modifying sites of sulfenylation with BTD. BTD had no impact on aggregation induced by low- or high-concentration of ADP or CRP, suggesting these physiologic activators alone do not generate sufficient H₂O₂ to promote cysteine sulfenylation. OxLDL/CD36 induced procoagulant phosphatidylserine externalization, assessed by flow cytometry using fluorophore-tagged annexin V, was also prevented by BTD. In conclusion, our studies show that platelet CD36 signaling in response to oxLDL induces intracellular H₂O₂ generation which in turn induces cysteine sulfenylation of Src family kinases to promote platelet activation. Cysteine sulfenylation by CD36 could potentially be targeted to reduce the risk for clinically significant thrombotic events while maintaining hemostasis.