Platelet CD36 Induces ERK5 Activation through a Redox-Regulated Signaling Pathway to Promote a Prothrombotic Phenotype

Atherothrombosis is a pathological event initiated by dysregulated platelet activation in the setting of unstable or ruptured atherosclerotic plaque, leading to myocardial infarction or stroke. Platelets express scavenger receptor CD36, a receptor of the innate immune system that recognizes endogenous danger signals, such as oxidized low-density lipoprotein (oxLDL), that are generated during the inflammatory and oxidative processes of atherosclerosis. Platelet CD36 induces a prothrombotic phenotype by lowering the threshold for platelet activation by low-dose agonists through multiple signaling pathways involving Src family kinases, mitogen-activated protein kinases (MAPK), Vav-family guanine nucleotide exchange factors, and a pathway that leads to the formation of reactive oxygen species (ROS). Although these signaling pathways contribute to the CD36-mediated prothrombotic phenotype, the mechanisms by which CD36 promotes thrombosis remain incompletely defined. ERK5 is a member of the MAPK family and is a redox sensor that is activated under oxidative stress conditions that can contribute to cardiovascular diseases. ERK5 was recently identified to be present and functional in platelets as a redox sensor that promotes infarct expansion in myocardial infarction. Since the role of ROS generated by CD36 in platelets is unclear, we tested the hypothesis that CD36 promotes thrombosis in atherogenic conditions by generating a redox-regulated signaling pathway requiring ERK5. Initial studies using HPLC to detect 2-hydroxyethidine, the specific product of the reaction between superoxide radical anion (O₂^●-) and hydroethidine, showed that CD36 induces a two-fold increase in O₂^●- in washed human platelets in response to oxLDL (p=0.01). We also showed that CD36 signaling generated hydrogen peroxide in human platelets, which is detected by the oxidation of the peroxide probe Amplex Red to the fluorescent product resorufin (p=0.05). We found that hydrogen peroxide has a functional role in CD36-enhanced platelet aggregation using polyethylene glycol-conjugated catalase (PEG-catalase), an enzyme catalyzing the decomposition of intracellular hydrogen peroxide to water. PEG-catalase inhibited platelet aggregation induced by oxLDL by 81.1 ± 4.3%. To determine the mechanism by which ROS promotes the CD36 signaling pathway, we stimulated human platelets with oxLDL and determined the relative phosphorylation level of ERK5 by immunoblot. We found that ERK5 was activated by oxLDL in a time- and dose-dependent manner through CD36. To investigate whether ROS can regulate ERK5 activation, we pretreated washed platelets with PEG-catalase before stimulation with oxLDL. ERK5 phosphorylation in the presence of PEG-catalase was inhibited, suggesting that hydrogen peroxide generated through CD36 signaling is critical for ERK5 activation. To test the functional relevance of ERK5 in the CD36-mediated prothrombotic phenotype, we used the small molecule pharmacologic inhibitors of ERK5 activation, BIX02188 and BIX02189, and found that pre-treatment of platelets with these compounds inhibited platelet activation, secretion, and aggregation in response to oxLDL by 50%-90%. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling via increased O₂^●- and hydrogen peroxide. This mechanism is mediated by increased activation of MAPK ERK5 to promote platelet activation and a prothrombotic phenotype.