CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a "pan" caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions.