Platelet CD36 Potentiates Thrombus Formation in Hyperlipidemic Conditions By Activating Redox Sensitive MAP Kinase ERK5

Atherosclerotic plaque instability is a pathological process that can lead to ischemic emergencies, such as myocardial infarction (MI) and stroke. Thrombosis in this context is promoted by circulating oxidized lipids present in LDL particles (oxLDL), which are generated during plaque formation. These particles are recognized by scavenger receptor CD36 present on platelets. CD36 binding to oxLDL lowers the threshold for platelet activation by recruitment and activation of Src kinases Fyn and Lyn, followed by signaling including generation of reactive oxygen species (ROS) through NADPH oxidase (Nox). Although these effectors are important for the prothrombotic properties of CD36, the downstream signaling that links CD36 to classic agonist-induced platelet activation pathways is incompletely defined. We hypothesize that platelet CD36 promotes thrombosis by generating specific ROS to modulate critical redox-sensitive signaling pathways in hyperlipidemia. Platelet MAP kinase ERK5 is a redox sensor that was shown to be required for optimal platelet activation in MI, a condition with greatly elevated ROS. We previously reported that oxLDL-CD36 signaling leads to the generation of the specific ROS superoxide radical anion (O₂^●-) and that O₂^●-/hydrogen peroxide (H₂O₂) are important mediators of platelet aggregation. Additionally, we reported that platelet ERK5 was activated by oxLDL in a CD36-dependent pathway requiring Src kinases, Nox and O₂^●-/H₂O₂ to promote platelet activation. We now report the function of platelet ERK5 in this context by performing an ex vivo microfluidic thrombosis assay in which mice whole blood is perfused over immobilized collagen. Platelet adhesion and accumulation were visualized in real time via mepacrine-tagged platelets. We found that stimulating whole blood from wild type C57Bl6 mice with oxLDL promoted platelet adhesion and accumulation by 12.9% compared to buffer stimulation (p=0.033). Whole blood from CD36 null mice showed indistinguishable platelet adhesion and accumulation when stimulated with oxLDL or buffer, suggesting CD36-dependency. We used the platelet ERK5 null mice, which was generated by crossing ERK5^flox mice with PF4-cre+ mice (ERK5^flox/PF4-cre+), and showed that platelet adhesion and accumulation by oxLDL was abrogated compared to control ERK5^flox mice. Additionally, the in vivo relevance of platelet ERK5 activation by CD36 was determined by performing a novel collagen-mediated murine thrombosis assay. This assay is dependent on syngeneic transplantation of the epigastric artery into the carotid artery, where the collagen-rich outer adventitial layer is exposed to blood flow, thus generating a thrombus. Platelets and fibrin accumulation were visualized in real time by Rhodamine 6G and fluorophore-tagged anti-fibrin antibody, respectively. We transplanted bone marrows from donor ERK5^flox mice or ERK5^flox/PF4-cre+ mice into recipient atherogenic apoE null mice and generated hyperlipidemia by feeding the mice a high fat diet (HFD). ApoE null mice with ERK5^flox bone marrows (apoE:ERK5^floxBM) on HFD developed rapid platelet accumulation with subsequent thromboembolisms compared to apoE null mice with platelet ERK5-null bone marrows (apoE:ERK5^flox/PF4-cre+BM) (p<0.01 at 10 min), demonstrating that this model is sensitive to detect a prothrombotic phenotype in diet-induced hyperlipidemia. Chow fed mice do not show potentiation of platelet accumulation in both apoE chimeras (p=0.56). Additionally, HFD-fed apoE:ERK5^floxBM showed accelerated fibrin accumulation compared to apoE:ERK5^flox/PF4-cre+BM, suggesting a role for CD36-ERK5 signaling in platelet procoagulant properties. Control diet-fed apoE null chimeras have indistinguishable fibrin accumulation kinetics. Subsequent studies to determine the mechanism for fibrin accumulation (via fluorophore-tagged Annexin V binding) showed that exposure of platelets to oxLDL induced dose-dependent phosphatidylserine (PS) exposure, a mechanism required for platelets to promote coagulation. Buffer or LDL controls had no effect. A CD36 blocking antibody, FA6, was able to abrogate oxLDL-mediated PS exposure compared to control antibody (p=0.041). These findings suggest that platelet CD36 potentiates thrombosis through a specific redox-regulated pathway requiring ERK5 and that ERK5 is a critical modulator of thrombosis in hyperlipidemic conditions.