Alox15 via H₂O₂ mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation

Thromboxane prostanoid (TP) receptors play an important role in platelet activation and hemostasis. Although TP receptors undergo palmitoylation within the platelet proteome, its impact on platelet functions and the underlying mechanisms have not been explored. To this end, we report for the first time that Alox15 (aka 12/15-lipoxygenase or 12/15-LOX) mediates TP receptor palmitoylation and its membrane trafficking, influencing platelet activation and hemostasis in response to 10,10-difluorothromboxane A2 (F₂-TXA2), a stable analog of thromboxane A2 (TXA2). We also found that activation of protein kinase Cθ (PKCθ) was required for F₂-TXA2-induced TP receptor palmitoylation downstream to 12/15-LOX. Furthermore, genetic deletion of 12/15-LOX, or pharmacological blockade of PKCθ activation or protein palmitoylation diminished platelet adhesion, aggregation, and spreading resulting in prolonged bleeding and delayed clotting and clot-retraction times. In addition, our findings reveal for the first time a novel role for xanthine oxidase (XO)-dependent H₂O₂ production in 12/15-LOX-mediated F₂-TXA2-induced PKCθ activation, TP receptor palmitoylation, and its membrane trafficking leading to platelet activation and hemostasis.