ALOX15 mediates thrombin-induced myristoylation, trafficking, and interaction of protease-activated receptors, leading to platelet activation and hemostasis

The role of 12/15-lipoxygenase (12/15-LOX; polyunsaturated fatty acid Lipoxygenase ALOX15) in platelet function is not known. Using knockout mice as well as pharmacological and molecular approaches, here we show for the first time that genetic deletion of 12/15-LOX suppresses thrombin-induced proteinase-activated receptor 4 (PAR4) myristoylation and trafficking, causing a reduction in its interaction with proteinase-activated receptor 3 (PAR3) in mouse platelets. We also found that 12/15-LOX, via activation of protein kinase C theta (PKCθ), mediates N-myristoyltransferase 1 (NMT1) phosphorylation, which modulates thrombin-induced PAR4 myristoylation, trafficking, and its interaction with PAR3, leading to PAR3/4 heterodimer formation in mouse platelets. In line with these observations, genetic deletion or inhibition of 12/15-LOX or pharmacological blockade of PKCθ or NMT1 attenuated thrombin-induced PAR4 myristoylation, trafficking, and its interaction with PAR3, leading to reduced platelet activation in mouse platelets. Similarly, mice with genetic deletion or inhibition of 12/15-LOX or blockade of PKCθ or NMT1 exhibited a prolonged bleeding time and delayed clotting and clot retraction times. Intriguingly, we found that Thrombin induces myristoylation, trafficking, and thereby dimerization of both PAR1 and PAR4 in human platelets, and inhibition of 15-LOX1, the human orthologue of murine 12/15-LOX, attenuated these effects. Following these observations, inhibition of 15-LOX1 blunted thrombin-induced platelet activation and clot retraction in humans. Thus, our findings unfold an essential role for 12/15-LOX in thrombin-induced platelet activation and hemostasis.

12/15‐lipoxigenase; hemostasis; myristoylation; protease‐activated receptor; protein kinase C.