Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A₂ (TxA₂) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB₂, a stable metabolite of TxA₂. TxB₂ production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80-90%. Ozagrel-treated blood also exhibited accumulation of PGD₂ and PGE₂, possibly resulting from the shunting of PGH₂ into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB₂ production during blood coagulation was completely lost, but 12-HHT production was reduced by 80-85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB₂ and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB₂. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo.