title: Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand.
abstract: 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.
authors.affiliation: Department of Medical Biochemistry, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Matsunobu
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Okuno
authors.forename: Chieko
authors.initials: C
authors.lastname: Yokoyama
authors.forename: Takehiko
authors.initials: T
authors.lastname: Yokomizo
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D044262:Prostaglandin H2 / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000378:metabolism*; D013929:Thromboxane B2 / Q000096:biosynthesis; D013930:Thromboxane-A Synthase / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D008024:Ligands; C412867:Ltb4r2 protein, mouse; D018102:Receptors, Leukotriene B4; D044262:Prostaglandin H2; C008033:12-hydroxy-5,8,10-heptadecatrienoic acid; D013929:Thromboxane B2; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D013930:Thromboxane-A Synthase
keywords: 12-HHT; TxAS; cyclooxygenase; eicosanoid; mass spectrometry