Activation of platelet 12-lipoxygenase (12-LO) generates large quantities of 12(S)- HETE, an oxidized metabolite of arachidonic acid. For decades, the function of this lipid was unknown. Recently, we demonstrated that platelets undergo oxidative fragmentation when exposed to nanomolar concentrations of 12(S)-HETE (JCI43:973, 2004). 12-LO activation occurs upon binding of human or rabbit anti-platelet GPIIIa to a specific platelet epitope GPIIIa49-66 (Cell106:551, 2001) which is regulated by a specific IgG conformation (JBC283: 3224, 2008). Fragmentation requires activation of the NADPH oxidase pathway downstream of the release of 12(S)-HETE since NADPH oxidase deficient (gp91 phox −/−) platelets fail to respond to exogenous 12(S)-HETE, whereas 12-LO−/− platelets are fully reactive. Since several lipid mediator receptors have been identified, including the leukotriene BLT1 and BLT2 receptors, we reasoned that the stereospecific action of the 12(S)-HETE was also likely mediated by a G-protein coupled receptor (GPCR). Recent data published in an abstract has suggested that some tumor cells might respond to 12(S)- HETE through the orphan GPCR, GPR31 (Guo, et al., IMPaCT, 2007). We searched for evidence of this receptor in platelets and in HUVEC. GPR31 mRNA was found in highly purified platelet and HUVEC preparations by RT-PCR. Further, incubation of gel filtered platelets with [³H]12(S)-HETE demonstrated saturation binding that was 30- fold above background. Binding of the radioligand was inhibited by a 100-fold molar excess of non-radioactive 12(S)-HETE, but was not effected by addition of 12(R)-HETE, a stereoisomer of the 12-LO product (n=3). In two experiments the K_d for 12(S)-HETE binding to platelets was ~20 nM with a B_max of ~16 fmol/10⁶ platelets. Oxidative platelet fragmentation induced by anti-GPIIIa49-66 Ab is a 12(S)-HETE-dependent process. We now show that this can be inhibited by 13(S)-HODE with an IC50 of ~400 nM (n=3). 13- HODE has been suggested to be a competitive inhibitor of 12(S)-HETE (Pidgeon, et al., Cancer Metastasis Rev26:503, 2007). CHO cells which do not express an endogenous 12-(S)-HETE receptor were transfected with a GPR31-bearing plasmid. [³H]12(S)-HETE binding to GPR31-CHO cells was dose-dependent and saturable while the radioligand did not bind to mock transfected cells. Radioligand binding to the GPR31-CHO cells was inhibited by both 13(S)-HODE as well as cold 12(S)-HETE with an IC50 of <50 nM. No inhibition was noted with 100-fold molar excess of the inactive stereoisomer, 12(R)- HETE. Employing a peptide phage display library with GPIIIa49-66 as bait, we have recently found 70% homology with a peptide within the C-terminal end of ADAMTS-18 (a Disintegrin Metalloproteinase with Thrombospondin Motifs which is present in endothelial cells). An active 45kD C-terminal fragment is formed from ADAMTS-18 after thrombin cleavage. This fragment binds to platelets and induces oxidative fragmentation via the activation of 12(S)-HETE synthesis (Blood110:127A, 2007). Fragmentation was inhibited by an anti-ADAMTS-18 Ab against the C-terminal end of the protein, as well as a C-terminal 18-mer peptide that mimics the platelet binding region of ADAMTS-18 (n=6). We next tested the ability of 12(S)-HETE to stimulate the release of ADAMTS-18 from HUVEC. Employing a highly sensitive immunoassay for ADAMTS-18, we observed a constitutive secretion of ADAMTS-18 that was enhanced ~15-fold with 12(S)-HETE (300 nM), but not by 12(R)-HETE (n=6). ADAMTS-18 secretion was also inhibited from 10- to 15-fold by 13-HODE (300 nM) as well …