Thromboxane synthase forms thromboxane (TX) A2 and 12(S)-hydroxyheptadeca-5(Z)-8(E)-10(E)-trienoic acid (HHT) at equimolar amounts. Twelve-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid (Oxo-HT) is the primary metabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive binding studies were performed to clarify its mode of action. Oxo-HT was prepared biosynthetically as well as chemosynthetically, purified and characterized by gas chromatography and mass spectrometry. Platelet activation was assessed by determination of shape change, aggregation, fibrinogen binding and P-selectin expression using optical aggregometry and flow cytometry. Oxo-HT 0.1 nM to 50 micro M did not induce platelet activation. Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo-HT inhibited platelet aggregation, fibrinogen binding and P-selectin expression induced by U46619 in a competitive manner. Schild analysis for U46619-induced fibrinogen binding and P-selectin expression revealed pA2 values of 6.1 and 6.6, respectively, which correspond to Kd values of approximately 0.8 micro M and 0.3 micro M, respectively. Oxo-HT also inhibited U46619 induced shape change (IC50 [approximately =] 10 micro M). However, Oxo-HT over a concentration range of 0.1-1 micro M enhanced the partial shape change induced by low concentrations of U46619. Thus Oxo-HT seems to possess a minimal agonistic potential, which alone is not sufficient to trigger a platelet activation but can enhance low levels of platelet activation. Oxo-HT blocked the binding of [(3) H]SQ 29548 in a concentration-dependent manner, whereas HHT did not displace [(3) H]SQ 29548. The Kd of Oxo-HT determined from competition binding studies was 7.7 micro M, about 10-25-fold higher than the apparent Kd determined by Schild analysis. This discrepancy might be due to a desensitization of the TXA2 receptor triggered by the minimal intrinsic activity of Oxo-HT. We conclude that Oxo-HT is a naturally occurring specific TXA2 receptor antagonist with minimal intrinsic activity. Oxo-HT may contribute to the regulation of TXA2-induced platelet activation in vivo.