CHARACTERIZATION OF THE THROMBOXANE SYNTHASE PATHWAY PRODUCT 12-OXOHEPTADECA-5(Z)-8(E)-1O(E)-TRIENOIC ACID AS A THROMBOXANE A(2) RECEPTOR ANTAGONIST WITH MINIMAL INTRINSIC ACTIVITY

Thromboxane synthase forms thromboxane (TX) A(2) and 12(S)-hydroxyhept adeca-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 m etabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive bind ing studies were performed to clarify its mode of action. Oxo-HT was p repared biosynthetically as well as chemosynthetically; purified and c haracterized by gas chromatography and mass spectrometry, Platelet act ivation was assessed by determination of shape change, aggregation, fi brinogen binding and P-selectin expression using optical aggregometry and now cytometry. Oxo-HT 0.1 nM to 50 mu M did not induce platelet ac tivation, Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo-HT inhibited platelet aggre gation, fibrinogen binding and P-selectin expression induced by U46619 in a competitive manner. Schild analysis for U46619-induced fibrinoge n binding and P-selectin expression revealed pA(2) values of 6.1 and 6 .6, respectively which correspond to K-d values of approximately 0.8 m u M and 0.3 mu M, respectively, Oxo-HT also inhibited U46619 induced s hape change (IC50 approximate to 10 mu M). However, Oxo-HT over a conc entration range of 0.1-1 mu M enhanced the partial shape change induce d by low concentrations of U46619. Thus Oxo-HT seems to possess a mini mal agonistic potential, which alone is not sufficient to trigger a pl atelet activation but can enhance low levels of platelet activation. O xo-HT blocked the binding of [H-3]SQ 29548 in a concentration-dependen t manner, whereas HHT did not displace [H-3]SQ 29548. The K-d of Oxo-H T determined from competition binding studies was 7.7 mu M, about 10-2 5-fold higher than the apparent K-d determined by Schild analysis. Thi s discrepancy might be due to a desensitization of the TXA(2) receptor triggered by the minimal intrinsic activity of Oxo-HT. We conclude th at Oxo-HT is a naturally occurring specific TXA(2) receptor antagonist with minimal intrinsic activity. Oxo-HT may contribute to the regulat ion of TXA(2)-induced platelet activation in vivo.