13-azaprostanoic acid (13-APA) acts as a direct antagonist of the human blood platelet thromboxane/endoperoxide receptor. Previous studies have suggested that arachidonic acid (AA) metabolites e.g. thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) can mobilize Ca2+ from Ca2+ accumulating vesicles derived from the platelet dense tubular system. In the present study we investigated the effect of 13-APA on this Ca2+ mobilization process. Platelet vesicles were prepared as previously described with the exception that oxalate was not incorporated into the vesicles as a precipitating anion. 45Ca uptake was determined by millipore filtration and isotope counting of the filter paper. 3H-sucrose was used as an index of extravesicle Ca2+ . TXA2 production by the vesicles was measured by radioimmunoassay of TXB2. Vesicles were incubated with 5 × 10-5M Ca2+ before addition of AA. Ca2+ uptake and TXB2 production were determined before and at 3, 9, and 16 mm following the addition of AA. It was found that the vesicles accumulated 10 nmoles of Ca2+ per mg of protein under steady state conditions. Addition of A A (2.5 × 10-5 M) resulted in the release of 19 ± 6.2%, 34 ± 4.8% and 42 ± 5.1% of accumulated Ca2+ at 3, 9 and 16 min respectively. Pretreatment of the vesicles with indomethacin (4 × 10-6M) completely inhibited AA- induced release and reduced TXB2 production by 82%. Pretreatment of the vesicles with 13-APA (2 × 10-5M) also completely inhibited Ca2+ release . In this case, however, there was no inhibition of TXA? synthesis, i.e. 56 ± 9 ng TXB? per mg protein in the control ana 64 ± 12 ng TXB2 per mg protein with 13-APA. These results confirm previous studies that AA must be metabolized to TXA2/PGH2 in order to release Ca2+ from isolated membrane vesicles. The finding that 13-APA blocks TXA2/ PGH2 stimulated release of Ca2+ suggests that this release process is mediated through a specific receptor interaction. (Supported by a grant in aid AHA 79895.)
Thromboxane A2 receptor in human blood platelet 2. Synthesis of photoaffinity labels
