thromboxane-a2 and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.

Topics: Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Cholic Acids; Detergents; Digitonin; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids, Unsaturated; Humans; Hydrazines; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

Topics: Animals; Aorta, Thoracic; Blood Platelets; Cell Membrane; Cells, Cultured; Cholic Acids; Detergents; Endothelium, Vascular; Humans; Iodine Radioisotopes; Kinetics; Muscle, Smooth, Vascular; Prostaglandins H; Radioligand Assay; Rats; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Solubility; Swine; Thromboxane A2

A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k-1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I]-PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation.

Topics: Blood Platelets; Cell Fractionation; Cholic Acids; Chromatography, Gel; Humans; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Solubility; Thromboxane A2