Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.     

Binding of an [125I] labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.     

Binding of a thromboxane A2/prostaglandin H2 agonist [3H]U46619 to washed human platelets

The binding characteristics of [3H]U46619 to washed human platelets were studied. [3H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a Kd of 11 +/- 4 nM (n = 4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a Kd of 20 +/- 7 nM and a Bmax of 9.1 +/- 2.3 fmole/10(7) platelets (550 +/- 141 binding sites per platelet) (n = 4). A number of compounds that act as either agonists or antagonists of the TXA2/PGH2 receptor were tested for their ability to inhibit the binding of [3H]U46619 to washed human platelets. The Kds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [3H]U46619 binds to the putative human platelet TXA2/PGH2 receptor.     

Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes

The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega-tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA-OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor.     

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.     

Isoelectric and mass characterization of human platelet thromboxane A2/prostaglandin H2 receptors

To further characterize the human thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor, preparative isoelectric focusing (IEF) was performed on solubilized platelet membranes. TXA2/PGH2 receptors, assayed by specific binding of the TXA2/PGH2 antagonist [125I]PTA-OH, were electrofocused at pH 5.6. Scatchard analysis of IEF fraction pH 5.6 revealed a 180-fold concentration of TXA2/PGH2 receptors (Bmax = 3650 +/- 228 pM/mg focused, 19 +/- 4 pM/mg unfocused) with no change in binding affinity (Kd = 47 +/- 7 nM focused, 36 +/- 14 nM unfocused). SDS-polyacrylamide gel electrophoresis of photoaffinity-labelled electrofocused receptors revealed concentration of specifically labelled proteins having molecular masses of 49,000 and 27,000 Daltons. These results suggest that the human platelet TXA2/PGH2 receptor has a pI of 5.6, molecular mass of 49,000 Daltons, and may exist as a dimer. Preparative IEF should prove useful in the eventual purification of this receptor.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thromboxane receptors in human kidney tissues.

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Photoaffinity labelling of a thromboxane A2/prostaglandin H2 antagonist binding site in human platelets

The diazonium salt of 9,11-dimethylmethano-11,12-methano-16-(4-aminophenoxy)13,14- dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 (PTA-POA) was synthesized and used as a photoaffinity ligand for the putative human platelet TXA2/PGH2 receptor. Incubation of human platelet membranes with the diazonium salt of PTA-POA followed by photolysis at 290 nm(hv) resulted in a 40% decrease in the specific binding of [125I]PTA-OH as measured in the radioligand binding assay. Co-incubation with a TXA2/PGH2 agonist followed by photolysis resulted in no decrease in specific binding. Incubation of the diazonium salt of PTA-POA with solubilized platelet membranes without photolysis followed by Scatchard analysis resulted in no change in the Kd for [125I]PTA-OH (38 nM) and the preparation which was incubated with the diazonium salt (42 nM). However, the Bmax for [125I]PTA-OH binding was reduced from 2.4 pmole/mg protein for control to 1.4 pmole/mg protein. These studies show that the diazonium salt of PTA-POA may be a useful photoaffinity ligand for human platelet TXA2/PGH2 receptors.     

Selective modulation of the human platelet thromboxane A2/prostaglandin H2 receptor by eicosapentaenoic and docosahexaenoic acids in intact platelets and solubilized platelet membranes.

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.     

Binding of a thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist to rabbit and pig heart membrane protein.

This study aimed at testing the hypothesis that the binding sites for TXA2/PGH2 are present and different in the heart as compared to platelets and blood vessels. Kinetic studies on the thromboxane binding to protein of membrane preparations from rabbit and pig hearts were carried out using [125I]-PTA-OH, a potent specific thromboxane receptor antagonist. The following points are stressed: 1. the binding sites to 125I-PTA-OH were shown to be present in the heart membrane whatever the adopted experimental conditions were i.e. the temperature (4 or 30 degrees C) used for incubation the protein fractions under study: either the 105,000 g supernatant or the 200,000 g supernatant of the solubilized pellet (105,000 g) the animal species: rabbit and pig 2. the radioligand binding was rapid, saturable and reversible 3. the kinetically determined Kd's were in the picomolar range--11 and 14 pM for the rabbit and pig heart membrane preparation respectively--instead of the nanomolar range found in other tissues of the nanomolar range found in other tissues--27-39 nM and 2 nM for human platelets and bovine artery endothelial cells respectively- using the same thromboxane receptor antagonist.     

Characterization of thromboxane A2/prostaglandin H2 receptors and histamine H1 receptors in cultured guinea-pig tracheal smooth-muscle cells.

We characterized thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors and histamine H1 receptors in Guinea-pig cultured tracheal smooth-muscle cells (TSMC). [3H]SQ 29,548 (a TXA2 antagonist)-binding sites were saturable and a high affinity with a dissociation constant of 6.2 +/- 0.60 nM (mean +/- S.E.) and a receptor density of 46 +/- 4.6 fmol/10(6) cells. [3H]SQ 29548 binding was completely inhibited by TXA2 mimetics or antagonists. Intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SQ 29548 binding. [3H]Mepyramine (a H1 antagonist)-binding sites were also present in TSMC. [3H]Mepyramine had a single class of low-affinity-binding sites with a dissociation constant of 2.6 +/- 0.081 microM and a receptor density of 10.6 +/- 0.11 nmol/mg protein. [3H]Mepyramine binding in TSMC membrane was inhibited by H1 antagonists, but not by H2 antagonists. The inhibition constants of mepyramine in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H1 antagonists. All these observations provide evidence that TXA2/PGH2 receptors, mepyramine-binding sites and/or H1 receptors are expressed in cultured TSMC.     

Platelet endoperoxide/thromboxane A2 ( ) receptors in patients with myeloproliferative disorders

In patients with myeloproliferative disorders (MPD) an altered sensitivity of platelets to antiaggregatory prostaglandins and to the endoperoxide analogue U 46619 has been found. In this study we examined U 46619-induced platelet aggregation and binding of the endoperoxide/thromboxane A2 (TXA2) receptor antagonist SQ 29548 in 11 patients with MPD and 11 healthy controls. Although platelet responsiveness to U 46619 was significantly enhanced (p less than 0.05) in MPD, binding affinity and binding capacity of the corresponding endoperoxide/TXA2 receptor were not altered (Bmax 0.67 +/- 0.20 vs. 0.58 +/- 0.14 pmol/10(9) platelets, Kd 0.41 +/- 0.11 vs. 0.55 +/- 0.09 nM). These data exclude the possibility that changes in the presentation of endoperoxide/TXA2 receptors are responsible for the enhanced platelet sensitivity to endoperoxides found in MPD.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

Synthesis and biological activity of 1-phenylsulfonyl-4-phenylsulfonylaminopyrrolidine derivatives as thromboxane a(2) receptor antagonists

The synthesis and biological activity of novel 1-phenylsulfonyl-4- phenylsulfonylaminopyrrolidine analogues are described. All compounds were produced through modification of the substituent formally corresponding to the 1,3-dioxane ring system and the omega-octenol side chain of thromboxane A(2) (TXA(2)), in reference to the structure of Daltroban. Several compounds were found to be potent TXA(2) receptor antagonists. Compound 51a was the most effective inhibitor of 9,11-epoxymethano PGH(2) (U-46619)-induced rat aortic strip contraction (IC(50)=0.48 nM).     

Synthesis and biological activity of 4-methyl-3,5-dioxane derivatives as thromboxane A2 receptor antagonists

The synthesis and biological activity of novel 4-methyl-3,5-dioxane analogues are described. All compounds were produced through modification of the substituent formally corresponding to the omega-octenol side chain of thromboxane A2 (TXA2), in reference to the structure of SQ29548. Several compounds were found to be potent TXA2 receptor antagonists. Compound 8b was the most effective inhibitor of 9,11-epoxymethano PGH2 (U-46619)-induced human platelet aggregation (IC50 = 7.4 nM).     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.