Pharmacologic characterization of human and canine thromboxane A2/prostaglandin H2 receptors in platelets and blood vessels: evidence for different receptors

The present study was undertaken to characterize thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in platelets and blood vessels. Both human and canine platelet aggregation and saphenous vein contractions were induced by the stable TXA2/PGH2 mimetics (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) and 9,11-epithio-11,12-methano-TXA2 (ONO-11113). ONO-11113 was a more potent agonist than U46619 in the human saphenous vein but less potent in the platelet. These agonists were equipotent in the canine platelet but ONO-11113 was more potent in the saphenous vein. Platelet aggregation and saphenous vein contraction induced by U46619 were blocked in a dose-dependent manner by the TXA2/PGH2 receptor agonists 9,11-dimethylmethano-11,12-methano-16-phenyl-13,14-dehydro-13-aza- 15 alpha beta-omega-tetranor-TXA2 (ONO-11120), 9,11-dimethylmethano-11,12-methano-16-(4-hydroxyphenyl)-13,14-d ihy dro-13-aza-15 alpha beta-omega-tetranor-TXA2 (PTA-OH), 9,11-dimethylmethano-11,12-methano-16-(4-methoxyphenyl)-13,14-d ihy dro-13-aza-15 alpha beta-omega-tetranor-TXA2 (PTA-OM), 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl-13,1 4-dihydro-13- aza-15 alpha beta-omega-tetranor-TXA2 (I-PTA-OH) and 9,11-dimethylmethano-11,12-methano-15-phenyl-13,14-dihydro-13-aza- 15 alpha beta-omega-pentanor-TXA2 [PTA-(omega-1)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to washed human platelets

A binding site for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist [125I]9,11-dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 to washed human platelets was studied. 9,11-Dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 competitively antagonized aggregation of washed human platelets induced by the TXA2/PGH2 mimetic U46619. A Schild analysis of the pharmacologic study revealed a pA2 of 8.08 and a slope of -1.12. The pA2 value yielded a Kd of 8 nM. The association rate constant (k1) for [125I]PTA-OH was 6.6 X 10(6)M-1 min-1 and the dissociation rate constant (k-1) was 1.82 X 10(-1), yielding a kinetically determined Kd (k-1/k1) of 27 nM. Scatchard analysis of [125I]PTA-OH binding to washed human platelets revealed one class of binding sites with a Kd of 21 +/- 5 nM and maximum binding of 42 +/- 6.4 fmol/10(7) platelets (N = 5) (2530 +/- 380 binding sites/platelet). Several TXA2/PGH2 receptor agonists and antagonists competed with [125I]PTA-OH for binding. For the four antagonists used in this study, the rank order of potency for displacing the ligand from its binding site correlated (r = 0.93) with the rank order of potency for their ability to inhibit U46619-induced aggregation in human platelet-rich plasma. The antiaggregatory prostaglandins prostaglandin F2 alpha, prostaglandin D2, and Iloprost also displaced the ligand, but only at concentrations considerably higher than that required to produce their pharmacologic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous desensitization of human platelet thromboxane A2/prostaglandin H2 receptors.

Desensitization of platelet thromboxane (TX)A2/prostaglandin (PG)H2 receptors was induced by incubating platelet-rich plasma with the stable PGH2 analog 11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM). Iloprost, a stable prostacyclin analog, was included in the incubation to prevent platelet activation. The TXA2 mimetic, [1S-1 alpha,2 beta(5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), was used to induce platelet aggregation, shape change and increases in intracellular free calcium. The EC50 values for I-BOP-induced rise in intracellular free calcium (control = 10.2 +/- 1.5 nM; desensitized = 79.4 +/- 22.4 nM, n = 6, P less than .05), aggregation (control = 15.8 +/- 2.4 nM; desensitized = 51.7 +/- 11.9 nM; P less than .05, n = 5) and shape change (control = 172 +/- 37 pM; desensitized = 350 +/- 60 pM; P less than .05, n = 7) were increased by the preincubation with U46619. Aggregation responses to thrombin and the calcium ionophore, ionomycin, were unaltered by the preincubation with U46619. Equilibrium binding studies at pH 7.4 revealed a decrease in the number of binding sites for the receptor antagonist 9,11-dimethylmethano-11,12- methano-16(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega- tetranor-TXA2 [125I]PTA-OH) (control = 3246 +/- 509 sites/platelet, desensitized = 2198 +/- 324 sites/platelet, n = 6, P less than .05) without a change in affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human erythroleukemia cells express functional thromboxane A2/prostaglandin H2 receptors

The human erythroleukemia (HEL) cell line is a cultured hematopoietic cell line reported to express platelet membrane glycoproteins and alpha-2 adrenergic receptors. The present studies were designed to determine if functional thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptors exist in HEL cells. Radioligand binding assays were performed using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist. Scatchard analysis revealed one class of binding sites for 1-PTA-OH with a Kd = 122 +/- 18 nM and maximum binding = 1.7 +/- 0.3 x 10(5) sites/cell. Competition for [125I]PTA-OH binding with the TXA2/PGH2 receptor agonists SQ26655 and U46619 revealed one class of binding sites for SQ26655 with a Kd = 17 nM and two classes of binding sites for for U46619 with a Kd = 45 nM for the high-affinity site and a Kd = 450 nM for the low-affinity site. Competition for [125I]PTA-OH by the steroisomeric TXA2/PGH2 receptor antagonists L657925 and L657926 revealed two classes of binding sites for the more potent L657925 with a Kd = 8 nM for the high-affinity site and a Kd = 400 nM for the low-affinity site whereas L657926 bound to one class of sites with a Kd = 380 nM. Stimulation of the TXA2/PGH2 receptor by SQ26655 and U46619 resulted in concentration-dependent increases in [Ca++], as measured by FURA-2 fluorescence, with EC50 values of 28 +/- 2 and 149 +/- 32 nM, respectively. I-PTA-OH, L657925 and L657926 antagonized this response to U46619 with IC50 values similar in rank potency to that seen in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an 125I-labeled thromboxane A2/prostaglandin H2 receptor agonist

Stable synthetic mimetics of thromboxane (TX) A2 and prostaglandin (PG) H2 have been synthesized and reported to stimulate platelets and vascular smooth muscle. The synthetic agonists induce aggregation of isolated platelets and contraction of vascular tissue. The tritiated agonists [3H]U46619 and [3H]U44069 have been used in radioligand binding studies to characterize platelet and vascular smooth muscle TXA2/PGH2 receptors, but have limited usefulness due to their low specific activities and variable specific binding. In an attempt to overcome these problems, we have synthesized a stable, high affinity, 125I-radiolabeled TXA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP). I-BOP induced shape change, increased intracellular free calcium concentrations and aggregated isolated human platelets (EC50 = 0.21 +/- 0.05 nM, n = 3; 4.1 +/- 1.1 nM, n = 4; 10.8 +/- 3 nM, n = 9, respectively). The kinetically determined Kd was 1.02 +/- 0.33 nM (kobs = 0.19 +/- 0.05 min-1, k-1 = 0.097 +/- 0.02 min-1, k1 = 0.119 +/- 0.03 min-1 M, n = 4). Equilibrium binding studies of [125I]BOP to isolated human platelets indicated one class of high affinity sites, Kd = 2.2 +/- 0.3 nM and a maximum binding of 0.028 +/- 0.002 x 10(-12) mol/10(7) platelets (1699 +/- 162 sites/platelet, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)     

7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid: a novel high-affinity radiolabeled antagonist for platelet thromboxane A2/prostaglandin H2 receptors.

A high-affinity thromboxane (TX)A2/prostaglandin (PG) H2 receptor antagonist, I-SAP [7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid] and its radiolabeled analog [125I]SAP (Mais et al., 1991) are characterized in the present study. I-SAP antagonized I-BOP ([1S-(1 alpha, 2 beta(5Z),3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2y l]-5'heptenoic acid) and U46619 [15S-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5Z,13E-dienoic acid)], two different TXA2/PGH2 mimetics, induced aggregation of washed human platelets in a similar manner (pA2 of 8.11 +/- 0.09, Kd = 7.8 nM, n = 3; pA2 = 8.01 +/- 0.05, Kd = 9.7 nM, n = 8, respectively). I-SAP also had agonistic activity, producing platelet shape change (EC50 = 9.7 nM +/- 0.6 nM at pH 7.4, n = 3) which was blocked by pretreatment of platelets with SQ29548 ([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), a TXA2/PGH2 receptor antagonist. Radioligand binding studies were performed with [125I]SAP using washed human platelets. Competition of three agonists and four antagonists for binding with [125I]SAP was determined. The compounds showed the appropriate rank order potencies, including stereoselective competition by a pair of stereoisomeric antagonists. In washed human platelets, the Kd for I-SAP was 468 +/- 49 pM and the maximum binding (Bmax) was 2057 +/- 156 sites/platelet at pH 7.4 (n = 6). The Bmax was significantly increased 49% to 3072 +/- 205 sites/platelet at pH 6.5 (P less than .01 but the Kd was unchanged (490 +/- 18 pM, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiplatelet activity of J78 (2-Chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), an antithrombotic agent, is mediated by thromboxane (TX) A2 receptor blockade with TXA2 synthase inhibition and suppression of cytosolic Ca2+ mobilization

We previously reported that J78 (2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, exhibited a potent antithrombotic effect, which might be due to antiplatelet rather than anticoagulation activity. In the present study, possible anti-platelet mechanism of J78 was investigated. J78 concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/ml), thrombin (0.05 U/ml), arachidonic acid (100 microM), and U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F(2); 1 microM), a thromboxane (TX) A(2) mimic, with IC(50) values of 0.32 +/- 0.01, 0.44 +/- 0.02, 0.50 +/- 0.04, and 0.36 +/- 0.02 microM, respectively. J78 also produced a shift to the right of the concentration-response curve of U46619, indicating an antagonistic effect on the TXA(2) receptor. J78 concentration-dependently inhibited collagen-induced arachidonic acid liberation. In addition, J78 potently suppressed TXA(2) formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner but had no effect on the production of PGD(2), indicating an inhibitory effect on TXA(2) synthase. This was supported by a TXA(2) synthase activity assay that J78 concentration-dependently inhibited TXB(2) formation converted from PGH(2). Furthermore, J78 was also able to inhibit the [Ca(2+)](i) mobilization induced by collagen or thrombin at such a concentration that completely inhibited platelet aggregation. Taken together, these results suggest that the antiplatelet activity of J78 may be mediated by TXA(2) receptor blockade with TXA(2) synthase inhibition and suppression of cytosolic Ca(2+) mobilization.     

Abnormal platelet response to thromboxane A2.

To determine the pathogenetic mechanism of a hereditary primary platelet release disorder, arachidonic acid metabolism via the cyclooxygenase pathway was investigated. The propositus' platelets exhibited defective release reaction and second-wave aggregation when stimulated by sodium arachidonate or U46619, a thromboxane A2 (TXA2) agonist. The lack of platelet response to U46619 suggested that the defect was beyond the thromboxane synthetase level. Furthermore, thromboxane B2 (TXB2) formation in the propositus' platelets (558.52 ng/10(8) platelets) was within the normal range (574.29 +/- SD 27.39 ng/10(8) platelets) and TXA2 formation appeared to be adequate for aggregating normal platelets. The results were indicative of an abnormal platelet response to TXA2. Failure of the propositus' platelets to aggregate in response to TXA2 formed in normal platelet-rich plasma induced by arachidonate confirmed this notion. To gain further insight, platelet cyclic (c) AMP content was determined. Prostacyclin induced a significant elevation of the propositus' platelet cAMP level comparable to normal values. U46619 suppressed prostaglandin I2-induced cAMP elevation in normal subjects but had no such effect in the patient. We conclude that the primary release disorder observed in this kindred is due to an abnormal platelet respnse to TXA2 possibly because of TXA2/PGH2 receptor abnormalities.     

Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A2/prostaglandin H2 receptor interaction

The mechanism by which the active metabolites of arachidonic acid (AA), i.e., thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2) induce platelet aggregation is not understood. Several reports have suggested that AA-stimulated aggregation is mediated by secreted ADP, whereas other studies have proposed that this response is ADP-independent. In the present report, we used the specific TXA2/PGH2 receptor antagonist, 13-azaprostanoic acid (13-APA), and the ADP antagonist, ATP, to examine the contribution of TXA2/PGH2 or secreted ADP to aggregation. We found that 13-APA, but not ATP, deaggregates platelets stimulated by AA or U46619 (a TXA2/PGH2 mimetic). In contrast, ADP-induced aggregation was reversed in response to ATP but not to 13-APA. These results suggest that TXA2/PGH2-stimulated aggregation is mediated through TXA2/PGH2 receptor occupation. Furthermore, secreted ADP does not appear to be required for maintenance of the AA-aggregation response.     

Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes. A radioligand binding study of human platelets.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA2/PGH2 mimetic [125I]BOP to washed human platelets (WP) was investigated. [125I]BOP was competitively displaced from its platelet binding site by stable TXA2/PGH2 analogues. Competition curves were shallow with Hill coefficients of -0.73 +/- 0.05 (P less than 0.001 different from unity) (90 +/- 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with Kd of 234 +/- 103 pM, Bmax of 0.7 +/- 0.3 pM/mg protein (180 +/- 87 sites/WP), and a lower affinity site with Kd of 2.31 +/- 0.86 nM, Bmax of 2.2 +/- 0.3 pM/mg protein (666 +/- 65 sites/WP). [125I]BOP association and dissociation kinetics gave a Kd of 157 pM without evidence of negative cooperativity. The EC50 for I-BOP-induced initial Ca2+ increase was 209 +/- 24 pM, shape change was 263 +/- 65 pM, and aggregation was 4.4 +/- 0.5 nM. Parallel binding studies using the TXA2/PGH2 receptor antagonist [125I]PTA-OH showed a single binding site. The rank order for TXA2/PGH2 analogues to displace [125I]PTA-OH was identical to that for [125I]BOP. These studies indicate that [125I]BOP binds to two distinct sites on human platelets that may represent platelet TXA2/PGH2 receptor subtypes. The close correlation of IC50 values for I-BOP-induced platelet shape change and aggregation with the two Kds for [125I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.     

In vitro characterization of a novel TXA2/PGH2 receptor ligand (S-145) in platelets and vascular and airway smooth muscle.

The stereoisomers of S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor ligand, were compared to TXA2/PGH2 receptor antagonists, SQ29548 and BM13505 in guinea pig platelets, aortas and trachea. Equilibrium binding assays in platelets yielded Kd values (nanomolar) for (+)-S-145 (0.57 +/- 0.04), (-)-S-145 (9.2 +/- 1.3), SQ29548 (11.1 +/- 0.70) and BM13505 (118 +/- 16). In aortas, the corresponding Kb values (nanomolar) were (0.014 +/- 0.002), (1.90 +/- 0.31), (16.8 +/- 3.3) and (142 +/- 29), respectively, whereas in trachea, the Kd values (nanomolar) were (0.019 +/- 0.004), (1.12 +/- 0.18), (1.94 +/- 0.30) and (18.99 +/- 2.59), respectively. S-145 stereoisomers elicited platelet shape change stereoselectively that was characterized by EC50 values 8 to 16-fold higher than the EC50 values for these ligands to block aggregation induced by TXA2/PGH2 mimetic, U44069. S-145 (+)- and (-)-isomers stereoselectively induced transient aortic contraction at concentrations 214,000- and 16,000-fold higher, respectively, than the corresponding Kb values in this tissue. S-145-induced platelet shape change and aortic contraction were inhibitable by low concentrations of SQ29548. We postulate that S-145 may elicit partial agonist activity in platelets and aorta via lower affinity for the active than inactive state of the TXA2/PGH2 receptor in those tissues. S-145 had no agonist activity in isolated trachea possibly indicating different TXA2/PGH2 recognition sites in aorta and trachea or a smaller preligand ratio of active to inactive TXA2/PGH2 receptors in trachea than in aorta.     

Castration reduces platelet thromboxane A2 receptor density and aggregability

BACKGROUND: Exogenously administered testosterone upregulates platelet thromboxane A2 (TXA2) receptors and increases aggregation response to thromboxane mimetics in healthy male volunteers. However, the biological impact of endogenous testosterone on platelet TXA2 receptor expression, especially in older men at risk of coronary artery disease, is unclear. AIM: To investigate the impact of reduction in circulating testosterone on platelet TXA2 receptor expression in older men. DESIGN: Cross-sectional case-control study. METHODS: We studied surgically and/or medically castrated men with prostate cancer (group A, n = 8, aged 71 +/- 8 years) and age-matched, uncastrated urology patients (group B, n = 7, aged 67 +/- 9 years). Plasma testosterone was measured by radioimmunoassay. Platelet TXA2 receptor expression was assessed by radioligand binding studies using radioactive 125I-BOP. Platelet aggregation responses to TXA2-mimetic I-BOP, and to thrombin, were also studied. RESULTS: Group A had significantly lower plasma testosterone than group B (16 +/- 5 ng/dl vs. 308 +/- 47 ng/dl, p<0.001). Platelet TXA2 receptor density (B(max)) but not affinity (K(d)) was lower in group A (0.50 +/- 0.12 vs. 1.01 +/- 0.17 pmol/mg protein, p = 0.03). Maximum platelet aggregation response to I-BOP (E(max)), but not sensitivity (EC50) was lower in group A (53 +/- 2% vs. 63 +/- 2%, p = 0.003 ANOVA). In vitro, high concentrations of hydroxyflutamide (100 microM) competitively inhibited U46619-induced platelet aggregation in washed platelets, without affecting the binding of 125I-BOP to platelet TXA2 receptors. DISCUSSION: Endogenous testosterone regulates platelet TXA2 receptor B(max) and the E(max) aggregation response to thromboxane mimetic I-BOP. Blockade of androgen receptors or inhibition of testosterone production may reduce platelet aggregation responses. Preliminary evidence suggests the presence of functional androgen receptors on human platelets, which may regulate TXA2 receptor expression.     

Platelet thromboxane A2 receptors in type I diabetes.

1. Human platelet thromboxane A2 receptor expression on the membrane surface is possibly dynamically regulated by changes either in the ligand concentration or in membrane fluidity. An increased thromboxane A2 production and a decreased membrane fluidity has been reported in diabetic patients. 2. In the present study the binding characteristics of platelet thromboxane A2 receptors have been investigated in nine diabetic patients (type I) and in 15 healthy control subjects by a radioligand-binding method using 9,11-dimethylmethane-11,12-methane-16-[3-125I-4-hydroxyphenyl]-13, 14- dihydro-13-aza-15-tetranor-thromboxane A2 as the radiolabelled ligand. 3. The maximum concentration of binding sites was 163 (SD 35) fmol/10(8) platelets (n = 15) with 987 (SD 209) receptors/platelet in controls, whereas in diabetic patients the maximum concentration of binding sites was 74.2 (SD 28) fmol/10(8) (n = 9) with 447 (SD 172) receptors/platelet (P less than 0.001). The dissociation constants were 18 (SD 4) nmol/l and 21 (SD 6) nmol/l (not significant) in control subjects and in diabetic patients, respectively. Glycated haemoglobin, which is reported to reduce membrane fluidity, was found to be negatively correlated (r = 0.60, P less than 0.05) with thromboxane A2 receptor number in the diabetic patient group. On the contrary, a positive linear correlation between the equilibrium dissociation constant and glycated haemoglobin was found in diabetic patients (r = 0.75, P less than 0.01).     

Endogenous vasoconstrictor prostanoids: role in serotonin and vasopressin-induced coronary vasoconstriction

A vasoconstrictor-induced prostacyclin (PGI2) production in a perfused rat heart was found, suggesting a mitigating role of PGI2 on coronary vasoconstriction. Treatment of the heart with cyclooxygenase inhibitors (aspirin or indomethacin) decreased PGI2 production by more than 90% and paradoxically reduced the vasoconstriction response. The attenuating effect of cyclooxygenase blockade suggested that endogenous prostanoids contribute to serotonin-, vasopressin- or U46619-induced vasoconstriction. Two prostaglandin (PG) H2/thromboxane A2 (TXA2) receptor antagonists, i.e., 13-azaprostanoic acid (13-APA) and SQ 29,548 were used to investigate putative endogenous vasoconstrictor prostanoids on the exogenously induced vasoconstriction. Retrogradely perfused (5-6 ml/min) rat hearts were rendered guiescent, yet responsive to stimuli, by local injection of lidocaine to the atrioventricular node. Changes in coronary vascular resistance (i.e., perfusion pressure at constant flow) were monitored and the cardiac effluent was collected for analysis of 6-keto PGF1 alpha (the stable metabolite of PGI2) as well as PGF2 alpha by radioimmunoassay. Three vasoconstrictors, i.e., serotonin, vasopressin and the TXA2/PGH2 analog U46619, as well as authentic PGD2, PGE2 and PGF2 alpha were infused. PGD2, PGE2 and PGF2 alpha exerted a dose-related coronary vasoconstriction, as did U46619, serotonin and vasopressin. Treatment with 13-APA (100 microM) or SQ 29,548 (100 nM) almost abolished U46619-induced vasoconstriction. The addition of PGH2/TXA2 receptor antagonists also significantly reduced the pressor effect of exogenously administered PGs, serotonin and vasopressin, with the exception that SQ 29,548 did not significantly antagonize PGE2-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thromboxane agonist (U46619) potentiates norepinephrine efflux from adrenergic nerves

The effect of the synthetic thromboxane/prostaglandin (PG) H2 agonist U46619 on the electrically stimulated rabbit isolated was deferens was examined to test for thromboxane influences on adrenergic nerves. U46619 effects on force generation, [3H] norepinephrine release and norepinephrine-induced contractions were assessed to determine the mechanism of action. U46619 maximally enhanced adrenergic force generation 135 +/- 24% at a concentration of 100 nM. U46619 potentiated maximal contractile effects of exogenously administered norepinephrine 16 +/- 4% and augmented [3H]norepinephrine release from electrically stimulated preparations 142 +/- 44%. A competitive thromboxane/PGH2 receptor antagonist, SQ29548, significantly shifted the concentration-response curve for U46619 to the right in a concentration-dependent manner and blocked U46619-induced tritium release. Thus, U46619 appears to potentiate neurotransmitter release by interacting with thromboxane/PGH2 receptors. Because SQ29548 did not prevent the potentiation of norepinephrine contractions by U46619, the postjunctional effect may be independent of thromboxane/PGH2 receptors. We interpret these results to be indicative of both pre- and postjunctional sites of action of U46619. The physiological importance of these thromboxane effects is unknown currently.     

Novel angiotensin II AT(1) receptor antagonist irbesartan prevents thromboxane A(2)-induced vasoconstriction in canine coronary arteries and human platelet aggregation

This study was conducted to investigate whether the novel orally active nonpeptide angiotensin II (Ang II) AT(1) receptor antagonist irbesartan interacts with the thromboxane A(2)/prostaglandin endoperoxide H(2) (TxA(2)/PGH(2)) receptor in canine coronary arteries and human platelets. Coronary artery rings were isolated from male dog hearts (n = 18) and isometric tension of vascular rings was measured continuously at optimal basal tension in organ chambers. Autoradiographic binding of [(3)H]SQ29,548, a TxA(2) receptor antagonist, in canine coronary sections was determined. Blood for platelet aggregation studies was collected by venous puncture from healthy human volunteers (n = 6) who were free of aspirin-like agents for at least 2 weeks. Vascular reactivity and platelet aggregation in response to the TxA(2) analogs U46619 and autoradioagraphic receptor binding to the TxA(2) receptor antagonist [(3)H]SQ29,548 were studied with and without irbesartan. The TxA(2) analog U46619 produced dose-dependent vasoconstriction in coronary rings (EC(50) = 11.6 +/- 1.5 nM). Pretreatment with irbesartan inhibited U46619-induced vasoconstriction, and the dose-response curve was shifted to the right in a dose-dependent manner. The EC(50) of U46619 was increased 6- and 35-fold in the presence of 1 and 10 microM of irbesartan without a change of maximal contraction. At 1 microM, irbesartan is 2-fold more potent than the AT(1) receptor antagonist losartan in the inhibition of U46619-induced vasoconstriction in canine coronary arteries. In contrast, neither AT(1) receptor antagonists (CV11974 and valsartan), the AT(2) receptor antagonist PD123319, nor the angiotensin converting enzyme inhibitor lisinopril had any effect on U46619-induced coronary vasoconstriction. Irbesartan did not change potassium chloride-induced vasoconstriction; however, irbesartan did inhibit the vasoconstriction mediated by another TxA(2)/PGH(2) receptor agonist prostaglandin F(2alpha) (PGF(2alpha)). Neither the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin had any effect on irbesartan's attenuation of U46619-induced vasoconstriction. Irbesartan specifically reversed U46619-preconstricted coronary artery rings with and without endothelium in a dose-dependent manner. Irbesartan at high concentrations significantly competed for [(3)H]SQ29,548 binding in canine coronary sections. U46619 stimulated dose-dependent human platelet aggregation of platelet-rich plasma. Preincubation with irbesartan significantly inhibited platelet aggregation in a concentration-dependent manner. In conclusion, the dual antagonistic actions of irbesartan by acting at both the AT(1) and TxA(2) receptors in blood vessels and platelets may overall enhance its therapeutic profile in the treatment of hypertension, atherosclerosis, and arterial thrombosis.     

Bacterial sepsis-induced decrease in lung vascular reactivity to 9,11-dideoxy-11a9a-epoxymethano-prostaglandin F2 alpha (U46619) in the rat

This study determined whether a sepsis-associated increase in cyclooxygenase products altered the pulmonary vascular response to the thromboxane A2 mimic, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2 alpha (U46619). Rats were anesthetized (50 mg/kg of sodium pentobarbital i.p.), and sepsis was induced by cecal ligation and puncture. Four hours later, pulmonary effluent immunoreactive thromboxane (iTXB2) levels were significantly increased (156.8%) and pulmonary vascular reactivity to U46619 (50-200 ng) was significantly (P less than .05) decreased compared to lungs from nonseptic controls. This decreased vascular reactivity was not seen in lungs from cecally ligated rats challenged with angiotensin II (5-200 ng). Sham surgery did not alter pulmonary iTXB2 synthesis nor did it result in a depressed vascular response to U46619. Rats pretreated with ibuprofen (15 mg/kg i.v.) did not show the sepsis-associated increase in iTXB2 levels nor was a decrease in pulmonary vascular reactivity to U46619 observed. These data indicate that a sepsis-associated increase in TXA2 and/or other cyclooxygenase products can alter the pulmonary vascular response to the TXA2 mimic, U46619.     

Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC(50), 7.9 and 17.1 microM, respectively), an effect abolished by 10 microM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC(50), 9.8 microM) and TXB(2) formation (-71% at 10 microM), and its potency increased in the presence of aortic rings (EC(50), 1.4 microM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA(2) formation, reduced by 2NTX-99 (10-40 microM): contraction, -28 and -47%, TXA(2) formation, -37 and -75.4%, respectively, with concomitant increase in PGI(2). 2NTX-99 (20-40 microM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 +/- 6.7 and -96 +/- 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 +/- 10 and -79.2 +/- 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA(2) receptor isophorm alpha receptor, 2NTX-99 did not compete with the ligand, [(3)H]SQ29,548 ([(3)H][1S-[1alpha,2beta(5Z),3beta,4alpha]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA(2) production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/microg/ml and a t(1/2) of 6 h were calculated, with a C(max) value of 31.8 +/- 8.2 microg/ml. An excellent correlation between plasma concentrations and TXA(2) inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential.     

Increased platelet aggregation due to chilling to 20 degrees C is not related to increased sensitivity to agonists

BACKGROUND: Previous studies suggested that chilled platelets have a greater sensitivity to agonists than do platelets aggregated at 37 degrees C. STUDY DESIGN AND METHODS: Washed platelets were aggregated at 20 or 37 degrees C with ADP (0-20 microM), arachidonic acid (0-200 microM), or the thromboxane mimetic U46619 (0-9 nM). RESULTS: Chilling caused a significant (p < or = 0.05) increase in spontaneous platelet aggregation (> 27% at 20 degrees C vs < 5% at 37 degrees C) and spontaneous dense granule release (> 0.01 nM of ATP at 20 degrees C vs. 0 nM of ATP at 37 degrees C), ADP and U46619 caused a significantly greater aggregation response and dense granule release at 20 degrees C, although there was no change in agonist sensitivity of platelets (effective dose of agonist necessary to induce 50% aggregation [ED50]: 1.00 +/− 0.35 microM ADP at 20 degrees C and 1.63 +/− 0.47 microM ADP at 37 degrees C; 8.26 +/− 3.65 pM U46619 at 20 degrees C and 18.97 +/− 4.82 pM U46619 at 37 degrees C). Platelets aggregated with arachidonic acid showed a significant decrease in aggregation and agonist sensitivity at 20 degrees C (ED50 118.7 +/− 44.4 microM) from those at 37 degrees C (ED50 25.6 +/− 7.2 microM), possibly as a result of the reduced enzymatic activity of cyclooxygenase and thromboxane synthase at the lower temperature. CONCLUSION: The data suggested that washed platelets chilled to 20 degrees C and aggregated are not more sensitive to agonists than are 37 degrees C controls, but rather that chilled platelets undergo greater spontaneous aggregation.