The role of thromboxane A2 in reperfusion injury

Thromboxane A2 (TXA2) receptor antagonists can limit infarct size in models of coronary occlusion and reperfusion, but it was unknown if these compounds can mitigate reperfusion injury. Anesthetized open chest dogs were subjected to left circumflex coronary (LCX) occlusion for 90 min. Two minutes before reperfusion, the dogs were given iv saline (0.9% NaCl) or the TXA2 antagonist SQ 29,548 (0.2 mg/kg + 0.2 mg/kg/hr). Reperfusion was instituted for 5 hr at which time infarct size was determined. Regional myocardial blood flow was determined before, during, and after occlusion. SQ 29,548 treatment resulted in a significant reduction in infarct size (57 +/- 7 and 34 +/- 8% of the left ventricular area at risk infarcted in the saline and SQ 29,548 groups, respectively). No differences in collateral flow during occlusion were observed between groups, but SQ 29,548 treatment resulted in a significantly higher subendocardial reperfusion flow (54 +/- 10 and 93 +/- 14 ml/min/100g for the saline and SQ 29,548 groups, respectively). Thus, TXA2 seems to play a role in exacerbating reperfusion injury and TXA2 receptor blockade may have potential as a mode of therapy for ischemia-reperfusion damage.     

The role of thromboxane A2 [TxA2] in liver injury in mice

The role of thromboxane A2 (TxA2) in CCl4-induced liver disease was investigated in mice. Significant elevation of TxB2 in the liver was observed 6 hours after the injection of CCl4. Administration of OKY-046, a selective TxA2 synthetase inhibitor (10 and 50 mg/kg) and ONO-3708, a TxA2 receptor antagonist, (0.5, 1 and 2 mg/Kg) suppressed the elevation of serum GOT and GPT levels and histopathological changes of the liver. In addition, OKY-046 inhibited the elevation of TxB2 in the liver. When U-46619, a stable TxA2 mimetic was injected i.v. into the mice, clear elevation of serum GOT and GPT levels and histopathological score of the liver were observed. These results suggest that TxA2 play a role for the onset of CCl4-induced liver injury in mice.     

Nitric oxide in CsA-induced hypertension: role of beta-adrenoceptor antagonist and thromboxane A2

Cyclosporine A (CsA) is an immunosuppressive agent, which also causes hypertension. The effect of CsA on vascular responses was determined in Sprague-Dawley rats and rat aortic rings. Male rats weighing 250-300 g were given either CsA (25 mg/kg/day) in olive oil or vehicle by intraperitoneal (ip) injection for 7 days. CsA administration produced a 42% increase (P < 0.001) in mean arterial pressure (MAP) which reached a plateau after 3 days. The level of both nitrate/nitrite (NO2/NO3), metabolites of nitric oxide (NO), decreased by 50% (P < 0.001), but the level of thromboxane A2 (TBXA2) increased by 75% (P < 0.001), in the urine. When 10(-9) M of CsAwas added acutely to intact aortic rings from untreated rats, NO2/NO3 production decreased by 83% (P < 0.011), but TBXA2 production increased by 86% (P < 0.001). The effects of CsA were reversed both in vivo and in vitro by pretreatment with propranolol (15 mg/kg/day ip), beta-adrenoceptor antagonist. There were no changes in MAP and tension in rats treated with prop alone. In addition, in aorta of rats that were treated with CsA ip for 7 days, CsA significantly activated protein kinase C (PKC) translocation. This suggests that PKC mediate, in part, CsA-induced hypertension. In summary, CsA inhibits endothelial NO formation, activate PKC, and increaseTBXA2 production, with resulting increase in MAP, and this changes can be overcome by pretreatment with propranolol.     

The role of protein kinase C in thromboxane A2-induced pulmonary artery vasoconstriction

In order to determine if protein kinase C (PKC) plays a significant role in the stimulant action of thromboxane A₂ (TxA₂) on pulmonary vascular smooth muscle, TxA₂-induced contractile responses were measured following inhibition of PKC. Rabbits were sacrificed and segments of the main trunk of the pulmonary artery were removed and placed within a temperature-controlled (37 °C) organ bath. Contractile responses that were evoked by a TxA₂ mimetic (U46,619, 0.5 µM) decreased by 27 and 35% following treatment with the PKC inhibitors, calphostin C (2 µM) and staurosporine (200 nM), respectively. These results account for the effect of the vehicle, DMSO, which was also found to have a concentration-dependent inhibitory effect on the U46,619-induced contractions. The effects of DMSO alone was subsequently subtracted from the previously measured responses to PKC inhibitors that were dissolved in DMSO to obtain effects attributable to the PKC inhibitor alone. It can therefore be concluded that inhibition of PKC results in partial attenuation of U46,619-induced responses supporting the hypothesis that activation of PKC plays a partial role in TxA₂-induced contraction of pulmonary arterial smooth muscle.     

A possible role of thromboxane A2 (TXA2) and prostacyclin (PGI2) in circulation.

Recently two local hormones, thromboxane A2 (TXA2) and prostacyclin (PGI2) have been discovered. These hormones are labile metabolites of arachidonic acid. TXA2 is generated by blood platelets, while PGI2 is produced by vascular endothelium. TXA2 is a potent vasoconstrictor. It also initiates the release reaction, followed by platelet aggregation. PGI2 is a vasodilator, especially potent in coronary circulation. It also inhibits platelet aggregation by virtue of stimulation of platelet adenyl cyclase. Common precursors for both hormones are cyclic endoperoxides PGG2 and PGH2, being formed by cyclooxygenation of arachidonic acid. This last enzymic reaction is more efficient in platelets than in vascular endothelium, and therefore the generation of PGI2 by vasuclar wall is accelerated by an interaction between platelets and endothelial cells. During this interaction platelets supply the endothelial PGI2 synthetase with their cyclic endoperoxides. The newly formed PGI2 repels the platelets from the intima. When PGI2 synthetase is irreversibly inactivated by low concentration of lipid peroxides, then the platelets are not rejected but stick to the endothelium, generate TXA2 and mature thrombi are formed. A balance between formation and release of PGI2, TXA2 and/or cyclic endoperoxides in circulation is of utmost importance for the control of intra-arterial thrombi formation and possibly plays a role in the pathogenesis of atherosclerosis.     

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.     

Production of platelet thromboxane A2 inactivates purified human platelet thromboxane synthase.

Human platelet thromboxane synthase was partially purified by DEAE-cellulose, Affi-Gel Blue, and Sephacryl S-300 chromatography to a specific activity of 259 nmol of thromboxane B2/min per mg. Thromboxane synthase retained 75-90% of its enzymic activity when bound to phenyl-Sepharose. The immobilized enzyme was inactivated at pH 3.0 and inhibited by 1-benzylimidazole and U-63,557A. The ability of the enzyme to produce thromboxane A2 from prostaglandin H2 was dramatically reduced by multiple additions of prostaglandin H2. Our data suggest that the production of thromboxane A2 by the enzyme is self-limiting and that the enzyme is inactivated during the reaction.     

Different calcium pools in human platelets and their role in thromboxane A2 formation.

Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.     

Evidence for a role of prostaglandin I2 and thromboxane A2 in the ductus venosus of the lamb

The prostaglandin (PG) endoperoxide, PGH2, and the thromboxane (TX) A2 analog, 9,11-epithio-11,12-methano-TXA2, were tested in vitro on the ductus venosus sphincter from fetal (premature and mature) and neonatal (1-day-old) lambs. PGH2 relaxed the indomethacin-contracted fetal ductus in a dose-dependent manner and its action was reduced after treatment with 15-hydroperoxyarachidonic acid. In contrast, reduced glutathione did not affect the PGH2 relaxation in the indomethacin-treated ductus, nor did it modify the response of the untreated ductus to constrictor stimuli. Unlike PGH2, the stable 9 alpha,11 alpha-epoxymethano-PGH2 analog contracted the vessel. Similarly, the TXA2 analog was a contractile agent, its action exceeding that of the PGH2 analog in potency and efficacy. The TXA2 analog was active on preparations from both premature (minimum 117 days gestation) and mature lambs, but a maximal effect was attained during the perinatal period. These results confirm the existence of a PG-mediated relaxing mechanism in the ductus venosus and suggest that the active compound is PGI2. This mechanism is likely responsible for keeping the ductus patent in the fetus. TXA2, formed within the liver parenchyma, is well suited for playing a role in postnatal closure of the vessel.     

Cell signalling through thromboxane A2 receptors

Thromboxane A2 receptors (TPs) are widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. Following the initial cloning of this receptor class from human placenta, the deduced amino acid sequence predicted seven-transmembrane spanning regions, four extracellular domains and four intracellular domains, making TP a member of the seven-transmembrane G-protein-coupled receptor (GPCR) super family. A single gene on chromosome 19p13.3 leads to the expression of two separate TP isoforms: TPalpha which is broadly expressed in numerous tissues, and a splice variant termed TPbeta which may have a more limited tissue distribution. Mutagenesis, photoaffinity labelling, and immunological studies have indicated that the ligand binding domains for this receptor may reside in both the transmembrane (TM) and extracellular regions of the receptor protein. In addition, separate studies have provided evidence that this receptor can couple to at least four separate G protein families. As a consequence, TP signalling has been shown to result in a broad range of cellular responses including phosphoinositide metabolism, calcium redistribution, cytoskeletal arrangement, integrin activation, kinase activation, and the subsequent nuclear signalling events involved in DNA synthesis, cell proliferation, cell survival and cell death. While activation of these different signalling cascades can all derive from TP stimulation, the relative signalling preference for a given cascade appears to be both tissue and cell specific. Finally, separate studies have indicated that TP signalling capacity can be both down-regulated by protein kinase activation and up-regulated by GPCR cross-signalling. Thus, the multitude of signalling events which derive from TP activation can themselves be modulated by endogenous cellular messengers.     

Effects of thromboxane A2 on lymphocyte proliferation

The main cyclooxygenase-dependent arachidonic acid derivatives produced by monocytes and macrophages have been shown to be thromboxane A2 and prostaglandin E2. The immunomodulatory effects of thromboxane A2 were examined using a specific thromboxane synthase inhibitor (dazoxiben), a thromboxane A2 analog (U46619), and a thromboxane A2 receptor blocker (BM13.177). Dazoxiben inhibited lymphocyte proliferation in response to mitogens (PHA and OKT3), but also reoriented cyclic endoperoxide metabolism towards the production of prostaglandin E2. Prostaglandin E2 has been shown previously to inhibit mitogen-induced lymphocyte proliferation. U46619, a stable thromboxane A2 analog, slightly enhanced lymphocyte responses to mitogens in the presence of dazoxiben and in the presence of a cyclooxygenase inhibitor (indomethacin). This occurred at concentrations of U46619 which are probably supraphysiological in view of the short half-life of natural thromboxane A2. Finally, the thromboxane A2 receptor blocker BM13.177 did not have any effect on mitogen-induced lymphocyte proliferation. It is concluded that thromboxane A2 has no or minimal modulatory effects on lymphocyte proliferative responses to mitogens and that the effect of thromboxane A2 synthase inhibition is rather due to reorientation of cyclic endoperoxide metabolism, resulting in increased prostaglandin E2 production.     

A solid-phase enzyme immunoassay of thromboxane B2

A solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and beta-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound beta-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2,3-Dinor- and 2,3,4,5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80% and the results correlated well with the values obtained by radioimmunoassay (r = 0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2,3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.     

Thrombocyte interaction with a collagen substrate: the role of thrombocyte-synthesized prostaglandin endoperoxides and thromboxane A2

The effects of (i) the exogenous arachidonic acid (AA), (ii) stable prostaglandin endoperoxide analogue--U46619, and (iii) cyclooxygenase inhibitor--aspirin on the interaction of platelets with a surface coated with fibrillar calf skin collagen were studied using scanning electron microscopy. AA and U46619 stimulate massive spreading of platelets (on the collagen substrate and formation of surface-bound multilayer (thrombi-like) aggregates. The stimulation of spreading and formation of thrombi-like aggregates by AA correlate with the thromboxane A2 (TXA2) synthesis in platelets. Unlike AA, U46619 induces these processes without transformation into TXA2 and stimulation of its synthesis in platelets. Cyclooxygenase inhibitor--aspirin prevents the AA-induced platelet spreading, formation of the surface-bound thrombi-like aggregates, and TXA2 synthesis. In the absence of soluble platelet inducers, aspirin inhibits the substrate-induced spreading, but doesn't affect the initial attachment of nonactivated platelets to the collagen substrate.     

Protection of atherogenesis in thromboxane A2 receptor-deficient mice is not associated with thromboxane A2 receptor in bone marrow-derived cells

In the previous study, we generated mice lacking thromboxane A2 receptor (TP) and apolipoprotein E, apoE(-/-)TP(-/-) mice, and reported that the double knockout mice developed markedly smaller atherosclerotic lesions than those in apoE(-/-) mice. To investigate the mechanism responsible for reduced atherosclerosis in apoE(-/-)TP(-/-) mice, we examined the role of TP in bone marrow (BM)-derived cells in the development of the atherosclerotic lesions. When we compared the function of macrophages in apoE(-/-) and in apoE(-/-)TP(-/-) mouse in vitro, there was no difference in the expression levels of cytokines and chemokines after stimulation with lipopolysaccharide. We then transplanted the BM from either apoE(-/-) or apoE(-/-)TP(-/-) mice to either apoE(-/-) or apoE(-/-)TP(-/-) mice after sublethal irradiation. After 12 weeks with high fat diet, we analyzed the atherosclerotic lesion of aortic sinus. When the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-) mice, the lesion size was almost the same as that of apoE(-/-) mice without BM transplantation. In contrast, when the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-)TP(-/-) mice, the lesion size was markedly reduced. These results indicate that the protection of atherogenesis in TP(-/-) mice is not associated with TP in BM-derived cells.