Inhibition of thromboxane A2 biosynthesis in human platelets by burimamide

1 Burimamide selectively inhibited the formation of thromboxane A2 from prostaglandin endoperoxides by human platelet microsomes in a dose-dependent manner (IC50 = 2.5 x 10(-5) M). Burimamide was found to be equipotent to imidazole as a thromboxane synthetase inhibitor. 2 Metiamide, cimetidine and a series of compounds either bearing a structural or pharmacological relationship to histamine caused little or no inhibition of thromboxane A2 biosynthesis by human platelet microsomes. 3 Burimamide (5 x 10(-4) to 2.3 x 10(-3) M) did not inhibit either the cyclo-oxygenase or the prostacyclin synthetase of sheep seminal vesicles or the prostacyclin synthetase of dog aortic microsomes. 4 Burimamide (2.5 x 10(-5) to 1.2 x 10(-4) M) inhibited sodium arachidonate-induced human platelet aggregation; the degree of inhibition was dependent upon the concentration of arachidonic acid used to aggregate the platelets.     

Phenothiazines inhibit collagen-induced thromboxane B2 synthesis and increase forskolin anti-aggregating effects in human platelets.

1. The aim of the present study was to investigate the effects of phenothiazine compounds chlorpromazine and trifluoperazine on human platelet response to collagen and sodium arachidonate. 2. The results demonstrated that phenothiazines inhibit collagen-induced platelet aggregation and thromboxane B2 synthesis in a dose-dependent way and increase the antiaggregating properties of forskolin. 3. Phenothiazines at high concentrations decreased the platelet aggregation in response to arachidonate, without a significant reduction of thromboxane B2 production. 4. The data provides evidence that phenothiazine effects could involve calmodulin-dependent enzymatic activities.     

Binding of thromboxane A2/prostaglandin H2 agonists to human platelets

The competition of [125I]-9, 11 dimethylmethano-11, 12 methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza 15 alpha beta-omega-tetranor-thromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 receptor antagonist, with a series of thromboxane A2/prostaglandin H2 (TXA2/PGH2) mimetics for binding to the putative TXA2/PGH2 receptor in washed human platelets was studied. The rank order potency for the series of mimetics to compete with [125I]-PTA-OH for binding was compared with their rank order potency for induction of platelet aggregation. The rank order potency for the mimetics to compete with [125I]-PTA-OH for binding was ONO-11113 greater than SQ-26655 greater than U44069 greater than U46619 = 9, 11-azo PGH2 greater than MB28767. This rank order potency was highly correlated with their rank order potency for inducing platelet aggregation (r = 0.992). Changes in the intra or extracellular concentrations of Na+ did not have a significant effect on the competition between U46619 and [125I]-PTA-OH for binding to the putative receptor. In summary, it appears that these TXA2/PGH2 mimetics activate human platelets through the putative TXA2/PGH2 receptor.     

Free Ca2+ requirements of agonist-induced thromboxane A2 synthesis in human platelets

In quin2-loaded human platelets ionomycin raised cytosolic free calcium to greater than 1 microM and generated less than 1 ng thromboxane. Collagen alone or in the presence of EPO92 generated up to 32 and 16 ng thromboxane respectively; in the latter case at calcium levels around 120 nM. Thrombin maximally raised calcium to greater than 1 microM and generated up to 27 ng thromboxane, although in the presence of 1 mM EGTA these calcium and thromboxane levels were reduced to 200 nM and 5 ng respectively.     

Further studies on the effects of epimers of thromboxane A2 antagonists on platelets and veins

The C-15 hydroxy epimers of three TXA2/PGH2 antagonists were resolved by HPLC and their pharmacology evaluated in canine saphenous veins and human platelets. For all three compounds, the epimers were equipotent in their ability to antagonize U46619 (1 microM) induced platelet aggregation. In contrast, one of the epimers for all three of the antagonists was significantly more potent than the other in antagonizing U46619 (10 nM) induced saphenous vein contractions. The data support the notion that the TXA2/PGH2 receptors in veins may be different from those in platelets.     

Competitive antagonism at thromboxane receptors in human platelets.

The inhibitory effects of three prostanoid analogues, EP 045, EP 092 and pinane thromboxane A2 (PTA2), on the aggregation of human platelets in vitro have been investigated. In diluted platelet-rich plasma (PRP), EP 045 (20 microM) and EP 092 (1 microM) completely inhibited irreversible aggregation responses to thromboxane A2 (TXA2), prostaglandin H2 (PGH2) and five chemically stable thromboxane mimetics, including 11,9-epoxymethano-PGH2 and 9,11-azo-PGH2. Reversible aggregation produced by the prostanoid analogue, CTA2, was also inhibited. The block of the stable agonist action was surmountable. In plasma-free platelet suspensions EP 045 and EP 092 were more potent antagonists. Schild analysis indicated a competitive type of antagonism for EP 045 (affinity constant of 1.1 X 10(7) M-1); the nature of the EP 092 block is not clear. Primary aggregation waves induced by ADP, platelet activating factor (Paf) and adrenaline were unaffected by EP 045 and EP 092, whereas the corresponding second phases of aggregation were suppressed. Aggregation and 5-hydroxytryptamine (5-HT) release induced by either PGH2 or 11,9-epoxymethano-PGH2 were inhibited in a parallel manner by EP 045. Inhibition of thromboxane biosynthesis is not involved in these effects. EP 045 and EP 092 did not raise adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the platelet suspensions. In plasma-free platelet suspensions PTA2 produced a shape change response which could be blocked by EP 045. PTA2, therefore, has a thromboxane-like agonist action. The block of the aggregatory action of 11,9-epoxymethano-PGH2 by PTA2 appears to be mainly due to competition at the thromboxane receptor. However, PTA2 produced a slight rise in cyclic AMP levels; this could be due to a very weak stimulant action on either PGI2 or PGD2 receptors present in the human platelet. Functional antagonism by PTA2 may therefore augment its thromboxane receptor blocking activity. The results are discussed in terms of (a) the specificity of antagonism produced by EP 045, EP 092 and PTA2, (b) the validity of affinity constant determinations for receptor antagonists when aggregation is the biological response, and (c) the characteristics of the human platelet thromboxane receptor in comparison with those of thromboxane receptors in smooth muscle.     

Induction of central signalling pathways and select functional effects in human platelets by beta-boswellic acid

We have recently shown that in polymorphonuclear leukocytes, 11-keto boswellic acids (KBAs) induce Ca2+ mobilisation and activation of mitogen-activated protein kinases (MAPK). Here we addressed the effects of BAs on central signalling pathways in human platelets and on various platelet functions. We found that beta-BA (10 microM), the 11-methylene analogue of KBA, caused a pronounced mobilisation of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal-regulated kinase (ERK)2, and Akt. These effects of beta-BA were concentration dependent, and the magnitude of the responses was comparable to those obtained after platelet stimulation with thrombin or collagen. Based on inhibitor studies, beta-BA triggers Ca2+ mobilisation via the phospholipase (PL)C/inositol-1,4,5-trisphosphate pathway, and involves Src family kinase signalling.Investigation of platelet functions revealed that beta-BA (> or =10 microM) strongly stimulates the platelet-induced generation of thrombin in an ex-vivo in-vitro model, the liberation of arachidonic acid (AA), and induces platelet aggregation in a Ca2+-dependent manner. In contrast to beta-BA, the 11-keto-BAs (KBA or AKBA) evoke only moderate Ca2+ mobilisation and activate p38 MAPK, but fail to induce phosphorylation of ERK2 or Akt, and do not cause aggregation or significant generation of thrombin.In summary, beta-BA potently induces Ca2+ mobilisation as well as the activation of pivotal protein kinases, and elicits functional platelet responses such as thrombin generation, liberation of AA, and aggregation.     

Effect of R and S enantiomers of naproxen on aggregation and thromboxane production in human platelets

The effects of R and S enantiomers of naproxen [(+)-6-methoxy-alpha-methyl- 2-naphthaleneacetic acid] were studied on platelet aggregation and on the production of thromboxane B2 from collagen-stimulated human platelets in order to determine the effect of each enantiomer in terms of cyclooxygenase inhibition. S-Naproxen caused inhibition of platelet aggregation in platelet-rich plasma and washed human platelets in a concentration-related fashion in the range 1-80 micrograms/L. A similar concentration-related suppression was noted for R-naproxen, but this inhibition was significantly less than that induced by S-naproxen for all concentrations except 1 micrograms/L. Similarly, both R- and S-naproxen (1-80 micrograms/L) caused a concentration-dependent suppression of thromboxane B2 production from platelet-rich plasma. These values were significant at all concentrations of drug (10-80 micrograms/L) except at 1 micrograms/L. Significant differences in thromboxane B2 production from washed human platelets were noted at concentrations of 10 and 25 micrograms/L. The findings support previous studies reported in the literature that S-naproxen is more active than R-naproxen. Our findings that S-naproxen is more active than R-naproxen on collagen-stimulated platelet aggregation and prostaglandin production suggest that the findings of greater activity of S isomer over the R isomer in animal models of inflammation may be a direct expression of the differential action on prostaglandin synthesis.     

Salicylic acid fails to inhibit generation of thromboxane A2 activity in platelets after in vivo administration to the rat.

Arachidonic acid-induced aggregation of rat platelets and the accompanying generation of thromboxane A2 activity were inhibited by aspirin, whereas 20 times higher doses of salicylic, gentisic and salicyluric acids were inactive. Salicylic acid administered to the rats before aspirin prevented the inhibition of the cyclo-oxygenase-mediated effects of arachidonic acid. These results do not support the hypothesis that the anti-inflammatory activity of salicylic acid is due to inhibition of prostaglandin systhetase (cyclo-oxygenase) by an unknown metabolite and indicate that salicylic acid displays an anti-inflammatory activity independent inhibition of prostaglandin biosynthesis.     

5HT对STA2介导的血小板聚集和解释反应的双重影响

目的：研究５－ＨＴ对ＳＴＡ２血小板聚集和释放反应的影响及可能的分子机制。方法：以透光法，介质中ＡＴＰ含量及荧光图像法评介血小板变形，聚集反应和［Ｃａ＾］ｉ水平。结果：（１）５－ＨＴ预处理可消除ＳＴＡ２的血小板变形，ＳＴＡ２ ０．３μｍｏｌ·Ｌ＾－１的聚集增强，１－２ｍｏｌ·Ｌ＾－１的聚集不变，释放反应抑制。（２）５－ＴＨ预处理增加ＳＴＡ２ ０．３μｍｏｌ·Ｌ＾－１的［Ｃａ＾２＋］ｉ降低３μｍｏｌ·     

Prostaglandin D2 interacts at thromboxane receptor-sites on guinea-pig platelets.

The anti-aggregatory prostanoid, prostaglandin D2 (PGD2) does not completely inhibit ADP-induced aggregation of guinea-pig platelets and thus produces a bell-shaped dose-inhibition curve. The nature of this bell-shaped curve has now been investigated in guinea-pig platelet-rich plasma. Two selective thromboxane receptor antagonists, 13-aza-prostanoic acid (13-AZA; 16-64.4 microM) and BM 13.177 (5.9-29.8 microM), converted PGD2 to a full inhibitor of aggregation in a dose-related manner. The putative platelet PGD2 receptor antagonist, N-0164 (75 microM) also converted PGD2 to a full inhibitor of platelet aggregation. In contrast to 13-AZA and BM 13.177, higher concentrations of N-0164 (380 and 760 microM) caused a dose-related rightward shift of the PGD2 dose-inhibition curve. The thromboxane receptor antagonism of N-0164 was confirmed in studies in which the dose-aggregation curve to U-46619, a thromboxane mimetic, was competitively antagonized with a pA2 value of 4.67 and a slope of 1.13, comparable to that of 13-AZA. The results show that N-0164 acts as both a platelet PGD2 and thromboxane-receptor antagonist in both human and guinea-pig platelet-rich plasma. The results further indicate that PGD2 can interact at thromboxane receptors in guinea-pig platelets.     

Subcellular localization of a thromboxane A2/prostaglandin H2 receptor antagonist binding site in human platelets

The subcellular localization of a binding site for the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) 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), was determined. Subcellular fractions of platelets were prepared by glycerol lysis or nitrogen cavitation, and were characterized by the use of enzymatic markers specific for plasma membranes, endoplasmic reticulum (dense tubular system), mitochondria, granules, and cytosolic constituents. The Kd and density of binding sites in the subcellular fractions were determined by Scatchard analysis of equilibrium binding data. The Kd and Bmax for [125I]-PTA-OH determined in the lysates were 49 +/- 11 nM and 4.1 +/- 1.7 pmol/mg protein respectively (N = 6). The Kd values were not significantly different in any of the fractions assayed. The binding sites were coenriched (4.5 +/- 0.66 fold) with the enzymatic markers for plasma membranes (3.7 +/- 0.5 fold) and dense tubular system (2.4 +/- 0.4 fold). The binding sites were not coenriched with markers for cytoplasmic constituents, mitochondria, or granules. The ability of the TXA2/PGH2 mimetic U46619 to compete with [125I]-PTA-OH for the binding site was also determined for the various subcellular fractions. The IC50 for U46619 was 5.4 +/- 1.2 microM in the lysate, and was not significantly different in the subcellular fractions. These data suggest that the binding site is the TXA2/PGH2 receptor described previously. These data are consistent with the notion that the putative TXA2/PGH2 receptor is localized in the plasma membranes and/or the dense tubular system.     

Reduction in the number of thromboxane receptors on human platelets after exposure to GR32191.

1. Exposure of human resuspended platelets in vitro for 30 min to the potent thromboxane A2 (TP)-receptor blocking drug GR32191, followed by its removal by dilution-dissociation, reduced the degree of subsequent binding to 2 nM [3H]-GR32191 by almost 50%. Exposure for longer periods (60 min) led to a further reduction. However, no change in the Kd of the radioligand was observed. 2. This effect of GR32191 could not be explained by persistent binding of drug to platelets since a dilution-dissociation stage, designed to remove all drug, was included prior to measurement of binding. 3. Using an alternative TP-receptor radioligand, [3H]-SQ29,548, to monitor receptor number, a reduction in Bmax was observed after GR32191 pre-treatment; the Kd value of the radioligand remained unchanged. 4. The effect was not a common property of TP-receptor blocking drugs since pre-exposure of platelets in vitro for 30 min to BM13.177 or SQ29,548 did not produce a fall in subsequent Bmax to [3H]-SQ29,548. 5. While the mechanism behind this apparent down-regulation of platelet TP-receptor is unknown, it may explain the long duration of action of GR32191 upon platelets in man which persists in the absence of detectable drug in the plasma.     

A rapid and simple assay for the study of thromboxane B2 synthesis by intact human platelets

Conversion of 1-¹⁴C-arachidonic acid (AA) to thromboxane B₂ (TXB₂) by human platelets was studied by using a new, simple technique. Organic solvent extraction was avoided by spotting aliquots of the reaction mixture directly on thin layer chromatography (TLC) plates. In this way it was possible to study the kinetic parameters of the formation of TXB₂. Moreover, the rapidity and simplicity of the assay should be particularly suitable when studying the possible relationships between thromboxane synthesis and aggregation function in human platelets, where a large number of determinations is required.     

Benzodiazepine analogues inhibit arachidonate-induced aggregation and thromboxane synthesis in human platelets.

1. Benzodiazepine analogues inhibit human platelet aggregation induced by arachidonate with an EC50 value of 0.68 microM for PK 11195, the most potent analogue used. 2. There was a highly significant correlation between the inhibition of arachidonate-induced aggregation and the affinity for the peripheral-type of benzodiazepine binding sites. 3. There was no significant correlation between the inhibition of the platelet activating factor (PAF)-induced aggregation and the binding to the peripheral-type of benzodiazepine binding sites. 4. The inhibition of platelet aggregation seems to result from the inhibition of arachidonic acid cyclo-oxygenation, since the synthesis of thromboxane and 12-hydroxy-heptadecatrienoic acid, both cyclo-oxygenase products, was reduced. 5. Our results suggest that peripheral-type of benzodiazepine binding sites on human platelets could be linked to cyclo-oxygenase.     

Antagonistic effects of losartan on thromboxane A2-receptors in human isolated gastroepiploic artery and saphenous vein.

In addition to its AT1-receptor antagonist activity, losartan has been shown to antagonize thromboxane A2 (TXA2)-induced contraction of animal vessels. We investigated for the first time in human isolated gastroepiploic artery (GEA) and saphenous vein (SV) the TXA2/PGH2-receptor antagonist activity of losartan in the presence of indomethacin (1 microM) and N(omega)-nitro-L-arginine (100 microg). Losartan at concentrations of > or =1 microM on GEA and from 10 microM on SV significantly shifted U46619-induced contractions to the right. In addition, 100 microM losartan decreased by 34% the amplitude of the contraction to U46619 on both GEA and SV. The potency of losartan for the TXA2 receptor was 50- and 80-fold lower than that for the AT1 receptor on human GEA and SV, respectively. This inhibitory effect of losartan appeared selective for angiotensin II and TXA2-induced contractions because 100 microM losartan did not modify either endothelin-1- or KCl-induced contraction in human SV, although a reduction of norepinephrine- and 5-hydroxytryptamine-induced contraction was observed in human GEA and SV, respectively. In conclusion, losartan is an antagonist of TXA2 receptor on human GEA and SV. However, this antagonist activity occurred for a relative high dose of losartan, suggesting that it contributes at a low level, if any, to its antihypertensive effect.     

Salicylic acid fails to inhibit generation of thromboxane A2 activity in platelets after in vivo administration to the rat

Arachidonic acid-induced aggregation of rat platelets and the accompanying generation of thromboxane A₂ activity were inhibited by aspirin, whereas 20 times higher doses of salicylic, gentisic and salicyluric acids were inactive. Salicylic acid administered to the rats before aspirin prevented the inhibition of the cyclo-oxygenase-mediated effects of arachidonic acid. These results do not support the hypothesis that the anti-inflammatory activity of salicylic acid is due to inhibition of prostaglandin synthetase (cyclo-oxygenase) by an unknown metabolite and indicate that salicylic acid displays an anti-inflammatory activity independent inhibition of prostaglandin biosynthesis.     

Effects of isosorbide dinitrate on thromboxane A2 synthesis in carbon dioxide exposed platelets

Isosorbide dinitrate (Nitorol R, 100 nmol/l) blocked an increase in thromboxane B2 concentration in human platelet-rich plasma caused by exposure to high CO2 tension, in the presence of arachidonic acid. Thromboxane B2 concentrations obtained at 1 or 2 mmol/l or arachidonic acid were not influenced by isosorbide dinitrate at low CO2 tension. Inhibitory action of isosorbide dinitrate on the thromboxane A2 synthesis appears to have a close relation to the site of CO2 action.     

Characterization of rat glomerular thromboxane A2 receptors: comparison to rat platelets.

This study was designed to characterize rat glomerular thromboxane A2 (TxA2) receptors and compare them to rat platelet TxA2 receptors. The radioligand binding characteristics of the receptors were characterized using [125I][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-2yl]-5-heptenoic acid ([125I]BOP), a TxA2 agonist. Equilibrium binding with [125I]BOP, as well as competitive binding assays between [125I]BOP and 13-azapinane TxA2 receptors antagonists, were performed in rat glomerular membranes (RGM) and washed rat platelets (WRP). [125I]BOP identified a single class of TxA2 receptor sites in glomerular membranes with a Kd of 318 +/- 55 pM and a Bmax of 260 +/- 62 fmol/mg protein (n = 14). [125I]BOP was displaced by the TxA2 agonist 15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid (U-46,619) (IC50 = 22 +/- 6 nM, n = 3), the antagonist SQ-29,548 (IC50 = 41 +/- 7 nM, n = 4), and stereoselectively by the antagonists (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,925) (IC50 = 0.27 +/- 0.04 nM, n = 3) and (+)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,926) (IC50 = 124 +/- 0 nM, n = 2). The ability of six 13-azapinane TxA2 antagonists to compete with [125I]BOP was evaluated. The rank orders for the 13-azapinanes showed no significant correlation between RGM and WRP.(ABSTRACT TRUNCATED AT 250 WORDS)