Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A₂ (TXA₂) and human vessel wall prostacyclin (PGI₂) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI₂-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA₂ generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI₂ release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF_1α. Minimum concentration of dipyridamole causing PGI₂ release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI₂ activity and to some extent by TXA₂ suppression. Stimulation of PGI₂ release by human vessels may not be seen in usual therapeutic concentrations.     

Regulation of microvascular prostacyclin and thromboxane with inhibitors or arachidonic acid metabolism

The levels of the stable degradation products of prostacyclin (PGI₂) and thromboxane A₂ (TXA₂): 6-oxo-prostaglandin F_1α(6-oxo-PGE_1α) and thromboxane B₂ (TXB₂) respectively were determined in the effluent of the rabbit epigastric skin flap after infusion of exogenous arachidonic acid. The blood to the flap passes through the microcirculation and thus the changes in eicosanoid biosynthesis in this part of the vasculature were recorded. The aim was to use inhibitors of arachidonic acid metabolism to increase the PGI₂/TXA₂ ratio. This may be potentially beneficial to ischaemic skin flaps by reducing platelet aggregation associated with damaged microvascular endothelium, overcoming vasospasm and increasing microvascular blood flow. Increased PGI₂/TXA₂ ratios (up to 5-fold) were best achieved using TXA₂ synthetase inhibitors such as dazoxiben hydrochloride. These were significantly more potent than the phosphodiesterase inhibitor dipyridamole, and the lipoxygenase inhibitor Bay g6575. No increase in blood flow was achieved. The cyclooxygenase inhibitor indomethacin did slow the blood flow at high concentrations (above 10⁻⁵ M), and inhibited both PGI₂ and TXA₂ synthesis. Approximately 2-fold higher concentrations of dazoxiben hydrochloride and dipyridamole were required to produce the same TXA₂ synthetase inhibition in the flap microvasculature compared with platelets .     

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A₂ biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10^?4 M, 1.75 × 10^?4 M, and 2 × 10^?3 M respectively. Their relative inhibitory potencies on thromboxane B₂ production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10^?3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B₂ formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A₂ biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.     

Enhancement of PGI2 formation by a new vasodilator, 2-nicotinamidoethyl nitrate in the coupled system of platelets and aortic microsomes

Exogenous arachidonate addition to the coupled system of platelets and aortic microsomes resulted in production of TXA₂ and PGI₂ (detected as the stable degradation products, TXB₂ and 6-keto PGF_1α, respectively). Imidazole, papaverine and dipyridamole increased PGI₂ and decreased TXA₂ in the coupled system. All of these agents inhibited TXA₂ formation by platelets from arachidonate. Nitroglycerin did not show any effect on PGI₂ and TXA₂ formation in the coupled system and on TXA₂ formation by platelets. In contrast with these compounds, in spite of showing no inhibitory effect on TXA₂ formation by platelets alone, 2-nicotinamidoethyl nitrate (SG-75) increased PGI₂ and decreased TXA₂ in the coupled system. It is suggested that SG-75 accelerated the conversion of PGH₂ to PGI₂ so that smaller amounts of TXA₂ was produced in the coupled system.     

Pharmacophore requirements in new series of pyridazinyl alkanoic acids, N-[(pyridazin-2-yl) alkyl] succinyl and glutaryl amides, inhibitors of thromboxane A2 biosynthesis

New series of 5-benzyl-6-methyl-4-oxo pyridazin-2-yl alkanoic acids, N-[(pyridazin-2-yl)alkyl] succinyl and glutaryl amides have been synthesized and evaluated in vitro as TXA₂ biosynthesis inhibitors. The experiments were carried out using arachidonic acid (32.8 μM) as a substrate and horse platelet microsomes as sources of TXA₂ synthase. The presence of TXB₂, a stable metabolite of TXA₂, was determined by RIA. The potency of active compounds (1.10⁻⁴ < IC 50 < 1.10⁻⁶ M) greatly depends on the length of the chain at the N-2 position on the pyridazine ring. Furthermore, enzyme inhibition in vitro is increased with the presence of a halogen atom on the aromatic moiety of the benzyl group at C-5. Compound 4f having a pentanoic side chain and a 4-fluoro benzyl moiety was the most active derivative with an IC₅₀ value of 6.69 × 10⁻⁶ M. Molecular modelling studies were done on all the synthesized pyridazinones and on prostaglandin H₂ (PGH₂) suggesting spatial features and volumes of TXA₂ synthase pharmacophore mode in these series of derivatives.     

The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses

We have investigated the presence of thromboxane A₂ (TXA₂) receptor associated with lipid rafts in human platelets and the regulation of platelet function in response to TXA₂ receptor agonists when lipid rafts are disrupted by cholesterol extraction. Platelet aggregation with TXA₂ analogs U46619 and IBOP was almost blunted in cholesterol-depleted platelets, as well as α_IIbβ₃ integrin activation and P-selectin exposure. Raft disruption also inhibited TXA₂-induced cytosolic calcium increase and nucleotide release, ruling out an implication of P2Y₁₂ receptor. An important proportion of TXA₂ receptor (40%) was colocalized at lipid rafts. The presence of the TXA₂ receptor associated with lipid rafts in platelets is important for functional platelet responses to TXA₂.     

Rat aortic strip as a bioassay tissue for thromboxane A2 and rabbit aorta contracting substance (RCS) released from guinea pig lung by bradykinin or anaphylaxis

Rat aortic strips and rabbit aortic strips were superfused in series with Krebs solution. Comparison of the sensitivity of the tissues to thromboxane A₂ (TXA₂), generated by mixing prostaglandin (PG)H₂ with human platelet microsomes (HPM), indicated that the rat aorta was just as sensitive an assay tissue as the rabbit aorta. Furthermore, it was equally selective in that it did not respond to low levels of the other metabolites of arachidonic acid. When the two tissues were used simultaneously to assay aortic contracting activity released from perfused guinea pig lung by bradykinin, both tissues detected activity that could be matched with similar known amounts of TXA₂. However, when ovalbumin was used to release aortic contracting activity from sensitized guinea pig lung, the amounts of TXA₂ needed to match the responses of the assay tissues often differed by 2–3 fold. This suggested that other substances, as well as TXA₂, released during anaphylaxis can affect the aortic strips and thus influence the bioassay of TXA₂. This discrepancy in assay of TXA₂ can be detected only when more than one assay tissue is used. In the series of experiments in which we used the assay tissues to detect TXA₂ released by bradykinin, we noted that bradykinin released more TXA₂ from unsensitized lungs than from sensitized ones. Although the significance of this observation remains unclear, it suggests that there are quantitative differences in the PG biosynthetic pathways induced by the sensitization process.     

Effects of free and macromolecular-bound TXA2 receptor antagonist BM 1..505 on U46619-induced platelet aggregation

The goal of this study was to synthesize a macromolecular probe of the TXA₂ receptor antagonist BM13.505 which is unable to penetrate the platelet membrane for localization and characterization of the TXA₂ receptor. The active NHS-ester of BM13.505 was synthesized and purified. It was used for covalent coupling of BM13.505 to bovine serum albumin, a macromolecular carrier. Inhibitory effects of free and macromolecular bound BM13.505 on aggregatory properties of U46619-stimulated platelets were measured and compared to TXA₂ generation in platelets, as determined by TXB₂ radioimmuno assay. No inhibitory effects of free and macromolecular-bound BM13.505 on ADP- or thrombin-induced platelet aggregation were observed. Equimolar concentrations of free or macromolecular bound BM13.505 inhibited U46619-induced platelet aggregation and TXA₂ generation with equal potency. IC₅₀-values for platelet aggregation inhibition by free and macromolecular bound BM13.505 were 64 nM and 96 nM respectively. It appears that the TXA₂ receptor ligand binding site is located close to the outer membrane surface of platelets. Interaction of macromolecular bound BM13.505 with the platelet thromboxane receptor does not depend on the availability of the free carboxyl residue in BM13.505. The method for coupling a TXA₂ receptor antagonist to a macromolecule will aid in constructing probes for the localization and characterization of the TXA₂ receptor.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid Two selective thromboxane synthetase inhibitors

Two selective thromboxane A₂ synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B₂ and prostaglandin E₂ accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B₂ formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H₂. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A₂ formation with an accompanying, parallel, suppression of the platelet aggregation.Imidazole selectively suppressed thromboxane A₂ formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H₂ on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A₂ formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A₂ in arachidonate mediated platelet aggregation.     

Imidazole: A selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymatic conversion of the endoperoxides (PGG₂ and PGH₂) to thromboxane A₂ by platelet microsomes (IC₅₀: 22 μg/ml; determined by bioassay). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of ¹⁴C-thromboxane B₂ from ¹⁴C-PHG₂. Several imidazole analogues and other substances were tested but only 1-methyl-imidadole was more potent that imidazole iteself. The use of imidazole of inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.     

Prostacyclin and thromboxane A2 release in isolated rat lungs

The influence of platelets and platelet membranes on the generation of prostacyclin (PGI₂) and thromboxane A₂(TXA₂) by isolated rat lung and porcine aortic endothelial cell, as measured by RIA of their stable end-producs, 6-oxo-PGF_1α and TXB₂ respectively, was studied. After introduction of either aspirin-treated platelets or membranes from aspirin-treated platelets to the perfusate, 1 5-fold increase in the amount of 6-oxo-PGF_1α and TXB₂ in the perfusate was observed. Treatment of the lung with aspirin produced a 50% reduction in the platelet-stimulated release of PGI₂ and TXA₂. Treatment of the lung with the phospholipase inhibitor, mepacrine, significantly reduced the platelet-stimulated release of PGI₂ and TXA₂. Incubation of endothelial cells with untreated platelet membranes did not alter the generation of PGI₂. These results suggest that platelet-stimulated release of PGI₂ and TXA₂ occurs via mechanical stimulation of phospholipase A₂, liberating arachidonic acid.     

Selective inhibition of thromboxane synthesis partially protected while inhibition of angiotensin II formation did not protect rats against acute renal failure induced with glycerol

Acute renal failure (ARF) was induced in 35 week-old conscious female Wistar rats, by intramuscular (IM) injection of glycerol. Intraperitoneal (IP) injection of imidazole, an inhibitor of thromboxane (TXA₂) synthesis, partially protected the animals against ARF. This protection was accompanied by a significant decrease in renal TXB₂ (the stable chemical metabolite of TXA₂) and a significant increase in renal 6-keto-PGF₁α (the stable chemical metabolite of PGI₂) synthesis. Intraperitoneal injection of captopril (SQ 14225) an angiotensin-converting-enzyme inhibitor, did not protect the animals against ARF. This lack of protection was accompanied by a significant increase in renal TXB₂ and a significant decrease in renal 6-keto-PGF₁α synthesis. The results suggest that: (a) the renin-angiotensin (R-A) system does not play a role, or has only a secondary one in the development of ARF; (b) thromboxane A₂ (the most potent vasoconstrictor and platelet aggregator agent known) is the preponderant agent responsible for the development of this pathological syndrome.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI₂ and TXA₂ were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI₂ and TXA₂ synthetase. 6-keto-PGF_1α and TXB₂, stable metabolites of PGI₂ and TXA₂ respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI₂ as well as TXA₂ from arachidonic acid. On the other hand, ischemia depressed both PGI₂ and TXA₂ synthetase activities of cardiac tissue: the depression was more pronounced on TXA₂ synthetase than on PGI₂ synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio indicating therefore that it can facilitate the formation of PGI₂. The post ischemic reperfusion of the heart counteracted the decrease in PGI₂ synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA₂ synthetase. However, the diminution in TXA₂ synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA₂ synthetase is more vulnerable than PGI₂ synthetase to a lack of oxygen and nutrients.     

Effect of single and repeated doses of a new nitroderivative of acetylsalicylic acid on platelet TXA2 production in rats

The effects of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO₂), on platelet TXA₂ synthesis after single and repeated doses in the rat were investigated. Compared to ASA, cumulative doses of ASA-NO₂ showed similar inhibitory effects on platelet TXA₂ synthesis and significant increases in nitrite/nitrate plasma concentrations l h after the last drug administration: 24 h later nitrite/nitrate plasma levels returned to the control values, while serum TXA₂ concentrations did not change. A time-course study after a single dose of ASA-NO₂ showed a significant inhibition of platelet TXA₂ production also 24 h after drug administration and a significant increase in nitrite/nitrate plasma levels until 10 h.     

Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized $\text{Ascaris suu}$ antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A₂, prostaglandin (PG) D₂ and PGI₂ from the PG endoperoxide intermediate, PGH₂, was assayed over a range of substrate concentrations from 3–200 uM. Synthesis of PGI₂ by trachealis microsomes was approximately 5-fold greater than that of TXA₂. PGI₂ and TXA₂ production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA₂ production predominated over PGI₂ by 9-fold, preparations from Ascaris- exposed animals synthesized 50% more TXA₂ than controls at PGH₂ concentrations of 25 uM and above, whereas synthesis of PGI₂ and PGD₂ were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA₂ and PGI₂ in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA₂ synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.     

Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species

The activity of prostacyclin (PGI₂), PGE₁ or PGD₂ as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD₂ was a weak inhibitor in dog, rabbit and rat plasma whereas PGE₁ and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE₁ or PGD₂. Compound N-0164 abolished the inhibition by PGD₂ of human platelet aggregation but did not inhibit the effects of PGE₁ or prostacyclin. The results suggest that prostacyclin and PGE₁ act on similar sites on platelets which are distinct from those for PGD₂.     

A stable analogue of throm☐ane A2, 9,11-epithio-11,12-methanothrom☐ane A2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture

Throm☐ane A₂ (TXA₂) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, throm☐ane B₂ (TXB₂). TXA₂ has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA₂ have not yet been proved because of its highly unstable nature.Recently, a chemically stable analogue of TXA₂, 9,11-epithio-11,12-methanothrom☐ane A₂ (STA₂), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E₂ (PGE₂), a well-known bone resorbing agent. STA₂ time-and dose-dependently stimulated the release of ⁴⁵Ca from prelabelled mouse calvariae. Both STA₂ and PGE₂ induced the accumulation of cAMP in mouse calvariae. The TXA₂, agonist. ONO-3708, inhibited STA₂-induced release of ⁴⁵Ca, TXB₂ induced neither bone resolor cAMP accumulation. When mouse marrow cells were cultured with STA₂ for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA₂ added. Again TXB₂ showed no effect on osteoclast-like cell formation. These results indicate a role for TXA₂ in some form of bone resorption.     

Metabolism and action of the prostaglandin endoperoxide PGH2 in rat kidney

Kidney membrane fractions metabolized [1-¹⁴C]PGH₂ to TXB₂, PGE₂, PGF_2α, PGD₂, 6-keto PGF_1α, and HHT. TXA₂, as measured by TXB₂, was enzymatically formed in cortex microsomes and was identified by thin layer chromatography and gas chromatography - mass spectrometry. PGH₂ caused a labile inhibition of cortical PGE₂-stimulated adenylate cyclase. PGE₂, PGF_2α, and PGD₂ are stimulators of cortical adenylate cyclase. The inability of two thromboxane synthetase inhibitors, imidazole and 9,11-azoprosta-5,13 dienoic acid, to block PGH₂ inhibition suggested that TXA₂ was not an obligatory intermediate in this process. Therefore, a potential function of cortical PGH₂ is inhibition of adenylate cyclase.     

The effect of imidazole and imidazole-analogues on bone resorption in vitro: A suggested role for thromboxane A2

The effects of imidazole, 1-methyl-imidazole and benzimidazole on bone metabolism $\text{in vitro}$ were investigated. The relative potencies of these compounds with respect to the inhibition of bone resorption was found to be comparable to their relative effectiveness as inhibitors of platelet microsome thromboxane synthetase activity. Since studies by others have shown that thromboxanes are produced by resorbing bone $\text{in vitro}$, these results suggest that the inhibition of bone resorption by imidazole is related to the inhibition of thromboxane A₂ formation. This could imply that thromboxane A₂ is an additional arachidonic acid oxidation product that is of importance in the regulation of bone metabolism.     

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone: A potent inhibitor of the blood platelet cyclo-oxygenase

Previous studies have demonstrated that 13-azaprostanoic acid (13-APA) is a potent and specific antagonist of thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) at the platelet receptor level. In the present study we evaluated the effects of a new azaprostanoid, 2-(6-carboxyhexyl) cyclopentanone hexylhydrazone (CPH), on human platelet function. This hydrazone was found to completely inhibit arachidonic acid (AA)-induced platelet aggregation at 1 uM CPH. On the other hand, CPH was not an effective inhibitor of PGH₂-induced aggregation. Furthermore, 100 uM CPH was completely ineffective in blocking platelet aggregation stimulated by adenosine diphosphate (ADP) or the stable prostaglandin endoperoxide analog U46619 (which presumably acts at the TXA₂/PGH₂ receptor). Measurement of platelet thromboxane B₂ (TXB₂) production demonstrated that the primary site-of-action of CPH is at the cyclo-oxygenase level. Thus, CPH inhibited TXB₂ formation from AA in a dose-dependent manner (0.1 uM–100 uM CPH)². In contrast, CPH blocked TXB₂ production from PGH₂ only at the highest CPH concentration tested, i.e., 100 uM. These results indicate that relative to 13-APA, addition of a second nitrogen at C₁₄ and a double bond between the 12- and 13- positions results in a loss of receptor activity but produces a high affinity for the platelet cyclo-oxygenase.