Nicotinic acid inhibits thromboxane synthesis in platelets

The formation of thromboxane A₂ by phospholipase A₂ in rat platelets is inhibited by nicotinic acid. The synthesis of PGE₂ and PGF_2α is increased.Nicotinic acid inhibits collagen-induced aggregation in rat platelets.     

The relationship between thromboxane and cytosolic calcium modifiers in rat platelets.

Platelet activity is controlled, in part, by cytosolic free ionized calcium concentration ([Ca++]i). Regulation of platelet thromboxane (TXB2) synthesis may be by regulation of [Ca++]i. Dietary linoleate is a regulator of TXB2 synthesis, therefore, it may act by influencing [Ca++]i. Aspirin is a regulator of TXB2 synthesis by inhibition of cyclooxygenase; ouabain and nifedipine are regulators of [Ca++]i. This study was conducted to determine whether these affectors of TXB2 synthesis and [Ca++]i cause associated responses. Male nonobese Zucker rats were fed diets supplying 30% of energy (en%) as fat. Dietary fat was a mixture of corn oil and beef tallow to provide 3.0, 4.5, 6.0, or 7.5 en% linoleic acid, with cholesterol added to provide equal cholesterol in all diets. Rats were fed for 30 days with 6 rats/diet. Isolated rat platelets were assayed for FA composition; the percentage of linoleic acid in platelet FA rose linearly with increasing dietary linoleate (r = 0.76, P less than 0.0001). Resting and thrombin-stimulated platelet [Ca++]i and TXB2 synthesis were measured in the presence or absence of extracellular calcium and aspirin, ouabain, or nifedipine. Aspirin caused reductions in both parameters; nifedipine blocked [Ca++]i, but did not affect TXB2; ouabain increased both. Changes induced by those modifiers of TXB2 and platelet [Ca++]i caused changes that were in the same direction for both. CaCl2 caused an increase in both and the [Ca++]i was correlated with the square root of the TXB2; without CaCl2 the two were negatively correlated; aspirin, ouabain, and nifedipine treatments resulted in no significant correlations. The results suggest that there is a common modifier of [Ca++]i and TXB2 synthesis.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO2) exposure on prostacyclin (PGI2) synthesis in the rat lung and thromboxane A2 (TXA2) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO2 for 1, 3, 5, 7 and 14 days. PGI2 synthesizing activity of homogenized lung decreased. The damage of PGI2 synthesizing activity reaches its maximum at 3 days. At 14 days, PGI2 synthesizing activity returned to the normal level. The activity of PGI2 synthetase decreased significantly. The formation of lipid peroxides due to NO2 exposure may cause the depression of PGI2 synthesizing activity of lung. On the other hand, platelet TXA2 synthesizing activity increased. This increased TXA2 synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO2 exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO2 exposure. These results indicate that the depression of PGI2 synthesizing activity of lung by NO2 exposure cause an increase in TXA2 synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO2 exposure.     

Interference with S and G2 phase progression by polyamine synthesis inhibitors

The distribution of Ehrlich ascites tumor cells in the cell cycle was studied by flow cytometry following treatment with α-methyl ornithine (αMO) or methylglyoxal-bis(guanylhydrazone) (MeGAG). αMO and MeGAG are potent inhibitors of ornithine decarboxylase (putrescine synthesis) and S-adenosyl-methionine decarboxylase (spermidine and spermine synthesis), respectively. The data show that these polyamine synthesis inhibitors produce significant cell cycle perturbations resulting in an accumulation of cells in S and G2. This fact suggests that a normal polyamine complement is essential for the progression through these phases of the cell cycle.     

Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells

Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A2. When primary cultures of human umbilical vein endothelial cells were incubated with 14C-arachidonic acid and the 14C-metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F1alpha and thromboxane B2, the degradation products of prostacyclin and thromboxane A2, respectively. Since platelets synthesize thromboxane A2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10(-5) M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F1alpha and thromboxane B2. The production of thromboxane B2 decreased in the passaged cells (207 +/- 44 pg/ml versus 65 +/- 12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F1alpha was measured in the passaged cells compared to the primary cultures (3159 +/- 356 pg/ml versus 1678 +/- 224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83 +/- 15 pg/ml versus 65 +/- 12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B2 and 6-keto-prostaglandin F1alpha decreased. In contrast, the thromboxane A2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F1alpha. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.     

Pharmacology of thromboxane synthetase inhibitors

Several selective and potent thromboxane synthetase inhibitors have now been described. In this paper I shall summarize their effects on platelet aggregation, endotoxin- and thrombin-induced pulmonary hypertension, the Forssman reaction-induced thrombocytopenia, and the rat tail vein bleeding time. Although any clinical utility of thromboxane synthetase inhibition remains to be demonstrated, it may be useful in conditions where vasoconstriction or vasospasm are involved, particularly where platelet activation is a contributing factor.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO₂) exposure on prostacyclin (PGIP₂) synthesis in the rat lung and thromboxane A₂ (TXA₂) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO₂ for 1, 3, 5, 7 and 14 days. PGI₂ synthesizing activity of homogenized lung decreased. The damage of PGI₂ synthesizing activity reaches its maximum at 3 days. At 14 days, PGI₂ synthesizing activity returned to the normal level. The activity of PGI₂ synthetase decreased significantly. The formation of lipid peroxides due to NO₂ exposure may cause the depression of PGI₂ synthesizing activity of lung. On the other hand, platelet TXA₂ synthesizing activity increased. This increased TXA₂ synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO₂ exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO₂ exposure. These results indicate that the depression of PGI₂ synthesizing activity lung by NO₂ exposure cause an increase in TXA₂ synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO₂ exposure.     

Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Effects of inhibitors of thromboxane synthesis on reactions of the cat bronchus in situ to prostaglandins

Cats were anesthetized by chloralose, relaxed by suxamethonium, and ventilated. The influence of 3 inhibitors of thromboxane synthesis, N.0096 (±α-benzyl-α-p-chlorobenzyl-4-hydroxyacetophenon phenylhydrogen-phosphonate), imidazole and dipyridamole were studied on bronchial reactions to prostaglandins E₂ and F₂α and arachidonic acid. During a bronchoconstriction maintained by infusion of 5-HT, N.0096 and imidazole dose-dependently potentiated the intensity and duration of the bronchodilator effect of PGE₂. This effect was maximal at a total dose of 17 mg/kg N.0096 or 25–50 mg/kg imidazole, but decreased after higher doses which are supposed to inhibit cyclo-oxygenase also. Bronchoconstrictor reactions to single injections of arachidonic acid were inhibited by N.0096 in the same dose range. Reactions to PGF_2α were unaltered by both drugs. Dipyridamole was devoid of a comparable activity.     

Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.     

Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain.

—Certain of the sulphur containing amino acids have been associated with synaptic transmission in the central nervous system. The enzymes involved in the synthesis of these putative neurotransmitter or modulator compounds have a different subcellular distribution in rat brain from those enzymes that catalyse the synthesis of other compounds in this pathway. Methionine adenosyltransferase and 5-methyltetrahydrofolate-homocysteine methyltransferase catalyse reactions that maintain the methylation functions of the pathway and are found in soluble fractions. Cystathionine β-synthase, cystathionase, cysteine dioxygenase and cysteine sulphinic acid decarboxylase catalyse the synthesis of those sulphur-containing amino acids implicated in neurotransmitter functions and these enzymes have both paniculate and soluble components. Serine hydroxymethyltransferase, which also has a particulate fraction in brain, is responsible for the synthesis of the neurotransmitter glycine, in addition to its role in the methionine-related metabolism of folate.     

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

The levels of the stable degradation products of prostacyclin (PGI2) and thromboxane A2 (TXA2): 6-oxo-prostaglandin E1 alpha (6-oxo-PGE1 alpha) 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 in vivo compared with platelets in vitro.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i 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 .     

Early increase in phosphatidyl choline synthesis by choline and transmethylation pathways in spreading fibroblasts

Phosphatidyl choline (PC) synthesis in trypsinized and reattaching fibroblasts during the spreading state was studied by incorporation of [14C]choline and [methyl-14C]methionine. The choline and phosphatidyl-ethanolamine (PE) transmethylation pathways were both transiently increased about 2-fold during the first 2 h after replating. Maximum increase appeared to be simultaneous with maximum spreading. Incorporation of [32P]orthophosphate showed that the increase in PC synthesis was specific and most probably related to establishment of cell-substrate adhesion sites.     

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC₅₀=100 nM) and thromboxane A₂ (TxA₂) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA₂ receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA₂ synthesis in human platelets.