Thromboxane A2 Synthase Activity Research Articles

Antiplatelet and antithrombotic effects of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo, in vivo, and in vitro.

BackgroundA species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo, in vivo, and in vitro antiplatelet and antithrombotic effects.MethodsEx vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation, serotonin release, thromboxane A2 (TXA2) production and its associated activities of enzymes [cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS)], arachidonic acid (AA) release and its associated phosphorylation of phospholipase Cβ3, phospholipase Cγ2 or cytosolic phospholipase A2, mitogen-activated protein kinase (MAPK) [p38 MAPK, extracellular signal-regulated kinase (ERK)], and blood coagulation time in rats were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. In vitro effects of WIB-801CE on cytotoxicity, and fibrin clot retraction in human platelets, and nitric oxide (NO) production in RAW264.7 cells or free radical scavenging activity were studied.ResultsCordycepin-enriched WIB-801CE inhibited ex vivo platelet aggregation, TXA2 production, AA release, TXAS activity, serotonin release, and p38 MAPK and ERK2 phosphorylation in collagen- and ADP-activated rat platelets without affecting blood coagulation. Furthermore, WIB-801CE manifested in vivo inhibitory effect on collagen plus epinephrine-induced pulmonary thromboembolism mice model. WIB-801CE inhibited in vitro NO production and fibrin clot retraction, but elevated free radical scavenging activity without affecting cytotoxicity against human platelets.ConclusionWIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. These were resulted from down-regulation of TXA2 production and its related AA release and TXAS activity, and p38 MAPK and ERK2 activation. These results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.

Water Extract from Rice Bran Fermented with Lactobacillus plantarum Hong Inhibits Thromboxane A2 Production Associated Microsomal Enzyme Activity in Human Platelets

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum Hong (RBLw), on activities of cyclooxygenase-1 (COX-1) and thromboxane A₂ synthase (TXAS), thromboxane A₂ (TXA₂) production associated microsomal enzymes and evaluated its the antiplatelet effect. RBLw, containing 13.5 mg of ferulic acid, dose-dependently inhibited ADP-induced platelet aggregation, and inhibited the production of TXA₂, an aggregation molecule. In addition, RBLw directly inhibited COX-1 activity in a dose-dependent manner, but not TXAS activity in platelet microsomal fraction having cytochrome c reductase (an endoplasmic reticulum marker enzyme) activity and expressing COX-1 (72 kDa) and TXAS (60.5 kDa) proteins. These results suggest that RBLw selectively inhibited the activity of COX-1 rather than TXAS to attenuate TXA₂ production in ADP-activated platelets. Thus, we demonstrate that RBLw might have direct COX-1 antagonistic function for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Furegrelate, a Thromboxane Synthase Inhibitor, Blunts the Development of Pulmonary Arterial Hypertension in Neonatal Piglets

The development of pulmonary arterial hypertension (PAH) in pediatric patients has been linked to the production of the arachidonic acid metabolite, thromboxane A2 (TxA2). The present study evaluated the therapeutic effect of furegrelate sodium, a thromboxane synthase inhibitor, on the development of PAH in a neonatal piglet model. Three-day-old piglets were exposed to 21 days of normoxia (N; 21% FIO2) or chronic hypoxia (CH; 10% FIO2). A third group of piglets received the oral TxA2 synthase inhibitor, furegrelate (3 mg/kg, 2 or 3 times daily) at the induction of CH. In vivo hemodynamics confirmed a 2.55-fold increase of the pulmonary vascular resistance index (PVRI) in CH piglets (104±7 WU) compared to N piglets (40±2 WU). The CH piglets treated twice daily with furegrelate failed to show improved PVRI, but furegrelate three times daily lowered the elevated PVRI in CH piglets by 34% to 69±5 WU and ameliorated the development of right ventricular hypertrophy. Microfocal X-ray computed tomography (CT) scanning was used to estimate the diameter-independent distensibility term, α (% change in diameter per Torr). Pulmonary arterial distensibility in isolated lungs of CH piglets (α=1.0±0.1% per Torr) was lower than that of N piglets (α=1.5±0.1% per Torr) indicative of vascular remodeling. Arterial distensibility was partially restored in furegrelate-treated CH piglets (α =1.2±0.1% per Torr) and microscopic evidence showing muscularization of small pulmonary arteries also was less prominent in these animals. Finally, isolated lungs of furegrelate-treated piglets showed lower basal and vasodilator-induced transpulmonary pressures compared to CH animals. These findings suggest that pharmacological inhibition of TxA2 synthase activity by furegrelate blunts the development of hypoxia-induced PAH in an established neonatal piglet model primarily by preserving the structural integrity of the pulmonary vasculature.

Antiplatelet Effect of NQ12: a Possible Mechanism Through the Arachidonic Acid Cascade

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC50 values of 0.71 ± 0.2, 0.82 ± 0.3, and 0.45 ± 0.1 µM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A2 receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA2 formation, but had no effect on the PGD2 production, indicating an inhibitory effect on TXA2 synthase, which was supported by a TXA2 synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA2 formation converted from PGH2. On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA2 synthase activity and TXA2 receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.

Antiplatelet Activity of Carnosic Acid, a Phenolic Diterpene from Rosmarinus officinalis

Carnosic acid is a major phenolic diterpene derived from Rosmarinus officinalis and has been reported to have antioxidant, antibacterial, anticancer, antiobese and photoprotective activities. This study investigated the antiplatelet activity of carnosic acid. carnosic acid significantly inhibited collagen-, arachidonic acid-, U46619- and thrombin-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 39+/-0.3, 34+/-1.8, 29+/-0.8 and 48+/-2.9 microM, respectively, while it failed to inhibit PMA-(a direct PKC activator) and ADP-induced platelet aggregation. In agreement with its antiplatelet activity, carnosic acid blocked collagen-, arachidonic acid-, U46619- and thrombin-mediated cytosolic calcium mobilization. accordingly, serotonin secretion and arachidonic acid liberation were also inhibited in a similar concentration-dependent manner. However, in contrast to the inhibition of arachidonic acid-induced platelet aggregation, carnosic acid had no effect on the formation of arachidonic acid-mediated thromboxane A2 and prostaglandin D2, thus indicating that carnosic acid has no effect on the cyclooxygenase and thromboxane A2 synthase activity. Overall, these results suggest that the antiplatelet activity of carnosic acid is mediated by the inhibition of cytosolic calcium mobilization and that carnosic acid has the potential of being developed as a novel antiplatelet agent.

An Antithrombotic Agent, NQ301, Inhibits Thromboxane A2 Receptor and Synthase Activity in Rabbit Platelets

In previous studies we have reported that NQ301, a synthetic 1,4-naphthoquinone derivative, displays a potent antithrombotic activity, and that this might be due to antiplatelet effect, which was mediated by the inhibition of cytosolic Ca(2+) mobilization in activated platelets. In the present study, the effect of NQ301 on arachidonic acid cascade in activated platelets has been examined. NQ301 concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen (10 microg/ml), arachidonic acid (100 microM) and U46619 (1 microM), a thromboxane A2 receptor agonist, with IC50 values of 0.60+/-0.02, 0.78+/-0.04 and 0.58+/-0.04 microM, respectively. NQ301 also produced a shift to the right of the concentration-effect curve of U46619, indicating a competitive type of antagonism on thromboxane A2/prostaglandin H2 receptor. NQ301 slightly inhibited collagen-induced arachidonic acid liberation. In addition, NQ301 potently suppressed thromboxane B2 formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner, but had no effect on the production of prostaglandin D2, indicating an inhibitory effect on thromboxane A2 synthase. This was supported by thromboxane A2 synthase activity assay that NQ301 concentration-dependently inhibited thromboxane B2 formation converted from prostaglandin H2. Moreover, NQ301 concentration-dependently inhibited 12-hydroxy-5,8,10,14-eicosatetraenoic acid formation by platelets that were exposed to arachidonic acid. Taken together, these results suggest that NQ301 has a potential to inhibit thromboxane A2 synthase activity with thromboxane A2/prostaglandin H2 receptor blockade, and modulate arachidonic acid liberation as well as 12-hydroxy-5,8,10,14-eicosatetraenoic acid formation in platelets. This may also be a convincing mechanism for the antithrombotic action of NQ301.

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4 ± 0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2 ± 0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0 ± 1.0 nM) and sulotroban (IC50 = 930 ± 42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 μM) and U-46619 (1 μM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272 ± 0.054 μM) and rat aorta (EC50 = 0.190 ± 0.002 μM) than (R)-BM-591 (EC50 of 9.60 ± 0.63 μM and 0.390 ± 0.052 μM, respectively). Moreover, at 1 μM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A2synthase inhibition and TXA2/PGH2receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC50values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A2(TXA2) formation were 0.17 ± 0.01, 0.23 + 0.03, 0.34 ± 0.02 and 0.12 ± 0.01 g/ml, respectively. [3H]-AA release induced by collagen (10 μg/ml) in rat platelet was decreased by onion compared to control (22.1 ± 2.13 and 5.2 ± 0.82% of total [3H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca2+concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA2synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA2mimetic, was inhibited by onion, indicating its antagonism for TXA2/PGH2receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA2synthase inhibition and TXA2/PGH2receptor blockade.

Nitric Oxide Modulates the Activity of the Hemoproteins Prostaglandin I2Synthase and Thromboxane A2Synthase

Nitric oxide modulates the activity of the hemoprotein isomerase enzymes that transform prostaglandin H2into prostaglandin I2and thromboxane A2. Two nitric oxide donors, 1-hexanamine, 6-(2hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-nitroso-hydrazine (MMNN) and 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, modulated prostaglandin I2synthase activity in a bidirectional manner. At moderate concentrations, they increased enzyme activity irreversibly and at higher concentrations they inhibited enzyme activity reversibly. We confirmed that these effects originated from nitric oxide. First, we showed that hemoglobin, a substance that sequesters nitric oxide, prevented both the activation and the inhibition of catalysis, stoichiometrically. Second, we showed that solutions depleted of nitric oxide had no effect on catalysis. Nitric oxide also modulated thromboxane A2synthase activity; however, its effects on thromboxane A2synthase differed from its effects on prostaglandin I2synthase in three ways: (i) It inhibited thromboxane A2synthase in a concentration-dependent manner. The IC50= 4.2 ± 0.8 μM MMNN corresponded to an IC50≅ 0.1–0.3 μM nitric oxide. (ii) It did not increase thromboxane A2synthase activity at any concentration tested. (iii) Its irreversible inhibition of thromboxane A2synthase contrasted with its reversible inhibition of prostaglandin I2synthase. Nitric oxide also inhibited cellular formation of thromboxane A2by intact platelets in a concentration-dependent manner. The IC50= 267 ± 26 μM MMNN corresponded to an IC50≅ 6–18 μM nitric oxide. We conclude that nitric oxide can modulate certain hemoprotein enzymes in the biosynthetic cascade that governs the formation of eicosanoid mediators of thrombosis and hemostasis.

Actions of SQ 29,548 on contractile responses and arachidonic acid metabolism of intrapulmonary arteries, in vitro.

This study was undertaken to investigate the influence of SQ 29,548, a thromboxane (TX) A2 receptor antagonist, on contractile responses and arachidonic acid (AA) metabolism of bovine intrapulmonary arterial (IPA) rings. The contractile responses to 5-hydroxytryptamine (5-HT), histamine, phenylephrine, and potassium chloride (KC1) were not significantly altered by 10(-8) M SQ 29,548 in either endothelium-intact or denuded IPA. The concentration of SQ 29,548 was chosen as it reduced the response to 10(-7) M U46619, a TXA2 mimetic, by 50%. AA metabolism by IPA produced more PGI2 whereas that by intrapulmonary vein (IPV) produced more PGE2. SQ 29,548 in concentrations of 10(-8) to 10(-5) M did not affect the activity of PGI2 synthase or GSH-dependent PGE2 isomerase in IPA or IPV microsomal fractions. No microsomal TXA2 synthase activity was detectable. SQ 29,548 had no effect on PGH synthase activity of IPA or IPV. The data indicate the presence of a TXA2-mediated contractile response in the IPA which is endothelium-independent and is selectively antagonized by SQ 29,548. The data further indicate that the contractile responses of IPA to 5-HT, histamine, phenylephrine, and KCl do not have a TXA2-mediated component. It is suggested that SQ 29,548 is a pharmacological probe to determine the role of TXA2 n pathophysiologic states in the pulmonary vascular bed and may be a therapeutic agent to treat pulmonary hypertensive disorders in which TXA2 is involved.

Inhibition of pulmonary thromboxane A2 synthase activity and airway responses by CGS 13080.

The effects of CGS 13080, a thromboxane (TXA2) synthase inhibitor, on airway responses to arachidonic acid (AA) were investigated in the anesthetized cat. Feline and human lung microsomal fraction exhibited prostaglandin I2 (PGI2, prostacyclin), and TXA2 synthase activities, and human platelet microsomal fractions exhibited TXA2 synthase activity. Cat and human lung microsomal fractions, but not human platelets, exhibited the presence of GSH-dependent PGE2 isomerase activity. CGS 13080 inhibited TXA2 synthase activity in all three microsomal fractions in a concentration-dependent manner. The increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance in response to AA were decreased significantly by CGS 13080. These data suggest that the bronchoconstrictor actions of AA are mediated in large part by the formation of TXA2. The data further indicate that cyclooxygenase products other than TXA2 are involved in the bronchoconstrictor response to AA since meclofenamate had greater inhibitory activity than did CGS 13080. Moreover, the effects of CGS 13080 were due to inhibition of TXA2 synthase rather than an effect on TXA2 receptors, since airway responses to the TXA2 mimic, U46619, were not altered. The present data show that CGS 13080 inhibits TXA2 synthase activity without altering cyclooxygenase, PGI2 synthase, or GSH-dependent PGE2 isomerase activities. The data further indicate that in vivo administration of CGS 13080 may selectively increase PGI2 synthase activity.

Differential effects of ibuprofen, indomethacin, and meclofenamate on prostaglandin endoperoxide H2 metabolism

Previous studies have suggested the possibility that the non-steroidal antiflammatory drug (NSAID), ibuprofen, may inhibit thromboxane (TX) A2 synthase activity in addition to inhibiting cyclooxygenase activity. Microsomal fractions isolated from the cat lung contain cyclooxygenase as well as prostacyclin (PGI2) synthase, TX synthase, and a GSH-dependent prostaglandin (PG) E2 isomerase activities. When [1-14C] PG endoperoxide H2 (PGH2) was used as substrate, ibuprofen, indomethacin, and meclofenamate exhibited differential effects on terminal enzyme activities. Ibuprofen, at concentrations up to 1 mM, had no effect on the activities of PGI2 synthase, TXA2 synthase of GSH-dependent PGE2 isomerase, whereas indomethacin selectively inhibited PGI2 synthase activity at 5 X 10(-4) M and 10(-3) M. Meclofenamate selectively inhibited TXA2 synthase activity at 5 X 10(-4) M and 10(-3) M. At concentrations of 5 X 10(-3) M, this selectivity was not observed, and indomethacin and meclofenamate decreased the formation of both 6-keto-PGF1 alpha and TXB2. These data indicate that the choice of NSAID and the concentration employed may specifically alter PGH2 metabolism. This action may affect the physiologic consequences of the exchange of PGH2 between cells. The data further indicate that indomethacin has the potential for use as a tool to specifically attenuate PGI2 synthase activity in vitro.

Prostacyclin and thromboxane A2 formation by atherosclerotic carotid artery: Comparison with normal aorta, saphenous vein, and platelets

Prostacyclin (PGI2) and thromboxane A2 (TxA2) formation by whole-tissue segments of nine carotid endarterectomy specimens (CES), five normal aortic specimens (NAS), six saphenous vein specimens (SVS), and four platelet samples were determined by incubation with 10 μmol/L 1-14C-radiolabeled prostaglandin endoperoxide H2 (PGH2), and in other experiments with and without 10 μmol/L of CGS 13080, a TxA2 synthase inhibitor. PGI2 formation (expressed as picomoles 6-keto-PGF1α/2-min incubation per sample) by nonatheromatous proximal intima of CES (307 ± 23, mean ± standard error) and distal intima of CES (260 ± 22) was not statistically different; however, it was greater than atheromatous transitional plaque (159 ± 13 pmol) (p < 0.01) and ulceration regions (140 ± 15 pmol) (p < 0.01) of CES, NAS (204 ± 16 pmol) (p < 0.01), and SVS (165 ± 9 pmol) (p < 0.01). TxA2 formation (expressed as picomoles TxB2/2-min incubation per sample) by CES ulceration (51 ± 2 pmol) was low but greater than proximal (17 ± 2 pmol) (p < 0.01), distal (19 ± 3 pmol) (p < 0.01), and transitional (23 ± 3 pmol) (p < 0.01) regions. TxA2 formation by NAS and SVS was not detected (less than 10 pmol). CGS 13080 inhibited TxA2 formation by CES below the limits of detection. Incubation of 1.9 × 105 intact platelets with 10 μmol/L of PGH2 formed a quantity of TxA2 equal to that of CES ulceration. Platelet homogenization reduced TxA2 formation (p < 0.01), demonstrating the sensitivity of TxA2 synthase activity to experimental conditions and the requirement for whole-tissue incubation with and without CGS 13080 to confirm its presence. Diminished PGI2 and minimal, but detectable, TxA2 formation by ulcerated regions of CES may partially explain platelet adherence and thrombogenicity at these foci because of imbalance between vasoregulatory eicosanoids.