Inhibition Of Thromboxane B2 Formation Research Articles

Abstract 34: High urinary thromboxane B2 associates with lethal prostate cancer in African American men and inversely correlates with aspirin use

Abstract Background: Systemic low-grade inflammation is a candidate risk factor for prostate cancer (PC) in African American (AA) men, potentially contributing to lethal PC development. We previously reported that regular aspirin use is associated with a decreased risk of advanced stage disease and disease recurrence in AA patients with PC. The cancer-preventive benefits of aspirin have been attributed to inhibition of the cyclooxygenase (COX) pathway. Thromboxane A2 (TXA2) is an eicosanoid produced by the COX1 enzyme in platelets. Aspirin can inhibit metastasis by inhibiting COX1 activity and TXA2 synthesis. Hence, we assessed the role of TXA2 in the development of lethal PC. Because TXA2 is unstable, we measured its corresponding primary metabolite, urinary thromboxane B2 (TXB2), to examine the relationship of TXA2 with PC. Method: TXB2 was measured in cases (977) and controls (1022) from the NCI-MD PC Case-Control study using a mass-spectrometry-based assay. TXB2 levels were modeled into low (≤median) and high (>median) levels. We calculated risk factor-adjusted odds ratios (ORs) and 95% confidence intervals (CI) using unconditional logistic regression, adjusted hazard ratios (HR) using Cox regression and sub hazard ratios (SHR) using the Fine-Gray model. Results: We observed increased TXB2 levels in cases and an inverse relationship between levels of TXB2 and aspirin use in both cases and controls. For cases who used aspirin, the odds of having high TXB2 were reduced when compared to non-users (OR 0.46 95%CI, 0.34-0.61). This observation remained significant when stratified by race/ethnicity (AA: OR 0.36 95%CI, 0.23-0.54; EA: OR 0.58 95%CI, 0.38-0.88), indicating significant inhibition of TXB2 formation by aspirin, most notably in AA men. Further analysis showed that high TXB2 is associated with a PC case status in AA men (OR 1.44 95%CI, 1.08-1.93) but not EA men (OR 1.07 95%CI, 0.83-1.93), indicating increased TXA2 synthesis in AA men with PC compared to AA men without the disease. Also, men with high TXB2 were more likely to be diagnosed with metastasis compared to men with low TXB2 (OR 4.27 95%CI, 1.48-12.29), indicating more aggressive disease for cases with high TXB2. Furthermore, high TXB2 was associated with all-cause mortality (HR 1.56 95%CI, 1.05-2.33) and PC mortality (HR 4.02 95%CI, 1.46-11.07; SHR 2.89 95%CI, 1.03-8.11) in AA men only. Our findings indicate a distinct association between TXA2 and PC outcomes in AA men. Conclusion: Aspirin use inversely associates with high TXB2. This is of clinical interest as we report that increased TXB2 associates with PC in AA men, with aggressive PC, and with PC death which disproportionally affects AA men. This is consistent with our previous findings that aspirin may reduce lethal PC in AA men and highlights the potential benefit of aspirin for prevention of lethal PC in this high-risk group of men through inhibition of TXA2 synthesis. Citation Format: Maeve Bailey-Whyte, Ginger Milne, Tsion Z. Minas, Tiffany H. Dorsey, Wei Tang, Cheryl J. Smith, Michael B. Cook, Clayton Yates, Stefan Ambs. High urinary thromboxane B2 associates with lethal prostate cancer in African American men and inversely correlates with aspirin use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 34.

Abstract PO-157: High urinary thromboxane B2 associates with aggressive prostate cancer and inversely correlates with aspirin use

Abstract Background: Systemic low-grade inflammation is a candidate risk factor for prostate cancer (PC) in African-American (AA) men potentially contributing to aggressive disease and mortality. Regular use of the anti-inflammatory drug aspirin is associated with decreased risk of advanced stage PC and increased disease-free survival in AA men. The chemopreventive benefits of aspirin have been attributed to inhibition of the cyclooxygenase (COX) pathway. Thromboxane A2 is an eicosanoid produced by the COX 1 enzyme in platelets. Aspirin inhibits cancer metastasis by inhibiting COX1 activity and thromboxane A2 synthesis. Hence, we assessed the role of thromboxane A2 in the development of lethal PC. Because thromboxane A2 is highly unstable, we measured its corresponding primary metabolite, urinary thromboxane b2 (TXB2), to examine the relationship of thromboxane A2 levels with PC. Method: TXB2 was measured in cases (977) and controls (1023) from the NCI-MD PC Case Control study using a mass-spectrometry-based assay. We estimated odds ratios (ORs) and 95% confidence intervals (CI) using unconditional logistic regression, with TXB2 levels modeled by quartiles. Multivariable models were adjusted for PC risk factors. Results: We observed an inverse relationship between aspirin use and levels of TXB2 in both cases and controls. For cases who used aspirin, the odds of having TXB2 urinary levels in the highest quartile (Q4) were significantly reduced when compared to non-users (adjusted OR = 0.29 95%CI 0.19-0.4) and this observation remained significant when stratified by race/ethnicity (AA men: OR 0.21 95%CI 0.11-0.38) (EA men: OR 0.34 95%CI 0.19-0.61), indicating significant inhibition of TXB2 formation by aspirin. The adjusted OR for PC was 1.42 (95%CI 1.09-1.85) for men in Q4 compared to Q1 (P trend 0.002). There was an association between TXB2 and PC in AA men (Q4 adjusted OR 1.95 95%CI 1.31-2.91, P trend <0.000) but not EA men (Q4 adjusted OR 1.04 95%CI 0.72-1.49, P trend 0.58), indicating increased thromboxane A2 synthesis in AA men with PC when compared to AA men without the disease. Also, men with Q4 levels of TXB2 were more likely to be diagnosed with metastasis compared to men with Q1 levels of TXB2 (adjusted OR 6.98 95%CI 1.51-32.32, P trend 0.014), indicating more aggressive disease for cases with the highest levels of TXB2. Conclusion: Aspirin use is protective against high levels of urinary TXB2. This is of clinical interest as we report increased urinary TXB2 associates with PC in AA men as well as with more aggressive PC which disproportionally affects AA men. These findings are consistent with our previous findings that aspirin inhibits aggressive disease in AA men and highlights the potential benefit of aspirin as a chemoprotective agent for this population through inhibition of thromboxane A2 synthesis. Citation Format: Maeve Kiely, Ginger Milne, Tsion Z. Minas, Tiffany H. Dorsey, Wei Tang, Cheryl J. Smith, Michael B. Cook, Clayton Yates, Stefan Ambs. High urinary thromboxane B2 associates with aggressive prostate cancer and inversely correlates with aspirin use [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-157.

Effects of chronic low-dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis.

Although early detection and treatment of colorectal cancer (CRC) have improved, it remains a significant health‐care problem with high morbidity and mortality. Data indicate that long‐term intake of low‐dose aspirin reduces the risk of CRC; however, the mechanisms underlying this chemopreventive effect are still unclear. Different mouse models for inflammation‐associated, sporadic, and hereditary CRC were applied to assess the efficacy and mechanism of low‐dose aspirin on tumor prevention. An initial dosing study performed in healthy mice indicates that aspirin at a dose of 25 mg/kg/d has a similar pharmacodynamic effect as low‐dose aspirin treatment in human subjects (100 mg/d). Chronic low‐dose aspirin treatment suppresses colitis‐associated and to a lesser extent spontaneous tumorigenesis in mice. Aspirin's antitumor effect is most pronounced in a preventive approach when aspirin administration starts before the tumor‐initiating genotoxic event and continues for the duration of the experiment. These effects are not associated with alterations in cell proliferation, apoptosis, or activation of signaling pathways involved in CRC. Aspirin‐induced reduction in tumor burden is accompanied by inhibition of thromboxane B2 formation, indicating reduced platelet activation. Aspirin treatment also results in decreased colonic prostaglandin E2 formation and tumor angiogenesis. With respect to colitis‐triggered tumorigenesis, aspirin administration is associated with a reduction in inflammatory activity in the colon, as indicated by decreased levels of pro‐inflammatory mediators, and tumor‐associated iNOS‐positive macrophages. Our results suggest that low‐dose aspirin represents an effective antitumor agent in the context of colon tumorigenesis primarily due to its well‐established cyclooxygenase inhibition effects.

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.

The preventive effect of ketoconazole on experimental metastasis from a human pancreatic carcinoma may be related to its effect on prostaglandin synthesis.

Arachidonic acid metabolites known to affect platelet function also interfere with tumor growth and metastases. The purpose of this study was to evaluate the anti-metastatic potential of ketoconazole, a thromboxane synthetase and 5-lipoxygenase inhibitor, on hepatic metastasis from a human pancreatic adenocarcinoma in nude mice and its effect on serum prostaglandin levels. The human pancreatic tumor cells (RWP-2) were injected intrasplenically in nude mice grouped into control, ketoconazole (270 microg), ketoconazole (360 microg), and ketoconazole (540 microg). The agent was administered intraperitoneally 30 min before and every 24 h after the tumor cell inoculation for 8 days. In a separate experiment thromboxane B2 (TxB2), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and 6-Keto-F1a (stable prostacyclin derivative) were measured on blood from controls, tumor bearing animals and animals bearing tumors treated with 270 microg of ketoconazole. Statistically significant differences were observed between the control and three-treatment groups on the reduction of liver tumor nodules (p < 0.001), and in the liver surface areas occupied by tumor (p < 0.001). The TxB2 levels decreased from 150.6 ng/mL in the tumor bearing to 104.8 ng/mL in the ketoconazole treated animals (p < 0.05). PGD2, PGE2 and 6-keto-F1a levels increased to 7.1 ng/mL, 8.3 ng/mL, and 13.6 ng/mL from 3 ng/mL, 5.8 ng/mL, and 0.02 ng/mL respectively (p < 0.001). These results indicate that ketoconazole significantly reduced hepatic metastases from the human pancreatic carcinoma RWP-2 in the nude mouse model, and inhibited thromboxane B2 formation, potentiating a concomitant redirection of platelet endoperoxide metabolism into PGD2, PGE2, and 6-keto-F1a. It is hypothesized that the changes in the arachidonic acid metabolism mediate the ameliorating effect of ketoconazole on experimental hepatic metastasis.

Inhibitory mechanisms of naloxone on human platelets.

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.

Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein Ib Antagonist From Venom of Crotalus atrox

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom ofCrotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 μg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 μg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 ± 0.1 × 10−7 mol/L, and its binding site was estimated to be 58,632 ± 3,152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 μg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 μg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 μg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein Ib Antagonist From Venom of Crotalus atrox

AbstractA potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom ofCrotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 μg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 μg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 ± 0.1 × 10−7 mol/L, and its binding site was estimated to be 58,632 ± 3,152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 μg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 μg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 μg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

Inhibition of blood platelet functions by cationic amphiphilic drugs in relation to their physico-chemical properties

The effects of bromadryl, dithiaden, chloroquine and propranolol on thrombin-stimulated rat platelet aggregation (measured turbidimetrically) and thromboxane B2 generation (detected by an RIA method) were compared with four selected physico-chemical parameters of these drugs. Platelet aggregation was inhibited in the rank order of potency: bromadryl > dithiaden > propranolol > chloroquine, which corresponded with the decrease in the net charge of the terminal methyl-(or ethyl-) groups in the side chain and with the increase of the dipole moment of drug molecules. On the other hand, the rank order of potency in which the drugs tested inhibited thromboxane B2 formation (chloroquine > dithiaden > bromadryl > propranolol) correlated well with the decline in molar refractivity of the drugs. No relationship was found between inhibitory effects of drugs and their partition coefficients. The results presented indicate that inhibition of platelet functions might consist of several types of drug-cell interactions, depend on the structure and physico-chemical properties of the drugs and cannot be estimated simply on the basis of partition coefficients.

Antiplatelet effect of demethyldiisoeugenol.

A semisynthetic chemical compound, demethyldiisoeugenol, concentration dependently inhibited platelet aggregation and ATP release stimulated by thrombin (0.1 U/mL), platelet-activating factor (2 ng/mL), arachidonic acid (100 microM), collagen (10 micrograms/mL), and U46619 (1 microM) in rabbit washed platelets. The IC50 values of demethyldiisoeugenol on the inhibition of platelet aggregation induced by these five agonists were 157.3 +/- 22.3, 156.2 +/- 12.9, 53.6 +/- 4.7, 54.5 +/- 3.9, and 87.7 +/- 3.2 microM, respectively. Demethyldiisoeugenol also inhibited thromboxane B2 formation induced by thrombin, platelet-activating factor, arachidonic acid, and collagen, and prostaglandin D2 formation was induced by arachidonic acid. The rising intracellular Ca2+ concentration stimulated by these five platelet aggregation inducers was inhibited by demethyldiisoeugenol, while formation of inositol monophosphate was unaffected. Platelet cAMP and cGMP levels were unchanged by demethyldiisoeugenol. It is concluded that demethyldiisoeugenol may directly inhibit both intracellular calcium mobilization and cyclooxygenase activity in platelets.

Antiplatelet and vasorelaxing actions of the acetoxy derivative of cedranediol isolated from Juniperus squamata.

The antiplatelet and vasorelaxing actions of 14-acetoxycedrol, an acetyl derivative of the sesquiterpene 8,14-cedranediol isolated from Juniperus squamata Hayata, were investigated in washed rabbit platelets and rat aorta, respectively. 14-Acetoxycedrol inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, platelet-activating factor (PAF), collagen, and thrombin. Prolongation of the incubation time of 14-acetoxycedrol with platelets did not cause further inhibition and the aggregability of the treated platelets could be restored after washing of the platelets. It inhibited thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, and thrombin in a concentration-dependent manner. The formation of inositol phosphate caused by collagen and PAF was inhibited by 14-acetoxycedrol, while that caused by thrombin was not affected. 14-Acetoxycedrol markedly inhibited the intracellular calcium rise caused by PAF, and slightly inhibited that caused by thrombin in quin-2/AM-load platelets. In rat thoracic aortae, 14-acetoxycedrol inhibited the high K+ (60 mM) and Ca2+ (0.03-3 mM) induced cumulative contractions in a concentration-dependent manner, while it did not affect the phasic and tonic contractions elicited by norepinephrine. The tonic contractions elicited by KCl (60 mM) and Bay K 8644 were also relaxed by 14-acetoxycedrol. It is concluded that the antiplatelet effect of 14-acetoxycedrol is due to the inhibition of thromboxane formation and phosphoinositides breakdown and the vasorelaxing action of 14-acetoxycedrol is due to inhibition of Ca2+ influx through the voltage-dependent Ca2+ channel.

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.

Protection by nitecapone against sodium taurocholate-induced damage to cultured gastric cells.

The goal of this study was to observe if nitecapone protected against taurocholate-induced damage in primary cultured rat gastric mucosal cells, as well as in a well-differentiated human gastric epithelial cell line (MKN 28). Prostaglandins were measured to analyze the protection mechanism. In primary rat gastric mucosal cell culture, nitecapone 125-250 microM protected the cells significantly against damage induced by sodium taurocholate, increasing cell viability by 31-38%. In the human gastric epithelial cell line, in which mitochondrial activity was measured as an indication of cell viability, nitecapone (62.5-250 microM) protected the cells against sodium taurocholate-induced damage by 12-20%. Prostaglandin E2, thromboxane B2, and 6-keto-prostaglandin F1 alpha measurements in the primary cultured rat gastric mucosal cells showed that nitecapone (125 microM and 250 microM) significantly stimulated prostaglandin E2 production (84.7% and 61.0%, respectively), and inhibited thromboxane B2 formation (50% at 250 microM), while the 6-keto-prostaglandin F1 alpha formation was unaffected. Nitecapone had no effect on prostaglandin E2 production in the MKN 28 epithelial cell line. Indomethacin or aspirin, at concentrations that did not affect cell viability, antagonized the stimulative effect of nitecapone on prostaglandin E2 formation in the primary cultured rat gastric mucosal cells. Although the prostaglandin E2 synthesis was blocked, nitecapone still protected against cell damage induced by taurocholate. These results demonstrated the direct and efficacious protection of nitecapone on gastric cell level and suggest that the "cytoprotection" by nitecapone against taurocholate may not be mediated through the mechanism of stimulated synthesis of prostaglandin E2.

Cyclooxygenase inhibitors blunt thromboxane action in human placental arteries by blocking thromboxane receptors.

The effects of cyclooxygenase inhibitors on thromboxane-mediated vasoconstriction in human placental arteries were studied in the isolated perfused fetoplacental cotyledon. The stable thromboxane agonist U-46619 caused a dose-related increase in perfusion pressure in the fetal side of the cotyledon. Meclofenamate (3.3 x 10(-5) M) significantly blunted the pressor response to U-46619, but not to angiotensin II, and inhibited thromboxane B2 formation in placental slices (IC50, 4.80 x 10(-8) M). The mechanism by which meclofenamate prevented thromboxane-induced vasoconstriction was studied using ligand-binding techniques in a membrane fraction prepared from placental cotyledons. Meclofenamate caused a dose-related inhibition of binding of the thromboxane receptor antagonist [3H]SQ 29548 with an IC50 of 2.61 x 10(-5) M. Scatchard analysis of equilibrium binding demonstrated that meclofenamate reduced the number of binding sites without altering the affinity of the receptor, suggesting a noncompetitive mechanism. Indomethacin also caused a dose-related inhibition of thromboxane binding (IC50, 3.27 x 10(-4) M). However, aspirin at a dose of 2.0 x 10(-3) M did not inhibit [3H]SQ 29548 binding. The data indicate that some cyclooxygenase inhibitors blunt thromboxane actions by interfering with binding at thromboxane receptor sites. These studies identify a new mechanism by which cyclooxygenase inhibition by some nonsteroidal anti-inflammatory drugs can prevent thromboxane action in fetoplacental blood vessels in vitro independent of reductions in thromboxane formation.

1,2-Dioctanoylglycerol but not 1-oleoyl-2-acetylglycerol inhibits agonist-induced platelet responses. Dependence of effects on extent of 45-kDa protein phosphorylation and agonist type.

1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro) and 1-oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets. 2. Pre-treatment (10-300 s) with Oco2Gro (15-60 microM) or PMA (16 nM) before addition of thrombin (0.2 U/ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular Ca2+ ([Ca2+]i) mobilisation and arachidonate/thromboxane B2 release. OleAcGro (62-125 microM) had no effect on thrombin-induced [Ca2+]i elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of Oco2Gro, PMA or OleAcGro on their own caused no rise in [Ca2+]i levels or arachidonate release. 3. Collagen (20 micrograms/ml) induced substantial arachidonate release without a detectable rise in [Ca2+]i. Pretreatment (10-300 s) with Oco2Gro (15-60 microM), PMA (16 nM) or OleAcGro (62 microM) before collagen addition or addition of these agents 30-60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2--2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of Oco2Gro or PMA. 4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations (less than 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5-15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 microM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with less than 2-min incubations) at sub-maximal agonist concentrations. 5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while Oco2Gro (60 microM) and PMA (16 nM) caused a 4--5-fold increase in 32P-labelling of this protein over a 5-min incubation period, OleAcGro (62-125 microM) caused a 1.5-fold increase in labelling which was only maintained for a 10--30-s period.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of cyclooxygenase activity and platelet aggregation by epoxyeicosatrienoic acids. Influence of stereochemistry.

Certain epoxyeicosatrienoic acids (EETs) that were not cyclooxygenase substrates were effective cyclooxygenase inhibitors. Both (+/-)-14,15-cis-EET and (+/-)-8,9-cis-EET inhibited purified enzyme at concentrations from 1 to 50 microM; (+/-)-11,12-cis-EET was ineffective at concentrations below 100 microM. For the case of 14,15-cis-EET, only the (14R,15S)-stereoisomer was active. Other isomers including (14S,15R)-cis-EET, (14R,15R)-trans-EET, (14S,15S)-trans-EET, and the erythro and threo vicinal 14,15-diols were inactive. In addition to their effects on isolated enzyme preparations, cyclooxygenase activity in platelet suspensions, reflected by thromboxane B2 formation, was also inhibited by (14R,15S)-cis-EET and (+/-)-8,9-cis-EET but not by the other isomers. Thus potency and stereospecificity requirements were maintained for cyclooxygenase within intact platelets. Unlike the stereospecific inhibition of the cyclooxygenase enzyme, platelet aggregation induced by arachidonic acid was inhibited by all EET isomers at concentrations from 1 to 10 microM with no evident stereospecificity. Inhibition of aggregation was not uniformly associated with inhibition of thromboxane B2 formation; ordinarily, these two parameters correlate closely. This dissociation was not maintained for another biochemical process involved in platelet activation. For instance, there was a uniform correlation between inhibition of phosphorylation of a 40-kDa platelet protein and inhibition of aggregation. Our results suggest that effects of EET may originate from either stereospecific or nonspecific mechanisms. Definition of such mechanisms may be important to appreciate any physiological relevance of these substances.

Inhibition by pesticides of prostaglandin formation in blood platelets.

Aggregation of human platelets was investigated after pre-incubation of platelet rich plasma with various pesticides of the carbamate type. Whereas N-methyl-carbamates (insecticides) inhibited the arachidonic acid induced aggregation, N-phenyl-carbamates (herbicides) had no effect. The influence of the different carbamates on aggregation coincided with their inhibition of thromboxane B2-formation. - DDE, a metabolite of the insecticide DDT, affected aggregation to a similar extent as N-methyl-carbamates. DDT, however, had no inhibitory activity.

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets.

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B2 both from [1-14C]arachidonic acid added to washed platelets and from [1-14C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin at 1 and 10 mg/ml inhibited the formation of thromboxane B2 from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B2 in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B2 (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B2 and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B2 formation by the elastin.