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Background RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), α-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles. Methods and results We studied two related patients with a RUNX1 mutation, easy bruising, and mild thrombocytopenia. Platelet aggregation and 14C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), α-granules (β-thromboglobulin) and AH-containing vesicles (β-glucuronidase, β-hexosaminidase, α-mannosidase) were normal or minimally decreased. Dense granules secretion on stimulation of gel-filtered platelets with thrombin and divalent ionophore A23187 (4-12 μmol L−1) were diminished. β-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14C -arachidonic acid into phospholipids and subsequent arachidonic acid release on thrombin activation was normal. Platelet thromboxane A2 production in whole blood serum and on thrombin stimulation of PRP was normal, suggesting that the defective secretion was not due to impaired thromboxane production. Conclusions These studies provide the first evidence in patients with a RUNX1 mutation for a defect in AH (lysosomal) secretion, and for a global defect in secretion involving all three types of platelet granules that is unrelated to a granule content deficiency. They highlight the pleiotropic effects and multiple platelet defects associated with RUNX1 mutations.

Whether high oxygen is harmful to the vascular function is unclear. The present study examined if high oxygen modifies vasodilator effect of cysteine via enhanced oxidative stress and thromboxane production. Rat mesenteric arteries with endothelium at 95 or 50 % oxygen were subjected to isometric force recordings, measurement of thromboxane B2 levels, determination of superoxide and peroxynitrite levels and evaluation of NADPH oxidase subunit protein expression, respectively. L-cysteine (0.01-3 mM) constricted or dilated arteries at 95 and 50 % oxygen, respectively. Thromboxane receptor antagonist SQ-29,548 (1 μM) abolished the constriction at 95 % oxygen. L-cysteine (3 mM) increased levels of thromboxane B2 in arteries upon 95 % oxygen application. L-cysteine relaxed arteries treated with superoxide inhibitor tiron (2 mM) or NADPH oxidase inhibitor gp91ds-tat (1 μM) irrespective of the oxygen concentration while ATP-sensitive K(+) channel inhibitor glibenclamide (1 μM) and cystathionine-γ-lyase (CSE) inhibitor DL-propargylglycine (10 mM) similarly abolished the relaxation. L-cysteine (3 mM) with 95 % oxygen augmented levels of superoxide as well as nitrotyrosine within the artery, concomitantly with enhanced membrane protein expression of NADPH oxidase subunit p47phox. The higher concentration of oxygen attenuates L-cysteine-induced vasodilation via superoxide production mediated by NADPH oxidase along with thromboxane A2 production, resulting in vasoconstriction. The increased levels of superoxide, as well as peroxynitrite, coexist with the impaired vasodilation related to ATP-sensitive K(+) channels and CSE. Higher oxygen with plasma cysteine may cause oxidative stress and vasoconstrictor prostanoid production in blood vessels.

Thromboxane A2 (TxA2) and the activation of its receptor have been shown to modulate vasoconstriction and platelet aggregation as well as dopaminergic and serotonergic signalling. Dopaminergic and serotonergic systems play a crucial role in the pathophysiology of schizophrenia and these systems are the main targets of antipsychotics (APs). As the first antipsychotic (AP) chlorpromazine (CPZ) has already been shown to reduce TxA2, we hypothesized that the AP clozapine and its metabolite N-desmethylclozapine (NDMC) might also influence TxA2 production. We measured levels of thromboxane B2 (TxB2), the metabolite of the very unstable molecule TxA2, in unstimulated and stimulated blood samples of 10 healthy female subjects in a whole blood assay using toxic shock syndrome toxin-1 (TSST-1) and monoclonal antibody against surface antigen CD3 combined with protein CD40 (OKT3/CD40) as stimulants. Blood was supplemented with the APs CPZ, clozapine or NDMC in one of four different concentrations. Additionally, thromboxane levels were measured in blood without the addition of APs under different stimulation conditions. Under TSST-1 as well as OKT3/CD40 stimulation, mean TxB2 concentrations were significantly (p < 0.05) decreased by clozapine over all applied concentrations. NDMC led to a decrease in TxB2 levels under unstimulated conditions as well as under TSST-1 stimulation. CPZ reduced TxB2 production at low concentrations under unstimulated and TSST-1- stimulated conditions. Clozapine, NDMC and CPZ possibly act on neurotransmitter systems via modulation of TxA2 or TxB2 production. Additionally, known side effects of APs such as orthostatic hypotension may be a result of changes in the concentrations of TxA2 or TxB2.

A goal of aspirin therapy is to inhibit thromboxane production and platelet aggregation without inhibiting endothelial production of the vasodilator and anti‐thrombotic prostacyclin. This study tested the hypothesis that extended‐release aspirin (NHP‐554C) would have increased selectivity for inhibition of basal and simulated thromboxane formation compared to immediate‐release aspirin (ASA). Thirty‐six healthy subjects were randomized to NHP‐554C or ASA groups. Within each group, subjects were randomized to 5‐day treatment with 81 mg/d, 162.5 mg/d and placebo in a crossover design in which treatment periods were separated by 2‐week washout. On the fifth day of treatment, 81 mg/d and 162.5 mg/d ASA reduced basal urinary excretion of the stable thromboxane metabolite 11‐dehydro‐thromboxane B2 62.3% and 66.2% and basal excretion of the stable prostacyclin metabolite 2,3‐dinor‐6‐keto‐PGF1α 22.8% and 26.5%, respectively, compared to placebo. NHP‐554C 81 mg/d and 162.5 mg/d reduced 11‐dehydro‐thromboxane B2 53% (P = 0.03 vs. ASA 81 mg/d) and 67.9% and 2,3‐dinor‐6‐keto‐PGF1α 13.4% and 18.5%, respectively. NHP‐554C 81 mg/d did not significantly reduce basal excretion of the prostacyclin metabolite. Both doses of ASA and NHP significantly reduced excretion of both thromboxane and prostacyclin metabolites following intravenous bradykinin. During NHP‐554C 162.5 mg/d, but not during ASA, bradykinin significantly increased urinary 2,3‐dinor‐6‐keto‐PGF1α. Nevertheless, 11‐dehydro‐thromboxane B2 and 2,3‐dinor‐6‐keto‐PGF1α responses to bradykinin were statistically similar during ASA and NHP‐554C. In conclusion, at doses of 81 and 162.5 mg/d immediate‐ and extended‐release aspirin selectively decrease basal thromboxane production. Both forms of aspirin decrease bradykinin‐stimulated thromboxane and prostacyclin production, but some stimulated prostacyclin production remains during treatment with NHP‐554C.

The prevalence of cardiovascular diseases in women increases sharply after menopause. In postmenopausal women, thromboxane production increases while prostacyclin decreases. Low dose aspirin reduces the production of both thromboxane and prostacyclin. The present study was an open-label clinical trial with two parallel groups of 15 premenopausal women and 15 postmenopausal women. Twenty-four hours urine was collected from each subject before and after aspirin 100 mg daily for 7 days. The concentration of thromboxane and prostacyclin was measured as their metabolites (11-dehydro-thromboxane B2 and 2,3-dinor-6-keto-prostaglandin-F1α) in urine using enzyme immunoassay methods. This study showed that aspirin significantly reduced thromboxane in both groups with significantly larger percentage reduction in postmenopausal women compared to premenopausal women (73.32 vs. 61.13%, p = 0.021). This study also showed that aspirin reduced prostacyclin significantly in both groups, but the percentage reduction between the groups was not significantly different. The decrease in the ratio of 11-dTXB2/2,3-dinor-6-keto-PGF1α should be compared to assess aspirin efficacy as an antithrombotic. Calculation of the ratio of 11-dTXB2/2,3-dinor-6-keto-PGF1α before aspirin consumption was higher in postmenopausal women than in premenopausal women. The decrease in 11-dTXB2/2,3-dinor-6-keto-PGF1α ratio by aspirin was greater in postmenopausal women than in premenopausal women (1.91 vs. 0.17; p = 0.022). It was concluded that aspirin reduced thromboxane and prostacyclin significantly in each group with significant 11-dTXB2 percentage reduction between groups and non-significant 2,3-dinor-6-keto-PGF1α percentage reduction between groups, but reduced the 11-dTXB2/2,3-dinor-6-keto-PGF1α ratio much larger in postmenopausal women compared to that in premenopausal women.

Growing evidence indicates that statins may reduce thromboxane A(2) synthesis and thrombin generation. We investigated the relationships between thromboxane production, thrombin generation, and oxidative stress in patients receiving aspirin before and after statin administration.An open-label study was conducted in 112 men, aged 54.4 ± 7.3 years, at an increased cardiovascular risk receiving aspirin (75 mg/d). Prior to and following a 3-month simvastatin treatment (40 mg/d), we evaluated circulating thromboxane B(2) (TXB(2)), inflammatory markers, 8-isoprostane, and prothrombin fragment 1.2 (F1.2), a marker of thrombin generation, which was also measured in blood collected every 60s at the site of standardized skin incisions.Subjects (n=28) with pretreatment TXB(2) concentrations in the highest quartile ("aspirin-resistant patients") were more frequently current smokers and had elevated C-reactive protein (CRP), interleukin-6, 8-isoprostane, shorter bleeding time, and increased F1.2 production in a model of microvascular injury, when compared with the 3 remaining quartiles (all, p0.001). Simvastatin decreased serum TXB(2) in the whole group (by 20%, p=0.0008). Patients in the highest quartile of the baseline TXB(2) had still higher posttreatment TXB(2), CRP, interleukin-6, and F1.2 formation following injury (all, p0.001). Simvastatin-induced change in TXB(2) correlated with the magnitude of changes in maximum levels and the velocity of F1.2 formation (all p0.001) but not with changes in inflammatory markers or lipid profile.The study shows that statins significantly reduce platelet TXA(2) formation in patients taking low-dose aspirin and this effect is associated with attenuated thrombin formation in response to vascular injury.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of pulmonary hypertension. ROS might mediate vascular responses, at least in part, by stimulating prostanoid production. Our goals were to determine whether the effect of ROS on vascular tone is altered in resistance pulmonary arteries (PRAs) of newborn piglets with chronic hypoxia-induced pulmonary hypertension and the role, if any, of prostanoids in ROS-mediated responses. In cannulated, pressurized PRA, ROS generated by xanthine (X) plus xanthine oxidase (XO) had minimal effect on vascular tone in control piglets but caused significant vasoconstriction in hypoxic piglets. Both cyclooxygenase inhibition with indomethacin and thromboxane synthase inhibition with dazoxiben significantly blunted constriction to X+XO in hypoxic PRA. X+XO increased prostacyclin production (70 ± 8%) by a greater degree than thromboxane production (50 ± 6%) in control PRA; this was not the case in hypoxic PRA where the increases in prostacyclin and thromboxane production were not statistically different (78 ± 13% versus 216 ± 93%, respectively). Thromboxane synthase expression was increased in PRA from hypoxic piglets, whereas prostacyclin synthase expression was similar in PRA from hypoxic and control piglets. Under conditions of chronic hypoxia, altered vascular responses to ROS may contribute to pulmonary hypertension by a mechanism that involves the prostanoid vasoconstrictor, thromboxane.

Although active smoking is known to enhance platelet thromboxane production, no data on passive smoking are available yet. In an 18 m3 room, the influence of single and repeated exposure to passive smoke for 60 minutes was assessed in nonsmokers and smokers. Smokers and nonsmokers were matched for sex and age. All the evaluated parameters (plasma TXB2, serum TXB2, malondialdehyde, 11-dehydro-TXB2, conversion of exogenous arachidonic acid to hydroxy-5,8,10-heptadecatrienoic acid, and TXB2) were higher in smokers than nonsmokers at baseline conditions, immediately and 6 hours after passive exposure to cigarette smoke. Repeated exposure of nonsmokers rendered their platelets more activated, so they became closer to the behavior of smokers. Contributing to the development of hemostatic imbalance, these results indicate that passive smoking may enhance thromboxane A2 release from the platelets.

This study sought to determine whether in vivo inhibition of thromboxane A2 (TXA2) action contribute to attenuate hepatic damage after bile duct ligation (BDL). Male Wistar rats were assigned to sham operation or BDL. At the time of operation, infusion pump with saline, ozagrel natrium (TXA2 synthase inhibitor), or SQ29548 (TXA2 receptor antagonists) was implanted in the abdominal cavity. Plasma alanine aminotransferase, aspartate aminotransferase, hyaluronic acid, and total bilirubin levels were measured at 4 days after the operation. The levels of plasma TXB2, a stable metabolite of TXA2, were significantly increased after BDL. Gene expression of TXA2 synthase was also significantly upregulated in the liver. Nonetheless, either an inhibition of TXA2 synthesis by ozagrel natrium or a blockade of TXA2 receptor by SQ29548 has no effect in every measured parameter related to hepatic function. These results indicated that despite a highly increased production in the liver, TXA2 is not directly related to the hepatic injury in BDL rats.

LDL-associated phospholipids (PLs) may be transferred into platelets. In this work, we characterized the role of VLDLs as PL donors. VLDL transferred radiolabeled PLs to platelets in a temperature- and concentration-dependent manner. LPL stimulated this process through its action on VLDL lipolysis, because it was abolished by tetrahydrolipstatin. LPL also stimulated the platelet production of thromboxane B2 (TXB2). Both LPL actions were inhibited in the presence of fatty acid-free albumin, suggesting that they were attributable to fatty acids generated during VLDL lipolysis. To study the relationship between PL transfers and platelet activation, we performed incubations in the presence of HDL, a physiological acceptor of PL released from VLDL. HDL antagonized the transfer of PL from VLDL to platelets but had no effect on the production of TXB2, suggesting that PL transfers were driven by platelet activation. Confirming this idea, thrombin stimulated both the production of TXB2 and the transfers of PL. In conclusion, VLDL can transfer PL to platelets. These transfers are stimulated by LPL and thrombin through their action on platelet activation. They might be enhanced in pathologies characterized by increased VLDL concentrations.

The antiplatelet effect of a new synthetic compound, 8,9-dimethoxyl-1-(4-methoxy-phenyl)-5,6-dihydro-pyrrolo[2,1-a]isoquinoline-2,3-dione (KW-7), was determined in rabbit platelets. KW-7 concentration-dependently prevented platelet aggregation caused by arachidonic acid, collagen, platelet-activating factor, and thrombin. KW-7 induced a substantial increase in cyclic AMP levels and a smaller increase in cyclic GMP levels in platelets. In platelet homogenates, KW-7 inhibited both cyclic AMP- and cyclic GMP-phosphodiesterase activities. The antiplatelet effect of KW-7 was reversed by SQ22536 (an inhibitor of adenylate cyclase) and H89 (an inhibitor of protein kinase A) but not by ODQ (an inhibitor of soluble guanylate cyclase). These data suggest that the antiplatelet effect of KW-7 is cyclic AMP-dependent, and is through inhibition of platelet phosphodiesterases. In addition, KW-7 inhibited arachidonic acid-stimulated thromboxane production; this effect was associated with an increase in prostaglandin D2 levels indicating KW-7 is also an inhibitor of thromboxane synthase. The dual inhibition of KW-7 on phosphodiesterase and thromboxane synthase might provide an attractive target in developing antiplatelet drugs.

Type 2 diabetes is characterized by increased plasma triglyceride levels and a fourfold increase in ischemic heart disease, but the mechanism is unclear. CD36 is a receptor/transporter that binds fatty acids of lipoproteins. CD36 deficiency has been linked with insulin resistance. There is strong evidence of in vivo interaction between platelets and atherogenic lipoproteins suggesting that atherogenic triglyceride-rich lipoproteins, such as VLDL, that are increased in diabetic dyslipidemia are important in this process. This study demonstrates that VLDL binds to the platelet receptor CD36, enhances platelet thromboxane A2 production, and causes increased collagen-mediated platelet aggregation. VLDL enhanced collagen-induced platelet aggregation by 1) shortening the time taken for aggregation to begin (lag time) to 70% of control (P = 0.001); 2) increasing maximum aggregation to 170% of control (P = 0.008); and 3) increasing thromboxane production to 3,318% of control (P = 0.004), where control represents platelets stimulated with collagen (100%). A monoclonal antibody against CD36 attenuated VLDL-enhanced collagen-induced platelet aggregation by 1) inhibiting binding of VLDL to platelets by 75% (P = 0.041); 2) lengthening lag time to 190% (P < 0.001); and 3) decreasing thromboxane production to 8% of control (P < 0.001). In support of this finding, platelets from Cd36-deficient rats showed no increase in aggregation, thromboxane production, and VLDL binding in contrast to platelets from rats expressing CD36. These data suggest that platelet Cd36 has a key role in VLDL-induced collagen-mediated platelet aggregation, possibly contributing to atherothrombosis associated with increased VLDL levels.

Phosphodiesterase 4 (PDE4) inhibitors have been shown to inhibit equine neutrophil function in vitro and may be of benefit in recurrent airway obstruction (RAO), an allergy-based respiratory disease characterized by inflammatory cell recruitment and activation within the lungs following exposure of susceptible horses to allergens in mouldy hay. The aim of this study was to evaluate the inhibitory effects of the PDE4 inhibitor, rolipram, in an in vitro assay of thromboxane (Tx) production. The assay was then used to monitor the activity of this compound in vivo in normal and RAO-affected horses. Rolipram and the structurally distinct PDE4 inhibitor, denbufylline, attenuated both lipopolysaccharide (LPS)-induced and unstimulated Tx production in blood from normal horses. Thromboxane production appeared to involve a calcium-dependent interaction between leucocytes and platelets (LPS-induced Tx production = 2.3 +/- 0.4, 4.5 +/- 1.1 and 20.8 +/- 3.6 ng/mL for platelets, leucocytes and blood, respectively) and rolipram-inhibited Tx production via an effect on leucocytes. Inhibition of ex vivo LPS induced Tx production was detected after intravenous administration of rolipram (5 microg/kg) to normal ponies. This dose did not significantly affect either lung function or neutrophil accumulation when administered to three horses with clinical signs of RAO. This study suggests that inhibition of Tx production in equine blood can be used to measure PDE4 activity. However, PDE4 inhibitors with improved therapeutic profiles are required for evaluation in RAO.

We and others have previously observed an imbalance in cytotrophoblast secretion of the vasoactive prostanoids prostacyclin and thromboxane A(2) in pre-eclampsia. To examine the effects of potential modulators of this imbalance, cytotrophoblasts isolated from normal and pre-eclamptic pregnancies were incubated in the presence of lipopolysaccharide, the calcium ionophore A23187, tumour necrosis factor alpha, or interleukin 1beta, with or without the cyclo-oxygenase inhibitor, indomethacin. Further incubations included the drugs tranylcypromine, a prostacyclin synthetase inhibitor (0.1, 10 microM ), or the thromboxane synthetase inhibitor, pirmagrel (0.001, 1 microM ). Results showed that cytotrophoblasts from pre-eclamptic pregnancies had increased thromboxane production and significant stimulation of prostacyclin production by lipopolysaccharide and calcium ionophore. Lipopolysaccharide stimulated thromboxane production in normal cytotrophoblasts, while indomethacin almost completely inhibited production of both prostanoids. Tranylcypromine mildly inhibited prostacyclin production in normal cytotrophoblasts only, whereas pirmagrel strongly inhibited thromboxane production in a dose-related manner, with reciprocal increase in prostacyclin production occurring in cytotrophoblasts from pre-eclamptic pregnancies. This study confirmed that cytotrophoblasts from pre-eclamptic women had increased thromboxane production and showed that pirmagrel, at the relatively low dose of 0.001 microM, was able to normalize the imbalance of thromboxane and prostacylin production and may therefore warrant further investigation as a treatment for pre-eclampsia.

Biomarker testing for efficacy of therapy is an accepted way for clinicians to individualize dosing to genetic and/or environmental factors that may be influencing a treatment regimen. Aspirin is used by nearly 43 million Americans on a regular basis to reduce risks associated with various atherothrombotic diseases. Despite its widespread use, many clinicians are unaware of the link between suboptimal response to aspirin therapy and increased risk for inferior clinical outcomes in several disease states, and biomarker testing for efficacy of aspirin therapy is not performed as routinely as efficacy testing in other therapeutic areas. This article reviews the clinical and laboratory aspects of determining whole-body thromboxane production, particularly as it pertains to efficacy assessment of aspirin responsiveness.

We investigated production of prostacyclin and the urinary ratio of thromboxane and prostacyclin in patients with rheumatoid arthritis. The prostacyclin production level was assessed according to the level of urinary 2,3-dinor-6-keto-prostaglandin F1 αmeasuring by gas chromatography/selected ion monitoring. In patients receiving medication, the prostacyclin level was lower and the thromboxane/prostacyclin ratio was greater compare with that of healthy volunteers. The prostacyclin level in patients without medication was approximately 4-fold higher than that of healthy volunteers and 8-fold higher than those of medicated groups. Although the ratio of the group without medication was similar to that of healthy volunteers, the urinary levels of each prostanoid were higher than those of other groups. Then, the ratios of groups receiving steroids were higher than that of other groups owing to high TX level. The present findings demonstrated that endogenous prostacyclin and thromboxane production increased in patients without medication, and prostacyclin production decreased with medication.

To examine the effects of altering the fatty acid (FA) composition of intravenous (IV) lipid emulsions on pulmonary vascular resistance (PVR) and thromboxane production, we studied three groups of newborn piglets after three days of either sow's milk (milk), or total parenteral nutrition (TPN) with either iv soy bean oil (SBO, 52% n-6 and 8% n-3 FA) or fish oil (FO, 5% n-6 and 51% n-3 FA) emulsions. At baseline, and during hypoxia at 20 min and 2 h, cardiac output (Q) was measured, PVR calculated and plasma levels of a prostacyclin metabolite (6-keto-PgF1α) and thromboxane B2 (TxB2) were measured. Fatty acid composition of the lung phospholipids was analyzed. There was an exaggerated increase in PVR and decrease in Q during prolonged hypoxia in the TPN-SBO group as compared with the other two groups. There was no difference in PVR and Q between the milk and TPN-FO groups. FA of lung phospholipids reflected the high dietary level of long chain n-3 FA in the TPN-FO group. However, no differences in plasma levels of 6-keto-PgF1α or TxB2 were found. Intravenous emulsions made from SBO reduced cardiac output and increased pulmonary vascular resistance in the hypoxic newborn piglet, whereas iv FO emulsions did not. When subjects with pulmonary hypertension are receiving TPN iv SBO may be detrimental; iv FO may be beneficial, giving similar responses as in a milk-fed subject.Key words: total parenteral nutrition, fish oil, pulmonary hypertension, lipid emulsion, fatty acids.Key words: total parenteral nutrition, fish oil, pulmonary hypertension, lipid emulsion, fatty acids.

Prostanoids have been proposed to play a major role in the regulation of uteroplacental blood flow. We examined the effect of hypoxia on the production of prostaglandin E(2)(PGE(2)) thromboxane B(2)(TXB(2)), and prostacyclin (measured as 6-keto-PGF(1alpha)) by human term trophoblast cells and villous placental explants. Explants (n=8) and purified trophoblast cells (n=5) were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. In trophoblast monolayer cultures, hypoxia attentuated PGE(2)production rates to 52+/-9.4 per cent (mean+/-sem, P0.05) but recovered to control rates within 48 h. In villous explants, PGE(2)production was significantly decreased after 48 and 72 h of hypoxia versus the normoxic control, accompanied by increased production of 6-keto-PGF(1alpha)to 173.9+/-26.7 per cent after 48 h. TXB(2)production was increased to 172.3+/-25.9 per cent and 653.2+/-135.7 per cent (P0.05) control after 48 and 72 h of hypoxia, respectively. These results were confirmed in villous explants (n=3) cultured in the presence of exogenous 10 microm arachidonic acid. Hypoxia had no significant effect on TXB(2)and 6-keto-PGF(1alpha)in trophoblast cells. In summary, our findings suggest that hypoxia could be responsible for abnormal profiles of prostanoid production commonly observed in women with pre-eclampsia. These results indicate a putative link between hypoxia and compromised placental perfusion.

TO examine the effect of plasma from preeclamptic women on production of the vasoactive substances prostacyclin, thromboxane, nitric oxide, and cyclic guano sine monophosphate (cGMP) by decidual endothelial cells; to determine any effects on cell growth and health; and to examine whether cells from preeclamptic women are activated compared with cells from normal women. Decidual endothelial cells from normal and preeclamptic women were incubated for 24 hours in media containing 10% plasma from preeclamptic women or matched normal women. Prostacyclin and thromboxane production was measured, as was nitric oxide and cGMP production after a further 45-minute generation period in 2% test plasma. Cell numbers and lactate dehydrogenase release were also determined. In plasma from preeclamptic women, cells grew significantly faster (P < .05), prostacyclin production was increased (P < .05), and lactate dehydrogenase release was reduced (P < .01). Production of thromboxane, nitric oxide, and cGMP was not significantly affected. Decidual endothelial cells from preeclamptic women had increased growth (P < .0001) and produced more prostacyclin (P < .05) and nitric oxide (P < .001) than normal decidual endothelial cells. There were highly significant correlations between prostacyclin and thromboxane production for incubations in plasmas from preeclamptic women and between background levels of prostacyclin in each plasma from preeclamptic women and the prostacyclin produced in incubations containing that plasma (P < .0001). We found that plasma from preeclamptic women contained a factor that stimulated endothelial cell growth and regulated production of related amounts of prostacyclin and thromboxane. The plasma level of this factor appeared to be related to background levels of prostacyclin. The results also indicated that decidual endothelial cells from preeclamptic women were in a relatively activated state.

The aim of this study was to investigate the effect of trans α-linolenic acid on platelet aggregation and blood haemostasis. A randomized, double blind dietary intervention trial was carried out with healthy male volunteers (n=88) in three European centers. After a 6-week washout period where subjects avoided foods containing all trans fats, subjects either continued for 6 weeks with a low trans diet or a diet where trans α-linolenic acid provided 0.6% of energy (supplied as oil, margarine, cheese, muffins, and biscuits). At the end of the washout period the intake of trans polyunsaturated fats was 58±115 mg/day; this increased in patients on the high trans diet by +1344±328 mg/day, compared with +10±67 mg/day in patients on the low trans diet (p trans α-linolenic acid in plasma cholesteryl esters was 0.26±0.20 on the high trans and 0.00±0.07% of fatty acids on the low trans diet (p trans diet was observed on platelet aggregation: collagen EC 50 high trans 157±100, low trans 152±90 ng/mL (NS); U44619 EC 50 high trans 81±61, low trans 59±27 nM (NS). The high trans diet did not affect platelet thromboxane production, fibrinogen levels, factor VII, activated factor VIIa, or plasminogen activator inhibitor activity. There were no center-specific differences in response to the high trans diet. A relatively high amount of trans α-linolenic acid for 6 weeks does not increase the risk of coronary heart disease by promoting platelet aggregation and blood coagulation.

Objective To study the influence of meloxicam, a cyclooxygenase-2 (COX-2) preferential nonsteroidal anti-inflammatory drug, on serum thromboxane and platelet function in healthy volunteers with use of the maximum recommended daily dosage of 15 mg/day. Methods This study used an open, randomized crossover design. Indomethacin (INN, indometacin) was given as a positive control for nonsteroidal anti-inflammatory drug–induced inhibition of platelet function. The following variables were recorded: thromboxane B2 serum concentrations by radioimmunoassay, platelet aggregation by whole blood aggregometry in response to collagen 1.1 μg/L and to arachidonic acid 0.35 mmol/L, and closure time with use of the PFA-100. Results Serum thromboxane B2 at baseline was 535 ± 233 nmol/L (mean ± SD) and was reduced for 95% by indomethacin to 26 ± 19 nmol/L (P < .001) and for 66% by meloxicam to 183 ± 62 nmol/L (P < .001). Maximal platelet aggregation in response to collagen at baseline was 18.7 ± 1.6 ohms (ω). It was reduced by indomethacin to 7.3 ± 4.5 ω (P < .001), but not by meloxicam (19 ± 2.5 ω). Platelet aggregation in response to arachidonic acid at baseline was 12.2 ± 2.0 ω. It was reduced by indomethacin in all subjects to 0 ω, but not by meloxicam (11 ± 2.4 ω). Closure time at baseline was 128 ± 24 seconds and was prolonged by indomethacin to 286 ± 38 seconds (P < .001). Meloxicam caused a minor prolongation of the closure time (141 ± 32 seconds; P < .05). Conclusion Meloxicam, 15 mg/day caused a major reduction of maximum thromboxane production but no reduction in collagen- or arachidonic acid–induced platelet aggregation and only minor increase of the closure time. Clinical Pharmacology & Therapeutics (1999) 66, 425–430; doi: 10.1053/cp.1999.v66.a101063

Clinical manifestations of mastocytosis are mediated, at least in part, by release of the mast cell mediators histamine and prostaglandin D2. It has been previously reported that in addition to prostaglandin D2, mast cells produce other eicosanoids, including thromboxane. Nonetheless, little information exists regarding the formation of other prostanoids in vivo. The most accurate method to examine the systemic production of eicosanoids in vivo is the quantitation of urinary metabolites. We previously developed a highly accurate assay employing mass spectrometry to measure a major urinary metabolite of thromboxane, 11-dehydro-thromboxane B2, in humans. We utilized this assay to quantitate thromboxane production in 17 patients with histologically proven mastocytosis. We report that thromboxane formation was significantly increased (2 SD above the mean) in at least one urine sample from 65% of patients studied. Of these, 91% of patients with documented systemic involvement had elevated thromboxane generation. In addition, endogenous formation of thromboxane was highly correlated with the urinary excretion of the major urinary metabolite of prostaglandin D2 (r = 0.98) and Ntau-methylhistamine (r = 0.91), suggesting that the cellular source of increased thromboxane in vivo could be the mastocyte. Enhanced thromboxane formation in patients with this disorder is unlikely to be of platelet origin as other markers of platelet activation, platelet factor 4 and beta-thromboglobulin, were not increased in three patients with marked overproduction of thromboxane. Furthermore, the recovery of 11-dehydro-thromboxane B2 excretion in two patients after the administration of aspirin occurred significantly more rapidly than the recovery of platelet thromboxane generation. These studies, therefore, report that thromboxane production is significantly increased in the majority of patients with mastocytosis that we examined and provide the basis to elucidate the role of this eicosanoid in disorders of mast cell activation.

Bolus i.v. injections of 1.2 N HCl elicit a rapid but transient pulmonary vasoconstriction in broiler chickens. In mammals, the pulmonary vasoconstrictive response to bolus acid injection depends on increased synthesis of thromboxane A2; however, the vascular responsiveness of domestic fowl to thromboxane previously had not been evaluated. In the present study, we tested the hypothesis that, if HCl triggers pulmonary vasoconstriction by stimulating thromboxane A2 synthesis in broilers, then bolus i.v. injections of the potent thromboxane A2 mimetic U44069 (9,11-dideoxy-9alpha,11alpha-epoxy-methanoprostaglandin++ + F2alpha; 1 micromol/mL; 0.5 mL injected volume) should trigger hemodynamic responses similar to those elicited by HCl (1.2 N; 1.5 mL injected volume). Both HCl and the thromboxane mimetic elicited twofold or greater increases in pulmonary vascular resistance, which in turn increased pulmonary arterial pressure by 50% despite concurrent reductions in cardiac output. The reductions in cardiac output were associated with reductions in stroke volume but not heart rate. The thromboxane mimetic also increased the total peripheral resistance, which minimized the reduction in mean systemic arterial pressure associated with the decrease in cardiac output. In contrast, HCl injections did not increase total peripheral resistance; consequently, the reduction in cardiac output caused the mean systemic arterial pressure to decrease by 30 mm Hg. Mannitol (2.5%; 1.5 mL) was injected i.v. as a volume control, and had no influence on any of the variables. This study provides the first direct evidence that thromboxane is a potent pulmonary vasoconstrictor in broilers, and provides support for the hypothesis that thromboxane mediates the pulmonary vasoconstrictive response to bolus i.v. injections of HCl. The differential response of the systemic vasculature to the thromboxane mimetic and HCl may indicate that cardiopulmonary responses to HCl injections are not mediated solely via thromboxane production. Alternatively, a direct dilatory effect of elevated hydrogen ion concentrations on the systemic vasculature may have counteracted any tendency for simultaneously evolved endogenous thromboxane to elicit systemic vasoconstriction.

Pre-exposure of THP-1 cells to low concentrations of endotoxin (lipopolysaccharide, LPS) down-regulates thromboxane (Tx) A2, an arachidonic acid (AA) metabolite, production in response to a subsequent LPS stimulation. To further delineate the mechanisms of LPS-induced down-regulation of TxA2, we examined expression of prostaglandin H synthase (PGHS)-2 mRNA, changes in PGHS activity, and content of PGHS-1 and -2. Pre-exposure to LPS (1 microg/mL for 18 h to desensitize cells) inhibits production of TxB2, the stable metabolite of TxA2, in response to secondary stimulation of LPS (10 microg/mL), when compared with LPS-stimulated naive cells (p < .05, n=5). LPS (10 microg/mL) induced expression of PGHS-2 mRNA at 1 and 2 h in naive cells, but this expression was decreased in the LPS-desensitized cells. However, exogenous AA (16 microM) or phorbol myristic acid (PMA), 3 microM) stimulated greater TxB2 production in the LPS-desensitized cells than in the naive cells (p < .05). Protein content of PGHS-1 and -2 were examined by Western blot analysis, using antibodies specific for PGHS-1 and PGHS-2. Densitometric analysis demonstrated a significant increase in PGHS-2 induction in LPS-stimulated naive cells (405+/-174%) over its respective basal group (p < .05, n=5). PGHS-1 was constitutively present, but there was no significant difference in quantity between naive and LPS-desensitized basal or LPS-stimulated groups. Thus, despite the reduction in expression of PGHS-2 mRNA, these composite data demonstrate that down-regulation of PGHS activity (assessed with exogenous AA or PMA) cannot be responsible for the inhibition of AA metabolism observed in LPS desensitization.

1. Myocardial thromboxane A2 production increases in patients with pacing-induced ischaemia and correlates with a decrease in myocardial lactate extraction. The release of myocardial thromboxane A2 before any lactate production was observed in patients with unstable angina. This study was proposed to clarify whether the early thromboxane A2 release contributed to the ongoing myocardial ischaemia and to determine which metabolites can be attributed to the thromboxane A2 release. Thirty-five patients with chest pain and positive treadmill exercise test underwent atrial pacing to the predicted maximal heart rate. The pacing was maintained at this peak rate for 10 min, then ceased. Blood samples of the ascending aorta and coronary sinus were drawn simultaneously at rest, at 2 and 10 min of peak-pacing, and 5 and 10 min after termination of the pacing; samples were used for analyses of lipid profiles, prostacyclin, thromboxane A2, lactate and lipid peroxides on plasma and low-density lipoprotein particles. 2. Twenty out of 35 patients who displayed pacing-induced ischaemia were documented by electrocardiographic evidence of ST depression >2 mm developing after 2 min of peak-pacing [ischaemic group, STΔ(+)]. They had (i) negative fractional lactate extraction; (ii) pacing-induced decreases of plasma thromboxane A2 levels in the coronary sinus blood (564 ± 57 versus 479 ± 47 ng/l, P < 0.05) at 2 min of peak-pacing; the data increased at 10 min of peak-pacing (564 ± 57 versus 620 ± 60 ng/l, P < 0.05), then returned to baseline levels at 5 and 10 min post-pacing; (iii) significantly increased lipid peroxides on low-density lipoprotein of the coronary sinus blood at 2 and 10 min of peak-pacing (each P < 0.001), as well as at 5 min post-pacing (P < 0.05); (iv) significant correlation between thromboxane A2 levels and lipid peroxides on low-density lipoprotein of the coronary sinus blood samples. 3. In STΔ(+) patients, myocardial thromboxane synthesis changed before lactate production and correlated with the increase of lipid peroxides on low-density lipoprotein of the coronary venous blood. This implies that lipid peroxides on low-density lipoprotein participate in thromboxane production and play a determinative role in pacing-induced ischaemia.

Abstract These studies have examined the effects of thrombin-related agonists in stimulating thromboxane production by human platelets. The results presented show that (1) the maximal response elicited by thrombin receptor agonist peptide (TRAP) stimulation was 40% to 50% of that seen with thrombin or the thrombin mutant Thrombin Quick I; (2) pretreatment of platelets with prostaglandin E 1 or genistein resulted in differential inhibition of thromboxane production in response to TRAP compared with either enzyme agonist; (3) an antibody to the thrombin receptor cleavage site that inhibits increases in intracellular [Ca 2+ ] only partially reduced thromboxane production in response to 5 nmol/L thrombin and 15 nmol/L Thrombin Quick I; (4) preincubation with 20 μmol/L TRAP resulted in desensitization to further stimulation by 100 μmol/L TRAP, but not by 100 nmol/L thrombin; and (5) the response to thrombin after TRAP desensitization was completely inhibited by the tyrosine kinase inhibitor genistein and was independent of an intracellular [Ca 2+ ] flux. The cumulative results may be explained by the existence of two proteolytically activated receptors that result in thromboxane production in response to thrombin. One is the thrombin receptor/substrate, PAR-1. Stimulation through the second receptor/substrate depends on a genistein-sensitive step, is independent of an intracellular Ca 2+ flux, and is initiated by a thrombin-activated receptor that does not depend on interaction with anion-binding exosite I, as previously indicated by the relative activity of Thrombin Quick I in stimulating platelet aggregation and thromboxane production. The proposed second thrombin receptor on platelets represents an additional member of the class of proteolytically activated receptors.

In pre-eclampsia, the ratio of prostacyclin:thromboxane production rate is decreased favouring the vasoconstrictive thromboxane. One of the rate-limiting steps in prostaglandin synthesis is cyclooxygenase (COX) activity. Therefore, we investigated the expression of COX-1 and COX-2 in human placenta and placental bed. Tissue specimens from the 29th to 40th week of pregnancy were obtained from Caesarean sections after uncomplicated and pre-eclamptic pregnancies before the onset of labour. COX-1 and COX-2 were localized immunohistochemically with the identification of positive cells by double immunofluorescence staining. The protein and mRNA levels were analysed by immunoblotting and quantitative reverse transcriptase-polymerase chain reaction. Expression of both COX-1 and COX-2 could be observed in placenta and placental bed. COX-1-like immunoreactivity was observed in most cell types with strongest staining in macrophages. Only macrophages, endothelium, vascular leiomyocytes and fibroblasts stained positively for COX-2. In placenta, COX-1 and -2 expression was unchanged after pre-eclampsia. In placental bed, protein and mRNA levels of COX-1 were increased in the pre-eclamptic group (P < 0.05), whereas COX-2 expression did not differ significantly from normal pregnancies. An increased expression of COX-1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed. A therapy with drugs inhibiting COX-1 might be beneficial in this condition.

Placental macrophages (Hofbauer cells) are located close to trophoblast cells and fetal capillaries, which makes them ideal candidates for involvement in regulatory processes within the villous core. Their production of various cytokines and prostaglandin (PG) synthesizing enzymes has previously been shown immunohistochemically. Hofbauer cells were isolated from human placenta after term deliveries by Ficoll and Percoll gradient centrifugation. Remaining trophoblast cells were removed with anti-epidermal growth factor (EGF)-receptor-coated Dynabeads followed by differential adherence. The identity of isolated cells was investigated by immunohistochemistry with anti-CD68, which showed that >90% cells were positive. After a 36 h recovery period in either 20% O2 or 5% O2, fresh medium was applied and PGE2 and thromboxane (TXA2) production analysed by enzyme immunoassay at 4, 8, and 24 h. PGE2 and TXA2 were both produced by placental macrophages with PGE2 synthesis being predominant. Concentrations of both could be stimulated by lipopolysaccharide with maximum effect after 24 h. Culture in low oxygen caused decreased PGE2 concentrations, whereas TXA2 production remained unchanged. In conclusion, the presented isolation protocol allows further study of Hofbauer cell function. This study also presents novel findings regarding the prostaglandin production of term Hofbauer cells under normal and hypoxic conditions.

The study addresses the relationship of plasma arachidonic acid and thromboxane production with the dietary compliance in treated phenylketonuric patients, whose vegan-like dietary pattern makes them a useful model to evaluate the effects of the near-total avoidance of animal fats. Thirteen treated phenylketonuric children were compared with twelve healthy controls for arachidonic acid intake, plasma fatty acids and platelet thromboxane B2 production, assessed as accumulation of this eicosanoid in serum. The calculated intake of arachidonic acid was lower in phenylketonurics than in controls and this was associated with lower levels in plasma lipids. Plasma arachidonic acid concentrations and serum thromboxane B2 levels correlated with the last 12 months phenylalanine levels, taken as negative indicator of dietary compliance. A direct relationship between plasma arachidonic acid concentration and thromboxane B2 production was observed only in phenylketonuric patients (r = 0.74, P = 0.01). While well-compliant PKU subjects have low arachidonic acid and thromboxane concentrations in plasma, the low compliance with animal food avoidance, evoking higher phenylalanine levels, results in elevation of both plasma arachidonic acid and serum thromboxane B2. This gives support to the hypothesis that the consumption of animal fats may affect the production of arachidonic acid-derived platelet eicosanoids.

The aims of the present studies were to examine the effects of fish oil (containing (n-3) polyunsaturated fatty acids) on myocardial thromboxane and prostacyclin production, superoxide dismutase (SOD) activity and malondialdehyde (MDA) production in the rat. Male rats were fed standard pellet diets and the same diets enriched with 7% (w/w) stabilized fish oil or 7% butter (saturated fat) for 2–6 wk. Myocardial production of thromboxane was lower in rats given fish oil than in those fed standard pellets (P < 0.01) or saturated fat (P < 0.05) and the prostacyclin/thromboxane ratio was higher than in rats fed standard pellets (P < 0.05). Myocardial SOD activity was higher in rats fed stabilized fish oil than in those given saturated fat (P < 0.05). Supplementation of the stabilized fish oil with extra vitamin E did not have any major effect on thromboxane and prostacyclin production or SOD activity. The percentage of arachidonic acid in the myocardial phospholipids was lower (P < 0.001) during fish oil than during saturated fat feeding, with no modifying effect of vitamin E supplementation. Feeding with the stabilized fish oil did not alter the myocardial α-tocopherol concentration, but the myocardial MDA concentration in vitro was higher (P < 0.01) than after feeding with saturated fat. Supplementation of the stabilized fish oil with extra vitamin E resulted in a higher α-tocopherol (P < 0.05) and lower MDA concentration (P < 0.05) in the myocardium compared to the unsupplemented fish oil. Plasma MDA concentration was not changed by fish oil feeding. In conclusion, fish oil feeding resulted in higher myocardial SOD activity and lower thromboxane production. These changes may be contributory mechanisms underlying the antiarrhythmic effect of fish oil. Copyright © 1996 Elsevier Science Inc.

In this study, we determined if vitamin D could inhibit oxidative stress-induced thromboxane production by placental trophoblasts. Trophoblast isolated from normal placentas were stimulated with CoCl2, a hypoxic mimicking agent, with or without pretreatment of 1,25(OH)2D3. Soluble phospholipase-A2, metabolites of thromboxane-A2 and prostacyclin, and 8-isoprostane were measured. Expression of cyclooxygenase-1 (COX-1), COX-2, and heme oxygenase-1 (HO-1) were determined. We found that pretreatment of trophoblasts with 1,25(OH)2D3 significantly reduced 8-isoprostane and the ratio of thromboxane-A2 to prostacyclin production, and blocked COX-2 expression induced by CoCl2. These results provide evidence of the beneficial effects of vitamin D on placental trophoblasts.

Metabolism of estradiol-17β to 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol contributes importantly to the vascular effects of estradiol-17β in several vascular beds. However, little is known about the role of estradiol-17β metabolites via the different estrogen receptors (ER-α/ER-β) on de novo endothelial prostacyclin and thromboxane production. We hypothesized that estradiol-17β and its metabolites, via ER-α or ER-β, can enhance the prostacyclin/thromboxane ratio through the classic phospholipase A 2 , cyclooxygenase-1, and prostacyclin synthase pathway in ovine uterine artery endothelial cells (UAECs) derived from pregnant (P-UAECs) versus nonpregnant (NP-UAECs) ewes. Western analyses showed higher expression of phospholipase A 2 , cyclooxygenase-1, and prostacyclin synthase in UAECs from the pregnant state, whereas thromboxane synthase was lowered in UAECs from the pregnant state. In UAECs from the pregnant state, estradiol-17β, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol and 4-methoxyestradiol concentration and time-dependently increased prostacyclin compared with controls. Prostacyclin increases in UAECs from the nonpregnant state were of a lower magnitude. Estradiol-17β and its metabolites stimulated higher prostacyclin/thromboxane ratios in UAECs from the pregnant state compared with UAECs from the nonpregnant state. Estradiol-17β–induced prostacyclin increases were abrogated by the antagonists SC-560 (cyclooxygenase-1), U-51605 (Prostacyclin synthase), ICI 182 780 (ICI; both ER-α/β), and 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α), but not by 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo[1,5-a]pyrim idin-3-yl]phenol (PHTPP; ER-β). Prostacyclin increases induced by its metabolites were abolished by SC-560 and U-51605, but unaltered by ICI, MPP, or PHTPP. Our findings demonstrate that estrogen via primarily ER-α and its metabolites via ER-independent mechanisms influence the de novo endothelial biosynthesis of prostacyclin, which may be important in the regulation of vascular tone. These findings also shed light on the complexities of estrogen signaling via its metabolism and the functional heterogeneity of the ERs.

To explore the mechanism(s) by which antiestrogens may protect against the development of cardiovascular disorders, we measured the production of vasodilatory, antiaggregatory prostacyclin (PGI2) and that of vasoconstrictive, proaggregatory thromboxane A2 (TxA2) before and after 6 months' use of antiestrogens in postmenopausal patients after operation for stage II breast cancer (n = 38). Urine samples were assayed by high performance liquid chromatography and radio-immunoassays for 2,3-dinor-6-ketoprostaglandin F1 alpha (= metabolite of PGI2, dinor-6-keto) and for 2,3-dinor-thromboxane B2 (= metabolite of TxA2, dinor-TxB2). In addition, in 35 of these 38 patients we assayed the capacity of platelets to produce thromboxane A2 during standardized blood clotting. The 4 patients using low-dose aspirin had low thromboxane production, and were excluded from further analysis of the data. An antiestrogen regimen consisting either of tamoxifen (n = 15) or of toremifene (n = 19) caused no changes in production of PGI2 or TxA2, or in their ratio, and in this regard, these antiestrogens behaved similarly. Hypertensive patients (n = 7) using different anti-hypertensive agents were characterized by reduced urinary out-put of dinor-6-keto (18.5 +/- 6.1 vs 35.5 +/- 18.5 ng/mmol, mean +/- SD, p < 0.05) and reduced platelet capacity to produce TxA2 (62.6 +/- 67.8 vs 134.6 +/- 75.6 ng/mL, p < 0.05). The patients (n = 15) who had used estrogen replacement therapy (ERT) up until diagnosis of breast cancer showed reduced dinor-TxB2 excretion (15.5 +/- 12.7 vs 29.9 +/- 20.9 ng/mmol, p < 0.05) before initiation of antiestrogens, and elevated dinor-6-keto output during the antiestrogen regimen (32.4 +/- 21.2 vs 22.7 +/- 8.7 ng/mmol, p = 0.07). Smokers (n = 6) had elevated dinor-TxB2 output before and during antiestrogen use. Thus we conclude that the cardiovascular protection provided by an antiestrogen regimen is unlikely to be mediated through vaso- and platelet active PGI2 and TxA2.

Platelet aggregation and thromboxane A 2 generation were studied in hypertensive pregnant women using normotensive non-pregnant and pregnant controls. In hypertensive pregnancy, adrenaline- and adenosine diphosphate-induced platelet aggregation was at the non-pregnant level and lower than in normotensive pregnancy. Collagen-induced aggregation was at a lower level in hypertensive pregnancy than in both control groups. Thromboxane generation during spontaneous clotting and in platelet-rich plasma did not differ between the three groups. However, thromboxane generation was low during aggregation induced by small collagen concentrations in hypertensive pregnancy, but at higher collagen concentrations the difference between the groups disappeared. During nifedipine treatment (10 mg t.i.d.), aggregation and thromboxane production in platelet-rich plasma induced by the three stimuli remained unaltered in hypertensive pregnancy, while thromboxane synthesis during spontaneous clotting was reduced. In nifedipine-treated non-pregnant controls, only EC 80 for adrenaline-induced aggregation decreased. In vitro, pharmacological concentrations of nifedipine inhibited platelet aggregation and thromboxane production. In conclusion, nifedipine reduces thromboxane generation in spontaneous clotting, without inhibiting platelet aggregation and thromboxane production in platelet-rich plasma in hypertensive pregnancy. Reduced aggregability of platelet ex vivo may reflect their continuous activation and desensitization in vivo in hypertensive pregnancy.

Cyclosporin A (CsA), a potent immunosuppressor used in organ transplants and autoimmune diseases, is associated with adverse effects in the kidney, liver, and nervous system. This drug was recently shown to stimulate platelet aggregation, to increase thromboxane synthesis, and to decrease vascular prostacyclin synthesis. In experimental cholestasis, thrombocyte function is altered. The present study was designed to assess the effects of CsA on platelet function in extrahepatic biliary obstruction (EBO) in rats. Cyclosporin or its excipient (Cremaphore El, polyethoxylated castor oil) did not modify collagen-induced platelet aggregation. In animals with EBO, platelet aggregation decreased by 50%. The administration of CsA (5 or 10 mg/kg) or Cremaphore El increased aggregation by 163%, 253% and 123% respectively. Thromboxane production increased by 119% after Cremaphore El was administered, but was not significantly modified by CsA. Cholestasis increased thromboxane synthesis by 48.4%, whereas CsA showed a direct dose-dependent effect, and excipient had no significant effect. Excipient inhibited the vascular synthesis of 6-keto-PFG1 alpha by 67.2%, as did 5 mg/kg (56.8%) and 10 mg/kg CsA (27.6%). EBO led to a nonsignificant increase in the vascular synthesis of 6-keto-PFG1 alpha. Cremaphore EI inhibited prostacyclin synthesis by 79%; inhibition by CsA was dose-dependent (31% at 5 mg/kg, 60% at 10 mg/kg). Our findings show that cholestasis enhances the effects of CsA on platelet aggregation and thromboxane/prostacyclin balance. These results may reflect the vascular effects of CsA, which in turn may be enhanced by cholestasis.

While active smoking is known to enhance platelet thromboxane production, no data on passive smoking is available yet. The influence of single and repeated exposure to passive smoke for 60 minutes in a 18 m3 room was assessed in non-smokers as compared to sex and age matched smokers. All the evaluated measures (malondialdehyde, plasma thromboxane B2, 11-dehydro-thromboxane B2, serum thromboxane B2, conversion of exogenous arachidonic acid to thromboxane B2 and to hydroxy-5, 8,10-heptadecatrienoic acid) were higher in smokers than non-smokers at baseline, immediately and 6 hours after passive exposure to cigarette smoke. Repeated exposure of non-smokers rendered their platelets more activated becoming close to the behaviour of smokers. These results indicate that passive smoking may activate thromboxane A2 release from the platelets, contributing to the development of hemostatic imbalance.

Many studies have found that familial hypercholesterolemia, a hyperlipoproteinemia associated with premature atherosclerosis, is characterized by enhanced platelet aggregation. This study was undertaken to measure the urinary excretion of the two main urinary thromboxane B2 (TXB2) metabolites (2, 3-dinor-TXB2 and 11-dehydro-TXB2) in 20 patients affected by familial hypercholesterolemia treated for one month with 40 mg/day of pravastatin (10 patients) in comparison to 10 normocholesterolemic subjects. After a run-in period, the type II A patients showed total cholesterol levels (296 +/- 32 mg/dL) significantly higher (P0.001) than those of control subjects (155 +/- 46 mg/dL). The urinary concentrations of 11-dehydro-TXB2 and 2,3-dinor-TXB2 also significantly differed (P0.001) between control group (1,463 +/- 1,440 and 386 +/- 447 pg/mg urinary creatinine) and treated patients (3,536 +/- 2,112 and 914 +/- 572 pg/mg urinary creatinine). At baseline there was a positive correlation between total cholesterol (TC) levels and urinary TXB2 metabolite concentrations (2,3-dinor-TXB2 r = 0.61, P0.02; 11-dehydro-TXB2, r = 48, P0.05), but not between low-density-lipoprotein cholesterol (LDL-C) and the urinary compounds. At the end of a four-week treatment. TC and LDL-C had decreased significantly from the baseline levels, by 27% and 30% in the fluvastatin group (P0.01) and by 23% and 31% in the pravastatin group (P0.01), with no significant difference between the two groups. After the two treatments with HMG-CoA reductase inhibitors, there was no statistically significant reduction of the urinary metabolite levels. In addition, the positive correlation seen at baseline between TC and TXB2 metabolites was no longer present. In accord with previous studies, we found a significant correlation between TC levels and TXB2 metabolites concentrations in type II A hypercholesterolemic patients. Although, short-term treatment with two statins reduced TC levels, it did not change the thromboxane metabolite excretion.

The prostanoid thromboxane A2 has been implicated to contribute to the pathogenesis of many cardiovascular diseases, including hypertension. To study the role of vascular thromboxane-prostanoid (TP) receptors in blood pressure regulation, we generated mice with cell-specific deletion of TP receptors in smooth muscle using Cre/Loxp technology. We crossed the KI SM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Tbxa2r gene ( Tp flox ). In KI SM22α-Cre + Tp flox/flox (TP-SMKO) mice, TP receptors were efficiently deleted from vascular smooth muscle cells. In TP-SMKOs, acute vasoconstrictor responses to the TP agonist U46619 were attenuated to a similar extent in both the peripheral and renal circulations. Yet, acute vascular responses to angiotensin II were unaffected at baseline and after chronic angiotensin II administration. Infusion of high-dose U46619 caused circulatory collapse and death in a majority of control mice but had negligible hemodynamic effects in TP-SMKOs, which were completely protected from U46619-induced sudden death. Baseline blood pressures were normal in TP-SMKOs. However, the absence of TP receptors in vascular smooth muscle cells was associated with significant attenuation of angiotensin II–induced hypertension and diminished vascular remodeling. This was also associated with reduced urinary thromboxane production after chronic angiotensin II. Thus, TP receptors in vascular smooth muscle cells play a major role in mediating the actions of thromboxane A 2 in TP agonist-induced shock, hypertension, and vascular remodeling of the aorta.

OBJECTIVE: We investigated the hypothesis that nonsteroidal antiinflammatory agents can influence the abnormal prostanoid production associated with antiphospholipid antibodies. We specifically assessed whether aspirin or indomethacin could eliminate the increased placental thromboxane production previously observed with immunoglobulin G fractions from patients with lupus anticoagulant without adversely affecting prostacyclin production. STUDY DESIGN: Immunoglobulin G fractions were prepared from the plasma of eight nonpregnant patients with antiphospholipid antibody syndrome and demonstrable lupus anticoagulant. Samples from each patient were then placed in incubation wells containing explains from normal term pregnancies and 10 −4 mol/L aspirin, 10 −7 mol/L indomethacin, or no added drug. Aliquots were removed all intervals up to 48 hours of incubation and assessed for placental prostacyclin and thromboxane production by radioimmunoassay of the stable metabolites prostaglandin F 1α and thromboxane B 2 . RESULTS: The addition of aspirin to wells contaiing immunoglobulin G from patients with lupus anticoagulant was associated with a significant decrease in thromboxane production compared with wells without added drug, but prostacyclin production was unaffected. In contrast, the addition of indomethacin also decreased thromboxane production significantly, but prostacyclin production was also diminished, so the ratio of thromboxane to prostacyclin was unaffected. CONCLUSION: These results support a role for the use of aspirin for antiphospholipid antibody-related pregnancy loss through a mechanism similar to that postulated for preeclampsia, namely, selective inhibition of thromboxane production.

Placentas obtained from women with preeclampsia produce more lipid peroxides and more thromboxane, but less prostacyclin, than normal. The tissue compartments within the placenta that are responsible for this are not known. The placenta is a heterogeneous tissue compartmentalized into trophoblast cells and villous core tissue that is comprised of stromal and vascular tissue. In this study we determined the placental compartments responsible for increased production of lipid peroxides and thromboxane in preeclampsia. Placentas were obtained from six normally pregnant women and seven women with preeclampsia. Trophoblast cells and villous core tissues were isolated and incubated in Dulbecco's Modified Eagle's Medium for 48 h. Samples were collected at 0, 2, 6, 16, 28, and 48 h of incubation and analyzed spectrophotometrically for lipid peroxides by a peroxide equivalent assay and for thromboxane and prostacyclin by RIA of their stable metabolites, thromboxane-B2 and 6-keto-prostaglandin-F1 alpha. Trophoblast cells isolated from preeclamptic placentas produced significantly more lipid peroxides (1972 +/- 502 vs. 1102 +/- 335 pmol/micrograms protein after 48 h of incubation), more thromboxane (328 +/- 57 vs. 153 +/- 53 pg/microgram at 48 h), and more prostacyclin (50 +/- 11 vs. 13 +/- 3 pg/microgram at 48 h, respectively) than trophoblast cells isolated from normal placentas. Villous core tissue isolated from preeclamptic placentas produced significantly more lipid peroxides (455 +/- 107 vs. 241 +/- 34 pmol/microgram) and more thromboxane (148 +/- 51 vs. 76 +/- 14 pg/microgram) than normal villous core tissue, but there was no difference in prostacyclin production (36 +/- 11 vs. 40 +/- 9 pg/microgram). Because of the increase in thromboxane production, the ratio of thromboxane to prostacyclin was higher in preeclamptic than normal villous core tissue (6.29 vs. 2.17). Comparison of production by different compartments within the placenta demonstrated that lipid peroxides and thromboxane were primarily produced by the trophoblast cells and stromal tissue, whereas prostacyclin was primarily produced by the vascular tissue. We conclude that increased placental production of lipid peroxides and thromboxane in preeclampsia originates from both the trophoblast cell and the villous core compartments. As the placenta secretes lipid peroxides, the trophoblast cells could be a source of increased lipid peroxides in the maternal circulation of women with preeclampsia. The increased ratio of thromboxane to prostacyclin in the villous core could be responsible for increased placental vasoconstriction.

Preeclampsia is associated with altered placental production of several end-products of cyclooxygenase activity. Thromboxane (TX) is one of these end-products, and trophoblast is a source of villous thromboxane. We cultured term trophoblast in the presence or absence of fibrin to study how differentiation and epithelial-matrix interactions regulate cyclooxygenase expression. The cellular trophoblast present during the first 24 h of culture on uncoated plastic produced TXB 2 , but little or no TX was produced in cultures grown longer than 24 h when differentiation into syncytial trophoblast occurred. Growth of cells on a fibrin matrix enhanced cellular trophoblast TX production five-fold. Medium containing 10 μmol/l arachidonic acid maximized thromboxane production in cells cultured for less than 24 h, regardless of growth surface, but this medium had little or no effect on TX production by cultures grown for more than 24 h. In contrast, exogenous arachidonic acid enhanced prostaglandin E 2 (PGE 2 ) production by both cellular and syncytial trophoblast. Cytochemical staining indicated that changes in cyclooxygenase content occurred with trophoblast differentiation. Western immunoblot analysis of cells cultured in the presence or absence of a fibrin matrix showed cyclooxygenase was induced under both growth conditions. Detectable cyclooxygenase protein disappeared beyond 24 h in cells grown on uncoated plastic. In contrast, cells grown beyond 24 h on fibrin showed sustained expression of cyclooxygenase by Western immunoblotting, and this enzyme protein expression correlated with increased PGE 2 production by the differentiated trophoblast. These studies indicate (a) cellular trophoblast produces TX, (b) syncytial trophoblast produces PGE 2 but little or no TX, (c) trophoblast-matrix interactions increase TX production, and (d) expression of cyclooxygenase is regulated during trophoblast differentiation in vitro and is modulated by a fibrin matrix. Altered in vitro placental TX production in preeclampsia may result from a response of cellular trophoblast to the increased fibrin deposition typical of placentas from preeclamptic women.

Since hyperglycemia is involved in the “aspirin resistance” occurring in diabetes, we aimed at evaluating whether high glucose interferes with the aspirin-induced inhibition of thromboxane synthesis and/or activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway in platelets. For this purpose, in platelets from 60 healthy volunteers incubated for 60 min with 5–25 mmol/L d-glucose or iso-osmolar mannitol, we evaluated the influence of a 30-min incubation with lysine acetylsalicylate (L-ASA; 1–300 μmol/L) on 1) platelet function under shear stress; 2) aggregation induced by sodium arachidonate or ADP; 3) agonist-induced thromboxane production; and 4) NO production, cGMP synthesis, and PKG-induced vasodilator-stimulated phosphoprotein phosphorylation. Experiments were repeated in the presence of the antioxidant agent amifostine. We observed that platelet exposure to 25 mmol/L d-glucose, but not to iso-osmolar mannitol, 1) reduced the ability of L-ASA to inhibit platelet responses to agonists; 2) did not modify the L-ASA–induced inhibition of thromboxane synthesis; and 3) prevented the L-ASA–induced activation of the NO/cGMP/PKG pathway. Preincubation with amifostine reversed the high-glucose effects. Thus, high glucose acutely reduces the antiaggregating effect of aspirin, does not modify the aspirin-induced inhibition of thromboxane synthesis, and inhibits the aspirin-induced activation of the NO/cGMP/PKG pathway. These results identify a mechanism by which high glucose interferes with the aspirin action.

The effect of zearalenone, a nonsteroidal mycotoxin with estrogenic activity, and of 17 beta-estradiol on prostacyclin and thromboxane production in human endothelial cells was investigated. Zearalenone stimulated prostacyclin production in low concentrations (10(-7) and 10(-8)M) and inhibited it at a higher concentration (10(-5)M). Estradiol alone in the concentration range 10(-5)-10(-8)M had no clear-cut effect on the prostacyclin production. The combination of both substances effected changes in prostacyclin production similar to that from zearalenone alone; with the exception of estradiol at a concentration of 10(-6)M which enhanced the effect of zearalenone. No distinct changes in the thromboxane production from the two substances could be found, either alone or in combination.

In the present study, we compared the blood pressure in the SHR-SP and in the spontaneously hypertensive rat (SHR) after dietary administration of fish oil from 4 to 17 weeks of age. The retarding influence of dietary fish oils on the development of hypertension was prominent in the SHR (26 mmHg) and not evident in the SHR-SP (8 mmHg). The enhanced development of blood pressure in both the SHR and the SHR-SP is characterised by an elevated maximum contraction in the mesenteric vascular bed to sympathetic nerve stimulation and to injected noradrenaline. In SHR, but not SHR-SP, this maximum contraction was significantly attenuated by dietary fish oil. Likewise, acetylcholine mediated relaxation of the isolated aorta was enhanced in preparations from the SHR but not the SHR-SP. These physiological changes were also associated with a change in the total n-3 polyunsaturated fatty acids (PUFAs) content in vascular tissue, which were inversely proportional to the prevailing blood pressure values seen in all three strains of rat receiving dietary fish oils. Platelet activated thromboxane production was equally depressed in WKY (Wistar Kyoto), SHR and SHR-SP rats. The results indicate that the blood pressure lowering effect of fish oil when administered during the period of development of hypertension is much greater in the SHR than it is in the SHR-SP. Furthermore the lowering of blood pressure by fish oil administration is related to a restoration of normal vascular contraction and normal vascular relaxation, but not related to a suppression of serum thromboxane production.

Postmortem studies have demonstrated that morbidly obese subjects, surprisingly, have less coronary atherosclerosis than obese subjects. However, the reasons for this apparent protection from atherosclerosis are not yet clear. Thromboxane A2, a marker of platelet activation, is greater in obese subjects than in lean subjects, and this might be a clue to their increased cardiovascular risk. However, data on thromboxane A2 in morbidly obese subjects are lacking; therefore, we hypothesized that lower levels of thromboxane A2 in morbidly obese subjects might play a role in their lower atherothrombotic burden. We measured the serum levels of thromboxane B2 (TxB2), a stable metabolite of thromboxane A2, high-sensitivity C-reactive protein (hs-CRP) and leptin in 17 lean subjects (body mass index [BMI] 22.9 ± 1.6 kg/m(2)), 25 obese subjects (BMI 32.6 ± 2.4 kg/m(2)), and 23 morbidly obese subjects (BMI 48.6 ± 7.1 kg/m(2)), without insulin resistance, diabetes, or overt cardiovascular disease. The serum TxB2 levels were lower in the lean subjects than in the obese subjects (p = 0.046) and in the morbidly obese subjects than in the lean and obese subjects (p = 0.015 and p0.001, respectively). In contrast, the hs-CRP and leptin levels were greater in the obese than in the lean subjects (hs-CRP, p0.001; leptin, p0.001) and in the morbidly obese subjects than in the lean subjects (p0.001 for both). Leptin was also higher in the morbidly obese subjects than in the obese subjects (p0.001). TxB2 negatively correlated with leptin and BMI. hs-CRP correlated with leptin, and both also correlated with waist circumference, BMI, and homeostasis model assessment of insulin-resistance. In conclusion, insulin-sensitive morbidly obese subjects had lower levels of TxB2 than the obese subjects and lean subjects, suggesting that reduced platelet activation could play a role in the paradoxical protection of morbidly obese subjects from atherosclerosis, despite the greater levels of leptin.

CsA nephrotoxicity in rats is associated with an increase in renal thromboxane production. Treatment with selective thromboxane synthase inhibitors or receptor antagonists improves renal function in these animal models. In humans, it is unclear whether intervention aimed at reducing the effects of thromboxane on the kidney will be clinically useful. However, we reported previously that thromboxane metabolite excretion is increased in CsA-treated renal allograft recipients with evidence of CsA toxicity and that 48-hr intravenous infusion of the selective thromboxane synthase inhibitor CGS 13080 improves renal function in such patients. We undertook the present study to determine the effect of more prolonged treatment with an oral thromboxane synthase inhibitor, CGS 12970, in renal transplant recipients taking CsA. We measured glomerular filtration rate and p-aminohippurate clearance before and after 4 weeks of treatment with CGS 12970 in 13 patients with renal allografts who had been treated with CsA for a mean 6.3 months and had mild renal insufficiency. Baseline serum creatinine was 1.8 +/- 0.3. Treatment with CGS 12970 resulted in 83% inhibition of urinary thromboxane B2 (TXB2), 93% inhibition of 2,3-dinor-TXB2, and 89% inhibition of 11-dehydro-TXB2, but no change in the urinary excretion of prostacyclin metabolites. However, suppression of urinary thromboxane metabolites to these levels did not significantly affect renal function. Glomerular filtration rate was 45 +/- 4 ml/min/1.73 m2 at baseline and 43 +/- 4 ml/min/1.73 m2 after 4 weeks of treatment with CGS 12970. Estimated renal plasma flow was 272 +/- 21 ml/min/1.73 m2 at baseline and 251 +/- 38 ml/min/1.73 m2 with thromboxane synthase inhibition. Thus, substantial suppression of thromboxane production with CGS 12970 did not improve renal function in CsA-treated renal allograft recipients.

Objective: We evaluated whether the production of prostacyclin and thromboxane by normal human placental tissue is consistently altered by incubation with immunoglobulin G fractions prepared from plasma of patients with lupus anticoagulant. Study Design: The immunoglobulin G fractions were prepared from eight patients with lupus anticoagulant and eight control patients. Doses of these fractions (3 mg, 7.5 mg, and 12 mg) were incubated with placental expiants obtained from normal pregnancies, and prostacyclin and thromboxane production was assessed over 48 hours. Results: Prostacyclin production was similar for placental tissue incubated with immunoglobulin fractions from control and lupus anticoagulant patients at all of the doses tested. Placental production of thromboxane was significantly increased with immunoglobulin fractions from lupus anticoagulant patients for all three doses ( p = 0.02). Conclusions: The immunoglobulin G fraction from patients with lupus anticoagulant consistently alters placental thromboxane production without affecting prostacyclin production. Increases in placental thromboxane production may contribute to antiphospholipid antibody-mediated pregnancy loss.

Based on our previous observations that linoleic acid is an important dietary requirement for the development of liver injury in the intragastric feeding rat model for alcoholic liver disease, we postulated that the conversion of linoleic acid to arachidonic acid and various eicosanoids could be important in the pathogenesis of liver injury. We showed that liver nonparenchymal cell production of thromboxane B2 and leukotriene B4 was higher in rats fed corn oil and ethanol (liver injury model) than in animals fed saturated fat and ethanol (no liver injury). In contrast, prostaglandin E2 levels were lower in the former group. The best correlate of pathologic changes was the plasma level of thromboxane B2. The importance of thromboxanes in alcoholic liver injury is further suggested by the fact that inhibition of thromboxane production is accompanied by amelioration of liver injury.

Placental lipid peroxides and thromboxane are abnormally increased in preeclampsia. Thromboxane is a potent vasoconstrictor of the placental vasculature. Peroxides stimulate cyclooxygenase (prostaglandin H synthase), and thereby could increase thromboxane, to cause vasoconstriction in the placenta. This study was performed to test the hypothesis that peroxides would produce vasoconstriction in the human placenta by stimulating thromboxane production.Isolated human placental cotyledons were perfused for 20-minute intervals with 100 mumol/L t-butyl hydroperoxide alone, and during and after perfusion with low-dose aspirin (5 x 10(-5) mol/L) (n = 6) or the thromboxane receptor blocker SQ 29,548 (n = 2). Krebs-Ringer-bicarbonate buffer gassed with 95% oxygen and 5% carbon dioxide was used for the perfusion buffer. Perfusion pressure was monitored continuously, and effluent flow rates were measured during each experimental treatment. Maternal and fetal effluent samples were analyzed for thromboxane B2 and 6-keto-prostaglandin F1 alpha.Compared with control Krebs-Ringer-bicarbonate buffer perfusion, peroxide perfusion significantly increased (p0.05) vascular resistance (14 +/- 2 vs 25 +/- 3 mm Hg.min/ml, mean +/- SE, respectively), thromboxane B2 secretion (fetal 0.20 +/- 0.04 vs 1.65 +/- 0.26 ng/min, maternal 4.8 +/- 1.5 vs 8.1 +/- 2.1 ng/min) and 6-keto-prostaglandin F1 alpha secretion (fetal 21 +/- 5 vs 60 +/- 1.8 pg/min, maternal nondetectable). Peroxide perfusion increased the thromboxane B2/6-keto-prostaglandin F1 alpha ratio threefold on the fetal side. Subsequent perfusion with aspirin significantly blocked the peroxide-induced vasoconstriction (13 +/- 1 mm Hg.min/ml during aspirin + peroxide) and the peroxide-induced increase in the secretion of thromboxane B2 (fetal 0.52 +/- 0.12 ng/min, maternal 2.0 +/- 0.3 ng/min) and 6-keto-prostaglandin F1 alpha (fetal 30 +/- 8 pg/min). After perfusion with aspirin the thromboxane B2/6-keto-prostaglandin F1 alpha ratio declined. When the thromboxane receptor blocker was used instead of aspirin, maternal and fetal secretion rates of thromboxane were still significantly increased by peroxide perfusion, but there was no change in perfusion pressure or vascular resistance.(1) Peroxide induces placental vasoconstriction coincident with increased secretion of thromboxane; (2) low-dose aspirin blocks both increased thromboxane secretion and vasoconstriction, whereas a thromboxane receptor antagonist allows increased thromboxane secretion but prevents peroxide induced vasoconstriction; (3) therefore, peroxide induces vasoconstriction by stimulating thromboxane synthesis.

The goal of the present study was to characterize the role of the endothelium in the 5-hydroxytryptamine (5-HT)-induced contraction of the rat basilar artery. Rat basilar artery segments were mounted in myographs to study their isometric tension development. 5-HT caused dose-dependent contractions that were minimally affected by endothelium removal. The dose-response curve obtained with the 5-HT1 receptor agonist, 5-carboxamidotryptamine (5-CT), was biphasic in arteries with endothelium; removal of the endothelium eliminated the first phase of the contraction. The 5-HT2 receptor antagonist, ketanserin (30 nM), shifted the dose-response curve to 5-HT to the right; in arteries with endothelium, the curve became biphasic. Ketanserin inhibited the second phase of the dose-response curve to 5-CT. The mixed 5-HT1/5-HT2 receptor antagonist, metergoline (30 nM), shifted the dose-response curve to 5-HT non-competitively to the right and depressed both phases of the dose-response curve to 5-CT. In basilar arteries with endothelium and treated with ketanserin, the thromboxane A2 receptor antagonist, ICI 192605 (1 microM), significantly decreased the responsiveness to 5-HT and the dose-response curve for 5-HT became monophasic. ICI 192605 and the thromboxane A2 synthase inhibitor, ridogrel (10 microM), both suppressed the first phase of the dose-response curve to 5-CT. These data indicate that both endothelial 5-HT1 and smooth muscle 5-HT2 receptors participate in the contractions caused by 5-HT in the rat basilar artery.(ABSTRACT TRUNCATED AT 250 WORDS)