TXB2 Production Research Articles

Effect of aspirin on renal disease progression in patients with type 2 diabetes: A multicenter, double-blind, placebo-controlled, randomized trial. The renaL disEase progression by aspirin in diabetic pAtients (LEDA) trial. Rationale and study design

The primary end point of the LEDA trial is to evaluate the 1-year decline of eGFR in T2DM patients treated or not with low-dose aspirin (100mg/d). Secondary end points will be the rapid decline in renal function, defined as a reduction of eGFR ≥5mL/min, and change of renal function class after 1-year follow-up. Furthermore, urinary excretion 11-dehydro-TxB2 will be related to renal function modifications. A phase 3 no-profit, multicenter, double-blind, randomized intervention trial of aspirin 100mg/dvs placebo (ClinicalTrials.gov Identifier: NCT02895113). All patients will be monitored at 6 and 12months after randomization to assess drug adherence and eGFR changes. The LEDA trial is the first double-blind, placebo-controlled, randomized clinical trial aimed at examining whether aspirin treatment may beneficially affect kidney function in patients with T2DM by reducing the annual eGFR decline. The trial will also examine whether the potential renoprotective effects of aspirin might be partly due to its inhibition of TxB2 production.

Crataegus orientalis Leaves and Berries: Phenolic Profiles, Antioxidant and Anti-inflammatory Activity

The present study was designed to define the phenolic content, antioxidant and anti-inflammatory activity of Crateagus orientalis Pall. ex M. Bieb., traditionally used by local people in southern parts of F.Y.R. Macedonia. The presence and content of 7 phenolics in ethanolic extracts of leaves and berries were studied using HPLC-DAD, where the most dominant compounds were hyperoside, isoquercitrin and chlorogenic acid. The leaf extract was more effective as a DPPH radical scavenger (IC₅₀ = 29.7 μg/g) than the berry extract, as well as in the relative reducing power on Fe³⁺. Anti-inflammatory potential was studied by means of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibitory activity; both extracts evinced activity. Furthermore, C. orientalis leaf extract showed a concentration dependent inhibition of COX-1 pathway products 12-HHT and TXB₂, reaching IC₅₀ values below the lowest applied concentration (68.9% and 55.2% of 12-HHT and TXB2 production inhibition, respectively, at concentration of 0.4 mg/mL). Although inhibitors such as acetylsalicylic acid and quercetin showed higher activity, this study demonstrates that the investigated extracts are potential anti-inflammatory agents.

Effects of a single bout of strenuous exercise on platelet activation in female ApoE/LDLR−/− mice

Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI2) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms.In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR−/− mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF1α, nitrite/nitrate, lipid profile, and blood cell count.Sedentary 3- and 7-month-old ApoE/LDLR−/− mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR−/− but not in WT mice, strenuous exercise partially inhibited TXB2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR−/− mice; however, only 7-month-old ApoE/LDLR−/− mice showed lower TXB2 production after exercise. In female 4–6-month-old ApoE/LDLR−/− but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF1α was observed. In turn, the pre- and post-exercise plasma concentrations of nitrite (NO2−) and nitrate (NO3−) were decreased in ApoE/LDLR−/− as compared to that in age-matched WT mice.In conclusion, we demonstrated overactivation of platelets in ApoE/LDLR−/− as compared to WT mice. However, platelet activation in ApoE/LDLR−/− mice was not further increased by strenuous exercise, but was instead attenuated, a phenomenon not observed in WT mice. This phenomenon could be linked to compensatory up-regulation of PGI2-dependent anti-platelet mechanisms in ApoE/LDLR−/− mice.

The lack of aspirin resistance in patients with coronary artery disease.

BackgroundAspirin resistance established by different laboratory methods is still a debated problem. Using COX1 specific methods no aspirin resistance was detected among healthy volunteers. Here we tested the effect of chronic aspirin treatment on platelets from patients with stable coronary artery disease. The expression of COX2 mRNA in platelets and its influences on the effect of aspirin was also investigated.MethodsOne hundred and forty four patients were enrolled in the study. The direct measurement of COX1 acetylation was carried out by monoclonal antibodies specific to acetylated and non-acetylated COX1 (acCOX1 and nacCOX1) using Western blotting technique. Arachidonic acid (AA) induced TXB2 production by platelets was measured by competitive immunoassay. AA induced platelet aggregation, ATP secretion and VerifyNow Aspirin Assay were also performed. COX2 and COX1 mRNA expression in platelets were measured in 56 patients by RT-qPCR.ResultsIn 138 patients only acCOX1 was detected, in the remaining six patients nacCOX1 disappeared after a compliance period. AA induced TXB2 production by platelets was very low in all patients including the 6 patients after compliance. AA induced platelet aggregation, secretion and with a few exceptions the VerifyNow Assay also demonstrated the effect of aspirin. Smoking, diabetes mellitus and inflammatory conditions did not influence the results. The very low amount of COX2 mRNA detected in 39 % of the investigated platelets did not influence the effect of aspirin.ConclusionsNo aspirin resistance was detected among patients with stable coronary artery disease. COX2 expression in platelets did not influence the effect of aspirin.

Mechanisms underlying uridine adenosine tetraphosphate-induced vascular contraction in mouse aorta: Role of thromboxane and purinergic receptors

Uridine adenosine tetraphosphate (Up4A), a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorders and induces aortic contraction through activation of cyclooxygenases (COXs). We and others demonstrated that activation of A1 or A3 adenosine receptors (ARs) results in vascular contraction via thromboxane (TX) A2 production. However, the mechanisms of Up4A-induced vascular contraction in mouse aorta are not understood. We hypothesize that Up4A-induced aortic contraction is through COX-derived TXA2 production, which requires activation of A1 and/or A3AR. Concentration responses to Up4A were conducted in isolated aorta. The TXB2 production, a metabolite of TXA2, was also measured. Up4A (10−9-10−5M) produced a concentration-dependent contraction >70%, which was markedly attenuated by COX and COX1 but not by COX2 inhibition. Notably, Up4A-induced aortic contraction was blunted by both TX synthase inhibitor ozagrel and TXA2 receptor (TP) antagonist SQ29548. Surprisingly, A3AR deletion had no effect on Up4A-induced contraction. Moreover, A1AR deletion or antagonism as well as A1/A3AR deletion potentiated Up4A-induced aortic contraction, suggesting a vasodilator influence of A1AR. In contrast, non-selective purinergic P2 receptor antagonist PPADS significantly blunted Up4A-induced aortic contraction to a similar extent as selective P2X1R antagonist MRS2159, the latter of which was further reduced by addition of ozagrel. Endothelial denudation almost fully attenuated Up4A-induced contraction. Furthermore, Up4A (3μM) increased TXB2 formation, which was inhibited by either MRS2159 or ozagrel. In conclusion, Up4A-induced aortic contraction depends on activation of TX synthase and TP, which partially requires the activation of P2X1R but not A1 or A3AR through an endothelium-dependent mechanism.

Synthesis and antiplatelet activity of antithrombotic thiourea compounds: biological and structure-activity relationship studies.

The incidence of hematological disorders has increased steadily in Western countries despite the advances in drug development. The high expression of the multi-resistance protein 4 in patients with transitory aspirin resistance, points to the importance of finding new molecules, including those that are not affected by these proteins. In this work, we describe the synthesis and biological evaluation of a series of N,N'-disubstituted thioureas derivatives using in vitro and in silico approaches. New designed compounds inhibit the arachidonic acid pathway in human platelets. The most active thioureas (compounds 3d, 3i, 3m and 3p) displayed IC50 values ranging from 29 to 84 µM with direct influence over in vitro PGE2 and TXA2 formation. In silico evaluation of these compounds suggests that direct blockage of the tyrosyl-radical at the COX-1 active site is achieved by strong hydrophobic contacts as well as electrostatic interactions. A low toxicity profile of this series was observed through hemolytic, genotoxic and mutagenic assays. The most active thioureas were able to reduce both PGE2 and TXB2 production in human platelets, suggesting a direct inhibition of COX-1. These results reinforce their promising profile as lead antiplatelet agents for further in vivo experimental investigations.

A high-fat plus fructose diet produces a vascular prostanoid alterations in the rat.

In the rat, a high-fat (HF) plus fructose (F) diet produces cardiovascular and metabolic alterations that resemble human metabolic syndrome. Prostanoids (PR), cyclo-oxygenase-derived arachidonic acid metabolites, have vasoactive properties and mediate inflammation. The aim of this study was to analyse the effect of a HF+F diet on blood pressure (BP), metabolic parameters and mesenteric vascular bed PR production in male Sprague-Dawley rats. Four groups were studied over 9 weeks (n = 6 each): control (C), standard diet (SD) and tap water to drink; F+SD and 10% w/v F solution to drink; HF 50% (w/w) bovine fat added to SD and tap water; and HFF, both treatments. PR were determined by HPLC. Blood pressure was elevated in all experimental groups. Triglyceridaemia, insulinaemia and HOMA-IR were increased in the F and HF groups. HF+F animals showed elevated glycaemia, insulinaemia, HOMA-IR and triglyceridaemia. F decreased the vasodilator prostanoids PGI2 and PGE2 in the mesenteric vascular bed. Body weight was not significantly altered. In HFF, production of PGE2 , PGF2 alpha and TXB2 was elevated. The increased BP in HF and HFF could be partly attributed to the imbalance in vascular PR production towards vasoconstrictors. On the other hand, this dietary modification could induce inflammation, which would explain the elevation of PGE2 . In the F group, hypertension could be related to decreased vasodilator PRs. The simultaneous administration of HF and F in the rat produces deleterious effects greater than observed when treatments are applied separately.

Pharmacokinetics and pharmacodynamics of three formulations of firocoxib in healthy horses.

The objectives of this study were to compare the pharmacokinetics and COX selectivity of three commercially available formulations of firocoxib in the horse. Six healthy adult horses were administered a single dose of 57 mg intravenous, oral paste or oral tablet firocoxib in a three-way, randomized, crossover design. Blood was collected at predetermined times for PGE2 and TXB2 concentrations, as well as plasma drug concentrations. Similar to other reports, firocoxib exhibited a long elimination half-life (31.07 ± 10.64 h), a large volume of distribution (1.81 ± 0.59L/kg), and a slow clearance (42.61 ± 11.28 mL/h/kg). Comparison of the oral formulations revealed a higher Cmax , shorter Tmax , and greater AUC for the paste compared to the tablet. Bioavailability was 112% and 88% for the paste and tablet, respectively. Maximum inhibition of PGE2 was 83.76% for the I.V. formulation, 52.95% for the oral paste formulation, and 46.22% for the oral tablet formulation. Pharmacodynamic modeling suggests an IC50 of approximately 27 ng/mL and an IC80 of 108 ng/ mL for COX2 inhibition. Inhibition of TXB2 production was not detected. This study indicates a lack of bioequivalence between the oral formulations of firocoxib when administered as a single dose to healthy horses.

Cyclooxygenase-2-dependent oxidative stress mediates palmitate-induced impairment of endothelium-dependent relaxations in mouse arteries

Palmitic acid, one of the saturated free fatty acids, impairs cardiovascular function as manifested by inducing vascular inflammation, apoptosis and over-production of reactive oxygen species (ROS) although the origin for ROS remains unclear. The present study investigated the cellular mechanisms underlying palmitate-induced impairment of endothelial function. Ex vivo treatment in tissue culture with palmitate concentration-dependently attenuated acetylcholine-induced endothelium-dependent relaxations, up-regulated the expression of cyclooxygenase-2 (COX-2) and elevated superoxide formation in mouse aortic endothelial cells (MAECs) measured by dihydroethidium (DHE) staining and electron paramagnetic resonance (EPR) spectroscopy. Superoxide scavengers, COX-2 inhibitor and thromboxane prostanoid (TP) receptor antagonist, but not COX-1 inhibitor reversed the harmful effects of palmitate. Furthermore, palmitate impaired acetylcholine-induced relaxations and raised superoxide in en face endothelium of aortas only from COX-1−/− mice but not from COX-2−/− mice. Palmitate increased the production and release of TXB2, a stable thromboxane A2 metabolite in mouse aortas, which was abolished by COX-2 inhibitor. Superoxide scavenger did not affect palmitate-induced up-regulated expression of COX-2 in MAECs. Both real time PCR and luciferase reporter gene assay confirmed COX-2 up-regulation in palmitate-treated MAECs and NF-κB was substantially involved in this up-regulation. The present study provides novel evidence that palmitate up-regulates COX-2 through NF-κB-dependent mechanism and resultant COX-2-associated oxidative stress impairs endothelium-dependent relaxations in mouse aortas.

Variation in thromboxane B2 concentrations in serum and plasma in patients taking regular aspirin before and after clopidogrel therapy

Dual antiplatelet therapy with aspirin and a P2Y12 antagonist is widely prescribed for the prevention of thrombotic events in patients with an acute coronary syndrome or undergoing percutaneous coronary intervention (PCI). It is recognised that there is inter-individual variation in the antiplatelet effects of both drugs. Recent data also suggest that P2Y12 antagonists can affect the response to aspirin. A direct indicator of the effect of aspirin on platelets is their ability to generate thromboxane, which if measured as the difference between the level of thromboxane B2 in serum and plasma ([TxB2]S-P) avoids the confounding effect of endogenous TxB2 production from other cells. We therefore analysed [TxB2]S-P as a measure of aspirin response in a group of 123 patients undergoing elective PCI before and after the introduction of clopidogrel. In a subgroup of 40 patients taking aspirin alone, we compared [TxB2]S-P and VerifyNow Aspirin for the assessment of aspirin response. There was a wide variation in plasma and serum TxB2 concentrations both before and after clopidogrel therapy but only 3.5% of patients had residual serum concentration of TxB2 > 10 ng/ml. There was a strong correlation between the pre and post clopidogrel levels of TxB2 (r ≥ 0.78; p = 0.001) and no significant difference in [TxB2]S-P. There was no correlation between the magnitude of response to clopidogrel response and the generation of thromboxane B2. Correlation between [TxB2]S-P and VerifyNow Aspirin was poor. We conclude that the use of a P2Y12 antagonist does not influence the effect of aspirin on the ability of platelets to generate thromboxane. Therefore, measurement of TxB2 levels in serum, after subtracting the contribution from plasma, provides a measure of the response to aspirin in patients taking dual antiplatelet therapy.

4-Methylcoumarin Derivatives with Anti-inflammatory Effects in Activated Microglial Cells

Inflammation contributes to the pathogenesis of neurodegenerative diseases and anti-inflammatory compounds may have a role in prevention or treatment of these pathologies. 4-Methylcoumarins are effective antioxidants with anti-inflammatory properties. In this study, the inhibitory effects of two 4-methylcoumarin derivatives, 7,8-dihydroxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DHEMC) and 7,8-diacetoxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DAEMC) were examined on the inflammatory processes induced by lipopolysaccharide (LPS) in activated primary rat microglial cultures. LPS-induced production of nitric oxide (NO, measured by Griess method) and other pro-inflammatory mediators, thromboxane (TX) B2 and prostaglandin (PG) E2 (both determined by radioimmunoassay (RIA)), as well as tumor necrosis factor (TNF)-α (determined by enzyme-linked immunosorbent assay (ELISA)) were inhibited in the presence of 100 µM DHEMC and DAEMC. DAEMC was able to significantly inhibit NO, TXB2 and TNF-α production also at 50 µM. Both compounds at 100 µM significantly lowered cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated microglial cells measured by Western blot, but only DAEMC showed an inhibitory effect on inducible nitric oxide synthase (iNOS) protein expression at 100 µM. In conclusion, our findings show that 4-methylcoumarin derivatives can modulate inflammatory pathways in microglial cells, probably by acting at the protein expression level.

Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A₂ (TxA₂) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB₂, a stable metabolite of TxA₂. TxB₂ production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80-90%. Ozagrel-treated blood also exhibited accumulation of PGD₂ and PGE₂, possibly resulting from the shunting of PGH₂ into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB₂ production during blood coagulation was completely lost, but 12-HHT production was reduced by 80-85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB₂ and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB₂. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo.

Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods

Aspirin, a commonly used antiplatelet agent, blocks platelet thromboxane A₂ (TXA₂) formation from arachidonic acid (AA) by acetylating platelet cyclooxygenase-1 (COX-1). Laboratory methods currently used to detect this antiplatelet effect of aspirin provide variable results. We have reported three methods that assess platelet COX-1 acetylation (inactivation) by aspirin and its direct consequences. The first and second assays use monoclonal anti-human-COX-1 antibodies that only detect acetylated (inactivated) COX-1 and active (non-acetylated) COX-1, respectively. The third method measures platelet production of TXB₂ (the stable metabolite of TXA₂) in vitro in response to AA. We compared the results of these three reference methods with other routinely used methods for assessing the functional consequences aspirin treatment. 108 healthy volunteers were treated with low-dose aspirin for 7 days. On day 7 following aspirin treatment COX-1 in the platelets was fully acetylated whereas only non-acetylated COX-1 was present in the day 0 platelets. Further, TXB2 production by day 7 platelets was completely blocked. The following tests were performed on the samples obtained from study participants before and after seven days of aspirin treatment: PFA-100 closure time with collagen/epinephrine cartridge, VerifyNow (VN) Aspirin Assay, platelet aggregation and ATP secretion using AA, ADP, epinephrine and collagen as agonists. Comparing the pre-treatment and day 7 values, methods that use AA as platelet agonist (AA-induced platelet aggregation/secretion and VN Aspirin Assay) showed high discriminative power. In contrast, results of the other tests showed considerable overlap between day 7 and day 0 values. Only assays that clearly distinguish between acetylated and non-acetylated platelet COX-1 are useful for establishing the antiplatelet effect of aspirin. The other tests are not suitable for this purpose.

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

AimsIn liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB2) agonists JWH-133 and GP 1a attenuate portal hypertension. Main methodsIn vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4weeks after bile duct ligation (BDL). Zymosan (150μg/ml, isolated liver perfusion) or LPS (4mg/kgb.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10mg/kgb.w.), GP 1a (2.5mg/kgb.w.) or ZnPP IX (1μM). Isolated KC were treated with Zymosan (0.5mg/ml) in addition to JWH-133 (5μM). The thromboxane (TX) B2 levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB2 were analyzed by Western blot or confocal microscopy. Key findingsJWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB2 and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB2 production following KC activation was attenuated by JWH-133 pre-treatment. SignificanceJWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.

Randomized trial to examine the effect of ASA dose or ASA dosing frequency on ASA resistance after coronary artery bypass graft surgery

Purpose: Low-dose ASA (81 mg daily) is administered after coronary artery bypass graft (CABG) surgery to reduce the risk of graft failure. There is transient ASA resistance after such surgery, which may compromise its effectiveness. Because such resistance may reflect the failure of once-daily, low-dose ASA to acetylate cyclo-oxygenase (COX)-1 and inhibit thomboxane A2 (TXA2) synthesis in the setting of the increased platelet turnover that occurs post-operatively, we performed a randomized trial to determine whether higher-dose once-daily or more frequent ASA dosing overcomes the resistance observed with usual ASA dosing. Methods: Adults undergoing CABG surgery were randomized to one of 3 ASA dosing regimens; 81 mg once-daily, 325 mg once-daily, or 81mg four times daily. ASA was started on the first post-operative day and continued until day 7 or hospital discharge. Using an immunoassay, levels of serum TXB2, the stable metabolite of TXA2, were measured daily before starting ASA. The primary outcome was the median TXB2 level on postoperative day 4. Results: A total of 100 patients undergoing CABG surgery (mean age 65, females 16%) were randomized. On day 4, median TXB2 level in the group receiving ASA 81mg once-daily was 10.7 ng/ml (Q1,Q3; 6.1,30.8 ng/ml). The median TXB2 levels were significantly lower in the groups randomized to ASA 325 mg once-daily and 81 mg four times daily; 3.6 ng/ml (Q1,Q3; 1.9,10.1 ng/ml) and 1.1 ng/ml (Q1,Q3; 0.5,2.4 ng/ml), respectively; P=0.001 and P<0.0001, respectively. Conclusions: ASA resistance after CABG surgery, defined as failure of ASA to suppress TXB2 formation, is attenuated by higher dose once-daily ASA and almost abolished by four times daily low-dose ASA. The efficacy of more frequent ASA dosing supports the concept that resistance reflects, at least in part, increased platelet turnover. Attenuation of TXB2 production by higher dose once-daily ASA raises the possibility that the susceptibility of megakaryocyte COX-1 to acetylation by ASA may be dose-dependent.

Enhanced uridine adenosine tetraphosphate-induced contraction in renal artery from type 2 diabetic Goto-Kakizaki rats due to activated cyclooxygenase/thromboxane receptor axis

The dinucleotide uridine adenosine tetraphosphate (Up4A), which has both purine and pyrimidine moieties, was reported as a novel endothelium-derived contracting factor. Recently, growing evidence has suggested that Up4A plays an important role in regulation of the cardiovascular function. We previously demonstrated that Up4A-induced vasoconstrictions are altered in arteries from DOCA-salt hypertensive rats. We have assessed responses to Up4A shown by renal arteries from type 2 diabetic Goto-Kakizaki (GK) rats (42-46weeks old) and identified the molecular mechanisms involved. Concentration-dependent contractions to Up4A were greater in renal arterial rings from the GK than age-matched control Wistar group. In both groups, the inhibition of nitric oxide synthase (with N (G)-nitro-L-arginine) increased the response to Up4A, whereas the inhibition of cyclooxygenase (COX) (with indomethacin) decreased the response. Specific inhibitors of COX-1 (valeroyl salicylate) and COX-2 (NS398), a thromboxane (TX) receptor (TP) antagonist (SQ29548), and P2 receptor antagonist (suramin) also decreased the response to Up4A. Protein expressions of COXs in renal arteries were greater in the GK than Wistar group. The production of TXB2 (a metabolite of TXA2) by Up4A did not differ between these groups. Concentration-dependent contractions to U46619, an agonist of the TP receptor, were greater in renal arteries from the GK than Wistar group. The expression of P2X1 and P2Y2 receptors did not differ between these groups. These results suggest that enhancement of the Up4A-induced contraction in renal arteries from GK rats may be attributable to the increased activation of COXs/TP receptor signaling.

Antithrombotic and antiplatelet activities of Soshiho-tang extract

BackgroundSoshiho-tang (SH; Chinese name, Xiao-Chai-Hu-Tang; Japanese name, Shosaiko-to) is a traditional Korean, Chinese, and Japanese medicine, which has been used to treat various conditions, including hepatitis, liver cirrhosis, and chronic and acute liver disease. SH consists of seven herbal components, of which Scutellaria baicalensis Georgi and Zingiber officinale Roscoe, are reported to have antithrombotic and antiplatelet activities. We investigated the antithrombotic activity of SH, including S. baicalensis and Z. officinale, as an integrative therapy.MethodsTo identify the antithrombotic activity of SH, we used a FeCl3-induced thrombus formation model. The mechanism of SH-mediated antithrombotic activity was assessed by determining platelet aggregation and coagulation times ex vivo, washed platelet aggregation, serotonin secretion, and thromboxane B2 formation.ResultsSH prolonged the occlusion time of thrombus formation when applied in a FeCl3-induced thrombus formation model. SH also inhibited collagen-induced platelet aggregation ex vivo in a concentration-dependent manner; however, it did not affect coagulation. Hence, to identify the antiplatelet effect of SH, we investigated washed platelet aggregations in vitro. SH significantly inhibited various agonist-induced platelet aggregations, and it completely inhibited serotonin secretion and thromboxane B2 formation.ConclusionsThe findings suggest that SH inhibited FeCl3-induced thrombus formation through antiplatelet activity, including inhibition of platelet aggregation, and serotonin and TXB2 production. Thus, SH may be useful as an integrative herbal formula for the treatment of thrombosis.

New direct and indirect methods for the detection of cyclooxygenase 1 acetylation by aspirin; the lack of aspirin resistance among healthy individuals

BackgroundAspirin is widely used in the prevention of acute atherothrombotic complications. It acetylates Ser529 residue in cyclooxygenase-1 (COX-1) and prevents thromboxane A2 (TXA2) formation from arachidonic acid (AA) in platelets. Laboratory methods used for the detection of aspirin effect provide inconsistent results. MethodsTwo new methods were developed for the direct and indirect detection of COX-1 acetylation by aspirin in 108 healthy volunteers treated daily with 100mg enteric-coated aspirin for 7days. Monoclonal antibodies were raised against acetylated and non-acetylated nonapeptides corresponding to the amino acid sequence of human COX-1 525–533 residues. Using Western blotting technique the antibodies clearly distinguished between acetylated and non-acetylated COX-1 in platelet lysate. The second method measures AA-induced TXB2 production of platelets in diluted platelet rich plasma. ResultsNo acetylated COX-1 was detected in platelets before aspirin treatment. At the same time antibodies raised against non-acetylated peptide gave intense reaction with COX-1 on the Western blot. In contrast, after 7days of aspirin treatment, with a single exception, only acetylated COX-1 could be detected in the platelet lysate. The non-responding volunteer showed full response to aspirin after controlled drug intake. In parallel experiments aspirin treatment for 7days practically completely inhibited AA-induced TXB2 production by platelets. ConclusionsChemical (“true”) aspirin resistance, if it exists, must be a rarity among healthy individuals. The new methods could be used for detecting the acetylation of COX-1 by aspirin in patients on preventive aspirin therapy and for evaluating methods routinely used for such purpose.

Phytochemical profile and biological activities of Allium carinatum Ssp. Carinatum L. extract

Members of genus Allium have been used for thousands of years for their medicinal properties and characteristic flavor. Chemical composition and biological activities of most Allium species, including A. carinatum ssp. carinatum L., are still unexplored. Therefore, the aim of this study was to evaluate phytochemical profile and biological activities of methanolic extract of A. carinatum ssp. carinatum. Chemical composition evaluation included the content of total phenolics, flavonoids and anthocyanins, quantitative LC-MS/MS and GC/MS analysis. Antioxidant activity was evaluated by LP, DPPH and NO assays, antimicrobial activity by disk diffusion and MIC assays, while anti-inflammatory activity was observed measuring the inhibitory potential on eicosanoids production. High contents of total phenolics, flavonoids and monomeric anthocyanins were found. The dominant phenolic compounds in the herb extract are ferulic acid, quercetine-3-O-Glc, kaempferol-3-O-Glc and rutin. Dimethyl-disulphide was detected as the only volatile compound. The extract suppressed production of 12-HETE, 12-HHT and TXB2, but not of PGE2, meaning that extract inhibits TXS and 12-LOX activity. Extract have not shown any antimicrobial activity towards nine bacterial strains, while antioxidant activity was weak compared with synthetic antioxidants.

Prasugrel inhibits platelet-leukocyte interaction and reduces inflammatory markers in a model of endotoxic shock in the mouse

Prasugrel, through its active metabolite, reduces atherothrombosis and its clinical manifestations by inhibiting platelet activation and aggregation. Platelets also contribute to inflammation through interaction with different classes of leukocytes. We investigated whether the inhibitory effect of prasugrel on platelets also counteract inflammatory responses. The effect of prasugrel active metabolite, R-138727, was investigated on platelet P-selectin expression, platelet adhesion to polymorphonuclear leukocytes (PMN) and monocytes (MN) and Mac-1 expression in PMN and MN, in vitro, in human cells. The ex vivo effect of prasugrel administration on P-selectin, thromboxane (TXB)2 formation, platelet-PMN conjugates and Mac-1 expression in PMN triggered by PAR-4 agonist peptide was examined in whole blood from healthy mice as well as from mice in which an acute inflammatory reaction was induced by treatment with endotoxin. The effect of prasugrel on inflammatory markers in endotoxin-treated animals was also tested in vivo. R-138727 inhibited agonist-stimulated expression of platelet P-selectin, platelet-PMN and platelet-MN adhesion and platelet-dependent Mac-1 expression in leukocytes. Addition of aspirin did not modify the inhibitory effect elicited by R-138727. Treatment of mice with prasugrel resulted in a profound inhibition of platelet P-selectin expression, TXB2 production, platelet-PMN adhesion and Mac-1 expression in PMN induced by ex vivo stimulation with PAR-4 agonist peptide of whole blood from healthy or endotoxin-treated mice. Measurement of markers revealed that prasugrel reduced TXB2 and tumour necrosis factor-α synthesis and increased nitric oxide metabolites in endotoxin-treated mice in vivo. In conclusion, prasugrel reduces platelet interactions with PMN and MN. Through these effects prasugrel may curb platelet-mediated inflammatory responses.