ABSTRACT
Platelets are key mediators of physiological hemostasis and pathological thrombosis, whose function must be carefully balanced by signaling downstream of receptors such as protease-activated receptor (PAR)4. Protein kinase C (PKC) is known to regulate various aspects of platelet function. For instance, PKCδ is known to regulate dense granule secretion, which is important for platelet activation. However, the mechanism by which PKCδ regulates this process as well as other facets of platelet activity is unknown. We speculated that the way PKCδ regulates platelet function may be because of the phosphorylation of tyrosine residues on PKCδ. We investigated phosphorylation of PKCδ following glycoprotein VI-mediated and PAR4-mediated platelet activation and found that Y311 is selectively phosphorylated when PAR4 is activated in human platelets. Therefore, we generated PKCδ Y311F knock-in mice, which are viable and have no gross abnormalities. However, PKCδY311F mice have significantly enhanced tail-bleeding times compared with WT littermate controls, which means hemostasis is interrupted. Furthermore, PKCδY311F mice exhibit longer time to carotid artery occlusion compared with WT control using a ferric chloride in vivo thrombosis model, indicating that the phosphorylation of PKCδ Y311 is prothrombotic. Washed platelets from PKCδY311F mice have reduced reactivity after stimulation with a PAR-4 agonist indicating its importance in platelet signaling. The phenotype observed in Y311F mouse platelets is because of reduced thromboxane generation, as an inhibitor of thromboxane generation equalizes the PKCδY311F platelet response to that of WT. Therefore, phosphorylation of PKCδ on Y311 is important for regulation of platelet function and specifically thromboxane generation, which reinforces platelet activation.
Subject(s)
Blood Platelets/metabolism , Protein Kinase C-delta/chemistry , Protein Kinase C-delta/metabolism , Thromboxanes/biosynthesis , Tyrosine/metabolism , Animals , Humans , Mice , Models, Molecular , Phosphorylation , Protein ConformationABSTRACT
Prostanoids are short-lived autocrine and paracrine signaling molecules involved in a wide range of biological functions. They have been shown to be intimately involved in many different disease states when their regulation becomes dysfunctional. In order to fully understand the progression of any disease state or the biological functions of the well state, a complete evaluation of the genomics, proteomics, and metabolomics of the system is necessary. This review is focused on the enzymology for the enzymes involved in the synthesis of the prostanoids (prostaglandins, prostacyclins and thromboxanes). In particular, the isolation and purification of the enzymes, their enzymatic parameters and catalytic mechanisms are presented.
Subject(s)
Prostaglandins/biosynthesis , Prostaglandins/metabolism , Prostaglandins/physiology , Humans , Prostaglandins I/biosynthesis , Prostaglandins I/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Thromboxanes/biosynthesis , Thromboxanes/metabolismABSTRACT
Imaging methods based on time-lapse microscopy are important tools for studying the dynamic events that shape thrombus formation upon vascular injury. However, there is a lack of methods to translate the vast amount of visual data generated in such experiments into quantitative variables describing platelet movements that can be subjected to systematic analysis. In this study, we developed experimental and computational protocols allowing for a detailed mathematical analysis of platelet movements within a developing thrombus. We used a flow chamber-based model of thrombosis wherein a collagen strip was used to initiate platelet adhesion and activation. Combining the use of a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways.
Subject(s)
Blood Platelets/physiology , Cell Movement/drug effects , Thrombosis/diagnostic imaging , Cells, Cultured , Computer Simulation , Diffusion Chambers, Culture , Humans , Microscopy, Fluorescence , Platelet Aggregation , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Receptors, Purinergic P2Y12/metabolism , Signal Transduction , Thrombosis/drug therapy , Thrombosis/pathology , Thromboxanes/biosynthesis , Time-Lapse ImagingABSTRACT
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 14 C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), α-granules (ß-thromboglobulin) and AH-containing vesicles (ß-glucuronidase, ß-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. ß-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14 C -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.
Subject(s)
Blood Platelet Disorders/genetics , Blood Platelet Disorders/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Haploinsufficiency , Hydrolases/metabolism , Adult , Arachidonic Acid/metabolism , Blood Platelet Disorders/blood , Blood Platelets/metabolism , Case-Control Studies , Child, Preschool , Female , Humans , Male , Mutation , Phospholipids/biosynthesis , Platelet Count , Secretory Vesicles/metabolism , Serotonin/metabolism , Thromboxanes/biosynthesisABSTRACT
RATIONALE: Autologous adipose-derived stromal cells (ASCs) offer great promise as angiogenic cell therapy for ischemic diseases. Because of their limited self-renewal capacity and pluripotentiality, the therapeutic efficacy of ASCs is still relatively low. Thromboxane has been shown to play an important role in the maintenance of vascular homeostasis. However, little is known about the effects of thromboxane on ASC-mediated angiogenesis. OBJECTIVE: To explore the role of the thromboxane-prostanoid receptor (TP) in mediating the angiogenic capacity of ASCs in vivo. METHODS AND RESULTS: ASCs were prepared from mouse epididymal fat pads and induced to differentiate into endothelial cells (ECs) by vascular endothelial growth factor. Cyclooxygenase-2 expression, thromboxane production, and TP expression were upregulated in ASCs on vascular endothelial growth factor treatment. Genetic deletion or pharmacological inhibition of TP in mouse or human ASCs accelerated EC differentiation and increased tube formation in vitro, enhanced angiogenesis in in vivo Matrigel plugs and ischemic mouse hindlimbs. TP deficiency resulted in a significant cellular accumulation of ß-catenin by suppression of calpain-mediated degradation in ASCs. Knockdown of ß-catenin completely abrogated the enhanced EC differentiation of TP-deficient ASCs, whereas inhibition of calpain reversed the suppressed angiogenic capacity of TP re-expressed ASCs. Moreover, TP was coupled with Gαq to induce calpain-mediated suppression of ß-catenin signaling through calcium influx in ASCs. CONCLUSION: Thromboxane restrained EC differentiation of ASCs through TP-mediated repression of the calpain-dependent ß-catenin signaling pathway. These results indicate that TP inhibition could be a promising strategy for therapy utilizing ASCs in the treatment of ischemic diseases.
Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Cell Differentiation/physiology , Endothelial Cells/metabolism , Receptors, Thromboxane A2, Prostaglandin H2/biosynthesis , Thromboxanes/biosynthesis , Adipocytes/drug effects , Adipose Tissue/cytology , Adipose Tissue/drug effects , Animals , Cell Differentiation/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Gene Knockdown Techniques , Humans , Mice , Stromal Cells/drug effects , Stromal Cells/metabolism , Vascular Endothelial Growth Factor A/pharmacology , beta Catenin/biosynthesisABSTRACT
PURPOSE: Obstructive sleep apnea increases the risk of cardiovascular diseases. Alternations in prostacyclin and thromboxane concentrations and balance could constitute one of mechanisms linking sleep apnea and cardiovascular events. Thus we aimed to assess the concentrations of 6-keto-prostaglandin F1α (6-keto-PGF1α) (metabolite of prostacyclin) and thromboxane B2 (TXB2) (metabolite of thromboxane A2) in urine and blood of obstructive sleep apnea patients and controls (snoring subjects with otherwise normal polysomnogram). MATERIAL AND METHODS: Overnight urine and morning blood samples were taken from subjects and controls at baseline and in sleep apnea group during continuous positive airway pressure (CPAP) treatment. Samples were analyzed using mass chromatography/gas spectrometry. RESULTS: We analyzed data from 26 obstructive sleep apnea subjects (mean apnea-hypopnea index 45.4±17.3) and 22 well-matched controls. At baseline sleep apnea patients, when compared to controls, have higher 6-keto-PGF1α in urine (0.89±0.15 vs 0.34±0.06, p=0.01) and blood (24.49±1.54 vs 19.70±1.77, p=0.04). TXB2 levels in urine and blood were not different across groups. CPAP treatment significantly decreased 6-keto-PGF1α in urine (0.92±0.17 vs 0.22±0.10, p=0.04), but not in blood. TXB2 levels during CPAP treatment did not change significantly. CONCLUSIONS: These results suggest augmented systemic prostacyclin production in obstructive sleep apnea patients, which potentially could constitute a protective mechanism against detrimental effects of sleep apnea.
Subject(s)
Epoprostenol/biosynthesis , Sleep Apnea, Obstructive/metabolism , Thromboxanes/biosynthesis , 6-Ketoprostaglandin F1 alpha/blood , 6-Ketoprostaglandin F1 alpha/urine , Adult , Aged , Case-Control Studies , Continuous Positive Airway Pressure , Female , Humans , Male , Middle Aged , Sleep Apnea, Obstructive/blood , Sleep Apnea, Obstructive/urine , Thromboxane B2/blood , Thromboxane B2/urineABSTRACT
Kisspeptin has been implicated in cardiovascular control. Eicosanoids play a crucial role in the activation of platelets and the regulation of vascular tone. In the present study, we investigated the effect of kisspeptins on eicosanoid synthesis in platelets and aorta in vitro. Platelets and aorta were isolated from Wistar-Kyoto rats. After preincubation with different doses of kisspeptin, samples were incubated with [1-(14)C]arachidonic acid (0.172 pmol/mL) in tissue culture Medium 199. The amount of labeled eicosanoids was measured with liquid scintillation, after separation with overpressure thin-layer chromatography. Kisspeptin-13 stimulated the thromboxane synthesis. The dose-response curve was bell-shaped and the most effective concentration was 2.5 × 10(-8) mol/L, inducing a 27% increase. Lipoxygenase products of platelets displayed a dose-dependent elevation up to the dose of 5 × 10(-8) mol/L. In the aorta, kisspeptin-13 induced a marked elevation in the production of 6-keto-prostaglandin F1α, the stable metabolite of prostacyclin, and lipoxygenase products. Different effects of kisspeptin on cyclooxygenase and lipoxygenase products indicate that beyond intracellular Ca(2+) mobilization, other signaling pathways might also contribute to its actions. Our data suggest that kisspeptin, through the alteration of eicosanoid synthesis in platelets and aorta, may play a physiologic and (or) pathologic role in the regulation of vascular tone.
Subject(s)
Eicosanoids/biosynthesis , Kisspeptins/physiology , Vasoconstriction/physiology , 6-Ketoprostaglandin F1 alpha/biosynthesis , Animals , Aorta/drug effects , Aorta/metabolism , Arachidonic Acid/metabolism , Blood Platelets/drug effects , Blood Platelets/metabolism , Dose-Response Relationship, Drug , Kisspeptins/pharmacology , Male , Rats , Signal Transduction/drug effects , Signal Transduction/physiology , Thromboxanes/biosynthesisABSTRACT
The present study describes the effects of atorvastatin on whole body synthesis of nitric oxide (NO), prostacyclin (PGI2), and thromboxane A2 (TxA2), on oxidative stress and nitrite/nitrate-related renal carbonic anhydrase (CA) activity in patients with type 2 diabetes mellitus (T2DM). A double-blind, randomized, placebo-controlled parallel-group trial (the DALI study group) on 217 patients with T2DM and dyslipidemia was performed. Urinary samples were collected before and after administration of a standard dose (10 mg/d, n=73), a maximal dose atorvastatin (80 mg/d, n=72) or placebo (n=72) for 30 weeks. Urinary nitrite and nitrate were measured to assess whole body NO synthesis. The urinary molar ratio of nitrate to nitrite (UNOxR) served as a measure of renal CA activity. Free radical- and cyclooxygenase (COX)-catalyzed lipid peroxidation was estimated by measuring urinary 8-iso-prostaglandin F2α (8-iso-PGF2α). In subgroups, systemic PGI2 and TxA2 synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-6-keto-prostaglandin F1α and 2,3-dinor-thromboxane B2, respectively. All biochemical parameters were measured by GC-MS and GC-MS/MS methods. T2DM patients had elevated levels of nitrate, nitrite, UNOxR, and 8-iso-PGF2α compared to healthy non-diabetic and normolipidemic subjects. Thirty-week treatment with atorvastatin (10 or 80 mg/d) did not significantly alter NO, PGI2, TxA2 and 8-iso-PGF2α synthesis and did not improve the renal reabsorption of nitrite which is considered an important reservoir of NO. Our study suggests that atorvastatin (10 or 80 mg/d) does not provide cardiovascular benefit beyond its cholesterol lowering effect in patients with T2DM.
Subject(s)
Atorvastatin/pharmacology , Diabetes Mellitus, Type 2/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Nitric Oxide/biosynthesis , Oxidative Stress/drug effects , Prostaglandins I/biosynthesis , Thromboxanes/biosynthesis , 6-Ketoprostaglandin F1 alpha/metabolism , Aged , Creatinine/metabolism , Dinoprost/analogs & derivatives , Dinoprost/urine , Dose-Response Relationship, Drug , Double-Blind Method , Female , Humans , Male , Middle AgedABSTRACT
Paracetamol (acetaminophen) is a widely used analgesic drug. It interacts with various enzyme families including cytochrome P450 (CYP), cyclooxygenase (COX), and nitric oxide synthase (NOS), and this interplay may produce reactive oxygen species (ROS). We investigated the effects of paracetamol on prostacyclin, thromboxane, nitric oxide (NO), and oxidative stress in four male subjects who received a single 3 g oral dose of paracetamol. Thromboxane and prostacyclin synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F(1α), respectively. Endothelial NO synthesis was assessed by measuring nitrite in plasma. Urinary 15(S)-8-iso-prostaglanding F(2α) was measured to assess oxidative stress. Plasma oleic acid oxide (cis-EpOA) was measured as a marker of cytochrome P450 activity. Upon paracetamol administration, prostacyclin synthesis was strongly inhibited, while NO synthesis increased and thromboxane synthesis remained almost unchanged. Paracetamol may shift the COX-dependent vasodilatation/vasoconstriction balance at the cost of vasodilatation. This effect may be antagonized by increasing endothelial NO synthesis. High-dosed paracetamol did not increase oxidative stress. At pharmacologically relevant concentrations, paracetamol did not affect NO synthesis/bioavailability by recombinant human endothelial NOS or inducible NOS in rat hepatocytes. We conclude that paracetamol does not increase oxidative stress in humans.
Subject(s)
Acetaminophen/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/drug effects , Nitric Oxide Synthase/metabolism , Oxidative Stress/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , Adult , Animals , Epoprostenol/biosynthesis , Hepatocytes/enzymology , Humans , Male , Middle Aged , Nitric Oxide/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/pharmacology , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type II/pharmacology , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type III/pharmacology , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Thromboxanes/biosynthesisABSTRACT
Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2ß1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response.
Subject(s)
Collagen/pharmacology , Enzyme Inhibitors/pharmacology , Isoleucine/analogs & derivatives , Lactones/pharmacology , Lipoprotein Lipase/antagonists & inhibitors , Platelet Activation/drug effects , Arachidonic Acid/metabolism , Aspirin/pharmacology , Blotting, Western , Humans , Isoleucine/pharmacology , Phosphorylation , Platelet Aggregation/drug effects , Thromboxanes/biosynthesis , p38 Mitogen-Activated Protein Kinases/metabolismSubject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Aspirin/therapeutic use , Coronary Artery Disease/drug therapy , Dipyrone/adverse effects , Platelet Aggregation Inhibitors/therapeutic use , Aged , Drug Interactions , Humans , Platelet Aggregation/drug effects , Thromboxanes/biosynthesisABSTRACT
Recent meta-analyses have indicated that patients with vascular disease demonstrated by laboratory tests to be aspirin or clopidogrel-resistant are at an increased risk of major vascular events. The suggested mechanisms of aspirin resistance include genetic polymorphism, alternative pathways of platelet activation, aspirin-insensitive thromboxane biosynthesis, drug interactions, or a low aspirin dose. Clopidogrel resistance is likely to develop as a result of a decreased bioavailability of the active metabolite, due to genetic variation or concomitant drug treatment. Additional work is required to improve and validate laboratory tests of platelet function, so that they may become useful tools for selection of the most appropriate antiplatelet therapy for an individual patient. Improvements in antiplatelet treatment strategies in the future should lead to a reduction in premature vascular events.
Subject(s)
Aspirin/pharmacology , Drug Resistance , Ticlopidine/analogs & derivatives , Age Factors , Aspirin/administration & dosage , Biological Availability , Clopidogrel , Drug Administration Schedule , Drug Interactions , Humans , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Function Tests , Polymorphism, Genetic , Sex Factors , Signal Transduction , Thromboxanes/biosynthesis , Ticlopidine/administration & dosage , Ticlopidine/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: Aspirin is the most commonly used antiplatelet treatment during the acute phase of cerebral ischemic events. It inhibits the production of thromboxane (TX) A(2), a powerful platelet activator. Despite this protection, early ischemic recurrences are frequent and considered clinical failures of this therapy. Only a few trials have focused on the use of antiplatelet therapy during this phase, and none has described the laboratory effect of the first dose of aspirin given after an ischemic cerebral event. However, this study may help clinicians to understand the mechanisms of early recurrences, and to design new therapeutic strategies, in particular for patients already treated with a daily dose of aspirin. METHOD: We studied laboratory parameters of the first 300-mg oral dose of aspirin given within 48 h after an ischemic cerebral event. Two blood samples were taken from all of the patients: the first during the third hour following aspirin intake (T1) and the second during the twenty-fourth hour (T2). For patients already treated with a daily dose of aspirin, a supplementary sample was taken before aspirin intake (T0). Platelet reactivity was studied on the basis of serum TXB(2) levels, a metabolite of TXA(2), and light transmission aggregometry after stimulation of platelet-rich plasma by arachidonic acid and by two concentrations of collagen, i.e. 2 µg/ml (Col2), dependent on the TXA(2) pathway, and 20 µg/ml (Col20), independent of the TXA(2) pathway. Results with Col2 were related to results with Col20 (Col2/20 ratio) to limit the impact of variations induced by the effects of preanalytical conditions. RESULTS: Fifty patients were included. TXB(2) values (p < 0.001) and relative values of the Col2/20 ratio (p = 0.037) were significantly higher at T2 compared to T1. For patients already treated with aspirin, TXB(2) levels (p < 0.001) were significantly lower at T1 compared to T0, and the Col2/20 ratio tended to decrease (p = 0.096). CONCLUSION: Platelet reactivity recovers within 24 h following the first 300-mg oral dose of aspirin during the acute phase of a cerebral ischemic event as demonstrated by an increase in TXB(2) levels, and the Col2/20 ratio, at T2 compared to T1. This would favor early ischemic recurrences. However, for patients already treated with aspirin, this dose is able to decrease TXB(2) levels and to complete the inhibition of the TXA(2) pathway, which shows the utility of this prescription in this case.
Subject(s)
Aspirin/therapeutic use , Brain Ischemia/drug therapy , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Acute Disease , Administration, Oral , Aged , Aspirin/administration & dosage , Blood Platelets/drug effects , Female , Humans , Male , Middle Aged , Platelet Aggregation Inhibitors/administration & dosage , Thromboxanes/biosynthesis , Time FactorsABSTRACT
Aspergillus fumigatus has been reported to produce prostaglandin (PG)-like substances. The molecular structure of these fungal eicosanoids is however still unknown. By using the gas chromatography-tandem mass spectrometry (GC-MS/MS) methodology we identified a number of eicosanoids that were formed upon incubation of the precursor arachidonic acid ethyl ester (AAE, 10 µM) with three strains of A. fumigatus. The eicosanoids identified include the prostaglandins (PG) PGE(2), 6-keto-PGF(1α) (the stable hydrolysis product of prostacyclin PGI(2)) and PGF(2α), the isoprostanes 15(S)-8-iso-PGF(2α) and 15(S)-8-iso-PGE(2), and thromboxane B(2) (TxB(2), the stable hydrolysis product of TxA(2)). These eicosanoids are identical with those produced by cyclooxygenases (COX) in humans. The biosynthesis of all of these eicosanoids could not be inhibited by the human COX inhibitors indomethacin (100 µM), acetylsalicylic acid (100 µM) or the non-selective human lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid (100 µM). By contrast, the selen-containing antioxidant ebselen (100 µM) was found to inhibit their synthesis. Our results suggest that mammals and fungi employ different eicosanoid biosynthesis pathways. As some of the detected eicosanoids are potent immunomodulators and bronchoconstrictors, they could play a possible role in pulmonary diseases associated with A. fumigatus infection.
Subject(s)
Aspergillus fumigatus/metabolism , Isoprostanes/biosynthesis , Prostaglandins/biosynthesis , Thromboxanes/biosynthesis , Arachidonic Acid/metabolism , Aspergillosis/metabolism , Aspergillus fumigatus/chemistry , Azoles/pharmacology , Eicosanoids/analysis , Eicosanoids/biosynthesis , Ether/metabolism , Gas Chromatography-Mass Spectrometry/methods , Immunoenzyme Techniques/methods , Isoindoles , Isoprostanes/analysis , Organoselenium Compounds/pharmacology , Prostaglandins/analysis , Tandem Mass Spectrometry/methods , Thromboxanes/analysisABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Leonotis leonurus L. (Lamiaceae) is used as a traditional medicine for a variety of ailments in South Africa. The diterpene marrubiin is the major product constituent in specimens of this plant occurring in South Africa. MATERIALS AND METHODS: Marrubiin isolated from South African specimens of L. leonurus in addition to an organic extract of L. leonurus were tested in vivo, ex vivo and in vitro for their anticoagulant, antiplatelet and anti-inflammatory activities. RESULTS: Marrubiin and the organic extract suppressed coagulation, platelet aggregation and inflammatory markers. For the coagulation markers it was found that the organic extract and marrubiin significantly prolonged activated partial thromboplastin time (APTT). Fibrin and D-dimer formation were drastically decreased. These findings were observed in an ex vivo model and an obese rat model. Chemokines enhance leukocyte recruitment to inflammatory sites. TNF-α and RANTES secretion were significantly reduced by the extract and marrubiin when determined in the obese rat model relative to the controls. Calcium mobilization and TXB(2) synthesis were suppressed by the extract and marrubiin. An in vitro model was used to elucidate the antiplatelet mechanism and it was found that the extract and marrubiin inhibited platelet aggregation by inhibiting the binding of fibrinogen to glycoprotein (GP) IIb/IIIa receptor in a concentration dependent manner. CONCLUSION: The findings reflect that marrubiin largely contributes to the extract's anticoagulant, antiplatelet and anti-inflammatory effects observed.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Anticoagulants/pharmacology , Diterpenes/pharmacology , Lamiaceae/chemistry , Plant Extracts/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Animals , Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/therapeutic use , Anticoagulants/isolation & purification , Anticoagulants/therapeutic use , Blood Coagulation/drug effects , Calcium/metabolism , Chemokine CCL5/metabolism , Diterpenes/isolation & purification , Diterpenes/therapeutic use , Dose-Response Relationship, Drug , Fibrin/biosynthesis , Fibrin Fibrinogen Degradation Products/biosynthesis , Fibrinogen/metabolism , Fibrinolytic Agents/isolation & purification , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/therapeutic use , Glycoproteins/metabolism , Inflammation/drug therapy , Inflammation/etiology , Inflammation/metabolism , Male , Obesity/complications , Obesity/drug therapy , Obesity/metabolism , Partial Thromboplastin Time , Phytotherapy , Plant Extracts/chemistry , Plant Extracts/therapeutic use , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/isolation & purification , Platelet Aggregation Inhibitors/therapeutic use , Rats , Rats, Wistar , Thromboxanes/biosynthesis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The evidence of an incomplete inhibition of platelet function by aspirin, despite therapeutic doses of the drug proved to be clinically effective are employed, was first reported in the '80s, in the frame of studies devoted to platelet turnover. Because inhibition of platelet aggregation by aspirin is irreversible, the return after an interval of time of the ability to form thromboxane by platelets in circulating blood should reflect the entry into the circulation of platelets whose cyclooxygenase activity has not been affected by aspirin. Based on this concept, the possibility of monitoring the entry of newly formed platelets into the circulation after aspirin ingestion was documented by measuring the return of thromboxane biosynthesis by platelets challenged in vitro by pairs of aggregating agents. The data obtained showed that platelets with intact cyclooxygenase activity could be detected into the circulation of control individuals as early as 4-6 h after aspirin ingestion, and at shorter time intervals in diabetic angiopathy. In the latter setting,the data allowed to conclude that "schedules of aspirin which may suffice in normals are not effective in patients with diabetic angiopathy, presumably because these patients have a high rate of entry of new platelets into the circulation".
Subject(s)
Aspirin/pharmacology , Blood Platelets/drug effects , Diabetic Angiopathies/drug therapy , Drug Resistance , Animals , Disease Models, Animal , Humans , Platelet Aggregation/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , Thromboxanes/biosynthesisABSTRACT
BACKGROUND: Strong P2Y(12) blockade, as can be achieved with novel anti-platelet agents such as prasugrel, has been shown in vitro to inhibit both ADP and thromboxane A(2) -mediated pathways of platelet aggregation, calling into question the need for the concomitant use of aspirin. OBJECTIVE: The present study investigated the hypothesis that aspirin provides little additional anti-aggregatory effect in a group of healthy volunteers taking prasugrel. STUDY PARTICIPANTS/METHODS: In all, 9 males, aged 18 to 40 years, enrolled into the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg until day 21. At day 8, aspirin 75 mg was introduced and the dose increased to 300 mg on day 15. On days 0, 7, 14 and 21, platelet function was assessed by aggregometry, response to treatments was determined by VerifyNow and urine samples were collected for quantification of prostanoid metabolites. RESULTS: At day 7, aggregation responses to a range of platelet agonists were reduced and there was only a small further inhibition of aggregation to TRAP-6, collagen and epinephrine at days 14 and 21, when aspirin was included with prasugrel. Urinary prostanoid metabolites were unaffected by prasugrel, and were reduced by the addition of aspirin, independent of dose. CONCLUSIONS: In healthy volunteers, prasugrel produces a strong anti-aggregatory effect, which is little enhanced by the addition of aspirin. The addition of aspirin as a dual-therapy with potent P2Y(12) receptor inhibitors warrants further investigation.
Subject(s)
Aspirin/pharmacology , Piperazines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Purinergic P2Y Receptor Antagonists/pharmacology , Thiophenes/pharmacology , Adolescent , Adult , Humans , Male , Prasugrel Hydrochloride , Thromboxanes/biosynthesisABSTRACT
BACKGROUND AND PURPOSE: Most patients at elevated cardiovascular risk receive long-term aspirin (ASA) anti-platelet treatment. The present study specifically addresses the pharmacological interactions between selective COX-2 inhibitors and ASA and the possible consequences for the thrombotic risk during long-term treatment. EXPERIMENTAL APPROACH: New Zealand white rabbits were fed a standard laboratory diet supplemented with 1% cholesterol (CON) for 12 weeks. Age-matched control rabbits were fed the same standard diet without addition of cholesterol (SD). Rabbits were randomly assigned to one of the following groups: rofecoxib (ROFE, 25 mg·kg⻹, bid), acetylsalicylic acid (ASA, 5 mg·kg⻹, bid) or a combination of both (ASA + ROFE). At the end of the feeding period, the severity of atherosclerotic plaque formation was assessed in the aorta. Thrombus formation was assessed in the left carotid artery using a modified Folts procedure. KEY RESULTS: Treatment of cholesterol-fed rabbits with ASA significantly reduced plaque formation. This reduction in lesion size was not observed in animals treated with the combination of rofecoxib and ASA. In the modified Folts model, treatment with either rofecoxib or ASA increased the total blood flow above that of untreated animals. This increase was statistically significant in the case of ASA, while cotreatment with rofecoxib abolished this ASA effect completely and reduced the total flow rate to the levels seen in untreated hypercholesterolaemic controls. CONCLUSIONS: COX-2 inhibition by rofecoxib attenuates the antithrombotic and anti-atherosclerotic effects of ASA during long-term treatment in cholesterol-fed rabbits.
Subject(s)
Aspirin/antagonists & inhibitors , Aspirin/pharmacology , Atherosclerosis/drug therapy , Cyclooxygenase 2 Inhibitors/pharmacology , Fibrinolytic Agents/pharmacology , Lactones/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Sulfones/pharmacology , Animal Feed , Animals , Aorta/drug effects , Aorta/pathology , Atherosclerosis/chemically induced , Atherosclerosis/genetics , Atherosclerosis/pathology , Cholesterol/administration & dosage , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Dinoprostone/biosynthesis , Drug Antagonism , Epoprostenol/biosynthesis , Female , Immunohistochemistry , Male , Rabbits , Thrombosis/chemically induced , Thrombosis/drug therapy , Thrombosis/genetics , Thrombosis/pathology , Thromboxanes/biosynthesisABSTRACT
BACKGROUND: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. OBJECTIVES: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. METHODS: S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. RESULTS: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 µM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. CONCLUSIONS: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.
Subject(s)
Blood Platelets/metabolism , Lysophospholipids/blood , Sphingosine/analogs & derivatives , Thromboxanes/biosynthesis , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Aspirin/pharmacology , Blood Platelets/drug effects , Cells, Cultured , Chromatography, Thin Layer , Humans , Receptors, Thromboxane/agonists , Sphingosine/blood , Thrombin/pharmacologyABSTRACT
An increase of adhesion and aggregation functions of platelets in vivo and in vitro was detected in 5-6-year-old children with scoliosis. These disorders were caused by hyperproduction of von Willebrand's factor in the vascular wall and intensification of thromboxane production in blood platelets. Activation of thromboxane formation is the main cause of platelet hyperactivity in children with scoliosis. Correction of platelet hemostasis may include pathogenetically substantiated complex of therapeutic exercises, swimming, and massage.