Presence Of Acetylsalicylic Acid Research Articles

Canine platelets express functional Toll-like receptor-4: lipopolysaccharide-triggered platelet activation is dependent on adenosine diphosphate and thromboxane A2 in dogs

BackgroundFunctional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A2 (TxA2). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS).ResultsWe discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B2 secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA2 pathway.ConclusionExpression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA2 pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis.

Combination of pharmacological and non-pharmacological approaches in patients with hypertensive encephalopathy and hemorheological disorders

objective. To study the impact of complex treatment (acetylsalicylic acid, hirudotherapy, phytotherapy) on the functional activity of thrombocytes in patients with hypertensive encephalopathy. design and methods. An open prospective study included 25 patients with hypertensive encephalopathy and dysfunction of thrombocytes activity. All patients took acetylsalicylic acid. A simultaneous course of hirudotherapy (10 sessions, 2,5 months; own patent of the Russian Federation № 2327494) and a course of phytotherapy (polycomponent infusion, for 2,5 months; own patent of the Russian Federation № 2542423) were conducted. Functional activity of thrombocytes was studied by the phase contrast microscope at baseline and after the treatment was completed. Results. Initially, 25 patients with hypertensive encephalopathy showed abnormal platelet functional activity in the presence of acetylsalicylic acid: a decrease in the number of intact platelets — discocytes in 9 patients; increased number of activated cells with discoquinocyte sprouts on the surface — in 18 people; active forms in 17 patients; platelet aggregates — in 15 patients; increased number of small aggregates per 100 free thrombocytes — 13 patients; large aggregates — in 2 patients. After hirudotherapy and herbal medicine were applied in addition to acetylsalicylic acid, complete normalization of platelet functional activity was achieved in 11 of 25 patients, and partial normalization — in 12 subjects. conclusions. The complex approach, including both drug treatment (acetylsalicylic acid) and the course of hirudotherapy and phytotherapy showed a positive impact on the intravascular platelet activity in patients with hypertensive encephalopathy and initial disturbances of the platelet functional activity. As a result, the number of intact forms of platelets increased, the number of early and late active forms (discoquinocytes, spheroechinocytes and bipolar cells) decreased. Also the number of platelet aggregates and small aggregates reduced. Such a multimodal approach can be implemented in aspirin resistance, a high risk of primary and recurrent vascular events. The method is safe and well tolerated by patients.

Calcium-Dependent Src Phosphorylation and Reactive Oxygen Species Generation Are Implicated in the Activation of Human Platelet Induced by Thromboxane A2 Analogs.

The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho–Rho kinase pathway independently of integrin αIIbβ3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50–1000 nM U46619 and/or 10 μM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbβ3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.

Flow‐injection chemiluminescence determination of acetylsalicylic acid based on its enhancing effect on the lucigenin–hydrogen peroxide system

A sensitive flow-injection chemiluminescence method for the determination of acetylsalicylic acid is described. It is based on the enhanced chemiluminescent emission of the alkaline lucigenin–H2O2 system by acetylsalicylic acid. The difference in chemiluminescent intensity of alkaline lucigenin–H2O2 in the presence of acetylsalicylic acid from that in the absence of acetylsalicylic acid was linear at acetylsalicylic acid concentrations in the range of 0.0029–47.37 μg/mL, with detection and quantification limits of 0.0011 and 0.0029 μg/mL, respectively. The correlation coefficient of the working curve was 0.9983. The relative standard deviation (n = 10) for 25 μg/mL acetylsalicylic acid is 1.95%. All experimental parameters were optimized. The method was successfully applied to the determination of acetylsalicylic acid in pharmaceutical preparations. The recovery results obtained by the method were satisfactory.

Quality assessment of platelet rich plasma during anti-platelet therapy

Platelet rich plasma (PRP) is being used with increased frequency in many surgical procedures for its known benefits of accelerated surgical wound site healing. Speculations in its efficacy in the presence of anti-platelet therapy have been proposed. To aid in defining a quality platelet rich plasma product in the presence of acetylsalicylic acid (ASA) and Plavix (clopidogrel bisulfate), we investigated three (3) groups (n = 18) of cardiac surgical patients receiving PRP. Platelet function test, platelet concentration, and quantification of growth factors (PDGF-bb and TGF-b1) were evaluated. Results showed no statistical evidence of decreased growth factors delivered to the surgical wound site in the presence of acetylsalicylic acid (ASA) and/or Plavix (clopidogrel bisulfate). Evidence in this pilot study supports the use of PRP for patients receiving Plavix and aspirin therapy without compromising the quantity of specific growth factors delivered to a wound site.

Mitochondrial Associated Yeast Flocculation -The Effect of Acetylsalicylic Acid

Mitochondrial function is generally accepted as important for expression of yeast flocculation. In this study, a correlation between mitochondrial activity and flocculation is demonstrated using the XTT reduction assay. The mitochondrial activity of strongly flocculent cells was higher than those of weakly flocculent cells and cells cultivated in the presence of acetylsalicylic acid. Furthermore, we show the first oxylipin-containing flocculation binding sites on yeast cell surfaces using scanning electron microscopy. We propose that in addition to zymolectin-mediated flocculation, oxylipin interactions may also play a role in yeast flocculation.

A study of the interaction between ropranolol and NSAIDs in protein binding by gel filtration method.

Drug protein binding phenomena can lead to some interesting drug-drug interactions when one drug displaces another in the binding site. Studies of protein binding are conducted by several methods including equilibrium dialysis, ultra-filtration and chromatographic methods. Gel filtration is a simple chromatographic method in protein binding studies. Propranolol binds to plasma proteins by 90%-95% in circulation system and other drugs with high protein binding may displace it. In this study protein binding of propranolol has been studied using gel filtration to Bovine Serum Albumin (BSA) alone and in the presence of Acetyl salicylic acid (ASA), Indomethacin and mefenamic acid has been studied using gel filtration method. The results indicated that ASA decreased protein binding of propranolol by 20% to BSA and other drugs did not displace propranolol from the binding site. Therefore, ASA may alter pharmacological effects of propranolol.

Acetylsalicylic acid decreases tau phosphorylation at serine 422

Tau protein pathology in Alzheimer's disease is characterized by the hyperphosphorylation of tau at some specific sites. One of these sites is serine 422 which modification has been correlated with a possible toxic effect of phosphotau in neural cells. In this work, we have found that in the presence of acetylsalicylic acid, at a concentration like that used for anti-inflammatory treatments, tau phosphorylation at serine 422 decreases.

Modified clotting properties of fibrinogen in the presence of acetylsalicylic acid in a purified system.

To assess how treatment with acetylsalicylic acid (ASA) alters the fibrin network structure, clotting was initiated in purified fibrinogen incubated with ASA by adding thrombin. Clotting time and maximum absorbance of the fibrin aggregation curve were used to demonstrate the potential of fibrin generation. The results showed that the clotting properties of fibrinogen decreased and that the affinity of plasminogen to fibrin or thrombin inhibition by antithrombin increased if plasminogen or antithrombin, respectively, were present in the reaction system. The effect of ASA varied in a dose dependent manner. It was concluded that ASA may directly or indirectly confer positive or negative effects on the stability of the fibrin clot and that the balance between these effects may be regulated by the ASA dose.

Low-molecular weight heparin restores in-vitro trophoblast invasiveness and differentiation in presence of immunoglobulin G fractions obtained from patients with antiphospholipid syndrome.

The present study was designed to investigate the effects of immunoglobulin G obtained from patients with antiphospholipid syndrome (APS) on in-vitro models of trophoblast invasiveness and differentiation. We tested the binding of affinity-purified immunoglobulin G to human primary trophoblast cells. These antibodies affected the invasiveness and differentiation of cytotrophoblast cells after binding to the cell surface. In addition, we determined whether the drugs used to treat APS might be able to restore the trophoblast functions. Low-molecular weight heparin, in a dose-dependent manner, significantly reduced the immunoglobulin G binding to trophoblast cells and restored in-vitro placental invasiveness and differentiation. No effect was observed in the presence of acetylsalicylic acid. These observations may help in understanding the role of these treatments in women with APS.

Storage of potato microplants in vitro in the presence of acetylsalicylic acid

Acetylsalicylic acid was investigated as an alternative medium supplement to mannitol for slow-growth in vitro storage of potato microplants. At 8°C, culture in the presence of either 100 μM acetylsalicylic acid or 4% mannitolretarded microplant stem growth, and required intervals between subcultures ranged from 8 to over 12 months, depending on the genotype. Several media were tested for recovery of clones from slow-growth storage, with similar efficiences of recovery achieved for microplants from either mannitol or acetylsalicylic acid media. Prolonged culture on acetylsalicylic acid had no adverse effects on the yield of minitubers on plantlets transferred to glasshouse cultivation. At 18°C also, intervals between subcultures could be prolonged to 5.5–6 months by culture on either mannitol or acetylsalicylic acid media. Microplants cultured on acetylsalicylic acid did not exhibit the frequent phenotypic abnormalities found in microplants on mannitol medium.

Does ethylene play a role in thidiazuron-regulated somatic embryogenesis of geranium (Pelargonium × hortorum bailey) hypocotyl cultures?

The accumulation of ethylene in headspace of hypocotyl cultures of geranium (Pelargonium × hortorum Bailey) and its possible role in thidiazuron-mediated somatic embryogenesis was investigated. The action of ethylene as determined by various ethylene synthesis and action inhibitors was varied. Silver nitrate (AgNo3), aminoethoxyvinylglycine (AVG), and silver thiosulphate (STS) had no significant influence on the embryogenic response, while 1-methylcyclopropene (1-MCP) applied during the initial 3 d of induction or the expression phase, significantly increased the number of somatic embryos formed. Thidiazuron-treated tissues accumulated large quantities of ethylene within 6 h of culture, but the levels decreased after 12 h and reached very low levels after 3 d in culture. In the presence of acetylsalicylic acid (ASA), the levels of ethylene decreased by 20 to 50% during the first 48 h of culture. Analysis of endogenous auxin, cytokinins, and abscisic acid (ABA) indicated possible interactions of ethylene with other phytohormones during the induction of somatic embryos on geranium hypocotyl explants. Thidiazuron (10 µM) increased, while ASA decreased the levels of endogenous auxin, cytokinins, and abscisic acid during this period of induction.

Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) tissue cultures

Thidiazuron (TDZ) effectively induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey) during only a 3-day period of induction. The presence of acetylsalicylic acid (ASA) during this period caused a two-fold increase in the number of somatic embryos and enhanced synchronization of embryo development compared to the TDZ treatment alone. Salicylic acid was ineffective in modulating similar embryogenic responses as ASA. The ASA-induced enhancement and synchronization of somatic embryogenesis could possibly be used as an experimental system to study the interplay of growth regulators in somatic embryogenesis.

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase‐dependent proliferation of human skin fibroblasts

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10−9 M) with baFGF (40 μg/ml) and EGF(0.01 μg/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E2 release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblast proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network. © 1996 Wiley-Liss, Inc.

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase-dependent proliferation of human skin fibroblasts.

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10(-9)M) with baFGF(40 micrograms/ml) and EGF(0.01 microgram/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E2 release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblasts proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network.

The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

Resin bead detection and spectrophotometric determination of oxyphenbutazone with p-dimethylaminocinnamaldehyde: Application to bulk drug and dosage forms

An analytical method for resin bead detection and spectrophotometric determination of oxyphenbutazone with p-dimethylaminocinnamaldehyde as a reagent is described. Acid hydrolysed product of oxyphenbutazone produced a red color product with p-dimethylaminocinnamaldehyde that absorbs maximally at 520 nm. The detection limit is 0.8 μg in resin phase and 4.0 μg in aqueous phase. Beer's law is obeyed in the concentration range of 0.4–4.0 μg/ml of oxyphenbutazone. The method is unaffected by the presence of acetylsalicylic acid, salicylamide, phenylbutazone and number of other excipients but paracetamol gives a positive reaction and novalgin being reductone interferes with the test, both should be removed by extraction with dilute sulfuric acid.

Platelet Activating Properties of Murine Monoclonal Antibodies to β2-Glycoprotein I

Previously developed murine monoclonal antibodies (MAbs) to human beta 2-glycoprotein I (beta 2 GPI), a plasma protein required for the binding of anti-phospholipid antibodies, were studied for anti-platelet reactivity and influence on platelet function. The six MAbs (IgG1 isotype) tested interacted with both intact and fixed platelets in a beta 2 GPI-dependent manner. Carbamylated beta 2 GPI was still recognized by MAbs but was unable to mediate platelet-antibody binding. MAbs induced aggregation and secretion responses of platelets in platelet-rich plasma (PRP) and whole blood, provided subthreshold concentrations of weak agonists (i.e. ADP or adrenaline) were added. When aggregation in PRP was evaluated by a counting technique instead of turbidometrically, the sole addition of MAbs led to a rapid fall in single platelets. Triggering gel-filtered platelets with MAbs together with beta 2 GPI, but not its carbamylated form, led to platelet activation after a lag time, as monitored by aggregometry, measurements of ATP and beta-thromboglobulin secretion and calcium mobilization. F(ab')2 fragments of one of the MAbs failed to activate platelets but inhibited the responses to the whole antibody. This process thus depends on MAbs binding to platelets through both Fab and Fc domains, as confirmed by the suppression of platelet responses upon pretreatment with the anti-Fc gamma RII MAb IV.3. Aggregation and secretion induced by MAbs plus beta 2 GPI did not require exogenous fibrinogen and were variably inhibited in the presence of acetyl salicylic acid, apyrase or Ca2+, depending on the concentrations used for the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro platelets/endothelial cells interactions in presence of acetylsalicylic acid at various dosages

Venous endothelium is able to release in vitro substances which modifies platelet aggregation. A vascular fragment incubated in Michaelis buffer (pH 7.30), aliquoted and tested on platelet-rich-plasma partially inhibits the aggregometry parameters. Addition of acetylsalicylic acid (ASA) at ultra low dose (0.1 nM final solution in the incubation tube) presents a reversed effect on this inhibition. To explain this phenomenon, 6- keto-PGF 1α and von Willebrand factor were dosed in the incubation media. After determination of an active level of 6-keto-PGF 1α (200 pg/100μl), 2 series were made: series 1 included the values below 200 pg/100μl incubation media, series 2 , the values above 200 pg/100μl incubation media. When the vascular fragment was incubated as described above, the results of aggregometry ratio for series 1 were: test A (without ASA): 0.84±0.18, test B 1 (with 0.1 nM of ASA):0.87±0.13. For series 2 , they became: test A: 0.75±0.27, test B 1: 0.93±0.16. Control was always: 1.00± 0.00. For the same groups, 6-keto-PGF 1α values were: for series 1 , test A:81±57, test B 1:81±60 pg/100μl incubation medium, for series 2 , test A: 596±495, test B 1: 383±263 pg/100 μl incubation medium. Analyses were also performed with 2 high doses of ASA (B 2: 10 5 nM and B 3: 10 6 nM final solution) in the same experimental conditions. In these groups, aggregation parameters were decreased (0.86±0.14 for 10 5 nM, 0.84±0.15 for 10 6 nM) as well as 6-keto-PGF 1α production (189±199 for 10 5 nM, 152±182 for 10 6 nM). For these two last ASA treatments, comparison of the results in groups set up according to the sensitive 6-keto-PGF 1α value (200 pg/100μl solution) showed no modification. So it seems that a certain reactive state, specific of ultra low dose treatment is necessary for the vascular endothelium to be sensitive at such treatment.

Plasmapheresis in a patient with severe asthma associated with auto‐antibodies to platelets

Although the pathogenesis of aspirin-sensitive asthma remains to be specified, it is known that in the presence of acetylsalicylic acid or non-steroidal anti-inflammatory drugs, platelets from aspirin-sensitive asthmatics have been described as generating cytocidal mediators that killed parasite targets such as Schistosoma mansoni larvae. Here we report, in a patient with corticosteroid-dependent asthma associated with aspirin sensitivity, the presence of circulating IgE antibodies against 55 kD and 68 kD platelet antigens. In addition, the serum from this patient was shown to contain a factor able to trigger the release of cytocidal mediators from his platelets as well as from normal individual platelets. This platelet stimulatory activity was presumably supported by IgE antibodies or immune complexes. After informed consent the patient was submitted to plasma exchanges. Plasma removal induced clinical improvement, anti-platelet antibody decrease, and the reduction of the platelet stimulatory activity. All clinical symptoms disappeared within 2 weeks. The disease remained quiescent for 2 months, and daily requirements for prednisone (20-5 mg), and beta-agonist (10-16 to 0-1 inhalations) could be kept at a low level follow-up. The plasma exchanges were delayed by 3 mg kg-1 azathioprine with the maintenance of clinical improvement. A relapse occurred after the arrest of immunosuppressive therapy with the reappearance of both asthma attacks and anti-platelet antibodies, as well as the increase of the platelet stimulatory activity.(ABSTRACT TRUNCATED AT 250 WORDS)