Inhibition Of Arachidonic Acid Research Articles

Anti-inflammatory medicinal plants and the molecular mechanisms underlying their activities

Background: Medicinal plant and plant products have shown tremendous potentials and are used beneficially in the treatment of inflammation and in the management of diseases with significant inflammatory components. Many medicinal plants employed as anti-inflammatory and antiphlogistic remedies lack the gastro-erosive side effects of non-steroidal anti-inflammatory drugs (NSAID) or the plethora of unwanted side effects associated with steroidal anti-inflammatory drugs. In order to harness and optimise the applications of these herbs in inflammatory diseases, there is a need to understand how these herbs produce their anti-inflammatory actions. Materials and Methods : This paper is a review of some anti-inflammatory herbs and their molecular mechanisms of action. A literature search and analysis of published manuscript was employed to x-ray research findings that show how medicinal plants produce anti-inflammatory activities. Results : Many studies have shown that anti-inflammatory activities of herbal extracts and herb-derived compounds are mainly due to their inhibition of arachidonic acid (AA) metabolism, cyclo-oxygenase (COX), lipo-oxygenase (LOX), pro-inflammatory cytokines, inducible nitric oxide, and transcription activation factor (NF-eB). Some anti-inflammatory medicinal herbs are reported to stabilize lysosomal membrane and some cause the uncoupling of oxidative phosphorylation of intracellular signalling molecules. Many have also been shown to possess strong oxygen radical scavenging activities. Conclusion: Most of the mechanisms by which anti-inflammatory medicinal plants act are related and many herbal products have been shown to act through a combination of these molecular pathways. Key words : Medicinal plants, antiinflammatory, mechanism of action, molecular pathways.

Effects of Angiotensin II Receptor Blockers on Metabolism of Arachidonic Acid <i>via</i> CYP2C8

Arachidonic acid (AA) is metabolized to epoxyeicosatrienoic acids (EETs) via cytochrome enzymes such as CYP 2C9, 2C8 and 2J2. EETs play a role in cardioprotection and regulation of blood pressure. Recently, adverse reactions such as sudden heart attack and fatal myocardial infarction were reported among patients taking angiotensin II receptor blockers (ARBs). As some ARBs have affinity for these CYP enzymes, metabolic inhibition of AA by ARBs is a possible cause for the increase in cardiovascular events. In this study, we quantitatively investigated the inhibitory effects of ARBs on the formation of EETs and further metabolites, dihydroxyeicosatrienoic acids (DHETs), from AA via CYP2C8. In incubations with recombinant CYP2C8 in vitro, the inhibitory effects were compared by measuring EETs and DHETs by HPLC-MS/MS. Inhibition of AA metabolism by ARBs was detected in a concentration-dependent manner with IC50 values of losartan (42.7 µM), telmisartan (49.5 µM), irbesartan (55.6 µM), olmesartan (66.2 µM), candesartan (108 µM), and valsartan (279 µM). Losartan, telmisartan and irbesartan, which reportedly accumulate in the liver and kidneys, have stronger inhibitory effects than other ARBs. The lower concentration of EETs leads to less protective action on the cardiovascular system and a higher incidence of adverse effects such as sudden heart attack and myocardial infarction in patients taking ARBs.

Effects of danshensu on platelet aggregation and thrombosis: in vivo arteriovenous shunt and venous thrombosis models in rats.

Danshensu, a type of dihydroxyphenyl lactic acid, is one of the most abundant active phenolic acids in the dried root of Salvia miltiorrhizae (Lamiaceae)—widely used traditional Chinese medicine. The effects of danshensu on platelet aggregation and thrombus formation in rats were examined using various methods. It was found that danshensu significantly reduced thrombus weight in 2 experimental thrombosis models; dose-dependent inhibition of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced platelet aggregation occurred in normal and blood stasis-induced rats; Danshensu also significantly mitigated blood viscosity, plasma viscosity and hematocrit levels. Moreover, danshensu significantly inhibited venous thrombosis-induced expression of cyclooxygenases-2 (COX-2) rather than cyclooxygenases-1(COX-1) in the venous walls, down regulated thromboxane B2 (TXB2) and up regulated 6-keto prostaglandin F1α (6-keto-PGF1α), normalizing the TXB2/6-keto-PGF1α ratio. In addition, danshensu did not induce gastric lesions and even had protective effects on aspirin-induced ulcer formation at doses as high as 60 mg/kg. These findings suggest that the antithrombotic and antiplatelet aggregation effects of danshensu are attributed to its highly selective inhibition of COX-2 and ability to normalize the thromboxane A2(TXA2)/prostacyclin(PGI2) balance. These findings suggest that danshensu have great prospects in antithrombotic and antiplatelet therapy.

Abstract 571: A Novel High-Throughput Whole Blood Immuno-Counting Assay is Useful in the Assessment of Platelet Responses to Antiplatelet Therapy

Background: The current gold standard in platelet function testing, light transmission aggregometry, is time- and labor-intensive, and uses platelet-rich plasma which makes it sub-optimal for high throughput testing. In order to reduce blood manipulation prior to platelet function testing and to study multiple platelet activation pathways simultaneously, we have developed a 96-well plate-based assay carried out in whole blood, where aggregation is measured as a decrease in the number of fluorescently-labeled single platelets by flow cytometry. Aim: To investigate whether a 96-well plate-based whole blood assay can be used to assess platelet function. Methods: Platelet function in response to 5 concentrations of lyophilized arachidonic acid (AA), ADP, collagen, epinephrine, TRAP, U46619, and ristocetin, was assessed in healthy volunteers (n=20) to establish normal ranges. The effect of antiplatelet drugs was assessed in vitro by incubation with aspirin (100 μM), cangrelor (1 μM) or both (n=20), and in patients on dual antiplatelet therapy (n=20). After addition of 40 μl of whole blood per well, the plate was shaken for 5 min at 1000 rpm at 37°C; a fixative solution (Platelet Solutions, Nottingham) was applied to stop platelet aggregation and allow analysis in a central laboratory. Fixed whole blood samples (stable for up to 9 days) were labeled with FITC-conjugated CD42a and assessed by flow cytometry. Aggregation was calculated as (Platelet count in vehicle-treated sample - Platelet count in agonist-stimulated sample) / Platelet count in vehicle-treated sample x 100. Results: Dose-response curves were readily assessable for all agonists and intra-individual variability was minimal in healthy volunteers (CV<10%). In vitro addition of aspirin alone resulted in inhibition of AA- and collagen-induced aggregation, whereas cangrelor induced a shift in dose-response to most agonists in addition to profound inhibition of ADP responses. In patients on dual antiplatelet therapy, the pattern of response was consistent with the results obtained with in vitro agents. Conclusions: A 96-well plate-based whole blood assay with a minimal blood volume requirement (<2 ml) could be used to provide a global portrait of platelet responses to antiplatelet agents.

Nutritional factors and aging in demyelinating diseases

Demyelination is a pathological process characterized by the loss of myelin around axons. In the central nervous system, oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Remyelination is a regenerative process by which myelin sheaths are restored to demyelinated axons, resolving functional deficits. This process is often deficient in demyelinating diseases such as multiple sclerosis (MS), and the reasons for the failure of repair mechanisms remain unclear. The characterization of these mechanisms and the factors involved in the proliferation, recruitment, and differentiation of oligodendroglial progenitor cells is key in designing strategies to improve remyelination in demyelinating disorders. First, a very dynamic combination of different molecules such as growth factors, cytokines, chemokines, and different signaling pathways is tightly regulated during the remyelination process. Second, factors unrelated to this pathology, i.e., age and genetic background, may impact disease progression either positively or negatively, and in particular, age-related remyelination failure has been proven to involve oligodendroglial cells aging and their intrinsic capacities among other factors. Third, nutrients may either help or hinder disease progression. Experimental evidence supports the anti-inflammatory role of omega-6 and omega-3 polyunsaturated fatty acids through the competitive inhibition of arachidonic acid, whose metabolites participate in inflammation, and the reduction in T cell proliferation. In turn, vitamin D intake and synthesis have been associated with lower MS incidence levels, while vitamin D-gene interactions might be involved in the pathogenesis of MS. Finally, dietary polyphenols have been reported to mitigate demyelination by modulating the immune response.

Temporal and combinatorial control of pro‐resolution eicosanoid formation in TLR4 primed, purinergic receptor stimulated macrophages

Macrophages are critical immune cells for initiating the innate immune response, partly through generation of pro‐inflammatory lipid mediators including eicosanoids. Using LC‐MS lipidomics we have previously characterized the effects of fish oil omega‐3 fatty acid supplementation on TLR4 and purinergic eicosanoid signaling in RAW264.7 and resident macrophages. Supplementation of EPA or DHA leads to inhibition of COX‐1 and COX‐2 arachidonic acid derived pro‐inflammatory prostaglandins, but not inhibition of 5‐LOX in these cells. Consequently, fish oil supplementation can increase production of 5‐LOX AA, EPA and DHA‐derived mono‐hydroxylated metabolites after purinergic stimulation. More recently, we have observed endogenous formation of the pro‐resolution mediator, 15‐epi lipoxin A4, which was dependent on long‐term TLR4 priming followed by P2X7 stimulation with ATP. While mostly trans‐cellular mechanisms have been proposed to explain the switch to pro‐resolution eicosanoid signaling, our finding suggests macrophages can do this through a novel, independent route that requires temporal and combinatorial receptor‐mediated control. We are now characterizing the effects of fish oil omega‐3 supplementation on this mechanism.

Inhibition of platelet-mediated arterial thrombosis and platelet granule exocytosis by 3′,4′-dihydroxyflavonol and quercetin

Flavonols are polyphenolic compounds with broad-spectrum kinase inhibitory, as well as potent anti-oxidant and anti-inflammatory properties. Anti-platelet potential of quercetin (Que) and several related flavonoids have been reported; however, few studies have assessed the ability of flavonols to inhibit exocytosis of different platelet granules or to inhibit thrombus formation in vivo. 3′,4′-Dihydroxyflavonol (DiOHF) is a flavonol which is structurally related to Que and has been shown to have greater anti-oxidant capacity and to improve the endothelial function in the context of diabetes and ischaemia/reperfusion injury. While the structural similarity to Que suggests DiOHF may have a potential to inhibit platelet function, no studies have assessed the anti-platelet potential of DiOHF. We therefore investigated platelet granule inhibition and potential to delay arterial thrombosis by Que and DiOHF. Both Que and DiOHF showed inhibition of collagen, adenosine diphosphate and arachidonic acid stimulated platelet aggregation, agonist-induced GPIIb/IIIa activation as demonstrated by PAC-1 and fibrinogen binding. While both flavonols inhibited agonist-induced granule exocytosis, greater inhibition of dense granule exocytosis occurred with DiOHF as measured by both ATP release and flow cytometry. In contrast, while Que inhibited agonist-induced P-selectin expression, as measured by both platelet surface P-selectin expression and upregulation of surface GPIIIa expression, inhibition by DiOHF was not significant for either parameter. C57BL/6 mice treated with 6 mg kg−1 IV Que or DiOHF maintained greater blood flow following FeCl3-induced carotid artery injury when compared to the vehicle control. We provide evidence that Que and DiOHF improve blood flow following arterial injury in part by attenuating platelet granule exocytosis.

The in vitro effects of verbascoside on human platelet aggregation

Preliminary in vitro and animal studies have shown that verbascoside, a phenolic compound, may have several favourable biological activities, including an influence on endothelial function and on platelet aggregation. We sought to evaluate the effects of verbascoside, biotechnologically produced from plant cell cultures, on human platelet aggregation (PA). The blood from 40 aspirin-naïve volunteers with at least one cardiovascular risk factor was preincubated in vitro with verbascoside (1 and 2mg/dL) and aspirin (100μM). The blood from 20 patients with a prior diagnosis of coronary heart disease who were chronically assuming aspirin was preincubated in vitro with verbascoside (1 and 2mg/dL). PA is measured with a light transmission aggregometry and multiplate analyzer. As compared to reference, preincubation with verbascoside resulted in a significant inhibition of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced PA (p<0.01 for both). Verbascoside 2mg/dL did not show a stronger effect as compared to verbascoside 1mg/dL (p=0.4). As expected, the in vitro addition of aspirin reduced AA induced PA (p<0.01), but not that induced by ADP (p=0.5). The addition of verbascoside to the blood of aspirin-treated patients did not improve the values of PA after AA stimulus (p=0.8), whereas it ensured a stronger inhibition after ADP stimulus (p<0.01). Verbascoside in vitro affects PA by mildly inhibiting aggregation, triggered both by ADP and AA. These preliminary data, while intriguing, require confirmation in subjects receiving verbascoside orally in order to determine whether these findings are clinically relevant.

Stilbenoids from Gnetum macrostachyum Attenuate Human Platelet Aggregation and Adhesion

Platelets play a critical role in pathogenesis of cardiovascular disorders and strokes. The inhibition of platelet function is beneficial for the treatment and prevention of these diseases. The phytochemical investigation of stilbenoids from Gnetum macrostachyum Hook. f. led to the isolation of trans-resveratrol (1), isorhapotigenin (2), gnetol (3), bisisorhapontigenin B (4), gnetin C (5), parvifolol A (6), latifolol (7) and gnetuhainin C (8). The isolated stilbenoids were evaluated for in vitro antiplatelet activities via agonist-induced platelet aggregation and static platelet-collagen adhesion assays using washed human platelets. Compounds 1, 2 and 3 were active in the inhibition of arachidonic acid (AA)-induced platelet aggregation. Compound 2 and its dimer, compound 4, were the most active stilbenoids in thrombin-induced platelet aggregation. Moreover, compounds 4, 5 and 6, tended to be more potent than monomeric and trimeric stilbenoids in a human platelet-collagen adhesion assay under static conditions. This is the first report of the antiplatelet activity of stilbenoids isolated from G. macrostachyum.

Зміни метаболізму в міокарді при ішеміїреперфузії та активації аденозинтрифосфатчутливих калієвих каналів

In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) it was investigated the changes of biochemical processes in the different areas of heart (intact, risk and necrotic zone) during intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible cardioprotective mechanisms of flocalin action at ischemia-reperfusion conditions: the preservation of sufficient levels of de novo (by cNOS) NO synthesis, an inhibition of de novo (by iNOS) and salvage (by NADH-dependent nitratreductase) NO synthesis, an inhibition of L-arginine degradation by arginase, an inhibition of oxidizing metabolism due to limitation of ROS and RNS generation, inhibition of free arachidonic acid and eicosanoids synthesis, inhibition of ATP and GTP degradations and, possibly, stimulation of protective haem degradation. These changes may prevent formation of toxic peroxynitrite and suggest the possibility of participating in flocalin-mediated cardioprotective effects of warning a mitochondrial permeability transition pore (MPTP) opening and inhibition of apoptosis and/or necrosis of cardiomyocytes induced by it.

Abstract 3645: Omeprazole, A Potent Cyp 2c19 Inhibitor, Does Not Alter The Pharmacokinetics Or Platelet Aggregation Of Asa And Dipyridamole In Combination

Antiplatelet therapies such as aspirin (ASA), clopidogrel or a combination of ASA and extended-release dipyridamole (ASA/DP) are recommended therapeutic options for reducing the risk of stroke in patients with prior stroke or TIA. The clinical benefit of clopidogrel is highly dependent on the bioactivation of clopidogrel to its active metabolite by CYP 2C19. Omeprazole (OME), a frequently used gastric acid suppressant, is a potent inhibitor of CYP2C19, and has been identified as having a clinically significant interaction with clopidogrel. It was unclear whether ASA/DP would be subject to the same interaction risk when concomitantly administered with OME. This study formally tests whether multiple daily doses of OME 80 mg alters the pharmacokinetics of dipyridamole or the inhibition of platelet aggregation (IPA) of ASA/DP. This study was performed in 60 healthy volunteers as a multiple-dose, open-label, randomized, crossover trial with 4 treatments within 2 treatment sequences. The endpoints included the steady-state Cmax and AUC of DP as well as IPA at 4 and 12 hours postdose. The mean plasma DP concentration time profile for all treatments is displayed in Figure 1 . The 90% confidence interval for DP Cmax and AUC was fully contained within 80-125% bounds indicating no clinically significant interaction between ASA/DP and OME. Moreover, concomitant administration of ASA/DP with OME had no effect on the inhibition of arachidonic acid induced platelet aggregation over the normal dosing interval at steady-state ( Table 1 ). There was no difference detected regardless of whether subjects were pre-treated with OME alone or ASA/DP and OME. In conclusion, concurrent administration of OME with ASA/DP does not alter the steady-state pharmacokinetics of DP nor the inhibition of platelet aggregation of ASA/DP, regardless of the order of treatment administration. All treatments were well tolerated. [ Table 1 .] Table Notes: A: ASA/DP alone B: ASA/DP + Omeprazole following ASA/DP alone C: Omeprazole alone D: ASA/DP + Omeprazole following Omeprazole alone

Left Ventricular Assist Device Induced Coagulation and Platelet Activation and Effect of the Current Anticoagulant Therapy Regimen

Abstract▪2271▪This icon denotes a clinically relevant abstractLeft ventricular assist devices (LVADs) are mechanical pumps used to enhance cardiac function in heart failure patients. Platelet and coagulation activation was quantified to identify if the platelet and clot inhibiting drug regimen is effective in the LVAD patients. Seven patients with implanted LVADs were evaluated monthly for 3 months; 5 healthy adults and 5 patients on warfarin with normal cardiac output served as controls. All subjects signed an informed consent document prior to inclusion in the study. Freshly collected blood was assayed by the following: Plateletworks platelet aggregation (PWs; Helena; Beaumont, TX); PlateletMapping whole blood coagulation and platelet activity (PM; Haemoscope; Niles, IL); and flow cytometry platelet P-selectin upregulation and GPIIb/IIIa receptor activation. All assays used arachidonic acid (AA), ADP, collagen, and thrombin associated receptor activated peptide (TRAP) platelet agonists. In addition, von Willebrand factor (vWF) antigenic levels (Zymutest; Hyphen Biomed, France) were evaluated on frozen stored citrated plasmas. PWs showed a greater inhibition of collagen (31.8 vs 7.1% inh; p=0.008) and AA (30.8 vs 8.2% inh; p=0.002) induced platelet aggregation in patients; ADP induced platelet aggregation was not different between groups (10.8 vs 6.1% inh; p=0.711). PM showed an inhibition trend of AA (36.8 vs 25.1% inh; p=0.491) and ADP (36.2 vs 15.4% inh; p=0.056) mediated coagulation/platelet activation but a greater maximum amplitude of the blood clot in patients compared to normals (69.1 vs 64.9 mm; p=0.012). PM showed no significant inhibition of coagulation (R p=0.719; K p=0.569; angle p=0.430). In patients more than normals, flow cytometry showed an inhibition trend of platelet activation induced by AA (P-selectin: 24.2 vs 31.3% (+), p=0.064; GPIIb/IIIa: 16.8 vs 20.6% (+), p=0.052) and collagen (GPIIb/IIIa: 15.7 vs 29.7% (+), p=0.004); however a similar P-selectin expression to collagen and P-selectin and GPIIb/IIIa receptors to ADP and TRAP agonists was observed. There was no difference in any platelet or coagulation function parameter over the 3 months. The vWF levels, although remaining within the normal range, tended to increase during the 3 month period in the LVAD patients (from 97.9 ± 16.0% to 111.2 ± 18.2% at 1 month and to 113.9 ± 17.4% at 3 months; normals 106.5 ± 11.2%). This study shows that coagulation and platelet activation caused by the LVAD pump and generated shear forces is suppressed only to a limited degree by 81 mg/day aspirin and the anticoagulant effect of 1.8 INR warfarin is not robust. Modifications of the anticoagulant therapy regimen may improve upon the procoagulant effect generated by the LVAD pump. Using such assays as those described could facilitate determination of an optimal dosing strategy, as well as include consideration to avoid any potential increase in risk of bleeding. Disclosures:No relevant conflicts of interest to declare.

Polysulfated Xanthones: Multipathway Development of a New Generation of Dual Anticoagulant/Antiplatelet Agents

A multipathway strategy was used to evaluate the in vitro and in vivo antithrombotic effects of a new synthetic family of sulfated small molecules. Polysulfated xanthonosides showed highly effective anticoagulation effects in vitro, both in plasma (clotting times) and in whole human blood (thromboelastography), as well as in vivo (ip administration, mice). Physicochemical properties were assessed for mangiferin heptasulfate (7), which showed high solubility and stability in water and in human plasma and no putative hepatotoxicity in vivo. Mangiferin heptasulfate (7) was found to be a direct inhibitor of FXa, while persulfated 3,6-(O-β-glucopyranosyl)xanthone (13) acted as a dual inhibitor of FXa (directly and by antithrombin III activation). By impedance aggregometry, compounds 7 and 13 exhibited the antiplatelet effect by inhibition of both arachidonic acid and ADP-induced platelet aggregation. Dual anticoagulant/antiplatelet agents, such as sulfated xanthonosides 7 and 13, are expected to lead to a new therapeutic approach for the treatment of both venous and arterial thrombosis.

Eugenol and n-3 Rich Garden Cress Seed Oil as Modulators of Platelet Aggregation and Eicosanoids in Wistar Albino Rats

Eugenol, the active principle of clove oil is a potent antioxidant and a well known anti-inflammatory agent. In an earlier study, we have reported the in vitro inhibition of human platelet aggregation and LTC4 forma- tion in rat PMNL cells. In the present study, an attempt has been made to monitor the in vivo and in vitro modula- tion of platelet aggregation and eicosanoids (TXB2, LTC4) by eugenol and a-linolenic acid (ALNA) rich garden cress seed oil in adult Wistar rats. Feeding of Garden cress oil (GCO) supplemented diet to rats for 8 weeks moder- ately inhibited platelet aggregation induced by ADP and arachidonic acid (AA) at 14 and 7% respectively and also TXB2 levels at 10 and 32% in lung and spleen tissues. Oral feeding of eugenol at 40 mg/kg body weigh t for one week to GCO fed rats showed 20-30% inhibition of ADP and AA induced platelet aggregation and also TXB2 levels by ~ 50% in spleen. In an in vitro study, eugenol at 15.0 and 45.0 � M produced 15 and 78% inhibition of LTC4 levels, 27 and 80% inhibition of TXB2 in lungs and also 20 and 72% inhibition of LTC4 levels in the washed platelets respectively. Eugenol and GCO showed synergistic effect against platelet aggregation and TXB2 levels in spleen and lung tissues. Conclusively, data obtained from the present study provide new insights into modulatory effect of eugenol and a-linolenic acid on platelet aggregation and eicosanoids

PGE2 Release from Tryptase‐Stimulated Rabbit Ventricular Myocytes is Mediated by Calcium‐Independent Phospholipase A2γ

Inflammation is associated with cardiovascular disease, including myocardial infarction, atherosclerosis, myocarditis and congestive heart failure. Mast cells have been implicated in inflammation, but their precise role in cardiac inflammation remains unclear. Mast cells contain a variety of pre-formed granule-associated mediators, including tryptase. We have previously demonstrated that the majority of the phospholipase A(2) (PLA(2)) activity in isolated rabbit ventricular myocytes is membrane-associated, calcium-independent and selective for plasmalogen phospholipids. We hypothesized that tryptase stimulation of rabbit ventricular myocytes would increase iPLA(2) activity, leading to increased arachidonic acid and prostaglandin E(2) (PGE(2)) release. Isolated rabbit ventricular myocytes were stimulated with tryptase and iPLA(2) activity, arachidonic acid and PGE(2) release were measured. Tryptase stimulation increased iPLA(2) activity after 5 min. Activation of iPLA(2) was accompanied by increased arachidonic acid and PGE(2) release in tryptase-stimulated myocytes. However no increase in platelet activating factor was observed with tryptase stimulation. To distinguish between different iPLA(2) isoforms in the myocardium, we pretreated ventricular myocytes with the (R)- and (S)-enantiomers of bromoenol lactone (BEL) to selectively inhibit iPLA(2)γ and β respectively. Pretreatment with (R)-BEL resulted in complete inhibition of tryptase-stimulated iPLA(2) activity, arachidonic acid and PGE(2) release, suggesting the iPLA(2)γ is the predominant myocardial isoform activated by tryptase. These studies demonstrate that PGE(2) release from tryptase stimulated rabbit ventricular myocytes is mediated primarily by iPLA(2)γ.

Dual anticoagulant/antiplatelet persulfated small molecules

A new series of persulfated compounds was synthesized and assayed for in vitro anticoagulant and antiplatelet activities, which may be useful in the treatment of both venous and arterial thrombosis. Persulfation of polyphenolic components of wine, coumarins and other structurally diverse small molecules was achieved with triethylamine-sulphur trioxide adduct. The derivatives were highly effective in increasing the APTT, being trans-resveratrol 3-ß-d-glucopyranoside persulfate (15) the most potent (APTT2 = 1.5 × 10−4 M), and were able to completely block the clotting process at the highest concentration. Compound 15 showed good stability in human plasma and anticoagulation effects in whole blood. trans-Resveratrol 3-ß-d-glucopyranoside persulfate (15) and a series of polysulfated oligoflavonoids (1–4) also exhibited antiplatelet activity by inhibition of arachidonic acid and ADP-induced platelet aggregation.

Aspirin Resistance

Aspirin is an inexpensive, readily available medication that reduces the risk of subsequent vascular disease by about 25% in patients with known occlusive vascular disease. Aspirin's beneficial effect is mediated via inhibition of arachidonic acid (AA) activation of platelets and is detected by demonstrating a decrease in platelet function and/or a decrease in prostaglandin metabolites. Patients who are assumed to be taking their aspirin, but who do not demonstrate an aspirin effect are labeled as, "aspirin resistant." This is an unfortunate designation as the vast majority of patients labeled as "aspirin resistant" are noncompliant. Noncompliance is demonstrated in multiple studies that use repeat testing for platelet inhibition in patients with an initial inadequate response to aspirin. When the test is repeated under condition where ingestion of the test aspirin is assured, the patients' platelets are inhibited. Instead of using the term "aspirin resistance," this review will use "inadequate response to aspirin." Patients with an inadequate aspirin response have an increased likelihood for subsequent vascular events. Detection and treatment of an inadequate aspirin response would be facilitated by the development of a bedside assay that uses whole blood, is technically simple, inexpensive, sensitive, specific, reproducible, and provides an answer in a few minutes. Future research in patients with an inadequate response to aspirin should focus on mechanisms to improve compliance, which should decrease their risk of future vascular events.

Differential Inhibition of Arachidonic Acid Mediated Aggregation of Platelets by Branded and Generic Argatroban Preparations

AbstractAbstract 5131Argatroban represents a parentral antithrombin agent which is used in the management of anticoagulation in heparin compromised patients. Its main mechanism of action is mediated via inhibition of thrombin and its generation. While its effect on platelet activation inhibition by thrombin have been reported, very little information on the effects of this drug on thromboxane formation and Arachidonic Acid mediated activation of platelets is available. Argatroban and its generic versions namely Slovastan, Argaron and Gartban, may modulate Arachidonic Acid mediated platelet aggregation and release processes. To test this hypothesis a branded version of Argatroban (Mitsubishi – Tanade, Tokyo, Japan) and three generic versions of Argatroban namely Slovastan, Gartban, and Slovastan were compared for their effects on Arachidonic Acid mediated aggregation of platelets in normal healthy male and female volunteers (and n = 4). Other agonists such as Epinephrine, Collagen, and ADP were also used. The effect of Arachidonic Acid on serotonin release was also measured using an Elisa technique for the measurement of serotonin. All of the generic and branded versions of Argatroban produce varying levels of the inhibition of the Arachidonic Acid mediated aggregation of platelets, ranging from the 24 to 36 percent in comparison to the control at a concentration of 1 mg/ mL (p value < 0.05). Interestingly all of the Argatrobans produced a relatively weaker inhibition of the Arachidonic Acid mediated aggregation of platelets 24–26 percent inhibition versus 36 percent at the final concentration of 1 mg/mL. No differences were noted in the aggregation profile of ADP, Collagen, and Epinephrine between the control and the Argatrboban at a final concentration of 1 mg /mL. No differences were noted between the generic and branded Argatroban on all of the other agonists induced aggregation. In the serotonin release assays, all of the generic and branded Argatroban produced a concentration dependent inhibition of serotonin release which was stronger with the branded version of Argatroban. These results indicate that besides the inhibition of thrombin Argatroban is capable of inhibiting platelet activation via other mechanisms. Moreover, the generic versions of Argatroban exhibit a weaker inhibition of Arachidonic Acid mediated platelet aggregation and release. These studies suggest that beside thrombin mediated aggregation Argatroban and its generic versions can modulate platelet activation and release reactions. Furthermore a difference is observed between the generic and branded product which may impact the safety inefficacy profile in these agents. Argatroban is used commonly in the management of patients with heparin induced thrombocytopenia where multiple mechanisms of platelet activation are contributory to the pathogenesis of this syndrome. Modulation of thrombanxane formation and platelet release mechanism by Argatroban may represent an additional mechanism of the clinical effects of this parentral antithrombin agent. Disclosures:No relevant conflicts of interest to declare.

Prolonged Exposure of Cortical Neurons to Oligomeric Amyloid-β Impairs NMDA Receptor Function Via NADPH Oxidase-Mediated ROS Production: Protective Effect of Green Tea (-)-Epigallocatechin-3-Gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.

PGE2 release from tryptase‐stimulated rabbit ventricular myocytes is mediated by calcium‐independent phospholipase A2γ

Inflammation is associated with several cardiovascular diseases and mast cells have been implicated in many of these, but their precise role remains unclear. We hypothesize that tryptase, a pre formed mast cell mediator, stimulates rabbit ventricular myocyte calcium independent phospholipase A2 (iPLA2) activity, resulting in increased arachidonic acid and prostaglandin E2 (PGE2) release. Tryptase stimulation (20 ng/ml) increased iPLA2 activity after 5 mins (3.56 ± 0.12 to 5.87 ± 0.31 nmol/mg protein/min, n=6, p&lt;0.01). This was accompanied by increased arachidonic acid (1.02 ± 0.12 to 5.02 ± 0.21%, n=6, p&lt;0.01) and PGE2 release (4.50 ± 0.30 to 16.70 ± 0.59 ng/mg protein, n=8, p&lt;0.01) in stimulated myocytes. To distinguish between different myocardial iPLA2 isoforms, we pretreated ventricular myocytes with the R‐ and S‐ enantiomers of bromoenol lactone (BEL, 5 μM, 10 mins) to selectively inhibit iPLA2γ and β respectively. (R)‐BEL pretreatment resulted in complete inhibition of tryptase‐stimulated iPLA2 activity, arachidonic acid and PGE2 release, suggesting the iPLA2γ is activated in response to tryptase. Pretreatment with (S)‐BEL had little effect on tryptase‐stimulated responses. Previous studies suggest that PGE2 may be cardioprotective. Thus tryptase release from activated mast cells may play a protective role in cardiac inflammation via iPLA2γ activation and increased PGE2 release from myocytes.