Platelet Cyclooxygenase Research Articles

Nonsteroidal Anti-Inflammatory Drugs and the Heart

Aspirin has been on the market for 115 years. Beginning with the marketing of indomethacin for the treatment of rheumatoid arthritis in 1963, at least 20 other nonsteroidal anti-inflammatory drugs (NSAIDs) with aspirin-like actions have been developed over the past 50 years,1 culminating with the introduction of a new class of selective inhibitors of cyclooxygenase (COX)-2, the coxibs, approximately 15 years ago.2 The NSAIDs represent the single most crowded family of drugs sharing the same therapeutic activities and mechanism of action, perhaps reflecting the unmet therapeutic need in the area of pain management and the large interindividual variability in response to these agents. NSAIDs provide symptomatic relief of pain and inflammation associated with a variety of human disorders, including the rheumatic diseases. Their shared therapeutic actions (ie, analgesic, anti-inflammatory, and antipyretic) are usually accompanied by mechanism-based adverse effects that can, at least in part, be attenuated as a function of individual pharmacokinetic or pharmacodynamic properties.1 Nuances in the tolerability of different NSAIDs had been described in the precoxib era on the basis of clinical trials of a few hundred patients treated for up to a few months with soft end points. More recently, however, important differences in safety have been demonstrated in head-to-head randomized comparisons of individual coxibs and 1 or more traditional NSAIDs that involved tens of thousands of patients treated for up to a few years with hard end points. Even more significantly, the interpretation of the effects of NSAIDs has been greatly enhanced by the availability, for the first time, of large, placebo-controlled trials that aimed to assess the potential for chemoprevention of colorectal cancer by rofecoxib or celecoxib. As a result of these developments, the whole field of NSAIDs has been illuminated during the past 15 years. Although epidemiological studies had previously associated …

Suboptimal Inhibition of Platelet Cyclooxygenase 1 by Aspirin in Systemic Lupus Erythematosus: Association With Metabolic Syndrome

Low-dose aspirin prevents platelet aggregation by suppressing thromboxane A2 (TXA2 ) synthesis. However, in some individuals TXA2 suppression by aspirin is impaired, indicating suboptimal inhibition of platelet cyclooxygenase 1 (COX-1) by aspirin. Because patients with systemic lupus erythematosus (SLE) have increased risk of thrombotic events, many receive aspirin; however, the efficacy of aspirin in SLE has not been determined. We examined the hypothesis that aspirin response is impaired in SLE. We assessed the effect of aspirin by measuring concentrations of the stable metabolite of TXA2 , serum thromboxane B2 (sTXB2 ), before and after treatment with daily aspirin (81 mg) for 7 days in 34 patients with SLE and 36 control subjects. The inability to suppress sTXB2 synthesis to <10 ng/ml represents suboptimal inhibition of platelet COX-1 by aspirin. Aspirin almost completely suppressed sTXB2 in control subjects to median 1.5 ng/ml (interquartile range [IQR] 0.8-2.7) but had less effect in patients with SLE (median 3.1 ng/ml [IQR 2.2-5.3]) (P = 0.002). A suboptimal effect of aspirin was present in 15% (5 of 34) of the patients with SLE but not in control subjects (0 of 36) (P = 0.023). Incomplete responders were more likely to have metabolic syndrome (P = 0.048), obesity (P = 0.048), and higher concentrations of C-reactive protein (CRP) (P = 0.018). The pharmacologic effect of aspirin is suboptimal in 15% of patients with SLE but in none of the control subjects, and the suboptimal response was associated with metabolic syndrome, obesity, and higher CRP concentrations.

Emerging challenges to the existing paradigm of cyclo‐oxygenase pathways in regulating vascular function

Emerging challenges to the existing paradigm of cyclo‐oxygenase pathways in regulating vascular function

Platelet thromboxane (11-dehydro-Thromboxane B2) and aspirin response in patients with diabetes and coronary artery disease.

Aspirin (ASA) irreversibly inhibits platelet cyclooxygenase-1 (COX-1) leading to decreased thromboxane-mediated platelet activation. The effect of ASA ingestion on thromboxane generation was evaluated in patients with diabetes (DM) and cardiovascular disease. Thromboxane inhibition was assessed by measuring the urinary excretion of 11-dehydro-thromboxane B2 (11dhTxB2), a stable metabolite of thromboxane A2. The mean baseline urinary 11dhTxB2 of DM was 69.6% higher than healthy controls (P = 0.024): female subjects (DM and controls) had 50.9% higher baseline 11dhTxB2 than males (P = 0.0004), while age or disease duration had no influence. Daily ASA ingestion inhibited urinary 11dhTxB2 in both DM (71.7%) and controls (75.1%, P < 0.0001). Using a pre-established cut-off of 1500 pg/mg of urinary 11dhTxB2, there were twice as many ASA poor responders (ASA "resistant") in DM than in controls (14.8% and 8.4%, respectively). The rate of ASA poor responders in two populations of acute coronary syndrome (ACS) patients was 28.6 and 28.7%, in spite of a significant (81.6%) inhibition of urinary 11dhTxB2 (P < 0.0001). Both baseline 11dhTxB2 levels and rate of poor ASA responders were significantly higher in DM and ACS compared to controls. Underlying systemic oxidative inflammation may maintain platelet function in atherosclerotic cardiovascular disease irrespective of COX-1 pathway inhibition and/or increase systemic generation of thromboxane from non-platelet sources.

Platelet Rna As a Novel Biomarker for the Response to Antiplatelet Therapy

9 ISSN 1479-6678 10.2217/FCA.13.90 © 2014 Future Medicine Ltd Future Cardiol. (2014) 10(1), 9–12 Antiplatelet agents, such as aspirin, or P2Y 12 inhibitors, such as clopidogrel, prasugrel or ticagrelor, are widely prescribed to prevent ischemic events [1]. The combination of aspirin and a P2Y 12 inhibitor is endorsed by international guidelines in patients at high risk of ischemic events, while aspirin monotherapy is the backbone of preventative therapy for patients with established vascular disease [2–6]. Despite convincing evidence of its effectiveness, the efficacy of aspirin in certain individuals may vary and recurrence rates are high [2]. For example, in patients with established cardio vascular disease or multiple risk factors who use aspirin, 15–20% will suffer a future cardiovascular event (myocardial infarction [MI] or stroke), hospitalization for revascularization or death over 28 months [7]. Similarly, patients with Type 2 diabetes, a powerful magnifier of cardiovascular risk, are at equivalent risk of a first MI as nondiabetics with a prior MI (each 19–20% over 7 years) [8]. Consequently, aspirin is recommended in patients with Type 2 diabetes, especially if other cardiovascular risk factors are present [2,9]. However, two randomized trials of low-dose aspirin as primary prevention in more than 3000 diabetics each failed to significantly reduce MI or stroke [10,11], thereby suggesting that the benefits of aspirin are not uniform and may fail in certain high-risk individuals. Further more, antiplatelet agents are accompanied by an increase in bleeding events [2,12], which may offset any potential benefits, particularly in low-risk populations. Therefore, certain individuals may fail to benefit from aspirin, and others may be exposed to an increased risk of bleeding. The vast majority of clinicians and researchers lack reliable methods of identifying individuals with inadequate or unanticipated responses to antiplatelet drugs, and thus the tools for tailoring antiplatelet therapy. This need is greatest in the primary care setting where aspirin is widely used, but no method to assess drug response is accessible. Platelet function tests are available in certain settings and capture the large interindividual variability in response to aspirin, despite complete suppression of aspirin’s putative target, platelet COX-1 [13–16].

Appropriate Assessment of the Functional Consequences of Platelet Cyclooxygenase-1 Inhibition by Aspirin in vivo

Chronic use of low dose aspirin (acetylsalicylic acid) is generally an effective and relatively low cost strategy for preventing and treating cardiovascular disease [1]. John Vane reported the inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs in 1971 by demonstrating a dose-dependent inhibition of prostaglandin synthesis by aspirin, sodium salicylate and indomethacin [2]. Thromboxane A2 (TxA2) was subsequently identified by Samuelsson’s group as the potent vasoconstrictor and platelet agonist the synthesis of which was inhibited by aspirin [3].

Mitigating the Cardiovascular and Renal Effects of NSAIDs: Table 1

Nonsteroidal anti-inflammatory drugs (NSAIDs) are principal pharmacologic agents for symptom relief in patients with arthritis and other inflammatory conditions. Cardiovascular risk is associated with all NSAIDs, excluding aspirin. Selective inhibition of cyclo-oxygenase-2 (COX)-2 could produce a relative reduction in endothelial production of prostacyclin, while leaving the platelet production of thromboxane A2 (TXA2 ) intact. It has been speculated that this imbalance of homeostatic prostanoids might increase the risk for thrombotic events. The goal of this review is to provide physicians guidelines to mitigate cardiovascular and nephrotoxicity of NSAIDs. We conducted a systematic literature review to determine what information is available to guide treatment decisions in this patient population. Selective inhibition of COX-2 could produce a relative reduction in endothelial production of prostacyclin, while leaving the platelet production of TXA2 intact. Increasing degrees of selectivity for COX-2 are associated with augmented cardiovascular risk, whereas increasing degrees of selectivity for cyclo-oxygenase-2 (COX-1) are associated with augmented gastrointestinal risk. Some NSAIDs (such as ibuprofen) can interfere with the cardioprotective effects of aspirin by competitively binding to COX-1 enzyme, resulting in increased TXA2 production Naproxen may differ from other NSAIDs in sustaining functionally important degrees of inhibition of platelet cyclooxygenase-1 activity throughout the dosing interval. It is of paramount importance to consider individual health factors when choosing therapy with NSAIDs.

Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.

CORRIGENDUM

Journal of Veterinary Emergency and Critical CareVolume 23, Issue 6 p. 670-670 CorrigendumFree Access CORRIGENDUM This article corrects the following: Evaluation of platelet aggregometry in dogs using the Multiplate platelet analyzer: impact of anticoagulant choice and assay duration Clara B. Marschner DVM, Annemarie T. Kristensen DVM, PhD, DACVIM, DECVIM, Eva H. Spodsberg DVM, Bo Wiinberg DVM, PhD, Volume 22Issue 1Journal of Veterinary Emergency and Critical Care pages: 107-115 First Published online: February 17, 2012 First published: 04 December 2013 https://doi.org/10.1111/vec.12126AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Evaluation of platelet aggregometry in dogs using the Multiplate platelet analyzer: impact of anticoagulant choice and assay duration. Clara B. Marschner, DVM; Annemarie T. Kristensen, DVM, PhD, DACVIM, DECVIM; Eva H. Spodsberg, DVM and Bo Wiinberg, DVM, PhD In the February 2012 issue (volume 22(1):107–115), on page 108 of the article entitled “Evaluation of platelet aggregometry in dogs using the Multiplate platelet analyzer: impact of anticoagulant choice and assay duration” by Drs. Marschner, Kristensen, Spodsberg and Wiinberg, there were some omissions that the authors would like to correct. Page 108, first column, first paragraph, fourth sentence should have read: “Furthermore, aggregation in the Multiplate takes place on surfaces, which is a major difference compared to methods such as Born aggregometry and single platelet counting in which platelets aggregate with each other in the liquid phase.§ Such spontaneous aggregation presumable happens only in severely ill patients, as coagulation and platelet aggregation in vivo usually only take place on surfaces such as damaged endothelium.§” Page 108, second column, first paragraph, second sentence should have read: “The term ‘multiplate” refers to the multiple electrodes in the disposable test cell, multiple channels of the instruments, as well as multiple test procedures available.§” The fourth sentence should have read : “The standard test cell uses a sample volume of only 300 μL of whole blood . For research use a mini test cell is available with sample volume of 175 μL, making it suitable for use in small animals.§” Page 108, second column, first paragraph, eighth sentence should have read: “According to the Multiplate manufacturer's website, the ‘Multiplate continuously records platelet aggregation as an increase in impedance, which is transformed into arbitrary aggregation units (AU) and plotted against time (Figure 1). Three parameters are calculated: the most important parameter being the area under the aggregation curve (AUC), which is affected by the total height of the aggregation curve as well as by its slope and is best suited to express the overall platelet activity. Two more parameters are calculated for research use: the ’aggregation’ is the height of the curve and the ’velocity’ is the maximum slope of the curve. The parameters calculated by the software include the mean values of the parameters determined with each curve.’§” Page 108, second column, second paragraph, second sentence should have read: “The manufacturer's website also states that ‘ADPtest: Adenosine diphosphate (ADP)-induced platelet activation sensitive to clopidogrel, prasugrel and other ADP receptor antagonists. ADPtest HS: High sensitivity ADPtest with co-stimulation of platelet aggregation with ADP and prostaglandin E1, especially for the sensitive detection of clopidogrel in heparinized blood. ASPItest: Cyclooxygenase dependent aggregation (using arachidonic acid [AA]), sensitive to aspirin, non-steroidal anti-inflammatory drugs (NSAIDs) and other inhibitors of platelet cyclooxygenase. TRAPtest: Platelet stimulation via the thrombin receptor (using TRAP-6), sensitive to IIbIIIa receptor antagonists in people, but demonstrated to work in dogs.9 COLtest: Collagen (COL)- induced aggregation. RISTOtest: vWF and GpIb dependent aggregation (using ristocetin as agonist). Control material: ASA Control (Aspirin (ASA) for in vitro analysis), GpIIbIIIa antagonist (for in vitro analysis), liquid quality control kit.’§” In addition, the figure legends for Figures 1 and 2 should also have stated: “Reproduced with permission from Verum Diagnostica GmbH, Munich, Germany” Reference § Mutiplate Homepage. Available at: http://multiplate.net/en/splashpage2.php. Accessed 18 October , 2013 Google Scholar Volume23, Issue6November/December 2013Pages 670-670 ReferencesRelatedInformation

Fatty acids and TxA2 generation, in the absence of platelet-COX-1 activity

Background and aimsOmega-3 fatty acids suppress Thromboxane A2 (TxA2) generation via mechanisms independent to that of aspirin therapy. We sought to evaluate whether baseline omega-3 fatty acid levels influence arachidonic acid proven platelet-cyclooxygenase-1 (COX-1) independent TxA2 generation (TxA2 generation despite adequate aspirin use). Methods and resultsSubjects with acute myocardial infarction, stable CVD or at high risk for CVD, on adequate aspirin therapy were included in this study. Adequate aspirin action was defined as complete inhibition of platelet-COX-1 activity as assessed by <10% change in light transmission aggregometry to ≥1 mmol/L arachidonic acid. TxA2 production was measured via liquid chromatography–tandem mass spectrometry for the stable TxA2 metabolite 11-dehydro-thromboxane B2 (UTxB2) in urine. The relationship between baseline fatty acids, demographics and UTxB2 were evaluated. Baseline omega-3 fatty acid levels were not associated with UTxB2 concentration. However, smoking was associated with UTxB2 in this study. ConclusionBaseline omega-3 fatty acid levels do not influence TxA2 generation in patients with or at high risk for CVD receiving adequate aspirin therapy. The association of smoking and TxA2 generation, in the absence of platelet COX-1 activity, among aspirin treated patients warrants further study.

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

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

Drug/drug interaction of common NSAIDs with antiplatelet effect of aspirin in human platelets

Nonsteroidal anti-inflammatory drugs (NSAIDs) may interfere with the anti-platelet activity of aspirin at the level of the platelet cyclooxygenase-1 (COX-1) enzyme. In order to examine the interference of common NSAIDs with the anti-platelet activity of aspirin the human platelet rich plasma from voluntary donors was used for arachidonic acid-induced aggregation and determination of thromboxane synthesis. Further, docking studies were used to explain the molecular basis of the NSAID/aspirin interaction. The experimental results showed that celecoxib, dipyrone (active metabolite), ibuprofen, flufenamic acid, naproxen, nimesulide, oxaprozin, and piroxicam significantly interfere with the anti-platelet activity of aspirin, while diclofenac, ketorolac and acetaminophen do not. Docking studies suggested that NSAIDs forming hydrogen bonds with Ser530, Arg120, Tyr385 and other amino acids of the COX-1 hydrophobic channel interfere with antiplatelet activity of aspirin while non interfering NSAIDs do not form relevant hydrogen bond interactions within the aspirin binding site. In conclusion, docking analysis of NSAID interactions at the COX-1 active site appears useful to predict their interference with the anti-platelet activity of aspirin. The results, demonstrate that some NSAIDs do not interfere with the antiplatelet action of aspirin while many others do and provide a basis for understanding the observed differences among individual non-aspirin NSAIDs.

PP140—The contribution of platelet glycoproteins (GPIA C807T AND GPIBA C-5T) and cyclooxygenase 2 (COX-2 G--765C) polymorphisms to platelet response in patients treated with aspirin

PP140—The contribution of platelet glycoproteins (GPIA C807T AND GPIBA C-5T) and cyclooxygenase 2 (COX-2 G--765C) polymorphisms to platelet response in patients treated with aspirin

Oral clopidogrel improves cutaneous microvascular function through EDHF-dependent mechanisms in middle-aged humans

Platelet P₂Y₁₂-ADP and COX-1 receptor inhibition with oral clopidogrel (CLO) and low-dose aspirin (ASA), respectively, attenuates reflex-mediated cutaneous vasodilation, but little is known about how these medications affect local vasodilatory signaling. Reactive hyperemia (RH) results in vasodilation that is mediated by sensory nerves and endothelium-derived hyperpolarization factors (EDHF) through large-conductance calcium-activated potassium channels, whereas slow local heating (LH) elicits vasodilation largely through the production of nitric oxide (NO). We hypothesized that CLO and ASA would attenuate locally mediated cutaneous vasodilation assessed by RH and LH (0.5°C/min). In a randomized, cross-over, double-blind placebo-controlled study, nine healthy men and women (56 ± 1 yr) took CLO (75 mg), ASA (81 mg), and placebo for 7 days. Skin blood flow was measured (laser-Doppler flowmetry, LDF) and cutaneous vascular conductance (CVC) was calculated (LDF/mean arterial pressure) and normalized to maximal CVC (%CVCmax: 43°C and 28 mM sodium nitroprusside). RH response parameters, including area under the curve (AUC), total hyperemic response (THR), and the decay constant tau (λ) were calculated. NO-dependent vasodilation during LH was assessed by calculating the difference in %CVCmax between a control site and an NO synthase-inhibited site (10 mM l-NAME: intradermal microdialysis). CLO augmented the AUC and THR (AUCclo = 3,783 ± 342; THRclo = 2,306 ± 266% CVCmax/s) of the RH response compared with ASA (AUCASA = 3,101 ± 325; THRASA = 1,695 ± 197% CVCmax/s) and placebo (AUCPlacebo = 3,000 ± 283; THRPlacebo = 1,675 ± 170% CVCmax/s; all P < 0.0001 vs. CLO). There was no difference in the LH response or calculated NO-dependent vasodilation among treatments (all P > 0.05). Oral CLO treatment augments vasodilation during RH but not LH, suggesting that CLO may improve cutaneous microvascular function.

Mechanism of platelet activation induced by endocannabinoids in blood and plasma

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB2-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

The contribution of platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms to platelet response in patients treated with aspirin

ObjectiveAspirin is an antiplatelet agent commonly used in treatment of patients with high risk to develop stroke and myocardial infarction. However, inter-individual variability regarding the inhibition of platelet function by aspirin is well documented. In this study, the correlation between platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms and antiplatelet response in patients treated with aspirin was investigated. MethodsJordanian adult patients (n=584) who are taking aspirin as an antiplatelet agent participated in the study. Platelet aggregation response was measured using Multiplate Analyzer® system. Polymerase chain reaction–restriction fragment length polymorphism assay (PCR–RFLP) was used for genotyping of the examined polymorphisms. ResultsAspirin resistance was found in 15.8% of patients. Response to aspirin was significantly associated with GPIba C-5T polymorphism (P<0.05). However, the GPIa C807T and COX-2G-765C polymorphisms were not related to aspirin resistance (P>0.05). ConclusionA considerable fraction of the Jordanian population is resistant to the antiplatelet effect of aspirin, which might be related to GPIba C-5T polymorphism.

Synthesis, Pharmacological Characterization, and Docking Analysis of a Novel Family of Diarylisoxazoles as Highly Selective Cyclooxygenase-1 (COX-1) Inhibitors

3-(5-Chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6), a known selective cyclooxygenase-1 (COX-1) inhibitor, was used to design a new series of 3,4-diarylisoxazoles in order to improve its biochemical COX-1 selectivity and antiplatelet efficacy. Structure-activity relationships were studied using human whole blood assays for COX-1 and COX-2 inhibition in vitro, and results showed that the simultaneous presence of 5-methyl (or -CF3), 4-phenyl, and 5-chloro(-bromo or -methyl)furan-2-yl groups on the isoxazole core was essential for their selectivity toward COX-1. 3g, 3s, 3d were potent and selective COX-1 inhibitors that affected platelet aggregation in vitro through the inhibition of COX-1-dependent thromboxane (TX) A2. Moreover, we characterized their kinetics of COX-1 inhibition. 3g, 3s, and 3d were more potent inhibitors of platelet COX-1 and aggregation than P6 (named 6) for their tighter binding to the enzyme. The pharmacological results were supported by docking simulations. The oral administration of 3d to mice translated into preferential inhibition of platelet-derived TXA2 over protective vascular-derived prostacyclin (PGI2).

Aspirin Half Maximal Inhibitory Concentration Value on Platelet Cyclooxygenase1 in Severe Type-2 Diabetes Mellitus is not Significantly Different from that of Healthy Individuals

It is implicated that diabetic patients are more resistant to aspirin therapy than patients with other diseases or healthy individuals. We evaluated the inhibitory effects of aspirin on aggregation and the cyclooxygenase activity of platelets of 10 patients with severe type-2 diabetes mellitis (DM) and compared the results with those of healthy individuals. Although platelet aggregation had a tendency to be more resistant to aspirin with the DM group, there was no significant difference in half maximal inhibitory concentration 50 values of aspirin on the cyclooxygenase activity between the patients with DM and healthy individuals. Thus, the residual platelet aggregability uninhibited by aspirin appears to be independent of the cyclooxygenase activity. Since adenosine diphosphate (ADP) receptor blocking almost completely inhibited the residual platelet aggregability, it is suggested that hyperreactivity to ADP is more prevalent in patients with DM.

Abstract 349: Measurement of Platelet Cyclooxygenase-1 Acetylation Quantifies Interaction of Nonsteroidal Anti-Inflammatory Drugs with Aspirin in vivo

Aspirin prevents heart attack and stroke by irreversibly acetylating serine-529 in human platelet cyclooxygenase-1 (COX-1) and thereby inhibiting the biosynthesis of thromboxane (Tx). We hypothesized that precise quantification of COX-1 acetylation would allow the direct detection of drug-drug interactions between reversible COX inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), and aspirin, which may abrogate its cardio-protective effect. We developed a stable isotope dilution liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) method for measurement of platelet COX-1 acetylation. Serine-529 acetylation reflected faithfully aspirin pharmacology determined with conventional markers (serum TxB2 formation, urinary excretion of 11-dehydro-TxB2, and arachidonic acid induced platelet aggregation), but showed lower technical (CV% 3.7±2.7) and inter-individual variability (CV% 3.7). Oral administration of 325 mg of plain aspirin resulted in complete acetylation of platelet COX-1 within 2 hours and sustained maximal acetylation (71 ± 3% of total COX-1 amount) for up to two days in health volunteers (n=8). Recovery from acetylation, which reflects platelet turn-over, occurred at a rate of 7.2 ± 0.2 % per day. We compared the potential of five NSAIDs to block aspirin from acetylating platelet COX-1 in healthy volunteers (n=7 for each NSAID). Single doses of naproxen (500 mg), ibuprofen (600 mg), and diclofenac (100 mg) but not celecoxib (200 mg) and acetaminophen (1000 mg) prevented complete COX-1 acetylation by 325 mg aspirin administered 2 hours after the NSAID. The competition of NSAIDs with aspirin was conditioned by NSAID pharmacokinetics, potency, and COX isoform selectivity, suggesting variability in the likelihood of the drug-drug interaction between compounds and between patients. Monitoring platelet COX-1 acetylation may have utility in the identification of drug interactions that may limit the cardio-protective effect of low dose aspirin.

207 PLATELET COX-1 SUPPORTS THE PRODUCTION OF BOTH PROSTANOIDS AND HETES

<h3>Backround</h3> Prostaglandins (PGs) and hydroxyeicosatetraeinoic acids (HETEs) are synthesized from arachidonic acid (AA), which is released from the membrane phospholipids of cells through the activity of cytosolic phospholipase A_2α (cPLA_2α). It is known that in platelets AA is metabolised through the enzymes cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) leading to the production of a range of eicosanoids. Here we have determined the enzyme sources of the major prostanoid and HETE products released by platelets following activation by selective platelet agonists. To better model platelet activation we used either platelet rich plasma or whole blood within a platelet aggregometer, warmed and stirred at 1200rpm, and assessed whether HETEs originate from platelets or other blood cells. Furthermore, to investigate how the production of these eicosanoids is affected by anti-platelet therapy we examined the effects of the cyclooxygenase inhibitor, aspirin (100 µM), with or without the addition of the P2Y₁₂ receptor blocker, prasugrel (3 µM). <h3>Methods</h3> Blood from healthy volunteers was collected into hirudin and incubated (30 min, 37°C) either intact or following preparation into platelet rich plasma (PRP) in the presence of collagen (30 µg/ml), TRAP-6 (30 µM) or vehicle, in a platelet aggregometer with stirring (1200rpm). Plasma was then separated from the samples and the levels of prostanoids and HETEs were determined by LC/MS/MS analysis. In some experiments, blood and PRP were pre-treated with aspirin (100 µM), prasugrel (3 µM) or aspirin+prasugrel. <h3>Results</h3> LC/MS/MS analysis of collagen- and TRAP-stimulated PRP and blood showed large increases in the levels of thromboxane (TX) A₂, prostaglandin (PG) E₂, and PGD₂, as well as 8-, 11-, 15-and 12-HETE compared to unstimulated samples. Interestingly, as well as inhibiting the production of TXA₂, PGE₂ and PGD₂, aspirin also strongly inhibited the productions of 11-HETE and 15-HETE by both PRP and blood. The production of 12-HETE was reduced by aspirin and prasugrel used in combination. <h3>Conclusions</h3> LC/MS/MS analysis demonstrated that platelets are the main source of both prostanoids (TXA₂, PGE₂ and PGD₂) and HETEs (8-, 11-, 15- and 12) in blood exposed to platelet activators. Interestingly, in addition to abolishing the production of prostanoids, aspirin also blocked the production of 11-HETE and 15-HETE in both PRP and blood, indicating these HETEs are also formed through the activity of platelet COX-1. Aspirin in combination with prasugrel reduced, but did not abolish, the production of 12-HETE, consistent with this drug inhibiting the processes of platelet activation but not directly inhibiting platelet 12-LOX. It is possible these data may well help provide explanations for the beneficial effects of anti-platelet doses of aspirin in a range of cardiovascular diseases and cancers.