Thromboxane Synthesis Research Articles

Nonsteroidal Anti-Inflammatory Drugs: Exploiting Bivalent COXIB/ TP Antagonists for the Control of Cardiovascular Risk.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are some of the most widely prescribed or dispensed over the counter analgesics and antipyretics that act by inhibiting prostaglandins and thromboxane synthesis. After the identification of a second isoform of COX, the pharmaceutical research focused on developing COX-2- selective drugs (COXIBs) considered as second generation NSAIDs that would retain the anti-inflammatory and analgesic activities of traditional NSAID without blunting the gastrointestinal cytoprotection sustained by COX1-derived products such as PGE2. However, while several clinical trials confirmed a gastrointestinal safer profile of COXIBs vs unselective COX inhibitors, increasing evidence for potential cardiovascular risk associated with COXIBs rapidly emerged. Today, there are no really safe NSAIDs to be used in chronic pain and anti-inflammatory treatments, as an adequate therapy associated with a minimal gastrointestinal damage and cardiovascular toxicity is yet to be developed. Here, we present evidences that combining the anti-aggregating and antiatherotrombotic activities of a thromboxane receptor antagonist with the antiinflammatory activity of a COXIB we could obtain a new multitarget drug providing protection against the harmful activities mediated by the COXIB component, yet exploiting its recognized therapeutic advantages as a gastrointestinal-safer anti-inflammatory drug. We also summarize recent progress achieved in this field of research and possible new strategies to obtain a new bivalent compound. This possible third-generation NSAID with a safer pharmacological profile, will have all the pharmacological characteristics for the long-term therapy of chronic disorders such as inflammatory diseases or selected forms of cancer.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation.

In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor. Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbβ3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation. These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

Dietary supplements and drugs for preeclampsia prophylaxis

Pre-eclampsia (PE) is a major cause of mortality and morbidity during pregnancy. Many different strategies to prevent PE have been investigated in randomized controlled trials, but none have been found to be widely effective and this is not surprising given the complexities in pathogenesis. Low-dose aspirin, diminishing platelet thromboxane synthesis, seems to prevent or delay PE. A meta-analysis of individual patient data from 32,217 women (PARIS collaborative group) asses that low-dose aspirin during pregnancy is associated with moderate reduction in the relative risk of PE (RR 0.90 95%CI 0.84–0.97), of birth before 34 weeks’ gestation (RR 0.90 95%CI 0.83–0.98), and of having a pregnancy with a serious adverse outcome (RR 0.90 95%CI 0.85–0.96). This effect seems to be more significant in women at high risk of developing PE. Regarding diet and nutrients, there does not appear to be any clear benefit for prevention of PE from routine dietary supplementation. There is high quality evidence that vitamin C and vitamin E are not effective for prevention or treatment of PE. Actually a meta-analyses found that supplementation with these antioxidants is associated with a slightly increased risk of gestational hypertension and premature rupture of membranes. Also there is not enough high-quality evidence to show that dietary magnesium or omega-3 fatty acids supplementation during pregnancy is beneficial. Calcium supplementation instead appears to reduce the risk of PE and the rare occurrence of the composite outcome ’death or serious morbidity’ specially in women with a low baseline calcium intake. Some studies assess that preeclamptic women may be deficient in nitric oxide, which mediates vasodilatation and inhibits platelet aggregation. For this reason NO donors, such as isosorbide dinitrate, glyceryl trinitrate and S-Nitroglutathione, were used in some trials as treatment in preeclamptic women. Despite the reduction in blood pressure and in uterine artery pulsatility index, these substances were rarely used in clinical practice because of adverse effects such as headache or preoxynitrite production. Also l -arginine, the substrate for synthesis of nitric oxide, has been investigated as a mean of preventing PE. Even though a dysfunction of the l -arginine-NO pathway that seems to be present in preeclamptic women, l -arginine load appears to be associated with increased nitric oxide (NO) production, hypotension and reduction of PE/eclampsia rate. Also in obese women there is a dysfunction of the l -arginine-NO pathway that causes a decrease of endothelial-dependent vasodilatation. In these women a l -arginine supplement could be helpful. Conclusion Aspirin proven to be of some help provided dose and timing are adequate. Impact on PE is limited, and its use is better in high-risk. Calcium supply could be helpful in population with low dietary intake. l -Arginine supplementation is worth to receive the same attention aspirin had also considering that biological activity seems wider. Actual evidences are more robust than the ones that allows wasting time and money for Vitamins C and E.

Response of human platelets to inhibition of thromboxane synthesis

Response of human platelets to inhibition of thromboxane synthesis

Erratum to: COXIBRAIN: results of the prospective, randomised, phase II/III study for the selective COX-2 inhibition in chronic subdural haematoma patients.

Chronic subdural haematomas (cSDHs) have shown an increasing incidence in an ageing population over the last 20 years, while unacceptable recurrence rates of up to 30 % persist. The recurrence rate of cSDH seems to be related to the excessive neoangiogenesis in the parietal membrane, which is mediated via vascular endothelial growth factor (VEGF). This is found to be elevated in the haematoma fluid and is dependent on eicosanoid/prostaglandin and thromboxane synthesis via cyclo-oxygenase-2 (COX-2). With this investigator-initiated trial (IIT) it was thought to diminish the recurrence rate of operated-on cSDHs by administering a selective COX-2 inhibitor (Celecoxib) over 4 weeks’ time postoperatively in comparison to a control group. The thesis of risk reduction of cSDH recurrence in COX-2-inhibited patients was to be determined in a prospective, randomised, two-armed, open phase-II/III study with inclusion of 180 patients over a 2-year time period in four German university hospitals. The treated- and untreated-patient data were to be analysed by Fisher’s exact test (significance level of alpha, 0.05 [two-sided]). After screening of 246 patients from January 2009 to April 2010, the study had to be terminated prematurely as only 23 patients (9.3 %) could be enrolled because of on-going non-steroid anti-rheumatic (NSAR) drug treatment or contraindication to Celecoxib medication. In the study population, 13 patients were treated in the control group (six women, seven men; average age 66.8 years; one adverse event (AE)/serious adverse event (SAE) needing one re-operation because of progressive cSDH (7.7 %); ten patients were treated in the treatment group (one woman, nine men; average age 64.7 years; five AEs/SAEs needing two re-operations because of one progressive cSDH and one wound infection [20 %]). Significance levels are obsolete because of insufficient patient numbers. The theoretical advantage of COX-2 inhibition in the recurrent cSDH could not be transferred into the treatment of German cSDH patients as 66.6 % of the patients showed strict contraindications for Celecoxib. Furthermore, 55 % of the patients were already treated with some kind of COX-2 inhibition and, nevertheless, developed cSDH. Thus, although conceptually appealing, an anti-angiogenic therapy with COX-2 inhibitors for cSDH could not be realised in this patient population due to the high prevalence of comorbidities excluding the administration of COX2 inhibitors.

GPVI and Thromboxane Receptor on Platelets Promote Proinflammatory Macrophage Phenotypes during Cutaneous Inflammation

Platelets are well known for their role in hemostasis but are also increasingly recognized for their supporting role in innate immune responses. Here, we studied the role of platelets in the development of peripheral inflammation and found that platelets colocalize with macrophages in the inflamed tissue outside of blood vessels in different animal models for cutaneous inflammation. Collagen-treatment of macrophages isolated from paws during zymosan-induced inflammation induced thromboxane synthesis through the platelet-expressed collagen receptor glycoprotein VI. Deletion of glycoprotein VI or its downstream effector thromboxane A2 receptor (TP) reduced zymosan-induced mechanical allodynia without altering macrophage recruitment or formation of macrophage/platelet complexes. Instead, macrophages in inflamed paws of glycoprotein VI- and TP-deficient mice exhibited an increased expression of anti-inflammatory markers and synthesized less proinflammatory mediators (prostaglandin E2 and IL6). TP expression on platelets was necessary to mediate increased prostaglandin E2 and IL6 synthesis, whereas TP expression on macrophages was sufficient to decrease the expression of the anti-inflammatory macrophage marker CD206, showing that TP activation on platelets and macrophages regulates different aspects of macrophage activation.

Effects of Aspirin on Endothelial Function and Hypertension.

Purpose of reviewEndothelial dysfunction is intimately related to the development of various cardiovascular diseases, including hypertension, and is often used as a target for pharmacological treatment. The scope of this review is to assess effects of aspirin on endothelial function and their clinical implication in arterial hypertension.Recent findingsEmerging data indicate the role of platelets in the development of vascular inflammation due to the release of proinflammatory mediators, for example, triggered largely by thromboxane. Vascular inflammation further promotes oxidative stress, diminished synthesis of vasodilators, proaggregatory and procoagulant state. These changes translate into vasoconstriction, impaired circulation and thrombotic complications. Aspirin inhibits thromboxane synthesis, abolishes platelets activation and acetylates enzymes switching them to the synthesis of anti-inflammatory substances.SummaryAspirin pleiotropic effects have not been fully elucidated yet. In secondary prevention studies, the decrease in cardiovascular events with aspirin outweighs bleeding risks, but this is not the case in primary prevention settings. Ongoing trials will provide more evidence on whether to expand the use of aspirin or stay within current recommendations.

Effects of Firocoxib, Flunixin Meglumine, and Phenylbutazone on Platelet Function and Thromboxane Synthesis in Healthy Horses.

Determine the effects of nonsteroidal anti-inflammatory drugs (NSAID) on platelet function and thromboxane synthesis immediately after drug administration and following 5 days of NSAID administration in healthy horses. Randomized cross-over study. Healthy adult horses (n=9; 6 geldings and 3 mares). Horses received either flunixin meglumine (1.1 mg/kg IV every 12 hours), phenylbutazone (2.2 mg/kg IV every 12 hours), or firocoxib (loading dose of 0.27 mg/kg IV on day 1, then 0.09 mg/kg IV every 24 hours for 4 days) for a total of 5 days. Blood samples were collected prior to drug administration (day 0), 1 hour after initial NSAID administration (day 1), and then 1 hour post-NSAID administration on day 5. Platelet function was assessed using turbidimetric aggregometry and a platelet function analyzer. Serum thromboxane B2 concentrations were determined by commercial ELISA kit. A minimum 14 day washout period occurred between trials. At 1 hour and 5 days postadministration of firocoxib, flunixin meglumine, or phenylbutazone, there was no significant effect on platelet aggregation or function using turbidimetric aggregometry or a platelet function analyzer. There was, however, a significant decrease in thromboxane synthesis at 1 hour and 5 days postadministration of flunixin meglumine and phenylbutazone that was not seen with firocoxib. Preoperative administration of flunixin meglumine, phenylbutazone, or firocoxib should not inhibit platelet function based on our model. The clinical implications of decreased thromboxane B2 synthesis following flunixin meglumine and phenylbutazone administration are undetermined.

Acetylsalicylic acid (ASA) - How much, how often, and when? A clinical-pharmacological perspective.

The dose of acetylsalicylic acid (ASA) commonly used in the prevention and treatment of arteriosclerotic angiopathies is equal to or less than 100 mg daily. This choice of dose is predominantly based on molecular-pharmacological findings showing an inhibition in synthesis of the prothrombotic thromboxane (TXB2) and an irreversible inhibition in blood platelet aggregation. However, an analysis of ASA dose-effect relationships for doses of 50 - 500 mg (PO and IV) shows that doses of ASA up to 100 mg daily produce only a small or moderate inhibition in collagen/epinephrine-induced platelet aggregation and have no significant effect on the important platelet factors, PF3 and PF4. Doses of ASA 300 - 500 mg, on the other hand, inhibit platelet aggregation almost completely and, in addition, produce a 50 - 70% inhibition in PF3 and PF4 lasting at least 24 hours. There is also evidence that doses of ASA above 100 mg daily markedly inhibit thromboxane synthesis for up to 24 hours and that doses of 500 mg daily produce a clinically relevant inhibition in platelet adhesion to vessel walls for up 72 hours and prevent procoagulatory shape changes for up to 12 hours. These findings suggest that a dose of ≥ 300 mg at intervals of 2 - 3 days would be more appropriate for primary and secondary prophylaxis of arteriosclerotic angiopathies and that the benefit-risk ratio would be greater because of the increased availability of mucoprotective prostaglandins, PGI2 (prostacyclin) and the gastroprotective, PGE2. Our viewpoint, predominantly based on findings with biomarkers, could serve as a basis for further randomized controlled studies.

Hypoxia and hyperoxia potentiate PAF receptor-mediated effects in newborn ovine pulmonary arterial smooth muscle cells: significance in oxygen therapy of PPHN.

Platelet‐activating factor (PAF) acting via its receptor (PAFR) is implicated in the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Effects of long‐term oxygen therapy on newborn lung are not well understood; therefore, we studied the effect of oxygen tension on ovine newborn pulmonary artery smooth muscle cells (NBPASMC). Our global hypothesis is that PPHN results from failure of newborn lamb pulmonary system to downregulate PAFR activity or to upregulate vasodilatory cyclic nucleotides (Cnucs) activity. NBPASMC from newborns 6–12 days old were studied in vitro at three different oxygen tensions (pO 2, [Torr]: hypoxia, <40; normoxia, 80–100; and hyperoxia, >100 Torr often clinically imposed upon newborns with PPHN). PAFR‐ and Cnucs mediated effects were determined. PAFR and PKA Cα mRNA expression as well as prostacyclin, thromboxane, cAMP production, and DNA synthesis was studied to assess PAFR‐mediated hypertrophy and/or hyperplasia. Hypoxia and hyperoxia increased specific PAFR binding. PAF treatment during hyperoxia increased PAFR gene, but decreased PKA‐Cα gene expression. Hypoxia and hyperoxia increased NBPASMC proliferation via PAFR signaling. Baseline prostacyclin level was ninefold greater than in fetal PASMC, whereas baseline thromboxane was sevenfold less suggesting greater postnatal cyclooxygenase activity in NBPASMC. PAF decreased, while forskolin and 8‐Br‐cAMP increased cAMP production. Decrease of PAFR effects by Cnucs indicates that normal newborn PA physiology favors vasodilator pathways to minimize PAF‐induced hypertrophy or hyperplasia. We speculate that failure of newborn lung to anchor downregulation of vasoconstrictors with upregulation of vasodilators leads to PPHN.

The Effects of Cyclosporine and Aspirin on Platelet Function in Normal Dogs.

BackgroundCyclosporine increases thromboxane synthesis in dogs, potentially increasing the thrombogenic properties of platelets.Hypothesis/ObjectivesOur hypothesis was that the concurrent administration of low‐dose aspirin and cyclosporine would inhibit cyclosporine‐associated thromboxane synthesis without altering the antiplatelet effects of aspirin. The objective was to determine the effects of cyclosporine and aspirin on primary hemostasis.AnimalsSeven healthy dogs.MethodsA randomized, crossover study utilized turbidimetric aggregometry and a platelet function analyzer to evaluate platelet function during the administration of low‐dose aspirin (1 mg/kg PO q24h), high‐dose aspirin (10 mg/kg PO q12h), cyclosporine (10 mg/kg PO q12h), and combined low‐dose aspirin and cyclosporine. The urine 11‐dehydro‐thromboxane‐B2 (11‐dTXB 2)‐to‐creatinine ratio also was determined.ResultsOn days 3 and 7 of administration, there was no difference in the aggregometry amplitude or the platelet function analyzer closure time between the low‐dose aspirin group and the combined low‐dose aspirin and cyclosporine group. On day 7, there was a significant difference in amplitude and closure time between the cyclosporine group and the combined low‐dose aspirin and cyclosporine group. High‐dose aspirin consistently inhibited platelet function. On both days, there was a significant difference in the urinary 11‐dTXB 2‐to‐creatinine ratio between the cyclosporine group and the combined low‐dose aspirin and cyclosporine group. There was no difference in the urinary 11‐dTXB 2‐to‐creatinine ratio among the low‐dose aspirin, high‐dose aspirin, and combined low‐dose aspirin and cyclosporine groups.Conclusions and Clinical ImportanceLow‐dose aspirin inhibits cyclosporine‐induced thromboxane synthesis, and concurrent use of these medications does not alter the antiplatelet effects of aspirin.

Escherichia coli induces platelet aggregation in an FcγRIIa‐dependent manner

The discovery of pathogen-recognition receptors such as Toll-like receptors on platelets has led to the emergence of the concept of platelets as important components of the host response to infection. Escherichia coli (E. coli)-mediated sepsis is a serious illness characterized by the occurrence of thrombocytopenia. Whereas there has been a wealth of research on platelet activation by Gram-positive bacteria, little is known about the mechanisms associated with Gram-negative bacteria-induced platelet activation with Gram-negative bacteria. To determine the mechanisms by which Gram-negative E. coli induces platelet aggregation. Induction of platelet aggregation with E. coli strain O157:H7 was tested in platelet-rich plasma (PRP), washed platelets, and serum depleted of complement factors. Platelet inhibitors (against αII b β3 , glycoprotein Ibα and FcγRIIa) were used. Platelet thromboxane synthesis was analyzed after E. coli stimulation. Cell binding assays were used to assess the ability of E. coli to support platelet adhesion. Trypsinization was used to determine the role of E. coli surface proteins. E. coli-induced aggregation in PRP was donor-dependent. E. coli O157:H7 induced aggregation with a lag time of 6.9 ± 1.3 min in an αII b β3 -dependent and FcγRIIa-dependent manner. Furthermore, this interaction was enhanced by the presence of complement, and was dependent on thromboxane synthesis. These results show E. coli to be a potent inducer of platelet aggregation.

Comparison of aspirin and indobufen in healthy volunteers

The aim of this study is to quantify the extent and recovery of platelet inhibition after administration of indobufen and aspirin in healthy volunteers. Indobufen inhibits platelet aggregation by reversibly inhibiting the platelet cyclooxygenase enzyme, thereby suppressing thromboxane synthesis. Twenty healthy volunteers completed the study and received aspirin (200 mg/day for 2 weeks) followed by a 4-week washout period and then indobufen (200 mg twice a day for 2 weeks). The percent (%) inhibition of platelet aggregation (IPA) was assessed using arachidonic acid (0.5 mg/ml) and adenosine diphosphate (5 µM) at 4, 12, 24 and 48 hours after last dose of each drug. IPA assessed using arachidonic acid as the agonist was similar at 4 hours after the last dose of indobufen (81.07 ± 9.36%) and aspirin (96.99 ± 0.29%, p = 0.10), but significantly lower at 12 hours (74.04 ± 9.55% vs. 97.94 ± 0.28%, p = 0.02), 24 hours (33.39 ± 11.13% vs. 97.48 ± 0.32%, p < 0.001) and 48 hours (14.12 ± 9.74% vs. 98.22 ± 0.31%, p < 0.001) after indobufen, compared to the relative values for aspirin. IPA assessed using adenosine diphosphate as the agonist was similar in the two groups at 4, 12 and 24 hours after the last dose, but significantly lower 48 hours after the last dose of indobufen, compared to the relative value for aspirin (1.98 ± 3.57% vs. 12.61 ± 2.71%, p = 0.002). Indobufen (200 mg twice a day) caused equivalent initial inhibition of platelet aggregation to aspirin (200 mg daily), and the anti-aggregation effect diminished faster than after aspirin.

The effect of kisspeptin on the regulation of vascular tone.

Kisspeptin has been implicated in cardiovascular control. Eicosanoids play a crucial role in the activation of platelets and the regulation of vascular tone. In the present study, we investigated the effect of kisspeptins on eicosanoid synthesis in platelets and aorta in vitro. Platelets and aorta were isolated from Wistar-Kyoto rats. After preincubation with different doses of kisspeptin, samples were incubated with [1-(14)C]arachidonic acid (0.172 pmol/mL) in tissue culture Medium 199. The amount of labeled eicosanoids was measured with liquid scintillation, after separation with overpressure thin-layer chromatography. Kisspeptin-13 stimulated the thromboxane synthesis. The dose-response curve was bell-shaped and the most effective concentration was 2.5 × 10(-8) mol/L, inducing a 27% increase. Lipoxygenase products of platelets displayed a dose-dependent elevation up to the dose of 5 × 10(-8) mol/L. In the aorta, kisspeptin-13 induced a marked elevation in the production of 6-keto-prostaglandin F1α, the stable metabolite of prostacyclin, and lipoxygenase products. Different effects of kisspeptin on cyclooxygenase and lipoxygenase products indicate that beyond intracellular Ca(2+) mobilization, other signaling pathways might also contribute to its actions. Our data suggest that kisspeptin, through the alteration of eicosanoid synthesis in platelets and aorta, may play a physiologic and (or) pathologic role in the regulation of vascular tone.

The Advanced Lipoxidation end Product Precursor Malondialdehyde Induces IL-17E Expression and Skews Lymphocytes to the th17 Subset.

Malondialdehyde (MDA) is a highly reactive endogenous product of thromboxane synthesis in the prostagland and lipid peroxidation by reactive oxygen species. Elevated MDA levels occur in diabetes and atherosclerotic plaques. The aim of this study was to examine the molecular mechanisms of MDA-induced IL-17E cytokine expression and its effect on T-cell differentiation. Real-time PCR, RT-PCR and ELISA were used to assess the expression of IL-17 family cytokines in Jurkat T-cells and human peripheral blood lymphocytes (PBLCs) from diabetic subjects. Luciferase reporter assays were used for the promoter activation study. Pharmacological inhibitors were used for signaling pathway experiments. FACS analyses were used to measure the Th1, Th2 and Th17 subset levels. MDA induced significant (2- to 3-fold; p < 0.01) generation of IL-17E mRNA in a dose- and time-dependent manner in Jurkat T-cells and PBLCs. Elevated IL-17E mRNA levels were found in the lymphocytes from diabetic subjects. The increased IL-17E protein and mRNA levels correlate well with serum MDA levels from diabetic patients. Transient transfection of plasmid containing the minimum IL-17E promoter region (pIL-17E-Luc) showed a significant (2-fold; p < 0.01) increase in luciferase activity. Pretreatment of lymphocytes with pharmacological inhibitors showed the involvement of antioxidant, NF-ƙB, p38MAPK, PKC and ERK signaling pathways. Quantification of the Th1, Th2 and Th17 cell population in PBLCs via FACS analyses revealed an increase in the Th17 subset. These results show that MDA transcriptionally upregulates the expression of IL-17E in lymphocytes and alters lymphocyte differentiation towards the pathogenic Th17 subset.

Abstract 12357: Enzyme Kinetic Model Reveals Novel Characteristics of Pharmacodynamic Interaction of NSAIDs and Aspirin at COX-1, Which Lead to High on Aspirin Platelet Reactivity (HAPR)

Introduction: Non-aspirin NSAIDs attenuate the antiplatelet action of aspirin leading to High on Aspirin Platelet Reactivity (HAPR). However, detailed features of this interaction are still unresolved. Both competitive as well as non-competitive interaction at the cyclooxygenase-1 (COX-1) enzyme have been proposed. Hypothesis: We examined whether competitive enzyme kinetics describe COX-1 inhibition by aspirin and HAPR and if they are compatible with results obtained experimentally. Methods: We have developed an enzyme kinetic model of the aspirin/NSAID interaction at COX-1 using mathematical modelling and have varied enzyme kinetic parameters to predict the occurrence and the extent of HAPR. Subsequently, the predictions obtained were experimentally verified with selected NSAIDs using light transmission aggregometry and immunoassay for platelet thromboxane (TX) synthesis. Results: Enzyme kinetic modelling predicted that only low- and medium-affinity NSAIDs (Ki ≥1 μM), but not high-affinity NSAIDs, interfere with COX-1 inhibition by aspirin. Furthermore, the interaction was predicted to be most extensive at high concentrations of arachidonic acid (1mM). Experimental results confirmed the model by showing that low or medium-affinity COX-1 inhibitors piroxicam, dipyrone and celecoxib (each n=5-10) largely prevented the inhibition of aggregation and platelet TX synthesis in vitro by 30 μmol/L aspirin whereas the high-affinity COX-1 inhibitor SC560 (n=6) did not interfere with aspirin at all. In accordance to the mathematical model, aspirin/NSAID interaction was more pronounced in presence of high (1mmol/L) versus low (0.3mmol/L) concentration of arachidonic acid. Hence, the kinetic model was validated experimentally. Conclusion: Both enzyme kinetic modelling and experimental results suggest competitive antagonism of NSAID and aspirin at COX-1 as a cause for HAPR, possibly resulting in cardiovascular events. Critical parameters that influence this interaction are drug concentrations and NSAID affinity. Interestingly, the concentration of arachidonic acid also influences the NSAID/aspirin interaction, suggesting that “fatty acid tone” and lipid metabolism may influence this unfavourable drug/drug interaction.

The prostaglandin synthase 2/cyclooxygenase 2 (PTGS2/ COX2) rs5277 polymorphism does not influence risk of colorectal cancer in an Iranian population.

The prostaglandin-endoperoxide synthase 2 [PTGS2, commonly known as cyclooxygenase-2 (COX-2)] is an enzyme induced by proinflammatory stimuli that is often overexpressed in malignant tissue and involved in the synthesis of prostaglandins and thromboxanes, regulators of processes such as inflammation, cell proliferation, and angiogenesis, all relevant for cancer development. We investigated whether a functional genetic polymorphism, rs5277, in COX-2 may have a risk-modifying effect on sporadic colorectal cancer in an Iranian population. We conducted a case-control study on 167 patients with colorectal cancer and 197 cancer-free controls in Taleghani Hospital in Tehran, Iran, between 2007 and 2011. Peripheral blood samples of both groups were processed for DNA extraction and genotyping of the COX-2 gene polymorphism (rs5277) using PCR-RFLP. RFLP results were confirmed by direct sequencing. Logistic regression analysis was performed to calculate the adjusted odds ratio (OR) and 95% confidence interval (95% CI). There was no significant difference in the distribution of COX-2 gene rs5277 polymorphism genotype and the allelic form, among CRC patients compared with the healthy control group (p: 0.867). Our results suggest that rs5277 polymorphism in COX2 could not be a good prognostic indicator for patients with CRC.

Enhanced chemopreventive effects of a hydrogen sulfide-releasing anti-inflammatory drug (ATB-346) in experimental colorectal cancer

Regular use of nonsteroidal anti-inflammatory drugs is associated with a significantly lower incidence of several types of cancer, particularly those affecting the gastrointestinal tract. However, the propensity of these drugs to cause ulcers and bleeding in the stomach and small intestine limits their utility for chemoprevention of cancer. In the present study, we evaluated the effectiveness of a novel hydrogen sulfide-releasing derivative of naproxen in reducing the incidence of pre-cancerous lesions (aberrant crypt foci) in mice treated with the carcinogen azoxymethane. Weekly administration of azoxymethane over a 4-week period resulted in formation of an average of ∼50 aberrant crypt foci in the colon. Twice-daily treatment with naproxen at high doses significantly reduced the number of aberrant crypt foci. However, a significantly greater effect was observed with ATB-346 (H2S-releasing naproxen) and it was also effective at much lower doses, where naproxen was ineffective. The H2S-releasing moiety of ATB-346 did not significantly affect the number of aberrant crypt foci, suggesting that both the inhibition of cyclooxygenase activity and release of H2S were necessary for the enhanced chemopreventative effect. ATB-346 suppressed colonic prostaglandin synthesis and whole blood thromboxane synthesis as effectively as naproxen, but did not induce any gastrointestinal injury. These results demonstrate that ATB-346 exerts superior chemopreventive effects to those of naproxen, while sparing the gastrointestinal tract of the injury normally associated with use of the parent drug. ATB-346 may therefore be an attractive agent for chemoprevention of colon cancer, and possibly of cancers in other tissues.

Two lipids in the diet, rather than cholesterol, are responsible for heart failure and stroke

Heart failure is one of the major causes of death throughout the world. For over 65 years, it has been thought that cholesterol in the plasma was responsible for heart failure. I have never believed that cholesterol causes heart failure. This perspective presents evidence that it is oxidized cholesterol and trans fat in the diet that are the causes. It is these two lipids in the diet, rather than cholesterol in the plasma, that are responsible for heart failure. The oxysterols change the structure of the coronary arteries so that they are more susceptible to calcification and increase the synthesis of thromboxane. The trans fatty acids inhibit prostacyclin synthesis that cause the blood to clot. I believe that removing these two lipids from the diet would prolong the lives of hundreds of thousands of people annually.

Over-expression of COX-2 mRNA in colorectal cancer

BackgroundCyclooxygenase-2 (COX-2, PTGS2) is an enzyme involved in the synthesis of prostaglandins and thromboxanes, which are regulators of biologic processes such as inflammation, cell proliferation and angiogenesis. COX-2 over-expression was reported in many (pre) malignant tissues, but data strongly vary and seem to depend on the methodology used.MethodsNormal colorectal mucosa and paired cancerous tissue from 60 patients with colorectal cancer was investigated for the levels of COX-2 mRNA by real-time quantitative Polymerase Chain Reaction (qPCR). COX-2 levels were expressed relative to either: tissue weight or levels of the housekeeping genes beta-2 microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).ResultsCOX-2 mRNA levels, normalized with respect to tissue weight or mRNA levels of the housekeeping genes B2M or GAPDH, were over-expressed in 80%, 70% and 40% of the colorectal tumor tissues, as compared to the paired adjacent normal colorectal mucosa samples, respectively. Highest mRNA COX-2 ratios tumor/normal were measured when expressed per mg tissue (mean ratio 21.6). When normalized with respect to the housekeeping genes B2M or GAPDH, mean tumor/normal ratios were 16.1 and 7.5, respectively.ConclusionExpression of COX-2 mRNA levels per mg tissue is most simple in comparison to normalization with respect to the housekeeping genes B2M or GAPDH. Levels of COX-2 mRNA are found over-expressed in almost 80% of the colorectal tumors, compared to paired adjacent normal colorectal mucosa, suggesting a role of COX-2 as a potential biomarker for cancer risk, whereas inhibitors of COX-2 could be of value in chemoprevention of colon cancer.