Thromboxane Formation Research Articles

Efficacy and speed of effect after the first dose of aspirin in children with congenital heart disease.

Many paediatric studies report that patients must be established on aspirin therapy for a minimum of 5 days to achieve adequate response. This is not always practical especially in critical settings. Prospective identification of patients that are unresponsive to aspirin sooner could potentially prevent thrombotic events. The aim of this study was to investigate prospectively if the first dose of aspirin is effective in decreasing platelet aggregation, and thromboxane formation and if this can be measured after 2 hours in paediatric cardiology patients. A secondary aim was to identify a cut-off for a novel marker of aspirin responsiveness the maximum amplitude with arachidonic acid, which could potentially dramatically reduce the blood volume required. Third, we aimed to prospectively identify potentially non-responsive patients by spiking a sample of their blood ex vivo with aspirin. The majority (92.3%) of patients were responsive, when measured 2 hours post first dose of aspirin. Non-response or inadequate response (7.7%) can also be identified at 2 hours after taking the first dose of aspirin. Additionally, we have shown a novel way to reduce blood sample volume requirements by measurement of the maximum amplitude with arachidonic acid as a marker of response, particularly for monitoring. These findings of rapid efficacy in the majority of patients offer assurance in a sound, practical way to attending clinicians, patients, and families.

Elucidation of the Role of TRPV1, VEGF-A, TXA2, Redox Homeostasis, and Inflammatory Cascades in Protection against Cold Injuries by Herbosomal-Loaded PEG-Poloxamer Topical Formulation.

High-altitude regions, cold deserts, permafrost regions, and the polar region have some of the severest cold conditions on earth and pose immense perils of cold injuries to exposed individuals. Accidental and unintended exposures to severe cold, either unintentionally or due to occupational risks, can greatly increase the risk of serious conditions including hypothermia, trench foot, and cold injuries like frostbite. Cold-induced vasoconstriction and intracellular/intravascular ice crystal formation lead to hypoxic conditions at the cellular level. The condition is exacerbated in individuals having inadequate and proper covering and layering, particularly when large area of the body are exposed to extremely cold environments. There is a paucity of preventive and therapeutic pharmacological modalities that have been explored for managing and treating cold injuries. Given this, an efficient modality that can potentiate the healing of frostbite was investigated by studying various complex pathophysiological changes that occur during severe cold injuries. In the current research, we report the effectiveness and healing properties of a standardized formulation, i.e., a herbosomal-loaded PEG-poloxamer topical formulation (n-HPTF), on frostbite. The intricate mechanistic pathways modulated by the novel formulation have been elucidated by studying the pathophysiological sequelae that occur following severe cold exposures leading to frostbite. The results indicate that n-HPTF ameliorates the outcome of frostbite, as it activates positive sensory nerves widely distributed in the epidermis transient receptor potential vanilloid 1 (TRPV1), significantly (p < 0.05) upregulates cytokeratin-14, promotes angiogenesis (VEGF-A), prominently represses the expression of thromboxane formation (TXA2), and significantly (p < 0.05) restores levels of enzymatic (glutathione reductase, superoxide dismutase, and catalase) and nonenzymatic antioxidants (glutathione). Additionally, n-HPTF attenuates oxidative stress and the expression of inflammatory proteins PGF-2α, NFκB-p65, TNF-α, IL-6, IL-1β, malondialdehyde (MDA), advanced oxidative protein products (AOPP), and protein carbonylation (PCO). Masson's Trichrome staining showed that n-HPTF stimulates cellular proliferation, and increases collagen fiber deposition, which significantly (p < 0.05) promotes the healing of frostbitten tissue, as compared to control. We conclude that protection against severe cold injuries by n-HPTF is mediated via modulation of pathways involving TRPV1, VEGF-A, TXA2, redox homeostasis, and inflammatory cascades. The study is likely to have widespread implications for the prophylaxis and management of moderate-to-severe frostbite conditions.

Abstract P4-03-02: Low-dose aspirin prescriptions and breast cancer recurrence: a Danish nationwide cohort study with up to 23 years of follow-up

Abstract Background Low-dose aspirin irreversibly inhibits the cyclooxygenase enzymes COX-1 and COX-2, which catalyze the formation of prostaglandins and thromboxanes, inhibiting platelet aggregation. Platelets are thought to play a role in tumorigenesis. As such, aspirin may have a beneficial effect on breast cancer prognosis, but results to date are conflicting. Objectives To evaluate the association between aspirin use and breast cancer recurrence up to 23 years after primary diagnosis. Methods We included all women diagnosed with non-metastatic breast cancer during 1996-2004 registered in the Danish Breast Cancer Group (DBCG) clinical database. We obtained information on aspirin prescriptions (&amp;gt;= 2 prescriptions filled between cancer diagnosis and the landmark) from the Danish National Prescription Registry. Information on early and late (&amp;gt;10 years after primary diagnosis) recurrence was obtained using the DBCG database (for recurrence within the first 10 years after diagnosis) and for late recurrence via a previously described and validated algorithm, drawing on data from the Danish National Patient Registry, the Danish Pathology Registry, the Danish Cancer Registry and a contralateral breast cancer database. We used Cox regression to compute crude and adjusted hazard ratios (HRs) with 95% confidence intervals (CI) employing landmark analyses starting follow-up at years 5, 10, 15, and 20 after primary diagnosis. We adjusted for potential confounders including calendar year of diagnosis, menopausal status, comorbidities, estrogen receptor status, clinical stage, grade, surgery type, chemo- and endocrine therapy, diagnostic codes for alcoholism and obesity, as well as co-medications (angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, statins, bisphosphonates, metformin, digoxin, hormone replacement therapy, non-steroidal anti-inflammatory drugs, and vitamin K antagonists). We followed patients until recurrence, death, second cancer, loss to follow-up, or 31 December 2018. Results Among 21,684 women with non-metastatic breast cancer, 4,902 experienced recurrence during 242,427 person-years of follow-up. Our landmark analyses showed a reduced hazard of recurrence in the 5-, 10-, and 15-year landmark cohorts (5-year landmark adjusted HR = 0.80, 95% CI = 0.68, 0.94; 10-year landmark adjusted HR = 0.84, 95% CI = 0.71, 1.00, 15-year landmark adjusted HR = 0.83, 95% CI = 0.61, 1.14). The 20-year landmark analysis revealed an adjusted HR of 1.09 (95% CI = 0.49, 2.46). Conclusions The potential anti-cancer effect of aspirin appears most pronounced in the first 15 years after breast cancer diagnosis. Funding Elisabeth Solmunde is funded by the Danish Cancer Society (R320-A18464-B5768) and the Independent Research Fund Denmark (1149-00013B). This work was supported by grants to Deirdre Cronin-Fenton from the Danish Cancer Society (“Knæk Cancer” R147-A10100). Citation Format: Elisabeth Solmunde, Rikke N Pedersen, Mette Nørgaard, Lene Mellemkjær, Søren Friis, Bent Ejlertsen, Thomas P. Ahern, Deirdre Cronin-Fenton. Low-dose aspirin prescriptions and breast cancer recurrence: a Danish nationwide cohort study with up to 23 years of follow-up [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-03-02.

Drug-Drug Interaction between Antiplatelet Therapy and Lipid-Lowering Agents (Statins and PCSK9 Inhibitors).

Lipid-lowering agents and antiplatelet drugs are guideline-recommended standard treatment for secondary prevention of acute thrombotic events in patients with increased cardiovascular risk. Aspirin is the most frequently used antiplatelet drug, either alone or in combination with other antiplatelet agents (P2Y12 inhibitors), while statins are first-line treatment of hypercholesterolemia. The well-established mode of action of aspirin is inhibition of platelet-dependent thromboxane formation. In addition, aspirin also improves endothelial oxygen defense via enhanced NO formation and inhibits thrombin formation. Low-dose aspirin exerts in addition anti-inflammatory effects, mainly via inhibition of platelet-initiated activation of white cells.Statins inhibit platelet function via reduction of circulating low-density lipoprotein-cholesterol (LDL-C) levels and a more direct inhibition of platelet function. This comprises inhibition of thromboxane formation via inhibition of platelet phospholipase A2 and inhibition of (ox)LDL-C-mediated increases in platelet reactivity via the (ox)LDL-C receptor (CD36). Furthermore, statins upregulate endothelial NO-synthase and improve endothelial oxygen defense by inhibition of NADPH-oxidase. PCSK9 antibodies target a serine protease (PCSK9), which promotes the degradation of the LDL-C receptor impacting on LDL-C plasma levels and (ox)LDL-C-receptor-mediated signaling in platelets similar to but more potent than statins.These functionally synergistic actions are the basis for numerous interactions between antiplatelet and these lipid-lowering drugs, which may, in summary, reduce the incidence of atherothrombotic vascular events.

Different forms of vitamin E and metabolite 13’-carboxychromanols inhibit cyclooxygenase-1 and its catalyzed thromboxane in platelets, and tocotrienols and 13’-carboxychromanols are competitive inhibitors of 5-lipoxygenase

Cyclooxygenase (COX-1 and COX-2)- and 5-lipoxygenase (5-LOX)-catalyzed biosynthesis of eicosanoids play important roles in inflammation and chronic diseases. The vitamin E family has four tocopherols and tocotrienols. We have shown that the metabolites of δ-tocopherol (δT) and δ-tocotrienol (δTE), i.e., δT-13’-carboxychromanol (COOH) and δTE-13’-COOH, respectively, inhibit COX-1/-2 and 5-LOX activity, but the nature of how they inhibit 5-LOX is not clear. Further, the impact of tocopherols and tocotrienols on COX-1/-2 or 5-LOX activity has not been fully delineated. In this study, we found that tocopherols and tocotrienols inhibited human recombinant COX-1 with IC50s of 1–12 µM, and suppressed COX-1-mediated formation of thromboxane in collagen-stimulated rat's platelets with IC50s of 8–50 µM. None of the vitamin E forms directly inhibited COX-2 activity. 13’-COOHs inhibited COX-1 and COX-2 enzyme activity with IC50s of 3–4 and 4–10 µM, respectively, blocked thromboxane formation in collagen- and ionophore-stimulated rats’ platelets with IC50s of 1.5-2.5 µM, and also inhibited COX-2-mediated prostaglandins in stimulated cells. Using enzyme kinetics, we observed that δT-13’-COOH, δTE-13’-COOH and δTE competitively inhibited 5-LOX activity with Ki of 1.6, 0.8 and 2.2 µM, respectively. These compounds decreased leukotriene B4 from stimulated neutrophil-like cells without affecting translocation of 5-LOX from cytosol to the nucleus. Our study reveals inhibitory effects of vitamin E forms and 13’-COOHs on COX-1 activity and thromboxane formation in platelets, and elucidates mechanisms underlying their inhibition of 5-LOX. These observations are useful for understanding the role of these compounds in disease prevention and therapy.

Garlic inhibitory effect on platelet activity induced by different agonists

&#x0D; Platelets are essential elements of human blood. In addition to their normal role, platelets are involved in causing myocardial infarction, stroke and other thrombotic disorders. Platelet activation in vivo, probably involves a combination of agonists. Garlic has beneficial effects due to its ability to inhibit platelet aggregation and thromboxane formation. The aim of this work was to evaluate the ability of garlic extracts to inhibit platelet aggregation induced by different agonists and their mixtures in different donors. Significant differences were found in platelet aggregation in response to each agonist (P ≤ 0.05). The highest antiaggregatory effect was observed with arachidonic acid and the lowest effect with collagen-arachidonic acid mixture. Interaction effects between donor and agonist (or mixtures) were detected. The study showed the potential of aqueous garlic extracts to prevent platelet aggregation induced by different agonist.&#x0D; Highlights&#x0D; &#x0D; Platelets play a central role in the progression of atherosclerotic lesions.&#x0D; Blood from nine non-smoker healthy donors was used for in vitro platelet aggregation study.&#x0D; Significant differences were found in platelet aggregation in response to each agonist.&#x0D; Aqueous garlic extracts could prevent platelet aggregation induced by different agonist.&#x0D;

Brain-Derived Neurotrophic Factor Induces Platelet Aggregation

Background: A large extra-cerebral pool of Brain-Derived Neurotrophic Factor (BDNF) is found in platelets. With concentrations reaching 100-1000 fold those of neurons, platelet-stored BDNF is hypothesized to play an important role in the vasculature.Aims: To investigate the effect of BDNF on platelets, alone and synergistically with traditional agonists.Methods: The presence of BDNF in platelets was confirmed by Western Blotting and confocal microscopy. The ability of platelets to release BDNF upon stimulation was assessed by ELISA on platelet releasates following stimulation by maximal concentrations of arachidonic acid, adenosine diphosphate (ADP), epinephrine, collagen, or thrombin-receptor activating peptide (TRAP). To investigate the effect of BDNF on platelet function, we used a recombinant BDNF protein (rBDNF, Peprotech) added exogenously to a preparation of washed platelets obtained from healthy volunteers. Aggregation was recorded by light transmission aggregometry over 20 minutes. Synergistic assays were used with priming (sub-threshold) concentrations of either collagen or TRAP. The dependence on secondary mediators was assessed by blocking either COX-1-dependent thromboxane formation with aspirin or the ADP-P2Y12 receptor with AR-C66096. Platelet aggregation was blocked with a GPIIbIIIa inhibitor (abciximab) to assess the contribution of outside-in signalling.Results: Western blotting showed a 14kD band for the washed platelet protein preparations and for rBDNF used as a positive control. BDNF appeared stored in granules on confocal microscopy, consistent with previous reports. BDNF in the platelet releasate was increased 5-fold following platelet aggregation induced by traditional agonists, as compared with baseline. Exogenous rBDNF had no effect on platelet aggregation at low doses (1 and 3 µg/ml), but induced complete biphasic platelet aggregation at a high dose (10 µg/ml). The secondary wave of platelet aggregation was abrogated in the presence of aspirin and AR-C66096, but they had no effect on the primary wave. Platelet aggregation was completely abolished in the presence of the GPIIbIIIa inhibitor abciximab. Synergistic studies were conducted with priming concentrations of rBDNF, collagen and TRAP which were insufficient to elicit platelet aggregation on their own. Incubation of platelets with rBDNF (3 µg/ml) potentiated the platelet responses to priming concentrations of both collagen and TRAP. Indeed, in the absence of BDNF, no platelet aggregation was seen at the concentrations used, whereas incubation with BDNF led to complete and irreversible platelet aggregation to priming concentrations of collagen and TRAP.Conclusion: Platelets contain BDNF within their granules, which they release upon platelet activation with traditional agonists. Stimulation of washed platelets with high concentrations of rBDNF induced platelets to aggregate; in the presence of low concentrations of rBDNF, platelet aggregation was potentiated in response to priming concentrations of traditional agonists. Platelet responses to rBDNF were partly dependent on secondary mediators, as inhibition of COX-1 and the ADP-P2Y12 receptor inhibited the secondary wave of aggregation. However, the primary wave of aggregation was unaltered, thereby suggesting that platelets either possess a BDNF receptor or a traditional receptor that can signal in the presence of BDNF. Further studies are needed to uncover which receptor is implicated in platelet responses to BDNF, and the physiological role of BDNF-induced platelet responses. DisclosuresNo relevant conflicts of interest to declare.

Study on the Mechanism of Atropine in the Treatment of Septic Shock

Atropine (atropinol) is a typical anticholinergic drug that competes for M receptors. Large doses of atropine can expand blood vessels, relieve small vasospasm, improve microcirculation as well as cardiovascular function, and inhibit thromboxane formation. It is an ideal drug for the treatment of septic shock that are worthy of clinical application. In recent years, studies have found that the main mechanisms of atropine's anti-infective shock include inhibiting calcium ion overload, improving microcirculation disorders, inhibiting thromboxane formation, reducing the release of inflammatory mediators, and inhibiting the release of glutamate. The research progress of the pharmacological effects and mechanisms of atropine in the treatment of septic shock is summarized, and it provides a basis for the in-depth study of atropine.

Beneficial Modulation of Lipid Mediator Biosynthesis in Innate Immune Cells by Antirheumatic Tripterygium wilfordii Glycosides.

Tripterygium wilfordii glycosides (TWG) is a traditional Chinese medicine with effectiveness against rheumatoid arthritis (RA), supported by numerous clinical trials. Lipid mediators (LM) are biomolecules produced from polyunsaturated fatty acids mainly by cyclooxygenases (COX) and lipoxygenases (LOX) in complex networks which regulate inflammation and immune responses and are strongly linked to RA. The mechanism by which TWG affects LM networks in RA treatment remains elusive. Employing LM metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry revealed striking modulation of LM pathways by TWG in human monocyte-derived macrophage (MDM) phenotypes. In inflammatory M1-MDM, TWG (30 µg/mL) potently suppressed agonist-induced formation of 5-LOX products which was confirmed in human PMNL and traced back to direct inhibition of 5-LOX (IC50 = 2.9 µg/mL). TWG also efficiently blocked thromboxane formation in M1-MDM without inhibiting other prostanoids and COX enzymes. Importantly, in anti-inflammatory M2-MDM, TWG (30 µg/mL) induced pronounced formation of specialized pro-resolving mediators (SPM) and related 12/15-LOX-derived SPM precursors, without COX and 5-LOX activation. During MDM polarization, TWG (1 µg/mL) decreased the capacity to generate pro-inflammatory 5-LOX and COX products, cytokines and markers for M1 phenotypes. Together, suppression of pro-inflammatory LM but SPM induction may contribute to the antirheumatic properties of TWG.

Persistent long-term platelet activation and endothelial perturbation in women with Takotsubo syndrome

BackgroundTakotsubo (TTS) syndrome is an acute cardiac condition characterized by transient and reversible left ventricle dysfunction that mainly affects postmenopausal women. Catecholamine burst is the most accredited mechanism underpinning TTS onset and leading to endothelial dysfunction and platelet activation. Even if the use of low dose acetylsalycilic acid (ASA) in this clinical setting is based on both clinical presentation and unfavorable long-term prognosis, its efficacy has been recently challenged. AimThis study was designed to assess endothelial function, residual thromboxane formation and platelet aggregation in TTS women on low-dose ASA treatment at long-term follow-up. MethodsTwenty-eight females with previously diagnosis of TTS syndrome were enrolled. Data were compared to those obtained from 23 coronary artery disease (CAD) women with a history of acute myocardial infarction, and 26 control subjects with no TTS or clinically evident CAD. Psychological and clinical profile were assessed in all study groups at the enrollment. Main metabolites involved in L-arginine/nitric oxide pathway, urinary prostacyclin, serum and urine thromboxane metabolites were measured by LCMS/MS methods. Thrombomodulin levels were quantified using an ELISA kit, and platelet aggregation, carried out on platelet rich-plasma, was induced by ADP or by epinephrine (EPI), norepinephrine (NORE) and TRAP-6, alone or in association with ADP and evaluated by Born’s method. ResultsIn TTS women an endothelial derangement, characterized by reduced citrulline production and increased thrombomodulin concentration, with no perturbation in prostacyclin levels, was evidenced. In addition, despite ASA treatment, TTS displayed a higher residual thromboxane formation, in parallel with an enhanced platelet response to compared to CAD. ConclusionsOur study highlighted the presence of endothelial perturbation in TTS patients even at long-term from the index event. The residual thromboxane production and platelet aggregation still leave open the question about the use of low dose ASA in this clinical setting.

Endothelial dysfunction and its role in the prevention, treatment and complications of critical conditions in obstetrics

Background. Preeclampsia (PE) remains the leading cause of maternal and perinatal morbidity and mortality. As of today, there is no treatment for PE, and the childbirth is the only way to completely eliminate this condition. However, early childbirth is associated with a high risk of fetal morbidity and mortality.&#x0D; Objective. To describe the role of endothelial dysfunction (ED) in the development of PE and the possibility of its correction; to evaluate the effectiveness of Tivortin (“Yuria-Pharm”) in the correction of uterine-fetal-placental dysfunction.&#x0D; Materials and methods. Analysis of literature sources on this topic; own PLACENTA study. The study involved 535 women with mild PE at 26-29 weeks of gestation. Group I received standard therapy, group II – standard therapy in combination with Tivortin (4.2 g per day for 7-10 days).&#x0D; Results and discussion. Until the fetus reaches viability, PE treatment is aimed at correcting hypertension, however, antihypertensive therapy does not have a beneficial effect on the condition of the kidneys and placental blood flow. Decreased blood flow in placenta leads to the syndrome of limited fetal development (SLFD) and multisystem ED. The development of PE is inextricably linked with the systemic damage to maternal endothelial cells and a further decrease in endothelium-dependent vasodilators, which promotes vasospasm and activates the coagulation cascade. PE is accompanied by the imbalance in the systems of prostacyclin and nitric oxide (NO), as well as thromboxane and endothelin. Superficial placentation with insufficient remodeling of the spiral arteries and impaired response to changes in blood flow is one of the main causes of PE. ED is the most studied pathogenetic mechanism of PE. NO has a potent vasodilating effect and is involved in inhibiting thromboxane formation, platelet aggregation and stimulating prostacyclin formation. NO-synthase of endothelial cells, the substrate of which is L-arginine, is the main enzyme of NO production. Disorders of vasodilation in pregnant women with PE can be eliminated by prescribing L-arginine. Literature data show that L-arginine is successfully used in the need to continue pregnancy, as well as to eliminate hypertension and correct SLFD. Canadian guidelines for the treatment of hypertensive complications of pregnancy state that L-arginine is able to improve uteroplacental blood flow. According to a study by J. Chen (2016), the inclusion of L-arginine contributes to both maternal and perinatal outcomes of pregnancy. The effect of L-arginine on fetal growth is mediated by promoting the production of creatine, skeletal muscle protein and growth hormone. L-arginine also promotes the synthesis of polyamines, which can stimulate the growth and development of placenta. Own studies have shown that the use of L-arginine (Tivortin, “Yuria-Pharm”) in the combined PE therapy had improved the growth rate of the fetus and its functional condition, which allowed to prolong pregnancy to the optimal term of childbirth, as well as improve the consequences of childbirth and adaptation of newborns in the early neonatal period. PE is not the only field of application of Tivortin: its intravenous administration in community-acquired pneumonia and pyelonephritis of pregnant women helps to quickly eliminate the pathological process. The results of the PLACENTA study found that Tivortin helped to improve uterine-fetal-placental blood flow and reduce the centralization of blood circulation. In the Tivortin group there was a smaller number of cesarean sections, a tendency to lower blood loss (by 19.5 %), a higher average score of newborns on the Apgar scale. Comparison of clinical and morphological changes showed that the administration of Tivortin in the late second and early third trimester of pregnancy reduces the likelihood of premature placental maturation by 2.1 %, halves the likelihood of placental necrosis, 10 times reduces the signs of hypoxia.&#x0D; Conclusions. 1. ED, caused by a decrease in NO, is the main link in the PE pathogenesis. 2. In the human body NO is produced from L-arginine. 3. The use of L-arginine (Tivortin) improves uterine-fetal-placental blood flow, growth rate and functional status of the fetus, reduces fetal distress and allows to prolong pregnancy to the optimal term of childbirth.

Thromboxane Synthesis and Platelet Protein Release During Simulated Extracorporeal Circulation

Platelet secretion induced by certain soluble aggregating agents is associated with thromboxane formation. Activation of platelets by artificial surfaces may involve similar biochemical pathways. Therefore, we monitored platelet release and formation of thromboxane B2. the stable end-product of thromboxane A2, during simulated extracorporeal circulation where contact between blood and synthetic surfaces is extensive. Fresh heparinized human blood was recirculated for 2 hr at 37°C at 1 liter/min in circuits (0.95 sqm) constructed of standard silicone rubber components (0.1 sqm) and a spiral coil membrane oxygenator (0.85 sqm). Within 2 min of recirculation, plasma levels of the platelet-specific protein, low-affinity platelet factor 4 (LA-PF4) rose from <1.5% to 11 % ±3% SEM of that which was released by triton X-100, indicating significant release of platelet α-granule contents. This occurred despite the absence of detectable thromboxane B2 in plasma (<0.5 pmole/ml). Between 2 and 15 min, plasma levels of LA-PF4 increased from 11% to 42% ± 3%, and plasma thromboxane B2 concentrations rose to 3.2 pmole/ml. Subsequently, thromboxane B2 levels continued to rise to 9.8 pmole/ml by 2 hr. Thromboxane B2 was not detected in blood from donors who had ingested aspirin, and the extent of platelet secretion was reduced by about 50% (25% at 2 hr). Thus, although a component of platelet granule release during extracorporeal recirculation appears to be associated with thromboxane synthesis, considerable release of LA-PF4 may occur by thromboxanβ-indβpen-dent pathways.

Role of Cyclooxygenase Pathway and Risk Associated with Non-Steroidal Anti-inflammatory Drugs Therapy in Cardiovascular Diseases

&lt;p&gt;Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase enzyme activity through different&lt;br /&gt;mechanisms and prevent inflammation. But they all have different risks associated with them. Some are associated with&lt;br /&gt;gastrointestinal bleeding and some are strongly allied with the cardiovascular risks. Cyclooxygenase enzyme regulates&lt;br /&gt;prostaglandin synthesis by converting arachidonic acid present at the sn-2 position of membrane phospholipids to&lt;br /&gt;prostaglandin H2. Prostaglandin H2 is the precursor of all prostaglandins. There are two isoforms of cyclooxygenase&lt;br /&gt;enzyme, cyclooxygenase-1 and cyclooxygenase-2 which differ in their active site due to an isoleucine to valine&lt;br /&gt;substitution at amino acid 523 in cyclooxygenase-2. Cyclooxygenase-1 is constitutively expressed in platelets&lt;br /&gt;where it helps in the formation of thromboxane whereas cyclooxygenase-2 is inductive form and is expressed in&lt;br /&gt;the endothelial cells due to shear stress and forms prostacyclins. Both thromboxanes and prostacyclins maintain&lt;br /&gt;the homeostasis of the vascular wall. During vascular injury prostacyclin production decreases as a result of which&lt;br /&gt;thromboxane synthesis increases in the platelets which leads to platelet aggregation. Although, being strongly&lt;br /&gt;associated with cardiovascular risks, NSAIDs are still prescribed to the patients to prevent pain according to their&lt;br /&gt;condition. So this review aims to summarise the mechanism of cyclooxygenase pathway, possible mechanism of&lt;br /&gt;action of NSAIDs and the risks of cardiovascular events associated with the use of NSAIDs.&lt;/p&gt;

Synergistic inhibitory effect of capsaicin and dihydrocapsaicin on in-vitro platelet aggregation and thromboxane formation.

: Capsaicinoids, including capsaicin (CAP) and dihydrocapsaicin (DHC), the pungent principles of pepper fruits, individually inhibit in-vitro platelet aggregation. However, their effects, when present together, are not known. The aims of this study were to compare the effects of CAP and DHC alone, and in combination in the ratio that they are found in chilies (∼60% CAP : 40% DHC), on in-vitro platelet aggregation, platelet count and thromboxane B2 (TXB2) formation. The effects of 12.5 and 6.25 μmol/l CAP and DHC individually, and in combination (CAP : DHC, 60 : 40) on arachidonic acid-induced, ADP-induced and collagen-induced aggregation, were investigated. Platelet count was determined preincubation and postincubation with CAP and DHC and in combination. TXB2 formation from platelets treated with arachidonic acid in the absence and presence of CAP and DHC individually, and in combination, was measured. Compared with control, CAP and DHC (12.5 μmol/l) inhibited arachidonic acid-induced aggregation by 23.2 and 25.3%, respectively (both P < 0.01). In combination, CAP and DHC exhibited further inhibition in arachidonic acid-induced aggregation (CAP : DHC, 3.75 : 2.5 μmol/l, 36.5%, P = 0.01; 7.5 : 5 μmol/l, 57.5%, P < 0.001), compared with control. Incubation of platelets with caspaicinoids did not significantly affect the platelet count. In addition, the CAP : DHC (7.5 : 5 μmol/l) combination significantly inhibited (P < 0.001) TXB2 formation, compared with the individual capsaicinoids. Capsaicinoids had no effect ADP-induced or collagen-induced aggregation. The combination of CAP and DHC produces a significantly greater inhibitory effect on arachidonic acid-induced platelet aggregation and subsequent TXB2 formation, compared with the individual capsaicinoids.

Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus formation

Phosphoinositide 3-kinase (PI3K) plays an important role in platelet function and contributes to platelet hyperreactivity induced by elevated levels of circulating peptide hormones, including thrombopoietin (TPO). Previous work established an important role for the PI3K isoform; p110β in platelet function, however the role of p110α is still largely unexplored. Here we sought to investigate the role of p110α in TPO-mediated hyperactivity by using a conditional p110α knockout (KO) murine model in conjunction with platelet functional assays. We found that TPO-mediated enhancement of collagen-related peptide (CRP-XL)-induced platelet aggregation and adenosine triphosphate (ATP) secretion were significantly increased in p110α KO platelets. Furthermore, TPO-mediated enhancement of thrombus formation by p110α KO platelets was elevated over wild-type (WT) platelets, suggesting that p110α negatively regulates TPO-mediated priming of platelet function. The enhancements were not due to increased flow through the PI3K pathway as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) formation and phosphorylation of Akt and glycogen synthase kinase 3 (GSK3) were comparable between WT and p110α KO platelets. In contrast, extracellular responsive kinase (ERK) phosphorylation and thromboxane (TxA2) formation were significantly enhanced in p110α KO platelets, both of which were blocked by the MEK inhibitor PD184352, whereas the p38 MAPK inhibitor VX-702 and p110α inhibitor PIK-75 had no effect. Acetylsalicylic acid (ASA) blocked the enhancement of thrombus formation by TPO in both WT and p110α KO mice. Together, these results demonstrate that p110α negatively regulates TPO-mediated enhancement of platelet function by restricting ERK phosphorylation and TxA2 synthesis in a manner independent of its kinase activity.

P 048 - Garcinia kola – African ethno medication with anti-atherosclerotic effects?

The plant Garcinia kola is used in African ethno medicine to treat various diseases. Anti-inflammatory effects are one of the several beneficial properties described for this plant. We hypothesized that garcinoic acid (GA), as one of the phytochemicals isolated from Garcinia kola, is responsible for these effects. Therefore, we investigated the capacity of GA to block the inflammatory response in LPS-activated murine macrophages and the inflammatory disease atherosclerosis. We found that the LPS-induced upregulation of the expression of iNos and Cox2 and the formation of respective signaling molecules nitric oxide, thromboxanes and prostaglandins was significantly diminished by GA. Further, application of GA (1 mg/kg, i.p.) affected the composition, but not the size of developed atherosclerotic plaques in aortic roots of ApoE-/-mice fed with high-fat Western-type diet. In brief, stability of the plaques was improved by increased collagen content and smaller necrotic core size under GA treatment. Based on these data we predict that GA blocks inflammatory response and plays a pivotal role in the formation of atherosclerotic plaques, in particular, affecting the degree of stability. If the proposed anti-atherosclerotic properties of GA are supported by further studies, this compound is a promising new therapeutic lead molecule against atherosclerosis and its complications.

Thromboxane Formation Assay to Identify High On-Treatment Platelet Reactivity to Aspirin

Platelet inhibition by aspirin is indispensable in the secondary prevention of cardiovascular events. Nevertheless, impaired aspirin antiplatelet effects (high on-treatment platelet reactivity [HTPR]) are frequent. This is associated with an enhanced risk of cardiovascular events. The current gold standard to evaluate platelet hyper-reactivity despite aspirin intake is the light-transmittance aggregometry (LTA). However, pharmacologically, the most specific test is the measurement of arachidonic acid (AA)-induced thromboxane (TX) B2 formation. Currently, the optimal cut-off to define HTPR to aspirin by inhibition of TX formation is not known. Therefore, in this pilot study, we aimed to calculate a TX formation cut-off value to detect HTPR defined by the current gold standard LTA. We measured platelet function in 2,507 samples. AA-induced TX formation by ELISA and AA-induced LTA were used to measure aspirin antiplatelet effects. TX formation correlated nonlinearly with the maximum of aggregation in the AA-induced LTA (Spearman's rho R = 0.7396; 95% CI 0.7208-0.7573, p < 0.0001). Receiver operating characteristic analysis and Youden's J statistics revealed 209.8 ng/mL as the optimal cut-off value to detect HTPR to aspirin with the TX ELISA (area under the curve: 0.92, p < 0.0001, sensitivity of 82.7%, specificity of 90.3%). In summary, TX formation ELISA is reliable in detecting HTPR to aspirin. The calculated cut-off level needs to be tested in trials with clinical end points.

Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation

Human platelet transformation into balloons is part of the haemostatic response and thrombus architecture. Here we reveal that in aggregates of platelets in plasma, ballooning in multiple platelets occurs in a synchronised manner. This suggests a mechanism of coordination between cells, previously unrecognised. We aimed to understand this mechanism, and how it may contribute to thrombus development. Using spinning-disc confocal microscopy we visualised membrane ballooning in human platelet aggregates adherent to collagen-coated surfaces. Within an aggregate, multiple platelets undergo ballooning in a synchronised fashion, dependent upon extracellular calcium, in a manner that followed peak cytosolic calcium levels in the aggregate. Synchrony was observed in platelets within but not between aggregates, suggesting a level of intra-thrombus communication. Blocking phosphatidylserine, inhibiting thrombin or blocking PAR1 receptor, largely prevented synchrony without blocking ballooning itself. In contrast, inhibition of connexins, P2Y12, P2Y1 or thromboxane formation had no effect on synchrony or ballooning. Importantly, synchronised ballooning was closely followed by a surge in microvesicle formation, which was absent when synchrony was blocked. Our data demonstrate that the mechanism underlying synchronised membrane ballooning requires thrombin generation acting effectively in a positive feedback loop, mediating a subsequent surge in procoagulant activity and microvesicle release.

Garlic: A potential source of pharmaceuticals and pesticides: A review

Garlic (Allium sativum) holds a unique therapeutic potential as it inhibits invasion of carcinoma, provides cardiovascular protection, lowering of cholesterol, blood pressure, anti-platelet activities, and thromboxane formation. It provides protection against atherosclerosis and associated disorders and helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. It shows hypolipidemic, anti-platelet, and procirculatory effects, and antimutagenic and antiproliferative properties. It prevents cold and flu symptoms through immune enhancement and exhibits anticancer and chemopreventive activities. The main active component of garlic is alliin (S-allyl cysteine sulfoxide), a potent antioxidant which shows cardioprotective and neuroprotective actions. Diallyl trisulfide, major garlic derivatives, could inhibit the cell proliferation by triggering either cell cycle arrest or apoptosis in a variety of cancer cells. Organosulfur compounds from garlic inhibit the growth of transplanted as well as spontaneous cancers in preclinical animal models without any adverse side effects. Garlic is a good source of anti-invasive, antioxidant, anti-inflammatory, immunomodulatory, chemopreventive, hepatoprotective, antimicrobial, apoptotic, cardioprotective, antidiabetic agents and shows insecticidal effects against lepidopteran, coleopteran, dipteran and homopteran insect pests. Hence, its constituents could be used to develop alternatives to conventional insecticides for control of serious fruit and vegetable pests. Garlic herbal preparations can reduce non-target exposure to hazardous insecticides and curb resistance development in insects. No doubt garlic based different ailments and concoctions can be used to alleviate a variety of health problems. Its various supplements contain a different concentration of organosulfur compounds are available commercially in market

Risk Factors for Nonplatelet Thromboxane Generation After Coronary Artery Bypass Graft Surgery.

BackgroundPersistent thromboxane (TX) generation while receiving aspirin therapy is associated with an increased risk of cardiovascular events. The Reduction in Graft Occlusion Rates (RIGOR) study found that aspirin‐insensitive TXA 2 generation, indicated by elevated urine 11‐dehydro‐TXB 2 (UTXB 2) 6 months after coronary artery bypass graft surgery, was a potent risk factor for vein graft thrombosis and originated predominantly from nonplatelet sources. Our goal was to identify risks factors for nonplatelet TXA 2 generation.Methods and ResultsMultivariable modeling was performed by using clinical and laboratory variables obtained from 260 RIGOR subjects with verified aspirin‐mediated inhibition of platelet TXA 2 generation. The strongest variable associated with UTXB 2 6 months after surgery, accounting for 47.2% of the modeled effect, was urine 8‐iso‐prostaglandin (PG)F 2α, an arachidonic acid metabolite generated nonenzymatically by oxidative stress (standardized coefficient 0.442, P<0.001). Age, sex, race, lipid therapy, creatinine, left ventricular ejection fraction, and aspirin dose were also significantly associated with UTXB 2 (P<0.03), although they accounted for only 4.8% to 10.2% of the modeled effect. Urine 8‐iso‐PGF 2α correlated with risk of vein graft occlusion (odds ratio 1.67, P=0.001) but was not independent of UTXB 2. In vitro studies revealed that endothelial cells generate TXA 2 in response to oxidative stress and direct exposure to 8‐iso‐PGF 2α.ConclusionsOxidative stress–induced formation of 8‐iso‐PGF 2α is strongly associated with nonplatelet thromboxane formation and early vein graft thrombosis after coronary artery bypass graft surgery. The endothelium is potentially an important source of oxidative stress–induced thromboxane generation. These findings suggest therapies that reduce oxidative stress could be useful in reducing cardiovascular risks associated with aspirin‐insensitive thromboxane generation.