Inhibition Of Platelet Cyclooxygenase Research Articles

High rates of high on-aspirin platelet reactivity in patients after transcatheter aortic valve implantation

Abstract Background Guidelines recommend aspirin monotherapy in patients after transcatheter aortic valve replacement (TAVR) without an indication for oral anticoagulation. The prevalence of high on-aspirin platelet reactivity (HAPR) varies from 1% to 34% and may be associated with long-term ischemic risk or subclinical valve thrombosis which occurs in 17-40% of the patients after TAVR. Purpose To determine the prevalence of HAPR and its association with subclinical valve thrombosis in patients undergoing TAVR using methods that directly indicate the degree of platelet cyclooxygenase inhibition. Methods Data were collected in a prospective, single-center, observational cohort study between October 2020 and June 2022. Patients were enrolled after TAVR, and platelet function was assessed during the hospital stay with light-transmittance platelet aggregation (LTA) and thrombelastography (TEG) platelet mapping. Platelet aggregation was induced with arachidonic acid (AA)-induced LTA (final concentration [fc] 1.64 mM) and TEG (fc 1.0mM). The maximum percent aggregation (%aggr) was recorded. According to the literature, HAPR was defined as >20% aggregation by LTA or >50% aggregation by TEG, respectively. All patients were under maintenance of 100 mg aspirin or received a loading dose of 300-600mg aspirin before measurement. 47 of 75 patients underwent computed tomography (CT) to detect valve thrombosis (VT) 6 months after TAVR. Results Data from 75 patients under maintenance of aspirin were analyzed. The median age was 82 years (interquartile range, IQR 79-85), and 52% were male. 12 patients (16%) had a history of stroke or TIA, and 7 (9%) had suffered a previous myocardial infarction. TEG and LTA were performed during the hospital stay between days 0 and 17 post-TAVR. Median AA-induced maximum aggregation by LTA was 17 % (interquartile range, IQR 13-38) and by TEG was 29 % (IQR 5-64). When determining platelet function using LTA, 33 patients (44%) had residual platelet function (i.e., %aggrLTA >20%) despite aspirin treatment. According to the TEG results, 27 patients (36 %) were identified as HAPR (i.e., %aggrTEG >50%). The agreement (Kappa=0.06) and correlation (r=0.32, Figure 1) between the two tests were low to moderate. 13 patients had HAPR in both methods. 19 of 47 patients (40%) had VT on CT 6 months after TAVI. 12 of these 19 patients (63%) with VT responded well to aspirin in the TEG and LTA, while 7 (37%) had HAPR in both tests. HAPR was not significantly associated with HALT when measured with TEG or LTA (OR 0.90 [95% CI 0.27.2.76], p=0.86 and 0.58 [95% CI 0.17-2.04], p=0.37, respectively; Table 1). Conclusion HAPR assessed by LTA and TEG is frequent in patients undergoing TAVR. In this exploratory study, HAPR was not significantly associated with the occurrence of HALT. Further studies are required to evaluate the long-term risk of leaflet thrombosis or thromboembolic events in patients with HAPR.

The specific deletion of cyclooxygenase-1 in megakaryocytes/platelets reduces intestinal polyposis in ApcMin/+ mice

Clinical and experimental evidence sustain the role of cyclooxygenase (COX)−1 in intestinal tumorigenesis. However, the cell type expressing the enzyme involved and molecular mechanism(s) have not been clarified yet. We aimed to elucidate the role of platelet COX-1 (the target of low-dose aspirin in humans) in intestinal tumorigenesis of ApcMin/+ mice, considered a clinically relevant model. To realize this objective, we generated an ApcMin/+ mouse with a specific deletion of Ptgs1(COX-1 gene name) in megakaryocytes/platelets (ApcMin/+;pPtgs1-/-mice) characterized by profound inhibition of thromboxane(TX)A2 biosynthesis ex vivo (serum TXB2; by 99%) and in vivo [urinary 2,3-dinor-TXB2(TXM), by 79%]. ApcMin/+ mice with the deletion of platelet COX-1 showed a significantly reduced number (67%) and size (32%) of tumors in the small intestine. The intestinal adenomas of these mice had decreased proliferative index associated with reduced COX-2 expression and systemic prostaglandin(PG)E2 biosynthesis (urinary PGEM) vs. ApcMin/+mice. Extravasated platelets were detected in the intestine of ApcMin/+mice. Thus, we explored their contribution to COX-2 induction in fibroblasts, considered the primary polyp cell type expressing the protein. In the coculture of human platelets and myofibroblasts, platelet-derived TXA2 was involved in the induction of COX-2-dependent PGE2 in myofibroblasts since it was prevented by the selective inhibition of platelet COX-1 by aspirin or by a specific antagonist of TXA2 receptors. In conclusion, our results support the platelet hypothesis of intestinal tumorigenesis and provide experimental evidence that selective inhibition of platelet COX-1 can mitigate early events of intestinal tumorigenesis by restraining COX-2 induction.

Physiologically based modelling of the antiplatelet effect of aspirin: A tool to characterize drug responsiveness and inform precision dosing.

A computational approach involving mathematical modeling and in silico experiments was used to characterize the determinants of extent and duration of platelet cyclooxygenase (COX)-1 inhibition by aspirin and design precision dosing in patients with accelerated platelet turnover or reduced drug bioavailability. To this purpose, a recently developed physiologically-based pharmacokinetics (PK) and pharmacodynamics (PD) model of low-dose aspirin in regenerating platelets and megakaryocytes, was used to predict the main features and determinants of platelet COX-1 inhibition. The response to different aspirin regimens in healthy subjects and in pathological conditions associated with alterations in aspirin PK (i.e., severely obese subjects) or PD (i.e., essential thrombocytemya patients), were simulated. A model sensitivity analysis was performed to identify the main processes influencing COX-1 dynamics. In silico experiments and sensitivity analyses indicated a major role for megakaryocytes and platelet turnover in determining the extent and duration of COX-1 inhibition by once-daily, low-dose aspirin. They also showed the superiority of reducing the dosing interval vs increasing the once-daily dose in conditions of increased platelet turnover, while suggested specific dose adjustments in conditions of possible reduction in drug bioavailability. In conclusion, the consistency of our model-based findings with experimental data from studies in healthy subjects and patients with essential thrombocythemia supports the potential of our approach for describing the determinants of platelet inhibition by aspirin and informing precision dosing which may guide personalized antithrombotic therapy in different patient populations, especially in those under-represented in clinical trials or in those associated with poor feasibility.

A closer look at endothelial injury-induced platelet hyperactivity and the use of aspirin in the treatment of COVID infection.

In this commentary, we make a case that the mechanism of COVID pathogenesis is related to virus-induced endothelial injury resulting in platelet activation and the formation of microthrombi both systemically and in cardiac and pulmnonary circulation which result in major causes of COVID morbidity and mortality. Aspirin by virtue of its irreversible inhibition of platelet COX-1, should reverse these platelet-induced pathogenic changes associated with COVID infection for the 6–9 day lifetime of the platelet. We also cite recent findings of a retrospective analysis that supports the use of low-dose (81 mg) aspirin to treat the symptoms associated with the early stages of COVID infection.

Association of Platelet Thromboxane Inhibition by Low-Dose Aspirin With Platelet Count and Cytoreductive Therapy in Essential Thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by enhanced platelet production and thrombotic complications. The inhibition of platelet cyclooxygenase (COX) activity by the standard once‐daily aspirin is mostly incomplete due to accelerated thrombopoiesis. The phase II Aspirin Regimens in EsSential thrombocythemia (ARES) trial has recently compared the efficacy of once‐ vs. twice‐ or three‐times daily low‐dose aspirin in inhibiting platelet thromboxane (TX) A2 production, as reflected by serum (s) TXB2 measurements. The present substudy characterized the determinants of the highly variable response to the standard aspirin 100 mg once‐daily regimen in fully compliant patients with ET and the effects of the experimental dosing regimens on response variability. By multivariable analysis, the platelet count (directly) and cytoreductive treatment (inversely) were significantly associated with sTXB2 values in 218 patients with ET. However, the platelet count positively correlated with sTXB2 in patients not being treated with cytoreductive drugs (ρ = 0.51, P < 0.01, n = 84), but not in patients on cytoreduction. Patients in the lowest sTXB2 quartile were older, more often on cytoreductive drugs, had lower platelet count and Janus‐Associated Kinase2 (JAK2)‐V617F allele frequency as compared with patients in the upper sTXB2 quartiles. After 2 weeks of a twice‐ or 3‐times daily aspirin regimen, the association between the platelet count and sTXB2 became similar in cytoreduced and non‐cytoreduced patients. In conclusion, the platelet count appears the strongest determinant of TXA2 inhibition by once‐daily low‐dose aspirin in ET, with different patterns depending of cytoreductive treatment. More frequent aspirin dosing restores adequate platelet inhibition and reduces interindividual variability, independently of cytoreduction.

Low-dose Aspirin prevents hypertension and cardiac fibrosis when thromboxane A2 is unrestrained

Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A2 in their pathophysiology remains unclear. We investigated the systemic TXA2 biosynthesis in vivo and gene expression of its receptor TP in 22 essential hypertension patients and a mouse model of salt-sensitive hypertension. The contribution of platelet TXA2 biosynthesis on enhanced blood pressure (BP) and overload-induced cardiac fibrosis was explored in mice by treating with low-dose Aspirin, resulting in selective inhibition of platelet cyclooxygenase (COX)-1-dependent TXA2 generation. In essential hypertensive patients, systemic biosynthesis of TXA2 [assessed by measuring its urinary metabolites (TXM) reflecting predominant platelet source] was enhanced together with higher gene expression of circulating leukocyte TP and TGF-β, vs. normotensive controls. Similarly, in hypertensive mice with prostacyclin (PGI2) receptor (IP) deletion (IPKO) fed with a high-salt diet, enhanced urinary TXM, and left ventricular TP overexpression were detected vs. normotensive wildtype (WT) mice. Increased cardiac collagen deposition and profibrotic gene expression (including TGF-β) was found. Low-dose Aspirin administration caused a selective inhibition of platelet TXA2 biosynthesis and mitigated enhanced blood pressure, cardiac fibrosis, and left ventricular profibrotic gene expression in IPKO but not WT mice. Moreover, the number of myofibroblasts and extravasated platelets in the heart was reduced. In cocultures of human platelets and myofibroblasts, platelet TXA2 induced profibrotic gene expression, including TGF-β1. In conclusion, our results support tailoring low-dose Aspirin treatment in hypertensive patients with unconstrained TXA2/TP pathway to reduce blood pressure and prevent early cardiac fibrosis.

Defining platelet response to acetylsalicylic acid: the relation between inhibition of serum thromboxane B2 and agonist-induced platelet aggregation.

Arachidonic acid (AA)-induced platelet aggregation (PA) and serum thromboxane B2 (TxB2) inhibition are widely used to indicate cyclooxygenase-1 activity and the antiplatelet effect of acetylsalicylic acid (ASA). Despite decades of investigations, the relation between these measurements remains unclear. We sought to evaluate the relation between AA-PA and serum TxB2 inhibition. We serially measured AA-PA (conventional aggregation), serum TxB2, plasma ASA and salicylic acid (SA) (liquid chromatography-mass spectrometry), and urinary 11-dehydro thromboxane B2 (u11-dh TxB2) (enzyme-linked immunosorbent assay) levels at 10 times over 24 hours in seventeen healthy volunteers receiving a single dose of 162mg chewed and swallowed ASA (n = 6), 50mg inhaled ASA (n = 6), or 100mg inhaled ASA (n = 5) (ClinicalTrials.gov Identifier: NCT04328883, April 1, 2020). Baseline variability was more pronounced with serum TxB2 (31-680ng/mL) as compared to maximal AA-PA (65-81%) and u11-dh TxB2 (1556-4440pg/mg creatinine). The relation between serum TxB2 inhibition and AA-PA was stepwise; after 30-40% inhibition of serum TxB2, AA-PA fell to < 5%. By receiver operating characteristic curve analysis using AA-PA < 5% to define aspirin responsiveness, serum TxB2 inhibition > 49% and u11-dh TxB2 < 1520 pg/mg creatinine met the definition. Our study demonstrates a non-linear relation between serum TxB2 inhibition and AA-PA. Aggregation was nil once TxB2 inhibition reached > 49%. Moreover, these results suggest that the definition of > 95% inhibition of serum TxB2 to indicate the level of platelet COX-1 inhibition needed for clinical efficacy may be overestimated and should be re-considered in future translational research investigations that attempt to link the clinical efficacy of ASA with a laboratory measurement cutoff.

Cyclooxygenase activity mediates colorectal cancer cell resistance to the omega-3 polyunsaturated fatty acid eicosapentaenoic acid

PurposeThe naturally-occurring omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and inexpensive, making it an attractive anti-cancer intervention. However, EPA has only modest anti-colorectal cancer (CRC) activity, when used alone. Both cyclooxygenase (COX) isoforms metabolise EPA and are over-expressed in CRC cells. We investigated whether COX inhibition increases the sensitivity of CRC cells to growth inhibition by EPA.MethodsA panel of 18 human and mouse CRC cell lines was used to characterize the differential sensitivity of CRC cells to the growth inhibitory effects of EPA. The effect of CRISPR-Cas9 genetic deletion and pharmacological inhibition of COX-1 and COX-2 on the anti-cancer activity of EPA was determined using in vitro and in vivo models.ResultsGenetic ablation of both COX isoforms increased sensitivity of CT26 mouse CRC cells to growth inhibition by EPA in vitro and in vivo. The non-selective COX inhibitor aspirin and the selective COX-2 inhibitor celecoxib increased sensitivity of several human and mouse CRC cell lines to EPA in vitro. However, in a MC38 mouse CRC cell tumour model, with dosing that mirrored low-dose aspirin use in humans, thereby producing significant platelet COX-1 inhibition, there was ineffective intra-tumoral COX-2 inhibition by aspirin and no effect on EPA sensitivity of MC38 cell tumours.ConclusionCyclooxygenase inhibition by non-steroidal anti-inflammatory drugs represents a therapeutic opportunity to augment the modest anti-CRC activity of EPA. However, intra-tumoral COX inhibition is likely to be critical for this drug-nutrient interaction and careful tissue pharmacodynamic profiling is required in subsequent pre-clinical and human studies.

A randomized double-blind trial of 3 aspirin regimens to optimize antiplatelet therapy in essential thrombocythemia

AbstractEssential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2–dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the twice-daily and thrice-daily regimens showed substantially reduced interindividual variability and lower median (interquartile range) values for sTXB2 (ng/mL) compared with the once-daily arm: 4 (2.1-6.7; n = 79), 2.5 (1.4-5.65, n = 79), and 19.3 (9.7-40; n = 85), respectively. Urinary PGIM was comparable in the 3 arms. Urinary TXM was reduced by 35% in both experimental arms. Patients in the thrice-daily arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75 to 100 once daily for cardiovascular prophylaxis appears to be largely inadequate in reducing platelet activation in the vast majority of patients with ET. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement with further reductions (EudraCT 2016-002885-30).

Profiling the eicosanoid networks that underlie the anti- and pro-thrombotic effects of aspirin.

Aspirin prevents thrombosis by inhibiting platelet cyclooxygenase (COX)-1 activity and the production of thromboxane (Tx)A2, a pro-thrombotic eicosanoid. However, the non-platelet actions of aspirin limit its antithrombotic effects. Here, we used platelet-COX-1-ko mice to define the platelet and non-platelet eicosanoids affected by aspirin. Mass-spectrometry analysis demonstrated blood from platelet-COX-1-ko and global-COX-1-ko mice produced similar eicosanoid profiles in vitro: for example, formation of TxA2, prostaglandin (PG) F2α, 11-hydroxyeicosatraenoic acid (HETE), and 15-HETE was absent in both platelet- and global-COX-1-ko mice. Conversely, in vivo, platelet-COX-1-ko mice had a distinctly different profile from global-COX-1-ko or aspirin-treated control mice, notably significantly higher levels of PGI2 metabolite. Ingenuity Pathway Analysis (IPA) predicted that platelet-COX-1-ko mice would be protected from thrombosis, forming less pro-thrombotic TxA2 and PGE2. Conversely, aspirin or lack of systemic COX-1 activity decreased the synthesis of anti-aggregatory PGI2 and PGD2 at non-platelet sites leading to predicted thrombosis increase. In vitro and in vivo thrombosis studies proved these predictions. Overall, we have established the eicosanoid profiles linked to inhibition of COX-1 in platelets and in the remainder of the cardiovascular system and linked them to anti- and pro-thrombotic effects of aspirin. These results explain why increasing aspirin dosage or aspirin addition to other drugs may lessen antithrombotic protection.

Unexpected effects of long-term treatment with acetylsalicylic acid on late phase of pulmonary metastasis in murine model of orthotopic breast cancer.

Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease.

Activated Platelets Induce Endothelial Cell Inflammatory Response in Psoriasis via COX-1.

Patients with psoriasis have impaired vascular health and increased cardiovascular disease (CVD). Platelets are key players in the pathogenesis of vascular dysfunction in cardiovascular disease and represent therapeutic targets in cardiovascular prevention. The object of this study was to define the platelet phenotype and effector cell properties on vascular health in psoriasis and evaluate whether aspirin modulates the platelet-induced phenotype. Approach and Results: Platelets from psoriasis patients (n=45) exhibited increased platelet activation (relative to age- and gender-matched controls, n=18), which correlated with psoriasis skin severity. Isolated platelets from psoriasis patients demonstrated a 2- to 3-fold (P<0.01) increased adhesion to human aortic endothelial cells and induced proinflammatory transcriptional changes, including upregulation of IL 8 (interleukin 8), IL1β, and Cox (cyclooxygenase)-2 Platelet RNA sequencing revealed an interferon signature and elevated expression of COX-1, which correlated with psoriasis disease severity (r=0.83, P=0.01). In a randomized trial of patients with psoriasis, 2 weeks of 81 mg low-dose aspirin, a COX-1 inhibitor, reduced serum thromboxane (Tx) B2 and reduced brachial vein endothelial proinflammatory transcript expression >70% compared with the no-treatment group (P<0.01). Improvement in brachial vein endothelial cell inflammation significantly correlated with change in serum TxB2 (r=0.48, P=0.02). In patients with psoriasis, platelets are activated and induce endothelial cell inflammation. Low-dose aspirin improved endothelial cell health in psoriasis via platelet COX-1 inhibition. These data demonstrate a previously unappreciated role of platelets in psoriasis and endothelial cell inflammation and suggests that aspirin may be effective in improving vascular health in patients with psoriasis. Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03228017.

Platelet-Specific Deletion of Cyclooxygenase-1 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice.

Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A2 production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA2 enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA2 receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA2 in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A2 in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD.

Aspirin blocks formation of metastatic intravascular niches by inhibiting platelet-derived COX-1/thromboxane A2.

Because metastasis is associated with the majority of cancer-related deaths, its prevention is a clinical aspiration. Prostanoids are a large family of bioactive lipids derived from the activity of cyclooxygenase-1 (COX-1) and COX-2. Aspirin impairs the biosynthesis of all prostanoids through the irreversible inhibition of both COX isoforms. Long-term administration of aspirin leads to reduced distant metastases in murine models and clinical trials, but the COX isoform, downstream prostanoid, and cell compartment responsible for this effect are yet to be determined. Here, we have shown that aspirin dramatically reduced lung metastasis through inhibition of COX-1 while the cancer cells remained intravascular and that inhibition of platelet COX-1 alone was sufficient to impair metastasis. Thromboxane A2 (TXA2) was the prostanoid product of COX-1 responsible for this antimetastatic effect. Inhibition of the COX-1/TXA2 pathway in platelets decreased aggregation of platelets on tumor cells, endothelial activation, tumor cell adhesion to the endothelium, and recruitment of metastasis-promoting monocytes/macrophages, and diminished the formation of a premetastatic niche. Thus, platelet-derived TXA2 orchestrates the generation of a favorable intravascular metastatic niche that promotes tumor cell seeding and identifies COX-1/TXA2 signaling as a target for the prevention of metastasis.

Genetic Signatures in Ischemic Stroke: Focus on Aspirin Resistance

Stroke is one of the leading causes of death. There has been compelling evidence that stroke has a genetic component. Genetic variants not only influence susceptibility to stroke but have also been found to alter the response to pharmacological agents and influence the clinical outcome of the disease. Stroke patients are treated with antiplatelet drugs like aspirin and clopidogrel to prevent a secondary stroke. In spite of the fact that many new antiplatelet drugs have been developed, aspirin is still considered as a golden standard for the antiplatelet therapy. Aspirin achieves its action by inhibiting platelet cyclooxygenase (COX) system involved in the formation of thromboxane A2 (TXA2). TXA2 triggers reactions leading to platelet activation and aggregation. This Non-steroidal anti-inflammatory drug (NSAID) acts by inhibiting this mediator. Despite the demonstrated benefits of aspirin, many patients develop secondary stroke or other vascular events, an observation that has led to the concept of aspirin resistance. Studies have demonstrated that adequate antiplatelet effects are not achieved in 5-45% patients suggesting that many individuals are aspirin resistant. Aspirin resistance is multifactorial in origin. A genetic component has also been suggested, and variants in more than a dozen genes involved in absorption, distribution, metabolism, excretion (ADME) and pharmacodynamics of aspirin have been shown to be responsible for aspirin resistance. In addition, the patients on aspirin treatment also face adverse drug reactions on account of genetic variation. The present review has been compiled with an aim to revisit all the studies related to genetic variation contributing to aspirin resistance as well as adverse drug reactions. The output of high throughput genomic technology like genome wide association studies and others has also been discussed.

Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling.

Lipopolysaccharide (LPS) from the cell envelope of Gram-negative bacteria is a principal cause of the symptoms of sepsis. LPS has been reported to modulate the function of platelets although the underlying mechanisms of LPS action in these cells remain unclear. Platelets express the Toll-like receptor 4 (TLR4) which serves as a receptor for LPS, although the potential role of TLR4 and associated cell signalling in controlling platelet responses to LPS has not been extensively explored. In this study, we therefore investigated the actions of LPS prepared from different strains of Escherichia coli on platelet function, the underlying signalling mechanisms, and the potential role of TLR4 in orchestrating these. We report that LPS increased the aggregation of washed platelets stimulated by thromboxane (U46619) or GPVI collagen receptor agonists, effects that were prevented by a TLR4 antagonist. Associated with this, LPS enhanced fibrinogen binding, P-selectin exposure and reactive oxygen species (ROS) release. Increase of ROS was found to be important for the actions of LPS on platelets, since these were inhibited in the presence of superoxide dismutase or catalase. The effects of LPS were associated with phosphorylation of Akt, ERK1/2 and PLA2 in stimulated platelets, and inhibitors of PI3-kinase, Akt and ERK1/2 reduced significantly LPS enhanced platelet function and associated ROS production. Furthermore, inhibition of platelet cyclooxygenase or the thromboxane receptor, revealed an important role for thromboxane A2. We therefore conclude that LPS increases human platelet activation through a TLR4-PI3K-Akt-ERK1/2-PLA2 -dependent pathway that is dependent on ROS and TXA2 formation.

Inhibition of the Biosynthesis of Prostaglandin E2 By Low-Dose Aspirin: Implications for Adenocarcinoma Metastasis.

Meta-analyses have demonstrated that low-dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the antiplatelet action of low-dose aspirin likely contributes to its antimetastatic effect. Cycloxooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low-dose aspirin also inhibits PGE2 biosynthesis. We show that low-dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (P < 0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells, its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells upregulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet-tumor cell aggregates. In conclusion, low-dose aspirin has a significant effect on extraplatelet cyclooxygenase and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low-dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells. Cancer Prev Res; 9(11); 855-65. ©2016 AACR.

Systems Pharmacogenomics Finds RUNX1 Is an Aspirin-Responsive Transcription Factor Linked to Cardiovascular Disease and Colon Cancer

Aspirin prevents cardiovascular disease and colon cancer; however aspirin's inhibition of platelet COX-1 only partially explains its diverse effects. We previously identified an aspirin response signature (ARS) in blood consisting of 62 co-expressed transcripts that correlated with aspirin's effects on platelets and myocardial infarction (MI). Here we report that 60% of ARS transcripts are regulated by RUNX1 – a hematopoietic transcription factor - and 48% of ARS gene promoters contain a RUNX1 binding site. Megakaryocytic cells exposed to aspirin and its metabolite (salicylic acid, a weak COX-1 inhibitor) showed up regulation in the RUNX1 P1 isoform and MYL9, which is transcriptionally regulated by RUNX1. In human subjects, RUNX1 P1 expression in blood and RUNX1-regulated platelet proteins, including MYL9, were aspirin-responsive and associated with platelet function. In cardiovascular disease patients RUNX1 P1 expression was associated with death or MI. RUNX1 acts as a tumor suppressor gene in gastrointestinal malignancies. We show that RUNX1 P1 expression is associated with colon cancer free survival suggesting a role for RUNX1 in aspirin's protective effect in colon cancer. Our studies reveal an effect of aspirin on RUNX1 and gene expression that may additionally explain aspirin's effects in cardiovascular disease and cancer.

Aspirin for cancer is no mere antiplatelet prototype. There is potential in its ancient roots

Aspirin (ASA), increasingly accepted as predominantly a cyclooxygenase (COX)-1 inhibitor, is a prodrug for salicylic acid (SA) which has no such activity. SA is widespread in nature, vital in plants, and present in drug free serum from animals and man. Evolutionary conserved SA receptors are found in human tissues.Very low doses of ASA will, on repeat dosing, produce near maximal platelet COX-1 inhibition. Evidence for cancer prophylaxis is based on ASA doses of at least 75mg/day.Pleiotropic mechanisms underlie low dose ASA’s undoubted efficacy in preventive medicine but the key barrier to its more widespread use is gastrointestinal toxicity.ASA/SA combination formulations may improve the current risk/benefit ratio of chemo-prophylactic preparations. There is well established methodology for, and should be few regulatory barriers to, their evaluation.

Aspirin prevents colorectal cancer metastasis in mice by splitting the crosstalk between platelets and tumor cells.

We investigated whether platelets prime colon cancer cells for metastasis and whether pharmacological inhibition of platelet function may prevent it. Coculturing HT29 human colon carcinoma cells with human platelets led to the induction of mesenchymal-like cancer cells characterized by downregulation of E-cadherin and upregulation of Twist1, enhanced cell mobility and a proaggregatory action on platelets. These changes were prevented by different antiplatelet agents, aspirin[an inhibitor of cyclooxygenase(COX)-1], DG-041[an antagonist of prostaglandin(PG)E2 EP3 receptor] and ticagrelor (a P2Y12 receptor antagonist). The injection of HT29 cells, exposed to platelets in vitro, into the tail vein of humanized immunodeficient mice led to higher incidence of lung metastasis compared to the injection of untreated HT29 cells. This effect was associated with enhanced systemic biosynthesis of thromboxane(TX)A2 and PGE2 in vivo. Platelet COX-1 inhibition by aspirin administration to mice prevented the increased rate of metastasis as well as the enhanced production of TXA2 and PGE2 induced by the in vitro priming of HT29 cells by platelets. In conclusion, targeting platelet COX-1 with low-dose aspirin exerts an antimetastatic action by averting the stem cell mimicry of cancer cells associated with enhanced proaggregatory effects induced by platelet-tumor cell interactions. These effects may be shared by other antiplatelet drugs.