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In the last decade, novel oral anticoagulants (NOACs) have emerged as prominent therapeutic options in non-valvular atrial fibrillation (NVAF). We analysed the clinical burden and the switching rate between all available NOACs, and their dosage change over a period of 5 years in a representative population of patients with NVAF aged between 70 and 75 years. Methods and Results: This is a retrospective observational study on administrative databases, covering approximately 6.2 million health-assisted individuals by the Italian National Health System (around 11% of the entire Italian residents). Out of 4640 NVAF patients treated with NOACs and aged 70–75 years in 2017, 3772 (81.3%) patients were still in treatment with NOAC up to 2021 and among them, 3389 (73.0%) patients remained in treatment with the same NOAC during 2017–2021. In fact, 10.2% of patients switched NOAC type and 10.3% changed the dose of the same NOAC. Overall, after switching, the dabigatran and rivaroxaban groups lost, respectively, 13.5% and 2.8% of patients, while apixaban and edoxaban resulted in a relative percentage increase of 6.8% and 44.6% of patients, respectively. By a logistic regression analysis, the treatment with rivaroxaban, apixaban, and edoxaban (respect to dabigatran) was associated with a significant risk reduction of switch of 57%, 68%, and 44%, respectively. On the other hand, several features of high risk were associated with dose reduction. Conclusions. In our 5-year analysis of a large administrative database, a switching among NOACs or a change in NOAC dosages occurred in around 20% of elderly patients with NVAF. The type of NOAC was associated with a high switching rate, while several characteristics of high risk resulted as predictors of dose reduction of NOACs. Moreover, a worsening trend of clinical conditions occurred in patients maintaining the same NOAC treatment across 2017–2021.

This real-world analysis conducted on administrative databases of a sample of Italian healthcare entities was aimed at describing the role of therapeutic pathways and drug utilization in terms of adherence, persistence, and therapy discontinuation in HIV-infected patients under antiretroviral therapies (ART) and Tenofovir Alafenamide (TAF)-based regimens on healthcare resource consumption and related direct healthcare costs. Between 2015 and 2019, adults (≥18 years) prescribed with TAF-based therapies were identified and characterized in the year prior to the first prescription (index-date) for TAF-based therapies and followed-up until the end of data availability. Overall, 2658 ART-treated patients were included, 1198 of which were under a TAF-based regimen. TAF-based therapies were associated with elevated percentages of adherence (83.3% patients with proportion of days covered, PDC > 95% and 90.6% with PDC > 85%) and persistence (78.5%). The discontinuation rate was low in TAF-treated patients, ranging from 3.3% in TAF-switchers to 5% in naïve. Persistent patients had lower overall mean annual healthcare expenditures (EUR 11,106 in persistent vs. EUR 12,380 in non-persistent, p = 0.005), and this trend was statistically significant also for costs related to HIV hospitalizations. These findings suggest that a better therapeutic management of HIV infection might result in positive clinical and economic outcomes.

This real-world analysis evaluated the clinical and economic burden of non-dialysis-dependent CKD patients with and without secondary hyperparathyroidism (sHPT) in Italy. An observational retrospective study was conducted using administrative databases containing a pool of healthcare entities covering 2.45 million health-assisted individuals. Adult patients with hospitalization discharge diagnoses for CKD stages 3, 4, and 5 were included from 1 January 2012 to 31 March 2015 and stratified using the presence/absence of sHPT. Of the 5710 patients, 3119 were CKD-only (62%) and 1915 were CKD + sHPT (38%). The groups were balanced using Propensity Score Matching (PSM). Kaplan-Meier curves revealed that progression to dialysis and cumulative mortality had a higher incidence in the CKD + sHPT versus CKD-only group in CKD stage 3 patients and the overall population. The total direct healthcare costs/patient at one-year follow-up were significantly higher in CKD + sHPT versus CKD-only patients (EUR 8593 vs. EUR 5671, p < 0.001), mostly burdened by expenses for drugs (EUR 2250 vs. EUR 1537, p < 0.001), hospitalizations (EUR 4628 vs. EUR 3479, p < 0.001), and outpatient services (EUR 1715 vs. EUR 654, p < 0.001). These findings suggest that sHPT, even at an early CKD stage, results in faster progression to dialysis, increased mortality, and higher healthcare expenditures, thus indicating that timely intervention can ameliorate the management of CKD patients affected by sHPT.

Background: Platelet–cancer cell interactions modulate tumor metastasis and thrombosis in cancer. Platelet-derived extracellular vesicles (EVs) can contribute to these outcomes. Methods: We characterized the medium-sized EVs (mEVs) released by thrombin-stimulated platelets of colorectal cancer (CRC) patients and healthy subjects (HS) on the capacity to induce epithelial-mesenchymal transition (EMT)-related genes and cyclooxygenase (COX)-2(PTGS2), and thromboxane (TX)B2 production in cocultures with four colorectal cancer cell lines. Platelet-derived mEVs were assessed for their size distribution and proteomics signature. Results: The mEV population released from thrombin-activated platelets of CRC patients had a different size distribution vs. HS. Platelet-derived mEVs from CRC patients, but not from HS, upregulated EMT marker genes, such as TWIST1 and VIM, and downregulated CDH1. PTGS2 was also upregulated. In cocultures of platelet-derived mEVs with cancer cells, TXB2 generation was enhanced. The proteomics profile of mEVs released from activated platelets of CRC patients revealed that 119 proteins were downregulated and 89 upregulated vs. HS. Conclusions: We show that mEVs released from thrombin-activated platelets of CRC patients have distinct features (size distribution and proteomics cargo) vs. HS and promote prometastatic and prothrombotic phenotypes in cancer cells. The analysis of platelet-derived mEVs from CRC patients could provide valuable information for developing an appropriate treatment plan.

A real-world analysis among the Italian population has been carried out to estimate the number of atrial fibrillation (AF) patients undergoing catheter ablation and to evaluate their clinical outcome and economic burden. A retrospective analysis on administrative Italian databases has been performed. Between January 2011 and December 2019, all patients diagnosed with AF were considered and those undergoing catheter ablation were identified. Overall, 3084 (3.54%) of AF patients with at least one catheter ablation were included (mean age 63.2, 67.3% males). A significant decrease in the use of AF-related medications and in hospitalizations, mainly related to AF and heart failure, was observed during the 3-year post-ablation period. The average total cost per patient during the 1-year before ablation period was significantly higher compared to the 1-year post-ablation cost (EUR 5248 vs. 4008, respectively; p < 0.001). After propensity score matching, the overall mortality of patients who underwent ablation was significantly lower compared to that assessed in patients not treated with the procedure (9.386/1000 vs. 23.032/1000 person-year, respectively; p < 0.001). Moreover, the mean total costs were significantly higher in patients who did not undergo ablation compared to those who received ablation (EUR 5516 vs. 4008, respectively; p < 0.001). This real-world data analysis shows that in Italy, although catheter ablation is performed in a minority of AF patients, it is associated with significantly better post-procedure clinical outcomes and a significant reduction in healthcare-related costs.

Anaemia is a uraemia-related complication frequently found in non-dialysis-dependent chronic kidney disease (ND-CKD) patients, with iron-deficiency anaemia (IDA) as the main underlying mechanism. Given the suboptimal anaemia management in ND-CKD patients with a co-diagnosis of IDA, this study evaluated the role of IDA therapy on clinical outcomes and healthcare resource consumptions in an Italian clinical setting. A retrospective observational real-world analysis was performed on administrative databases of healthcare entities, covering around 6.9 million health-assisted individuals. From January 2010 to March 2019, ND-CKD patients were included and diagnosed with IDA in the presence of two low-haemoglobin (Hb) measurements. Patients were divided into IDA-treated and untreated, based on the prescription of iron [Anatomical-Therapeutic Chemical (ATC) code B03A] or anti-anaemia preparations (ATC code B03X), and evaluated during a 6-month follow-up from the index date [first low haemoglobin (Hb) detection]. IDA treatment resulted in significantly decreased incidence of all cause-related, cardiovascular-related, and IDA-related hospitalizations (treated vs. untreated: 44.5% vs. 81.8%, 12.3% vs. 25.3%, and 16.2% vs. 26.2%, respectively, p < 0.001). A healthcare direct cost estimation showed that overall mean expenditure per patient reduced by 47% with IDA treatment (5245€ vs. 9918€, p < 0.001), mainly attributable to hospitalizations (3767€ vs. 8486€, p < 0.001). This real-life analysis on Italian ND-CKD-IDA patients indicates that IDA therapy administration provides significant benefits in terms of patients’ clinical outcomes and healthcare cost savings.

Valentina Perrone,1 Chiara Veronesi,1 Melania Dovizio,1 Domenica Daniela Ancona,2 Margherita Andretta,3 Fausto Bartolini,4 Arturo Cavaliere,5 Alessandro Chinellato,6 Andrea Ciaccia,7 Mariarosaria Cillo,8 Adele De Francesco,9 Nicola Enieri,6 Fulvio Ferrante,10 Simona Gentile,11 Cataldo Procacci,2 Loredana Ubertazzo,12 Adriano Vercellone,13 Domenico Lucatelli,14 Michela Procaccini,14 Luca Degli Esposti1 1CliCon S.r.l., Società Benefit-Health, Economics & Outcomes Research, Bologna, Italy; 2Dipartimento Farmaceutico, ASL BAT, Andria, Italy; 3UOC Assistenza Farmaceutica Territoriale, Azienda ULSS 8 Berica, Vicenza, Italy; 4Dipartimento Farmaceutico, USL Umbria 2, Terni, Italy; 5Farmaceutica Aziendale, ASL Viterbo, Viterbo, Italy; 6Unità Operativa Farmacia Ospedaliera, ULSS 3 Serenissima, Mestre, Italy; 7ASL Foggia, Foggia, Italy; 8Dipartimento Farmaceutico, ASL Salerno, Salerno, Italy; 9Azienda Ospedaliero-Universitaria “Mater Domini”, Catanzaro, Italy; 10ASL Frosinone, Frosinone, Italy; 11Direzione Generale per la Salute, Campobasso, Italy; 12ASL Roma 4, Civitavecchia, Roma, Italy; 13ASL Napoli 3 sud, Napoli, Italy; 14Angelini Pharma, Roma, ItalyCorrespondence: Valentina Perrone, CliCon S.r.l., Società Benefit-Health, Economics & Outcomes Research, Via Murri 9, Bologna, 40137, Italy, Tel +39 3450316494, Email valentina.perrone@clicon.itPurpose: A retrospective analysis was conducted to estimate the number of patients with focal epilepsy and drug-resistant epilepsy (DRE) and their characteristics, the therapeutic patterns, the consumption of health resources in a real-world Italian setting.Patients and Methods: A retrospective study was carried out on the administrative databases of a sample of Italian Health Departments, covering approximately 8.7 million health-assisted individuals. All adult patients with at least one hospitalization for focal epilepsy and an electroencephalogram (between 01/2010 and 12/2019), and at least one prescription of antiseizure medication (ASM) (between 01/2011 and 12/2018) were included in the study. Patients with at least two treatment failures and treated with a subsequent ASM were considered DRE.Results: Overall, 1897 patients with focal epilepsy (mean age 56 years, 47% male) were identified, of which 485 (25.6%) with DRE (mean age 53 years, 43% male). Among patients with focal epilepsy and DRE, respectively, 48% and 54% had essential hypertension, 23.4% and 26.6% had cardiovascular disease, and 46.3% and 62.1% had peptic ulcer/prescription of gastric secretion inhibitors. During follow-up, patients with focal epilepsy maintained first-line treatment for 53.9 months; among these, 52% passed to the second-line, and 485 (25.6% of the total) began third-line treatment. In patients with focal epilepsy, the mean cost was € 4448 (of which € 1410 were epilepsy-related), and in DRE patients total expenditures averages € 5825 (of which € 2165 were epilepsy-related). In both patients with focal epilepsy and DRE, hospitalizations represented the most impacting item of expenditure.Conclusion: The present analysis conducted in a setting of Italian clinical practice has shown that 25% of patients with focal epilepsy were resistant to antiepileptic treatments. Furthermore, these results showed that health-care costs for the management of epileptic patients were mainly accountable for the costs related to the disease-management and to hospitalizations.Keywords: epilepsy, real-world evidence, clinical practice, pharmacoutilization, healthcare resource consumption

Background: The results of Aspirin prevention of colorectal adenomas in patients with familial adenomatous polyposis (FAP) are controversial. Methods: We conducted a biomarker-based clinical study in eight FAP patients treated with enteric-coated low-dose Aspirin (100 mg daily for three months) to explore whether the drug targets mainly platelet cyclooxygenase (COX)-1 or affects extraplatelet cellular sources expressing COX-isozymes and/or off-target effects in colorectal adenomas. Results: In FAP patients, low-dose Aspirin-acetylated platelet COX-1 at Serine529 (>70%) was associated with an almost complete inhibition of platelet thromboxane (TX) B2 generation ex vivo (serum TXB2). However, enhanced residual urinary 11-dehydro-TXB2 and urinary PGEM, primary metabolites of TXA2 and prostaglandin (PG)E2, respectively, were detected in association with incomplete acetylation of COX-1 in normal colorectal biopsies and adenomas. Proteomics of adenomas showed that Aspirin significantly modulated only eight proteins. The upregulation of vimentin and downregulation of HBB (hemoglobin subunit beta) distinguished two groups with high vs. low residual 11-dehydro-TXB2 levels, possibly identifying the nonresponders and responders to Aspirin. Conclusions: Although low-dose Aspirin appropriately inhibited the platelet, persistently high systemic TXA2 and PGE2 biosynthesis were found, plausibly for a marginal inhibitory effect on prostanoid biosynthesis in the colorectum. Novel chemotherapeutic strategies in FAP can involve blocking the effects of TXA2 and PGE2 signaling with receptor antagonists.

This Italian retrospective study aimed to analyze the pharmaco-utilization of anti-VEGF drugs and health care costs among patients with wet age-related macular degeneration (wAMD) or other ocular diseases. A retrospective analysis was performed on administrative databases of Italian entities covering approximately six million individuals. Across January 2010–December 2017, patients aged ≥50 years with a prescription of intravitreal anti-VEGFs were included as “wAMD” patients [by wAMD hospitalization or intravitreal injections] or as “other ocular diseases” patients [by hospitalization for other ocular disorders or intravitreal injections, with concomitant diabetes diagnosis or dexamethasone treatment]. The date of first matching of inclusion criteria was index-date. wAMD-cohort. Overall, 3879 patients were included; at index-date, 82.2% were treated with Ranibizumab, 15.8% with Aflibercept, and 2% with Pegaptanib. During the follow-up, the mean/annual anti-VEGF prescription [3.6 (first-year)–0.8 (third-year)] and the total expenditure [5799.84 € (first-year)–3212.84 € (third-year)] decreased. Other ocular diseases-cohort. Overall, 2646 patients were enclosed; 85.9% were treated with Ranibizumab, 13.5% with Aflibercept, and 0.6% with Pegaptanib. During the follow-up, the mean/annual anti-VEGF prescription [3.3 (first-year)–0.5 (third-year)] and the total cost [7196.83 € (first-year)–5162.68 € (third-year)] decreased. This observational study highlighted a decline in anti-VEGF prescriptions over time in both cohorts, suggesting a trend of under-treatment that could worsen the patients’ clinical outcomes and increase health care resource consumption.

Platelet-type lipoxygenase (pl12-LOX), encoded by ALOX12, catalyzes the production of the lipid mediator 12S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12S-HpETE), which is quickly reduced by cellular peroxidases to form 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12S-HETE). Platelets express high levels of pl12-LOX and generate considerable amounts of 12S-HETE from arachidonic acid (AA; C20:4, n-6). The development of sensitive chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods has allowed the accurate quantification of 12S-HETE in biological samples. Moreover, advances in the knowledge of the mechanism of action of 12S-HETE have been achieved. The orphan G-protein-coupled receptor 31 (GPR31) has been identified as the high-affinity 12S-HETE receptor. Moreover, upon platelet activation, 12S-HETE is produced, and significant amounts are found esterified to membrane phospholipids (PLs), such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), promoting thrombin generation. Platelets play many roles in cancer metastasis. Among them, the platelets' ability to interact with cancer cells and transfer platelet molecules by the release of extracellular vesicles (EVs) is noteworthy. Recently, it was found that platelets induce epithelial-mesenchymal transition(EMT) in cancer cells, a phenomenon known to confer high-grade malignancy, through the transfer of pl12-LOX contained in platelet-derived EVs. These cancer cells now generate 12-HETE, considered a key modulator of cancer metastasis. Interestingly, 12-HETE was mainly found esterified in plasmalogen phospholipids of cancer cells. This review summarizes the current knowledge on the regulation and functions of pl12-LOX in platelets and cancer cells and their crosstalk.Novel approaches to preventing cancer and metastasis by the pharmacological inhibition of pl12-LOX and the internalization of mEVs are discussed.

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.

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE

Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A

Ion channels are transmembrane proteins that play important roles in cell function regulation modulating ionic cell permeability. In megakaryocytes and platelets, regulated ion flows have been demonstrated to modulate platelet production and function. However, a relatively limited characterization of ion channel expression and function is available in the human megakaryocyte-platelet lineage.We analyzed the expression and function of the large-conductance calcium and voltage-activated potassium channel KTo investigate the functionality of KIn megakaryocytes, KThe present study reveals new mechanisms in platelet formation and activation, suggesting that targeting K

Background Ion channels are transmembrane proteins that play important roles in cell function regulation modulating ionic cell permeability. In megakaryocytes and platelets, regulated ion flows have been demonstrated to modulate platelet production and function. However, a relatively limited characterization of ion channel expression and function is available in the human megakaryocyte-platelet lineage. Objective We analyzed the expression and function of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 (also known as Maxi-K, BK, slo1) in human megakaryocytes and platelets. Methods To investigate the functionality of Kca 1.1, we exploited different agonists (BMS-191011, NS1619, NS11021, epoxyeicosatrienoic acid isoforms) and inhibitors (iberiotoxin, penitrem A) of the channel. Results In megakaryocytes, Kca 1.1 agonists determined a decreased proplatelet formation and altered interaction with the extracellular matrix. Analysis of the actin cytoskeleton demonstrated a significant decrease in megakaryocyte spreading and adhesion to collagen. In platelets, the opening of the channel Kca 1.1 led to a reduced sensitivity to agonists with blunted aggregation in response to ADP, with an inhibitory capacity additive to that of aspirin. The Kca 1.1 agonists, but not the inhibitors, determined a reduction of platelet adhesion and aggregation onto immobilized collagen underflow to an extent similar to that of aspirin and ticagrelor. The opening of the Kca 1.1 resulted in cell hyperpolarization impairing free intracellular calcium in ADP-stimulated platelets and megakaryocytes. Conclusions The present study reveals new mechanisms in platelet formation and activation, suggesting that targeting Kca 1.1 channels might be of potential pharmacological interest in hemostasis and thrombosis.

Neointima hyperplasia is a crucial component of restenosis after coronary angioplasty. We have hypothesized that enhanced generation of platelet-derived thromboxane (TX)A2 in response to vascular damage plays a critical role in neointimal hyperplasia and that antiplatelet agents may mitigate it. In cocultures of human platelets and coronary artery smooth muscle cells (CASMC), we found that platelets induced morphologic changes and enhanced the migration of CASMC. The exposure of platelets to Aspirin [an inhibitor of cyclooxygenase (COX)-1] reduced the generation of TXA2 and prevented the morphological and functional changes induced by platelets in CASMC. Platelet-derived TXA2 induced COX-2 and enhanced prostaglandin (PG)E2 biosynthesis in CASMC, a known mechanism promoting neointimal hyperplasia. COX-2 induction was prevented by different antiplatelet agents, i.e., Aspirin, the TP antagonist SQ29,548, or Revacept (a dimeric soluble GPVI-Fc fusion protein). The administration of the novel antiplatelet agent Revacept to C57BL/6 mice, beginning three days before femoral artery denudation, and continuing up to seven days after injury, prevented the increase of the systemic biosynthesis di TXA2 and reduced femoral artery intima-to-media area and the levels of markers of cell proliferation and macrophage infiltration. Revacept might serve as a therapeutic agent for percutaneous coronary angioplasty and stent implantation.

This Italian real-world data analysis evaluated the pharmaco-utilization of calcimimetics, cinacalcet or etelcalcetide, and the economic burden of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD) patients. From 1 January 2010 to 30 June 2020, adult patients with: (i) ≥1 prescription of etelcalcetide or cinacalcet, (ii) ≥3 hemodialysis/week, and (iii) without parathyroidectomy, were included. Based on the drug firstly prescribed, patients were allocated into etelcalcetide- and cinacalcet-treated cohorts, and the propensity score matching (PSM) methodology was applied to abate potential cohorts’ unbalances. Overall, 1752 cinacalcet- and 527 etelcalcetide-treated patients were enrolled. In cinacalcet- and etelcalcetide-treated patients, respectively, the most frequent comorbidities were hypertension (75.3% and 74.4%), diabetes mellitus (21.0% and 21.3%), and cardiovascular disease (18.1% and 13.3%, p < 0.01). In covariate-balanced cohorts, the treatment adherence and persistence rates were significantly higher in the etelcalcetide-treated (80.1% and 62.7%, respectively) vs. cinacalcet-treated cohort (62.3% and 54.7%, respectively). After PSM, the total costs for the management of cinacalcet- and etelcalcetide-treated patients, respectively, averaged EUR 23,480 and EUR 22,958, with the disease-specific drug costs (EUR 2629 vs. EUR 2355, p < 0.05) and disease-specific hospitalization costs (EUR 1241 vs. EUR 855) in cinacalcet- and etelcalcetide-treated patients. These results showed that, in etelcalcetide-treated patients, a higher treatment adherence and persistence was found, with disease-specific costs savings, especially those related to drugs and hospitalizations.

Platelets contribute to several types of cancer through plenty of mechanisms. Upon activation, platelets release many molecules, including growth and angiogenic factors, lipids, and extracellular vesicles, and activate numerous cell types, including vascular and immune cells, fibroblasts, and cancer cells. Hence, platelets are a crucial component of cell–cell communication. In particular, their interaction with cancer cells can enhance their malignancy and facilitate the invasion and colonization of distant organs. These findings suggest the use of antiplatelet agents to restrain cancer development and progression. Another peculiarity of platelets is their capability to uptake proteins and transcripts from the circulation. Thus, cancer-patient platelets show specific proteomic and transcriptomic expression patterns, a phenomenon called tumor-educated platelets (TEP). The transcriptomic/proteomic profile of platelets can provide information for the early detection of cancer and disease monitoring. Platelet ability to interact with tumor cells and transfer their molecular cargo has been exploited to design platelet-mediated drug delivery systems to enhance the efficacy and reduce toxicity often associated with traditional chemotherapy. Platelets are extraordinary cells with many functions whose exploitation will improve cancer diagnosis and treatment.

RationaleThe development of inhibitors of microsomal prostaglandin (PG)E2 synthase-1 (mPGES-1) was driven by the promise of attaining antiinflammatory agents with a safe cardiovascular profile because of the possible diversion of the accumulated substrate, PGH2, towards prostacyclin (PGI2).ObjectivesWe studied the effect of the human mPGES-1 inhibitor, AF3485 (a benzamide derivative) on prostanoid biosynthesis in human whole blood in vitro. To characterize possible off-target effects of the compound, we evaluated: i)the impact of its administration on the systemic biosynthesis of prostanoids in a model of complete Freund's adjuvant (CFA)-induced monoarthritis in rats; ii) the effects on cyclooxygenase (COX)-2 expression and the biosynthesis of prostanoids in human monocytes and human umbilical vein endothelial cells (HUVECs) in vitro.MethodsProstanoids were assessed in different cellular models by immunoassays. The effect of the administration of AF3485 (30 and 100 mg/kg,i.p.) or celecoxib (20mg/kg, i.p.), for 3 days, on the urinary levels of enzymatic metabolites of prostanoids, PGE-M, PGI-M, and TX-M were assessed by LC-MS.ResultsIn LPS-stimulated whole blood, AF3485 inhibited PGE2 biosynthesis, in a concentration-dependent fashion. At 100μM, PGE2 levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB2 inhibition (40.56 ± 5.77%). AF3485 administration to CFA-treated rats significantly reduced PGE-M (P < 0.01) and TX-M (P < 0.05) similar to the selective COX-2 inhibitor, celecoxib. In contrast, AF3485 induced a significant (P < 0.05) increase of urinary PGI-M while it was reduced by celecoxib. In LPS-stimulated human monocytes, AF3485 inhibited PGE2 biosynthesis with an IC50 value of 3.03 µM (95% CI:0.5–8.75). At 1μM, AF3485 enhanced TXB2 while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB2. At 100 μM, maximal inhibition of the two prostanoids was associated with the downregulation of COX-2 protein by 86%. These effects did not involve AMPK pathway activation, IkB stabilization, or PPARγ activation. In HUVEC, AF3485 at 100 μM caused a significant (P < 0.05) induction of COX-2 protein associated with enhanced PGI2 production. These effects were reversed by the PPARγ antagonist GW9662.ConclusionsThe inhibitor of human mPGES-1 AF3485 is a novel antiinflammatory compound which can also modulate COX-2 induction by inflammatory stimuli. The compound also induces endothelial COX-2-dependent PGI2 production via PPARγ activation, both in vitro and in vivo, which might translate into a protective effect for the cardiovascular system.

Platelets are involved in the development and progression of cancer through several mechanisms. Platelet activation at the site of tissue damage contributes to the initiation of a cascade of events which promote tumorigenesis. In fact, platelets release a wide array of proteins, including growth and angiogenic factors, lipids and extracellular vesicles rich in genetic material, which can mediate the induction of phenotypic changes in target cells, such as immune, stromal and tumor cells, and promote carcinogenesis and metastasis formation. Importantly, the role of platelets in tumor immune escape has been described. These lines of evidence open the way to novel strategies to fight cancer based on the use of antiplatelet agents. In addition to their ability to release factors, platelets are able of up-taking proteins and genetic material present in the bloodstream. Platelets are like ‘sentinels’ of the disease state. The evaluation of proteomics and transcriptomics signature of platelets and platelet-derived microparticles could represent a new strategy for the development of biomarkers for early cancer detection and/or therapeutic drug monitoring in cancer chemotherapy. Owing to the ability of platelets to interact with cancer cells and to deliver their cargo, platelets have been proposed as a ‘biomimetic drug delivery system’ for anti-tumor drugs to prevent the occurrence of off-target adverse events associated with the use of traditional chemotherapy.

Several pieces of evidence support the role of activated platelets in the development of the chronic inflammation-related diseases, such as atherothrombosis and cancer, mainly via the release of soluble factors and microparticles (MPs). Platelets and MPs contain a repertoire of proteins and genetic material (i.e., mRNAs and microRNAs) which may be influenced by the clinical condition of the individuals. In fact, platelets are capable of up-taking proteins and genetic material during their lifespan. Moreover, the content of platelet-derived MPs can be delivered to other cells, including stromal, immune, epithelial, and cancer cells, to change their phenotype and functions, thus contributing to cancer promotion and its metastasization. Platelets and MPs can play an indirect role in the metastatic process by helping malignant cells to escape from immunological surveillance. Furthermore, platelets and their derived MPs represent a potential source for blood biomarker development in oncology. This review provides an updated overview of the roles played by platelets and MPs in cancer and metastasis formation. The possible analysis of platelet and MP molecular signatures for the detection of cancer and monitoring of anticancer treatments is discussed. Finally, the potential use of MPs as vectors for drug delivery systems to cancer cells is put forward.

We studied the influence of cardiovascular (CV) risk factors, previous CV events, and cotreatments with preventive medicines, on residual platelet thromboxane (TX)B2 production in 182 patients chronically treated with enteric coated (EC)-aspirin (100 mg/day). The response to aspirin was also verified by assessing arachidonic acid-induced platelet aggregation and urinary 11-dehydro-TXB2 levels. Residual serum TXB2 levels exceeded the upper limit value for an adequate aspirin response in 14% of individuals. This phenomenon was detected at 12 hours after dosing with aspirin. The coadministration of statins (mostly atorvastatin) was an independent predictor of residual serum TXB2 levels, and the percentage of patients with enhanced values was significantly lower in statin users vs. nonusers. We provide evidence in vitro that atorvastatin reduced residual TXB2 generation by increasing the extent of acetylation of platelet COX-1 by aspirin. In conclusion, the coadministration of statins may counter the mechanisms associated with reduced bioavailability of aspirin detected in some individuals with CV disease.

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.

The prevention of cancer development and its progression is an urgent unmet medical need. Novel knowledge on the biology of cancer has evidenced that genetic changes occurring within cancer cells contribute, but are not sufficient, for tumor promotion and progression. The results of clinical studies and experimental animal models have suggested pursuing new avenues for the prevention of cancer development in the early stages, by using drugs that modulate platelet responses and those interfering with the synthesis and action of the mediators of inflammation. In fact, malignant tumors often develop at sites of chronic injury associated with platelet activation and chronic inflammation. In this review, we cover the evidence supporting this hypothesis and the rationale for the pharmacological treatment with antiplatelet agents, including low-dose aspirin, and antiinflammatory drugs to curb tumorigenesis and malignant progression. The evidence for a chemopreventive effect of low-dose aspirin against colorectal cancer (CRC) has been recently found appropriate by the U.S. Preventive Services Task Force, which recommends the use of the drug for primary prevention of cardiovascular disease and CRC.

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B2 production; ASA selectively affected TXB2 production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB2 while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y12 blocker) affected platelet aggregation associated with profound inhibition of TXB2 generation. P2Y12 receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.

The most recognized mechanism of aspirin (acetylsalicylic acid, ASA) action, at therapeutic dosing, is the inhibition of prostanoid biosynthesis through the acetylation of cyclooxygenase (COX)-isozymes (COX-1 at serine-529 and COX-2 at serine-516). Whether aspirin, also when given at the low-doses recommended for cardiovascular prevention, reduces the risk of colorectal cancer by affecting COX-2 activity in colorectal adenomatous lesions is still debated. We aimed to develop a direct biomarker of aspirin action on COX-2 by assessing the extent of acetylation of COX-2 at serine-516 using the AQUA strategy, enabling absolute protein quantitation by liquid chromatography-mass spectrometry. We compared the extent of acetylation and the inhibition of prostanoid biosynthesis by ASA using human recombinant COX-2 (hu-COX-2), the human colon cancer cell line HCA-7, isolated human monocytes stimulated with LPS (lipopolysaccharide) or human intestinal epithelial cells stimulated with interleukin (IL)-1β. Hu-COX-2 exposed in vitro to an excess of ASA was acetylated by approximately 40–50% associated with the inhibition of COX-2 activity by 80–90%. In the three cell-types expressing COX-2, the extent of COX-2 acetylation and reduction of prostaglandin (PG) E2 biosynthesis by ASA was concentration-dependent with comparable EC50 values (in the low μM range). The maximal % acetylation of COX-2 averaged 80%, at ASA 1000 μM, and was associated with a virtually complete reduction of PGE2 biosynthesis (97%). In conclusion, we have developed a proteomic assay to evaluate the extent of acetylation of COX-2 at serine-516 by aspirin; its use in clinical studies will allow clarifying the mechanism of action of aspirin as anticancer agent.

Platelet activation is the first response to tissue damage and, if unrestrained, may promote chronic inflammation-related cancer, mainly through the release of soluble factors and vesicles that are rich in genetic materials and proteins. Platelets also sustain cancer cell invasion and metastasis formation by fostering the development of the epithelial-mesenchymal transition phenotype, cancer cell survival in the bloodstream and arrest/extravasation at the endothelium. Furthermore, platelets contribute to tumor escape from immune elimination. These findings provide the rationale for the use of antithrombotic agents in the prevention of cancer development and the reduction of metastatic spread and mortality. Among them, low-dose aspirin has been extensively evaluated in both preclinical and clinical studies. The lines of evidence have been considered appropriate to recommend the use of low-dose aspirin for primary prevention of cardiovascular disease and colorectal cancer by the USA. Preventive Services Task Force. However, two questions are still open: (i) the efficacy of aspirin as an anticancer agent shared by other antiplatelet agents, such as clopidogrel; (ii) the beneficial effect of aspirin improved at higher doses or by the co-administration of clopidogrel. This review discusses the latest updates regarding the mechanisms by which platelets promote cancer and the efficacy of antiplatelet agents.

Background and Purpose Hyperglycaemic memory describes the progression of diabetic complications during subsequent periods of improved glycaemia. We addressed the hypothesis that transient hyperglycaemia causes aberrant COX-2 expression in HUVEC in response to IL-1β through the induction of long-lasting epigenetic changes involving microRNA-16 (miR-16), a post-transcriptional modulator of COX-2 expression. Experimental Approach Studies were performed on HUVEC collected from women with gestational diabetes mellitus (GDM) (dHUVEC) and normal women (nHUVEC). Key Results In dHUVEC treated with IL-1β, the expression of COX-2 mRNA and protein was enhanced and generation of prostanoids increased (the most abundant was the promitogenic PGF2α). COX-2 mRNA was more stable in dHUVEC and this was associated with miR-16 down-regulation and c-Myc induction (a suppressor of miR expression). dHUVEC showed increased proliferation in response to IL-1β, which was prevented by a COX-2 inhibitor and PGF2α receptor antagonist. Comparable changes in COX-2 mRNA, miR-16 and c-Myc detected in dHUVEC were produced in nHUVEC exposed to transient high glucose and then stimulated with IL-1β under physiological glucose levels; superoxide anion production was enhanced under these experimental conditions. Conclusions and Implications Our results describe a possible mechanism operating in GDM that links the enhanced superoxide anion production and epigenetic changes, associated with hyperglycaemic memory, to endothelial dysfunction through dysregulated post-transcriptional control of COX-2 gene expression in response to inflammatory stimuli. The association of conventional therapy for glycaemic control with agents affecting inflammatory responses and oxidative stress might lead to a more effective prevention of the complications associated with GDM.

The mechanism of action of low-dose aspirin in the prevention of colorectal cancer (CRC) remains largely hypothetical. We aimed to compare the effects of low-dose aspirin (100 mg/day for 7 days) given to 40 individuals undergoing CRC screening on the extent of cyclooxygenase (COX)-1 acetylation at serine-529 (AceCOX-1), in blood platelets vs. colorectal mucosa, at 7 (group 1) and 24 h (group 2) after dosing. A significantly (P < 0.01) lower %AceCOX-1 was detected in colonic and rectal mucosa (average 64%) vs. platelets (average 75%) in both groups. This effect was associated with an average 46% (P < 0.01) and 35% (P < 0.05) reduction in prostaglandin (PG) E2 levels and phosphorylated S6 (p-S6) levels, respectively. Rectal mucosal levels of p-S6/S6 significantly (P < 0.01) correlated with PGE2 . These findings demonstrate that low-dose aspirin produces long-lasting acetylation of COX-1 and downregulation of p-S6 in human colorectal mucosa, an effect that may interfere with early colorectal carcinogenesis.

A growing body of evidence supports the central role of platelets in early events of tumorigenesis and metastasis. Activated platelets, in response to tissue damage, induce a proinflammatory program involving the aberrant expression of cyclooxygenase (COX)-2, which leads to increased tissue concentrations of the proinflammatory and protumorigenic prostaglandin E2. The central role of platelet activation in cancer development is sustained by the analysis of clinical studies with aspirin showing an anti-cancer efficacy by the drug, even at the low doses used for the prevention of atherothrombosis. Low-dose aspirin acts as an antiplatelet agent by causing an irreversible inactivation of platelet COX-1 activity and the synthesis of thromboxane A2. Further experimental and clinical studies are ongoing to confirm the central role of platelets in the development of inflammation and cancer. The corroboration of this hypothesis will open new opportunities for the prevention and treatment of cancer. In addition to the possible use of traditional antithrombotic agents, the recent identification of novel molecular determinants involved in the cross-talk between platelets and other cellular player of tumorigenesis and metastasis has led to the suggestion of novel therapeutic strategies in oncology.

Platelets are anucleated cells that circulate in the blood as sentinels of tissue integrity. In fact, they are rich in a plethora of proteins and other factors stored in different granules which they selectively release upon stimulation. Moreover, platelets synthesize a vast number of lipids and release various types of vesicles, including exosomes which are rich in genetic material. Platelets possess a central function to interact with other cell types, including inflammatory cells and cancer cells. Recent findings have enlightened the capacity of platelets to induce changes in the phenotype of cancer cells which acquire invasiveness thus enhancing their metastatic potential. Thus, it has been hypothesized that targeting the platelet may represent a novel strategy to prevent the development and progression of cancer. This is supported by the efficacy of the antiplatelet agent low-dose aspirin. Studies are ongoing to verify whether other antiplatelet agents share the anticancer effectiveness of aspirin.

Morphine is mainly transformed to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in the liver. Glucuronidation is also performed by rat brain homogenates and UDP-glucuronosyltransferases (UGTs) are present in the brain. Here we investigated the possibility that microglia transforms morphine into its metabolites M3G and M6G. Primary cultures of neonatal rat microglia were incubated for different intervals of time in basal conditions or with different concentrations of morphine. The following measures were performed on these cultures and/or in the medium: (i) morphine as well as M3G and M6G concentrations; (ii) levels of mRNA coding for UGT1A1, UGT1A6, UGT1A7, and UGT2B1 as well as their protein levels; (iii) released prostaglandin (PG)E2 and nitrite concentrations. Results show that in basal conditions morphine and M3G are produced by microglia; accordingly, these cells expressed UGT1A1, UGT1A6 and UGT1A7, but not UGT2B1. When cultures were exposed to different concentrations of exogenous morphine, M6G was also synthesized. This shift in the glucuronidation was associated with variations in the expression of UGT isozymes. In particular, UGT1A7 expression was rapidly upregulated and this event was translated into enhanced protein levels of UGT1A7; lesser effects were exerted on UGT1A1 and UGT1A6. Upon prolonged exposure to morphine, microglial cell UGT expression returned to baseline conditions or even to reduced levels of expression. Morphine exposure did not affect the synthesis of both PGE2 and nitrites, ruling out a generalized priming of microglia by morphine. In conclusion, this study suggests that morphine glucuronides found in the cerebrospinal liquor upon peripheral morphine administration may at least in part be brain-born, reconciling the conceptual gap between the high hydrophilic features of morphine glucuronides and their presence beyond the blood-brain barrier.

A growing body of evidences suggests that aspirin and nonaspirin nonsteroidal anti-inflammatory drugs (NSAIDs) cause anticancer effects, mainly in the colorectum. At therapeutic doses, these agents act through the inhibition of cyclooxygenase (COX)-1 and COX-2, and, in turn, they affect the biosynthesis of prostanoids. Interestingly, the cancer chemopreventive effect was also found with low-dose aspirin, which is used to prevent cardiovascular disease for its preferential inhibitory effect on platelet COX-1. This finding led to hypothesize that the antiplatelet effect of aspirin is involved in its cancer chemopreventive action. In fact, it has been proposed that activated platelets, in response to tissue damage, may induce a pro-inflammatory program in tumor microenvironment involving enhanced biosynthesis of COX-2-dependent prostaglandin E2, a pro-inflammatory and protumorigenic prostanoid. Selective inhibitors of COX-2 (named coxibs) significantly reduce the risk of recurrent colorectal adenomas, but they may enhance the risk of cardiovascular events, thus precluding their use as anticancer agents. Importantly, the inhibition of platelet function by low-dose aspirin my also prevent tumor metastasis. In fact, within the circulatory system, the formation of platelet aggregates surrounding tumor cells will support tumor cell survival and protection from immune elimination and promote their arrest at the endothelium and extravasation. Aspirin and other NSAIDs are able to affect proliferation and to induce apoptosis of colon cancer cells in vitro, independently from their inhibitory effect on COX-dependent prostanoid biosynthesis, at very high concentrations, often in the millimolar range, that are not reached in the systemic circulation, at therapeutic doses.