The role of respiratory function tests in infants with stridor: diagnosis at glance and follow-up.

BACKGROUND: Recently, the development of advanced, noninvasive methods has allowed the study of respiratory function even in uncooperative infants. To date, there is still little data on the application of this technique in infants with suspected airway obstruction. THE AIMS OF OUR STUDY WERE: - To evaluate the role of respiratory function testing (PFR) in the diagnosis and follow-up of infants with stridor - To evaluate the differences between patients with inspiratory stridor and expiratory stridor. - To evaluate the concordance between PFR and endoscopy. METHODS: We enrolled infants aged < 1 year with a diagnosis of inspiratory and/or expiratory chronic stridor and a group of healthy controls. For each patient we performed PFR at diagnosis (T0) and for cases at follow-up, at 3 months (T1), 6 months (T2), 12 months (T3). At T0, all patients were classified according to a clinical score, and at follow-up, stature-ponderal growth was assessed. When clinically indicated, patients underwent bronchoscopy. RESULTS: We enrolled 48 cases (42 diagnosed with inspiratory stridor and 6 expiratory stridor) and 26 healthy controls. At T0, patients with stridor had increased inspiratory time (p < 0.0001) and expiratory time (p < 0.001) than healthy controls and abnormal curve morphology depending on the type of stridor. At T0, patients with expiratory stridor had a reduced Peak expiratory flow (p < 0.023) and a longer expiratory time (p < 0.004) than patients with inspiratory stridor. We showed an excellent concordance between PFR and endoscopic examination (k = 0.885, p < 0.0001). At follow-up, we showed a progressive increase of the respiratory parameters in line with the growth. CONCLUSIONS: PFR could help improve the management of these patients through rapid and noninvasive diagnosis, careful monitoring, and early detection of those most at risk.

Extracellular Vesicles Induce Nuclear Factor-κB Activation and Interleukin-8 Synthesis through miRNA-191-5p Contributing to Inflammatory Processes: Potential Implications in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Extracellular vesicles (EVs) play a pivotal role in a variety of physiologically relevant processes, including lung inflammation. Recent attention has been directed toward EV-derived microRNAs (miRNAs), such as miR-191-5p, particularly in the context of inflammation. Here, we investigated the impact of miR-191-5p-enriched EVs on the activation of NF-κB and the expression of molecules associated with inflammation such as interleukin-8 (IL-8). To this aim, cells of bronchial epithelial origin, 16HBE, were transfected with miR-191-5p mimic and inhibitor and subsequently subjected to stimulations to generate EVs. Then, bronchial epithelial cells were exposed to the obtained EVs to evaluate the activation of NF-κB and IL-8 levels. Additionally, we conducted a preliminary investigation to analyze the expression profiles of miR-191-5p in EVs isolated from the plasma of patients diagnosed with chronic obstructive pulmonary disease (COPD). Our initial findings revealed two significant observations. First, the exposure of bronchial epithelial cells to miR-191-5p-enriched EVs activated the NF-kB signaling and increased the synthesis of IL-8. Second, we discovered the presence of miR-191-5p in peripheral blood-derived EVs from COPD patients and noted a correlation between miR-191-5p levels and inflammatory and functional parameters. Collectively, these data corroborate and further expand the proinflammatory role of EVs, with a specific emphasis on miR-191-5p as a key cargo involved in this process. Consequently, we propose a model in which miR-191-5p, carried by EVs, plays a role in airway inflammation and may contribute to the pathogenesis of COPD.

Unveiling the Cutting-Edge Impact of Polarized Macrophage-Derived Extracellular Vesicles and MiRNA Signatures on TGF-ß Regulation within Lung Fibroblasts.

Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-ß production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-ß in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-ß production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-ß in fibroblasts, with potential regulation by miRNA-425-5p.

Sharenting: characteristics and awareness of parents publishing sensitive content of their children on online platforms.

BACKGROUND: The term "sharenting" describes the increasingly popular habit of parents to share photos, videos, or other information regarding their children on their social profiles, through online platforms. It is highly likely that many parents are posting content about their underage children online with little knowledge of the risks associated with this practice. This study aims to investigate whether variables such as parents' age, gender, marital status, occupation and educational level influence the practice of sharing child-related content and the degree of awareness. METHODS: We performed a pilot cross-sectional study, based on an anonymous questionnaire. The questionnaire was administered to parents of underage children attending the pediatric outpatient clinic of the Umberto I Hospital, Sapienza University, in Rome, Italy, by researchers, through the google forms platform; qualitative variables were generated on excel sheets and a statistical analysis was performed on SPSS Ibm-statistics using the chi-square test. RESULTS: Two hundred twenty-eight parents of children under 18 years of age completed the questionnaire (82% mothers, 18% fathers); 98% of the respondents used social media and 75% of them published their children's related content online. Thirty-one percent of the compilers started their practice of sharenting in the first 6 months of life of their child. Our analysis showed that compared to parents who do not post online, parents who usually post online their children are significantly more likely to be partial employees or unemployed (p = 0,002), with lower educational level (p = 0,05), younger (less than 35 years of age (p = 0,01)) and have a higher number of followers (p < 0,001). Finally, 93% of the compilers were not aware of the current legislation and of the risks related to the practice of sharenting. CONCLUSIONS: Pediatricians, healthcare assistants and preventive healthcare professionals should play a central role in alerting parents and families to the risks of sharenting; the results of our study could draw their attention to the increasing practice of sharenting and make healthcare professionals active part in the protection of children.

Rectus sheath block added to parasternal block may improve postoperative pain control and respiratory performance after cardiac surgery: a superiority single-blinded randomized controlled clinical trial.

BACKGROUND: The population undergoing cardiac surgery confronts challenges from uncontrolled post-sternotomy pain, with possible adverse effects on outcome. While the parasternal block can improve analgesia, its coverage may be insufficient to cover epigastric area. In this non-blinded randomized controlled study, we evaluated the analgesic and respiratory effect of adding a rectus sheath block to a parasternal block. METHODS: 58 patients undergoing cardiac surgery via median sternotomy were randomly assigned to receive parasternal block with rectus sheath block (experimental) or parasternal block with epigastric exit sites of chest drains receiving surgical infiltration of local anesthetic (control). The primary outcome of this study was pain at rest at extubation. We also assessed pain scores at rest and during respiratory exercises, opiate consumption and respiratory performance during the first 24 hours after extubation. RESULTS: The median (IQR) maximum pain scores (on a 0-10 Numeric Rate Scale (NRS)) at extubation were 4 (4, 4) in the rectus sheath group and 5 (4, 5) in the control group (difference 1, p value=0.03). Rectus sheath block reduced opioid utilization by 2 mg over 24 hours (IC 95% 0.0 to 2.0; p<0.01), reduced NRS scores at other time points, and improved respiratory performance at 6, 12, and 24 hours after extubation. CONCLUSION: The addition of a rectus sheath block with a parasternal block improves analgesia for cardiac surgery requiring chest drains emerging in the epigastric area. TRIAL REGISTRATION NUMBER: NCT05764616.

Rethinking food policy framework to tackle childhood obesity in the Italian context.

Pediatric obesity has been described by the World Health Organization as 1 of the most serious public health challenges of the 21st century. Projections of increasing burdens of pediatric obesity and its related diseases on society highlight the need for urgent and substantial action. Many scientific and public debates about the prevention of childhood obesity are centered around simple dichotomies presenting a single-level solution. In contrast, efficient prevention programs should overcome these overly simplistic dichotomies and proceed in the early years of life within the family environment and the whole society, throughout one's lifetime. Food policies have the potential to counteract pediatric obesity by creating healthy food environments. However, the current food policies approach lacks monitoring indicators to assess short- and long-term impact, and is not well integrated into regional, national, and cross-cutting initiatives. Therefore, redesigning and rethinking food policy strategies and goals is an important opportunity to address childhood obesity, safeguard the planet, and contribute to economic and social prosperity.

SARS-CoV-2-specific mucosal immune response in vaccinated versus infected children.

The anti-COVID-19 intramuscular vaccination induces a strong systemic but a weak mucosal immune response in adults. Little is known about the mucosal immune response in children infected or vaccinated against SARS-CoV-2. We found that 28% of children had detectable salivary IgA against SARS-CoV-2 even before vaccination, suggesting that, in children, SARS-CoV-2 infection may be undiagnosed. After vaccination, only receptor-binding domain (RBD)-specific IgA1 significantly increased in the saliva. Conversely, infected children had significantly higher salivary RBD-IgA2 compared to IgA1, indicating that infection more than vaccination induces a specific mucosal immune response in children. Future efforts should focus on development of vaccine technologies that also activate mucosal immunity.

Suppressor of cytokine signaling-3 expression and its regulation in relation to inflammation in Chronic Obstructive Pulmonary Disease.

Background: The family of Suppressor of Cytokine Signaling (SOCS) acts as a controller of the duration and intensity of cytokine function by negatively regulating the JAK-STAT signaling pathway. SOCS' role in inflammatory diseases in animal models is well demonstrated. However, its role in the development of human disease is still under investigation. SOCS3 plays an important role in tumor development where its downregulation has been implicated in the pathogenesis of various solid tumors such as triple-negative breast cancer. Aim: The aim of this work was to study (1) the expression of SOCS3 in smokers' lungs and its relation to the degree of inflammation and (2) SOCS3 regulation by microRNA (miRNA) in alveolar-macrophage (AM)-derived extracellular vesicles (EVs) in bronchoalveolar lavage (BAL). Methods: Group A: 35 smokers' [19 with COPD (SC) and 16 without COPD (S)] and 9 nonsmokers (NS); SOCS3, TNFα in AM, and CD8+ T cells were quantified by immunohistochemistry, in lung tissue. Group B: additional 9 SC, 11 S, and 5 NS; AM-EVs expressing SOCS3 (CD14+SOCS3+) and SOCS3 suppressors miRNA-19a-3p and 221-3p in EVs were quantified by flow cytometry and PCR, in BAL. Results: The percentage of SOCS3+ AM was higher in SC [68 (6.6-99)%] and S [48 (8-100)%] than in NS [9.6 (1.9-61)%; p = 0.002; p = 0.03] and correlated with % of TNFα+AM (r = 0.48; p = 0.0009) and CD8+ T cells (r = 0.44; p = 0.0029). In BAL, the CD14+SOCS3+ EVs/µL were increased in SC [33 (21-74)] compared to S [16 (8-37); p = 0.03] and NS [9 (7-21); p = 0.003]. Conversely, miRNA-19a-3p and miRNA-221-3p expression were increased in S when compared to SC [19 (2-53) vs. 3 (0.6-8); p = 0.03 and 3 (0.005-9.6) vs. 0.2 (0.08-0.7); p = 0.05]. Conclusions: The suppressor function of SOCS3 in COPD seems to be overridden by other factors and does not follow the animal-model paradigm. Expression of SOCS3 in BAL macrophage-derived EVs might be useful to assess the degree of inflammation and possible progression of COPD. Downregulation of SOCS3, by miRNA, in smokers without COPD might contribute to the risk of developing cancer in these patients.

Respiratory Syncytial Virus Bronchiolitis Before and After COVID-19 Pandemic: Has the Immunity Debt Been Paid Off?

BACKGROUND: Our aim was to hypothesize that the COVID-19 pandemic influenced the characteristics of viral bronchiolitis by comparing the last 3 epidemics with 3 pre-COVID-19 epidemics in infants hospitalized with bronchiolitis. METHODS: We prospectively enrolled 637 consecutive infants (median age 3.0 ± 2.1 months, 58.5% males), hospitalized for bronchiolitis during 6 consecutive annual epidemic seasons from 2017 to 2023. All parents of the children were given a structured anamnestic questionnaire. A nasopharyngeal aspirate was tested for 15 respiratory viruses. As measures of severity, we evaluated the O 2 supplementation and the admission at the pediatric intensive care unit. RESULTS: A total of 166 were hospitalized with bronchiolitis in 2017-2018, 97 in 2018-2019, 69 in 2019-2020, 0 in 2020-2021, 129 in 2021-2022 and 176 in 2022-2023. Taking together the 332 bronchiolitis cases hospitalized during the 3 prepandemic seasons, they peaked between December and January; after the flat curve in 2020-2021, the cases of bronchiolitis peaked in November 2021 and in December 2022. While the 2021-2022 season registered a less severe clinical presentation, O 2 supplementation and pediatric intensive care unit admissions increased in 2022-2023 with respect to the prepandemic seasons ( P < 0.001). CONCLUSIONS: This study represents an important scientific demonstration of the impact of primary prevention measures on the epidemiology of viral infections; their fluctuations were related to the intensity of restrictive measures and to the changing trend of respiratory viruses. It is essential to predict the real temporal trend of bronchiolitis not to leave high-risk children uncovered and to guide hospitals to maintain a high level of readiness.

Outcomes of postnatal systemic corticosteroids administration in ventilated preterm newborns: a systematic review of randomized controlled trials.

Introduction: Prolonged mechanical ventilation, commonly used to assist preterm newborns, increases the risk of developing bronchopulmonary dysplasia (BPD). In recent decades, studies have demonstrated that systemic corticosteroids play a significant role in the prevention and management of BPD. In this systematic review of randomized controlled trials (RCTs), we evaluated the association between the administration of systemic corticosteroids in preterm infants and its long-term outcomes, such as neurodevelopment, growth, extubation rate, and related adverse effects. Methods: We conducted an electronic search in Medline, Scopus, and PubMed using the following terms: "premature infants" and "corticosteroids." We considered all RCTs published up to June 2023 as eligible. We included all studies involving preterm newborns treated with systemic corticosteroids and excluded studies on inhaled corticosteroids. Results: A total of 39 RCTs were evaluated. The influence of steroids administered systemically during the neonatal period on long-term neurological outcomes remains unknown, with no influence observed for long-term growth. The postnatal administration of systemic corticosteroids has been found to reduce the timing of extubation and improve respiratory outcomes. Dexamethasone appears to be more effective than hydrocortisone, despite causing a higher rate of systemic hypertension and hyperglycemia. However, in the majority of RCTs analyzed, there were no differences in the adverse effects related to postnatal corticosteroid administration. Conclusion: Dexamethasone administered during the neonatal period appears to be more effective than hydrocortisone in terms of respiratory outcomes; however, caution should be taken when administering dexamethasone. Data derived from current evidence, including meta-analyses, are inconclusive on the long-term effects of the administration of systemic steroids in preterm infants or the possibility of neurodevelopmental consequences.

Programmes to manage food selectivity in individuals with autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex group of neurodevelopmental disorders characterised by impaired social communication and restricted interests/repetitive behaviours. In this regard, sensory processing difficulties and delayed oral motor skills often predispose individuals with ASD to food selectivity (FS). It is usually associated with repetitive eating patterns that can lead to multiple malnutrition conditions. The objective of this narrative review is to present an overview about the existing nutritional interventions aiming at promoting a healthy eating pattern and addressing food selectivity among individuals with ASD. Regarding the interventions targeting nutrition education, the majority of the analysed studies failed to demonstrate their effectiveness. On the other hand, many educational interventions involving taste or cooking sessions, as well as behavioural interventions for FS, demonstrated effective results. Moreover, multidisciplinary in tailoring such programmes, including psychology speech therapy and nutritional skills, is acknowledged as a key approach.

An overview of the nutritional status of childbearing age women, children and adolescents living in a rural area of Madagascar: preliminary results of the Tany Vao project.

OBJECTIVE: To describe the food consumption, nutrition knowledge and nutritional assessment of childbearing age women and their children, living in rural villages in Madagascar. The results presented are related to the Tany Vao research study. DESIGN: A cross-sectional pilot study. SETTING: The study was carried out in Ampanitosoha village on Nosy Mitsio island in Madagascar. PARTICIPANTS: 32 women (14-49 years) and 36 children and adolescents (2-17 years). RESULTS: 70 % of the women lacked nutrition knowledge and did not reach the Minimum Dietary Diversity Index for Women cut-off. The median BMI was 21·1 kg/m2 but 55·2 % of the women exceeded the cut-off for waist-to-hip ratio, 51·7 % for waist-to-height ratio and 81·2 % for mid-upper arm circumference (MUAC). Almost all had adequate intake of energy, protein and carbohydrates, while 27·6 % had excessive fat intake and 75·9 % of added sugars. Over half of the women did not meet the micronutrients Reference Daily Intake (RDI). For children, the MUAC z-score was lower for boys than for girls (P-value = 0·041). CONCLUSIONS: These results underline the importance of increasing women's nutritional knowledge to promote healthy pregnancy and lactation. Moreover, it is fundamental to provide people living in rural areas with sustainable tools to improve dietary diversity and support long-term health.

Acute Bronchiolitis: The Less, the Better?

BACKGROUND: Acute bronchiolitis is a viral infection of the lower respiratory tract affecting infants aged under 12 months, variably presenting with respiratory distress, diffuse crackles and inflammatory wheezing. The main causative agent is Respiratory Syncytial Virus (RSV). The diagnosis is clinical and treatment mainly supportive. Despite the availability of more than 30 international guidelines, consistent management recommendations are lacking and considerable variability in patients' care persists among different providers. OBJECTIVE: To review and describe current knowledge about epidemiology, physiopathology, clinic, diagnosis and management of acute bronchiolitis, with particular emphasis on updated evidence and future perspectives in terms of treatment and prevention. METHODS AND RESULTS: We searched Cochrane for systematic reviews and PubMed for scientific articles published in the last 10 years, using a combination of the following search terms: "bronchiolitis", "respiratory syncytial virus", "epidemiology", "risk factors", "severity", "diagnosis", "clinic", "diagnostic imaging", "management", "asthma", "wheezing", "bronchodilator", "steroids", "hypertonic saline", "oxygen", "blood gas analysis", "HHHFNC", "rehydration", "enteral feeding", "parenteral hydration", "prevention", "vaccine" and "COVID-19 or SARS-CoV2". We accordingly performed a deep and extensive selection of the most updated and considerable literature on the matter, summarizing the most significant evidence concerning all aspects of acute bronchiolitis (epidemiology, clinic, diagnosis, management and prevention). Furthermore, we examined references and available guidelines from UK, USA, Canada, Italy and Spain. Results are extensively discussed below. CONCLUSION: Although acute bronchiolitis has been a widely known disease for decades, its therapeutic approach remained unchanged and essentially limited to respiratory and metabolic support. Despite the abundance of studies, there is no significant evidence concerning therapeutic alternatives (e.g. steroids, inhaled hypertonic solution), which are therefore not recommended. According to most recent data, "acute bronchiolitis" definition encompasses a plethora of different clinical entities related to each subject's genetic and immune predisposition. Therefore, future research should focus on the precise characterization of such subcategories in order to individualize therapeutic management and ensure the most appropriate evidence-based care.

A Single Center Observational Study on Clinical Manifestations and Associated Factors of Pediatric Long COVID.

Children with SARS-CoV-2 are mostly mild symptomatic, but they may develop conditions, such as persisting symptoms, that may put them at greater risk of complications. Our aim was to evaluate the frequency and the presence of risk factors for persisting COVID-19 symptoms in children. We carried out a prospective observational study of the clinical manifestation of Long COVID at the Department of Maternal Infantile Science of a tertiary University hospital in Rome. We included 697 children (0-18 years), with previous SARS-CoV-2 infection. Children and parents were asked questions regarding persistent symptoms of COVID-19. Children with symptoms 30 days after initial diagnosis were 185/697 (26.4%). Moreover, 81/697 (11.6%) patients presented symptoms 90 days after the diagnosis. Thirty-day-persisting symptoms were mostly present in children with anosmia, atopy, asthenia, and cough in the acute phase compared with the asymptomatic children 30 days after infection. After 90 days, symptoms described were mainly neurological (47/697 children, 6.7%), and headache (19/697; 2.7%) was the most frequent manifestation. In conclusion, a relatively large proportion of the patients reported persisting symptoms that seem to be related to the symptom burden and to the atopy. Ninety days after the infection, most of the children had recovered, showing that long-term effects are not frequent. Limitations of the study include the single-center design and the lack of a control group.

Head-to-Head Comparison of Tissue Factor-Dependent Procoagulant Potential of Small and Large Extracellular Vesicles in Healthy Subjects and in Patients with SARS-CoV-2 Infection.

The relative contribution of small (sEVs) and large extracellular vesicles (lEVs) to the total plasma procoagulant potential is not yet well defined. Thus, we compared total and TFpos-sEVs and -lEVs isolated from healthy subjects and COVID-19 patients during the acute phase of the infection and after symptom remission in terms of (1) vesicle enumeration using nanoparticle tracking assay, imaging flow cytometry, and TF immunofluorescence localization in a single-vesicle analysis using microarrays; (2) cellular origin; and (3) TF-dependent Xa generation capacity, as well as assessing the contribution of the TF inhibitor, TFPI. In healthy subjects, the plasma concentration of CD9/CD63/CD81pos sEVs was 30 times greater than that of calceinpos lEVs, and both were mainly released by platelets. Compared to lEVs, the levels of TFpos-sEVs were 2-fold higher. The TF-dependent Xa generation capacity of lEVs was three times greater than that of sEVs, with the latter being hindered by TFPI. Compared to HSs, the amounts of total and TFpos-sEVs and -lEVs were significantly greater in acute COVID-19 patients, which reverted to the physiological values at the 6-month follow-up. Interestingly, the FXa generation of lEVs only significantly increased during acute infection, with that of sEV being similar to that of HSs. Thus, in both healthy subjects and COVID-19 patients, the TF-dependent procoagulant potential is mostly sustained by large vesicles.

Extracellular Vesicles in Pulmonary Hypertension: A Dangerous Liaison?

The term pulmonary hypertension (PH) refers to different conditions, all characterized by increased pressure and resistance in the pulmonary arterial bed. PH has a wide range of causes (essentially, cardiovascular, pulmonary, or connective tissue disorders); however, idiopathic (i.e., without a clear cause) PH exists. This chronic, progressive, and sometimes devastating disease can finally lead to right heart failure and eventually death, through pulmonary vascular remodeling and dysfunction. The exact nature of PH pathophysiology is sometimes still unclear. Extracellular vesicles (EVs), previously known as apoptotic bodies, microvesicles, and exosomes, are small membrane-bound vesicles that are generated by almost all cell types and can be detected in a variety of physiological fluids. EVs are involved in intercellular communication, thus influencing immunological response, inflammation, embryogenesis, aging, and regenerative processes. Indeed, they transport chemokines, cytokines, lipids, RNA and miRNA, and other biologically active molecules. Although the precise functions of EVs are still not fully known, there is mounting evidence that they can play a significant role in the pathophysiology of PH. In this review, after briefly recapping the key stages of PH pathogenesis, we discuss the current evidence on the functions of EVs both as PH biomarkers and potential participants in the distinct pathways of disease progression.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

BACKGROUND: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. METHODS: The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. RESULTS: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. CONCLUSIONS: Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.