ABSTRACT
Background: A considerable proportion of patients do not fully recover from COVID-19 infection and report symptoms that persist beyond the initial phase of infection: This condition is defined long-COVID-19 syndrome (LCS). LCS can involve lungs as well as several extrapulmonary organs, including the cardiovascular system. The risk and 1-year burden of cardiovascular diseases (CVD) is increased in COVID-19 survivors, even in subjects at low risk of CVD. Recently, we documented that acute COVID- 19 infection induces altered platelet activation state characterized by a prothrombotic phenotype and by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens. No data are yet available on the contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients. Purpose(s): To study platelet activation status, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients enrolled at 6 months after resolution of the acute phase (6mo-FU), compared to acute COVID-19 infection patients. Method(s): 6mo-FU COVID-19 patients (n=24) with established LCS were enrolled at Centro Cardiologico Monzino. Residual pulmonary impairment was assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows- CT scan evaluation. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were assessed by flow cytometry;pTGC by calibrated automated thrombogram. 46 patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison. Result(s): Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-fold (1.5% [1.2-2.9] vs 2.4% [1.6-5.7]) and 2-fold (217/mul [137-275] vs 435/mul [275-633]) lower at 6mo-FU compared to acute phase, being comparable to HS. pTGC behaved similarly. At 6mo-FU, the MV profile, in terms of total number and cell origin, returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression (6.9% [3-13.5] vs 11.7% [5.2-18.9]) and PLA formation (35.5% [27.4- 46.8] vs 67.7% [45.7-85.3]) was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed (r=-0.423;p=0.02). Conclusion(s): These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients.
ABSTRACT
Background: Coronavirus-2 (SARS-CoV- 2) infection causes a sustained prothrombotic state driven by a massive Tissue Factor (TF) expression in circulating platelets, leukocytes and microvesicles (MVs). Whether also circulating small extracellular vesicles (sEVs), in addition to large MVs (MVs), can contribute to this hypercoagulable scenario through TF expression is not yet known, mainly due to methodological issues in detecting and sizing the smallest vesicles. Aim(s): To characterize TF expression and activity in circulating sEVs, compared to that of MVs, of COVID-19 patients during acute phase infection and after symptom remission. Method(s): MVs and sEVs were isolated by plasma differential centrifugation from 10 COVID-19 patients enrolled at acute phase infection (T0) and at six-month- follow- up (T1). Ten healthy subjects (HS) were analyzed as controls. sEVs were counted by Nano Tracking Assay. In sEVs TF expression was analyzed within CD9/CD63/CD81pos events by imaging flow cytometry (IFC) and ExoViewTM microarray, while TF activity by FXa generation assay. TF expression and activity in MVs were evaluated for comparison. Result(s): By IFC analysis COVID-19 patients at T0 have about 1.5-and 4-fold higher number of TFpos-sEVs and -MVs, respectively, compared to HS, with a trend toward reduction to physiological levels at T1. By microarray analysis sEVs behaved similarly (36 +/- 12 and 25 +/- 10 TFpos-spots at T0 and T1, respectively;p = 0.0281). sEVs-associated TF is functionally active thus able to partially support FXa formation as sEVs preincubation with TF-neutralizing antibody reduced FXa generation by ~30%. However, although sEVs number is significantly higher compared to that measured for MVs (~600-fold in HS), functional activity of sEV is one-third lower compared to that of MVs. Conclusion(s): These data suggest that, in COVID-19 patients, the altered procoagulant phenotype could also be supported by TF carried by sEVs, although their functional activity is significantly lower than that of MVs.
ABSTRACT
Background: Long-COVID- 19 syndrome (LCS) is defined as symptoms persisting beyond initial phase of infection. Among them, pulmonary fibrotic damage remains in 25-30% of COVID-19 patients at 3-6 month-follow- up. We documented that acute COVID-19 patients have massive platelet activation characterized by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens, and by a prothrombotic phenotype. No data are currently available on contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients. Aim(s): To characterize platelet activation, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients at 6-month- follow- up (6mo-FU) compared to acute COVID-19 infection patients. Method(s): Twentyfour 6mo-FU COVID-19 patients with established LCS defined according to their residual pulmonary impairment assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows- CT scan evaluation were enrolled. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were evaluated by flow cytometry;pTGC by calibrated automated thrombogram. Fortysix patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison. Result(s): Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-and 2-fold lower at 6mo-FU compared to acute phase, being comparable to HS, as well as pTGC. At 6mo-FU, the MV profile (total number and derived from different cells) returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression and PLA formation was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed. Conclusion(s): These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients.
ABSTRACT
Background: Sustained platelet activation, thrombosis, vascular damage, fibrotic response as well as inflammatory overload are typical features of COVID-19 pathology. Common denominator in these processes is leukotrienes (LTs). Elevated levels of LTE4 have been detected in bronchoalveolar lavage of COVID-19 patients so the use of LT receptor antagonists as a potential therapeutic for COVID-19 patient treatment has been hypothesized. A first phase III randomized double-blind clinical trial testing montelukast in COVID-19 patients has been indeed proposed. Aim(s): To investigate whether montelukast affects the expression of the major markers of platelet activation such as tissue factor (TF), P-selectin, as well as the formation of platelet-leukocyte aggregates and microvesicle (MV) release observed in COVID-19 syndrome. Method(s): Blood from healthy subjects (HS;n = 4-6) was plasma-depleted and reconstituted with plasma pools (n = 3) from COVID-19 patients (4 patients/pool) or from the same HS blood donors. To assess the effect of montelukast on cell activation, blood from HS was preincubated for 30 minutes with the drug. Circulating cell-associated TF expression, platelet activation markers, and MV release were analyzed by flow cytometry. Result(s): Plasma from COVID-19 patients significantly increased (4-fold) the number of TF+-and P-selectin+- platelets of HS recapitulating the platelet activation status of COVID patients. Montelukast prevented platelet activation induced by plasma from COVID-19 patients and it reduced the formation of circulating monocyte-and granulocyte-platelet aggregates, decreasing the number of those TF+ by 4-times. Finally, it completely inhibited the release of TF+ circulating MVs, reducing by more than 2-times those derived from platelets. Conclusion(s): Our data indicate that leukotrienes contribute to sustain platelet activation occurring in the COVID-19 patient, which can however be prevented by treatment with montelukast. Until results from ongoing trials will be available, our data provide the molecular basis by which the drug may be effective in the treatment of COVID-19.
ABSTRACT
Introduction: The literature shows that in Italy community pharmacists and patients are not active enough in reporting adverse drug reactions (ADRs) [1-3]. To improve these replies it has been set up VigiNetWork project (September 2020-September 2022): it is a multiregional (Emilia-Romagna and Veneto Region) pharmacovigilance (PV) project funded by the Italian Medicines Agency, joining pharmacists and citizens in pharmacovigilance activities within the community pharmacies. Objective(s): To develop a pharmacovigilance network among community pharmacies, to promote spontaneous ADRs reporting by pharmacists and patients and to improve pharmacists' knowledge on drug safety. Method(s): The project has been promoted through social networks, webinars, local newspapers, and the Category Professional Associations. An information leaflet explaining its characteristics, a free access to two e-learning courses and a personal access to a website (www.vigirete.it) have been made available to enrolled pharmacists. A similar leaflet has been developed for patients. To evaluate the effectiveness of VigiNetWork we assessed the number of enrolled community pharmacies/pharmacists and of ADR reports submitted by them, their participation to e-learning courses and their visits to the website. Result(s): From September 2020 to March 2022 a total of 669 and 371 community pharmacies were enrolled respectively in Emilia- Romagna and Veneto Region (Table 1). A total of 339 and 516 (about 90% of total reports from community pharmacists) ADR reports were submitted from the pharmacists of VigiNetWork respectively in Emilia-Romagna and Veneto Region and an increase in comparison to the previous 18 months was observed. The impact of the project in patient reporting was presumed, but not assessable. In our study period 40% and 58% of enrolled pharmacists registered for distance learning courses respectively in Emilia-Romagna and Veneto Region, even if only about 5% of them completed the training in both Regions. The website reached 2113 (86% of enrolled pharmacists) unique visitors. Conclusion(s): Our project showed a great attention of community pharmacists to PV, increased also due to COVID-19 pandemic. The total number of submitted reports, even if limited, represented almost the totality of all reports from community pharmacists in both Regions and their interest in our website contents was appreciable. More efforts could be done to continue these activities in daily practice. (Table Presented).
ABSTRACT
Introduction: The literature shows that in Italy community pharmacists and patients are not active enough in reporting adverse drug reactions (ADRs) [1-3]. To improve these replies it has been set up VigiNetWork project (September 2020-September 2022): it is a multiregional (Emilia-Romagna and Veneto Region) pharmacovigilance (PV) project funded by the Italian Medicines Agency, joining pharmacists and citizens in pharmacovigilance activities within the community pharmacies. Objective: To develop a pharmacovigilance network among community pharmacies, to promote spontaneous ADRs reporting by pharmacists and patients and to improve pharmacists' knowledge on drug safety. Methods: The project has been promoted through social networks, webinars, local newspapers, and the Category Professional Associations. An information leaflet explaining its characteristics, a free access to two e-learning courses and a personal access to a website (www.vigirete.it) have been made available to enrolled pharmacists. A similar leaflet has been developed for patients. To evaluate the effectiveness of VigiNetWork we assessed the number of enrolled community pharmacies/pharmacists and of ADR reports submitted by them, their participation to e-learning courses and their visits to the website. Results: From September 2020 to March 2022 a total of 669 and 371 community pharmacies were enrolled respectively in Emilia-Romagna and Veneto Region (Table 1). A total of 339 and 516 (about 90% of total reports from community pharmacists) ADR reports were submitted from the pharmacists of VigiNetWork respectively in Emilia-Romagna and Veneto Region and an increase in comparison to the previous 18 months was observed. The impact of the project in patient reporting was presumed, but not assessable. In our study period 40% and 58% of enrolled pharmacists registered for distance learning courses respectively in Emilia-Romagna and Veneto Region, even if only about 5% of them completed the training in both Regions. The website reached 2113 (86% of enrolled pharmacists) unique visitors.Conclusion: Our project showed a great attention of community pharmacists to PV, increased also due to COVID-19 pandemic. The total number of submitted reports, even if limited, represented almost the totality of all reports from community pharmacists in both Regions and their interest in our website contents was appreciable. More efforts could be done to continue these activities in daily practice.