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AbstractIn contrast to red blood cells, platelets float rather than sediment when a column of blood is placed in the gravitational field. By the analogy of erythrocyte sedimentation (ESR), it can be expressed with the platelet antisedimentation rate (PAR), which quantitates the difference in platelet count between the upper and lower halves of the blood column after 1 h of 1 g sedimentation. Venous blood samples from 21 healthy subjects were analyzed for PAR. After a 1-h sedimentation, the upper and lower fractions of blood samples were analyzed for platelet count, mean platelet volume (MPV), immature platelet fraction (IPF), and high-fluorescence IPF (H-IPF). The mechanisms behind platelet flotation were explored by further partitioning of the blood column, time-dependent measurements of platelet count and comparison with ESR. The structure and function of the platelets were assessed by electron microscopy (EM) and atomic force microscopy (AFM), and platelet aggregometry, respectively. Platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation. The area under the curve (AUC) of the whole blood adenosine diphosphate (ADP) aggregation curves showed significant differences between the upper and lower samples (p

Abstract Background Progressive cardiac fibrosis is the central aspect of the myocardial involvement in systemic sclerosis (SSc). We hypothesized that circulating biomarkers of the cardiac fibrosis may be useful in the early diagnosis of the cardiac manifestation in this disease. Thus, we investigated the potential correlations between the levels of galectin-3, soluble suppression of tumorigenicity-2 (sST2) and the echocardiographic markers of the myocardial mechanics in SSc patients. Methods Forty patients (57.3 ± 13.7 years, 36 female) were investigated. In addition to the conventional echocardiography, tissue Doppler and speckle tracking-derived strain techniques were used to assess the function of both ventricles and atria. To estimate the correlations between galectin-3 and sST2 levels and the echocardiographic variables, partial correlation method was used with age as correcting factor. Results In age adjusted analysis galectin-3 level showed significant correlation with left ventricular global longitudinal strain (r = 0.460, p = 0.005); grade of left ventricular diastolic dysfunction (r = 0.394, p = 0.013); septal e’ (r = − 0.369, p = 0.021); septal E/e’ (r = 0.380, p = 0.017) and with the grade of mitral regurgitation (r = 0.323, p = 0.048). No significant correlation was found between sST2 levels and the echocardiographic variables. Conclusions Galectin-3 levels, but not sST2 levels show significant correlation with the parameters of the left ventricular systolic and diastolic function. Galectin-3 may be a useful biomarker for the screening and early diagnosis of SSc patients with cardiac involvement.

Introduction Precapillary pulmonary hypertension (PH) implies a worse prognosis in myeloproliferative neoplasms (MPN). N-terminal pro-B-type natriuretic peptide (NT-proBNP) is elevated in cardiopulmonary involvement. In MPN patients with precapillary PH, elevated vascular endothelial growth factor (VEGF) values, but in left heart (LH) disease patients, decreased values were reported. Our aim was to determine whether a combination of NT-proBNP and VEGF is suitable for the detection of the precapillary forms of PH in MPN patients. Material and methods Eighty-one MPN patients were investigated. Pulmonary hypertension was defined as Doppler-derived systolic pulmonary artery pressure (sPAP) ≥ 40 mm Hg. Patient groups with cardiopulmonary involvement (precapillary PH, PH due to LH disease, left ventricular ejection fraction < 50%, atrial fibrillation) or LH disease (PH due to LH disease, left ventricular ejection fraction < 50%, atrial fibrillation) were identified. Results In 9 patients PH was associated with LH disease. In 2 patients precapillary PH was found with extremely high NT-proBNP values. NT-proBNP significantly correlated with sPAP (r = 0.550; p < 0.001). NT-proBNP ≥ 466 pg/ml was the best predictor of cardiopulmonary involvement (AUC: 0.962, sensitivity: 86.7%, specificity: 93.9%). No correlation was found between VEGF levels and sPAP values. VEGF ≤ 431 pg/ml was the best predictor of LH disease (AUC: 0.609, sensitivity: 76.9%, specificity: 62.7%). Conclusions NT-proBNP levels reflect cardiopulmonary involvement with high accuracy, but the combination of NT-proBNP and VEGF is not suitable for the detection of precapillary PH as the diagnostic power of VEGF is limited. Highly elevated NT-proBNP levels may suggest precapillary PH but further investigation is necessary for the exclusion of LH disease or atrial fibrillation.

Introduction: Exploration of novel and effective antiplatelet strategies for the secondary prevention of ischemic stroke is utmost. Some platelet derived microparticles (PMVs) in convalescent stroke subjects were found to be predictive for the next vascular event. Patients with high-on-treatment platelet reactivity (HTPR) had a significantly higher risk for ischemic stroke. Here, we aimed to explore associations among circulating microparticles and responsivness to antiplatelet (clopidogrel) therapy. Methods: A total of 18 patients on clopidogrel therapy due to secondary stroke prevention were rospectively recruited into this study. Twenty age-matched healthy subjects served as controls. Flow cytometric measurements of microparicles (MVs) and data analysis were performed on Beckman-Coulter FC-500 cytometer with CXP software. Besides, platelet aggregometry data were revealed. Both measurements were performed in whole blood and from the lower and upper blood fractions separated after 1-hour gravity sedimentation by the analogy with erythrocyte sedimentation rate. Results: The total number of circulating MVs, and particularly the platelet derived CD42+ and PAC-1+ were significantly higher in post-stroke patients (p < 0.001). The platelet aggregation in the whole blood (area under the curve, AUC) showed a significant negative correlation with the total number of MPs in the lower blood sample after 1-hour gravity sedimentation (r = –0.650, p = 0.005). Next, we analyzed associations among MPs and aggregometry data obtained from clopidogrel responders and non-responders. Both, area under the curve (AUC) and velocity in the whole blood showed opposite correlation with the total number of MVs in the lower blood sample after 1-hour gravity sedimentation. Importantly, a significant negative correlation was observed for the velocity (r = –0.801, p = 0.005), but not for the AUC in responders. Platelet derived CD42+ and PAC-1+ MVs showed positive correlations with neutrophils in the lower blood sample (p = 0.008 and p = 0.006 respectively). Conclusions: Circulating MVs may allow to monitor the response to antiplatelet therapy in post-stroke patients. In addition, the link between platelet derived MVs and neutrophil granulocytes might become therapeutic targets in the future.

Background: The effect of post-vaccination adverse events on immunogenicity is unknown. We aimed to explore relationship between post-vaccination adverse reactions and antibody levels during 6-month follow-up. Methods: Blood was serially drawn from healthcare workers after the second dose of BNT162b2 mRNA vaccine (Day 12, 30, 60, 90, 120, 150, and 180) and anti-SARS-CoV-2 spike IgG (S-IgG) levels were measured. Following each vaccine dose, volunteers completed a questionnaire regarding adverse reactions (symptomatic vs. asymptomatic groups). Results: A total of 395 subjects received the second dose of the vaccine. The main results were as follows: (i) fever after the 2nd dose was independently associated with the median S-IgG level at all follow-up time points; (ii) significantly higher S-IgG levels were observed in the symptomatic group of patients without prior COVID-19 infection throughout the entire follow-up period; (iii) prior COVID-19 positivity resulted in higher S-IgG levels only in the asymptomatic group from Day 90 of the follow-up period; (iv) both prior COVID-19 disease with asymptomatic status and symptomatic status without prior COVID-19 infection resulted in similar S-IgG antibody levels; (v) significantly lower serum S-IgG levels were observed in smokers. Conclusion: Fever may play an important role in the post-vaccination immune response in the long term.

Background: Long COVID is a condition characterized by long-term sequelae persisting after the typical convalescence period of COVID-19. Previous reports have suggested the role of an unsatisfactory immune response and impaired viral clearance in the pathogenesis of long COVID syndrome. We focused on potential associations between post-vaccination changes of antibody titers and the severity of long COVID symptoms and factors influencing the state of remission observed in patients with long COVID after vaccination. Methods: The severity of long COVID symptoms and serum anti-SARS-CoV-2 spike (S-Ig) and nucleocapsid (NC-Ig) levels were assessed in 107 post-COVID subjects at two time points: at baseline, and 17–24 weeks later. Besides, vaccination status was also assessed. Symptoms were evaluated based on the Chalder fatigue scale (CFQ-11) and visual analogue scale (VAS). Results: Serum level of S-Ig and NC-Ig at baseline were significantly higher in the patients with non-severe fatigue than those with severe fatigue, and this difference remained significant at follow-up in the case of NC-Ig. NC-Ig level above median was as an independent predictor for complete remission at follow-up. The difference in NC-Ig levels in subgroup analyses (severe fatigue vs. non-severe fatigue; complete remission vs. incomplete remission or progression) was found to be significant only in patients who received vaccination. Conclusions: The immune response against the SARS-CoV-2 nucleocapsid may play a more important role than the spike in the course of long-term COVID syndrome.

High rates of thrombosis are present in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Deeper insight into the prothrombotic state is essential to provide the best thromboprophylaxis care. Here, we aimed to explore associations among platelet indices, conventional hemostasis parameters, and viscoelastometry data. This pilot study included patients with severe COVID-19 (n = 21) and age-matched controls (n = 21). Each patient received 100 mg aspirin therapy at the time of blood sampling. Total platelet count, high immature platelet fraction (H-IPF), fibrinogen, D-dimer, Activated Partial Thromboplastin Time, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor activity, plasminogen, and alpha2-antiplasmin were measured. To monitor the aspirin therapy, a platelet function test from hirudin anticoagulated whole blood was performed using the ASPI test by Multiplate analyser. High on-aspirin platelet reactivity (n = 8) was defined with an AUC > 40 cut-off value by ASPI tests. In addition, in vitro viscoelastometric tests were carried out using a ClotPro analyser in COVID-associated thromboembolic events (n = 8) (p = 0.071) nor the survival rate (p = 0.854) showed associations with high on-aspirin platelet reactivity status. The platelet count (p = 0.03), all subjects. COVID-19 patients presented with higher levels of inflammatory markers, compared with the controls, along with evidence of hypercoagulability by ClotPro. H-IPF (%) was significantly higher among non-survivors (n = 18) compared to survivors (p = 0.011), and a negative correlation (p = 0.002) was found between H-IPF and plasminogen level in the total population. The platelet count was significantly higher among patients with high on-aspirin platelet reactivity (p = 0.03). Neither the ECA-A10 (p = 0.008), and ECA-MCF (p = 0.016) were significantly higher, while the tPA-CFT (p < 0.001) was significantly lower among patients with high on-aspirin platelet reactivity. However, only fibrinogen proved to be an independent predictor of hypofibrinolysis in severe COVID-19 patients. In conclusion, a faster developing, more solid clot formation was observed in aspirin ‘non-responder’ COVID-19 patients. Therefore, an individually tailored thromboprophylaxis is needed to prevent thrombotic complications, particularly in the hypofibrinolytic cluster.

Introduction: A modified platelet function test (mPFT) was recently found to be superior compared to impedance aggregometry for selection of post-stroke patients with high on-treatment platelet reactivity (HTPR). We aimed to explore some peripheral blood cell characteristics as predictors of recurrent ischemic episodes. The predictive value of mPFT was also assessed in a cohort followed up to 36 months regarding recurrent ischemic vascular events.Methods: As a novelty, not only whole blood (WB), but after 1-h gravity sedimentation the separated upper (UB) and lower half blood (LB) samples were analyzed including neutrophil antisedimentation rate (NAR) in 52 post-stroke patients taking clopidogrel. Area under the curve (AUC, AUCupper and AUClower, respectively) was separately measured by Multiplate in the WB, UB and LB samples to characterize ex vivo platelet aggregation in the presence of ADP. Next, the occurrence of vascular events (stroke, acute coronary syndrome, ACS) were evaluated during 36-month follow-up.Results: A total of 11 vascular events (stroke n = 5, ACS n = 6) occurred during the follow-up period. The AUCupper was significantly higher in patients with recurrent stroke compared to those with uneventful follow-up (p = 0.03). The AUCupper with a cut-off value ≥70 based on the mPFT, was able to predict all stroke events (p = 0.01), while the total vascular events were independently predicted by NAR with a sensitivity of 82% and specificity of 88%.Conclusions: A combination of NAR reflecting the inflammatory state and AUCupper indicating HTPR may provide a better prediction of recurrent ischemic events suggesting a better selection of patients at risk, thus providing an individually tailored vascular therapy.

Background: It is known that adverse reactions following SARS-CoV-2 vaccinations show a positive correlation with the subsequent antibody titer. However, it is not clear how the adverse reactions following the booster vaccination are related to the antibody levels that can be measured after a 3rd dose. The primary goal of this study was to investigate whether the adverse reactions following the booster vaccination show a correlation with subsequent antibody levels. Methods: Adverse reactions occurring within 7 days after the 3rd vaccination were recorded and the anti-SARS-CoV-2 spike protein immunoglobulin (Ig) level in the venous blood was measured on post-vaccination 14th, 60th and 120th days. Results: A total of 218 volunteers were included in the study. Main findings: (i) The adverse reactions that appeared after the booster dose did not show a positive correlation with the subsequent antibody level, except a correlation in the case of fever; (ii) there were more symptomatic patients in the group receiving heterologous booster vaccine, (iii) fever after the 2nd dose was independently associated with a reduction in the likelihood of COVID-19 positivity after the booster dose. Conclusion: No adverse reactions, but fever showed a correlation with the antibody level after the booster SARS-CoV-2 vaccine.