Introduction Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. Materials and methods Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. Results and conclusions All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.
The aim of the study was to determine the molecular genetic prognostic criteria for the severity of the course pneumonia based on the analysis of the association of genetic polymorphism in toll-like receptors with the severity of NETosis.Materials and Methods. The study included 38 patients with the main diagnosis of community-acquired pneumonia with a severe course. All the patients underwent standard clinical laboratory examinations, computed tomography of the thoracic organs, microbiological examination of blood and tracheobronchial aspirate. The level of neutrophilic extracellular traps (NETs) in blood smears was determined on the 1 st -2 nd and 5 th -7 th days of hospitalization. Genotyping of rs5743551 (TLR1), rs5743708 (TLR2), and rs4986790 (TLR4) polymorphic loci was performed by pyrosequencing.Results. The level of NETs on the 1 st day of admission was statistically significantly lower in heterozygous and homozygous carriers of rs4986790 (TLR4) polymorphism (AG and GG genotypes) compared with patients with the wild-type genotype (AA genotype) (p<0.05). When comparing the number of NETs with genotypes for rs5743708 (TLR2) and rs5743551 (TLR1) polymorphisms, no statistically significant correlation was found (p>0.05). The study of the NET level in dynamics demonstrated a decrease in the NETosis activity of neutrophils during the first week of hospitalization (p<0.05). The presence of the G allele in the patient's genotype for rs5743551 (TLR1) polymorphism increases the risk of a poor outcome of the disease (p<0.0001) (OR=20.3; 95% CI (4.3-135.0)). Conclusion.The obtained data suggest that level of NETs is a marker of the activity of neutrophils which are closely related to the studied genetic polymorphisms, and affects the prognosis of the pneumonia outcome.
Background: This paper demonstrates the use of optical diagnostic methods to assess the dynamic skin changes observed in acute and chronic exposure to ultraviolet (UV) radiation in vivo. Methods: Firstly, in order to initiate photoaging (chronic UV exposure), animals (n = 40) were divided into two groups: chronic UV exposure (n = 30), and control (n = 10; without irradiation). Photoaging in animals was induced by chronic repeated exposure to UVA radiation three times per week, for 12 weeks continuously, while the UV dose increased stepwise over the course of the experiment (55 minimal erythema doses (MED) in total). Laser fluorescence spectroscopy (LFS), optical tissue oximetry (OTO), laser Doppler flowmetry (LDF), and optical coherence tomography (OCT) of the shaved dorsum skin were performed regularly, once per week until the conclusion of the study. At 0, 5, and 12 weeks of the experiment, histological examination of animal tissues using hematoxylin/eosin and Masson’s trichrome staining was performed. At the second stage, erythema was induced in mice (n = 15) by acute UV exposure at high doses. The colorimetric assay of the image from a digital RGB camera was used to evaluate the erythema index. Results: The tissue content index ηcollagen of collagen was appropriate for the characterization of skin photoaging. Significant differences (p < 0.05) in ηcollagen were found between the control and photoaging groups from the 5th to the 9th week of the experiment. In addition, the rate of collagen degradation in the control group was about half that of the photoaging group. This marker allows the differentiation of photo- and chronoaging. OCT revealed the main optical layers of the skin in compliance with the histological pattern. The analysis of the RGB camera images provided visualization of the acute skin reaction to UV radiation. Conclusions: This study demonstrates the applicability of optical methods for the quantitative assessment of acute and chronic skin effects of UV exposure in vivo.
Rationale: An important element of antiviral defense in the pathophysiology of COVID-19 is the innate cell immunity including polymorphonuclear neutrophils prone to netotic transformation. Neutrophils can be not only a marker of acute infection, but, being a source of neutrophil extracellular traps (NET), can play a key role in the development of thrombotic complications leading to acute respiratory insufficiency in COVID-19.Aim: To determine the diagnostic and prognostic value of NET levels in patients with COVID-19.Materials and methods: We monitored NET levels in peripheral blood of 34 patients with COVID-19 (mean age, 67 ± 15.8 years), admitted to MONIKI hospital. The control group consisted of 54 healthy volunteers (mean age, 52 ± 11.5 years). Whole blood samples of 2 μL each were used for the preparation of monolayer smears (Giemsa stain) and calculation of at least 200 cell structures including native intact and transformed neutrophils (MECOS-C2 microscope, Medical computer systems).Results: Patients with COVID-19 had higher NET levels, compared to those in healthy controls: 14.5% (2.9–28.6%) vs. 5.0% (1.8–11.9%, p < 0.0001). The patients who were on non-invasive respiratory support (23.5%) had a NET level of 12% (8.1– 22.3%), whereas those on invasive mechanical ventilation (17.6%) had a 1.5-fold higher NET level of 17.9% (12.3–28.2%) (p < 0.05). In the patients who died (11.8% of the cases), the NET level amounted to 19% (16.5–26%, p < 0.05). Monitoring of blood NET levels was performed in 9 patients from the day of admittance to the day of their transfer to the intensive care unit / discharge / death. It was shown that a decrease of NET levels mirrors an improvement of the patient’s clinical condition and efficacy of his/hers treatment. On the opposite, an increase of NET levels can indicate a deterioration and risk of unfavorable course.Conclusion: We have identified some pathophysiological mechanisms in COVID-19, related to the neutrophil compartment. Patients with coronavirus infection are characterized by high NET levels which is at least 3-fold higher than that in healthy volunteers. This indicates an abnormality in immune host defense and development of an inadequate inflammatory response. An increase of NET in whole blood smears of more than 16% can be a criterion of an unfavorable prognosis of the disease course and the risk of death.
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