ABSTRACT
Background: Neutrophil extracellular traps (NETs) release is the one of the main mechanisms behind hypercoagulability and disease severity in severe acute respiratory syndromes. The identification of drugs capable of inhibiting this pathological mechanism is mandatory. Aim(s): Neutrophil extracellular traps (NETs) release is the one of the main mechanisms behind hypercoagulability and disease severity in severe acute respiratory syndromes. The identification of drugs capable of inhibiting this pathological mechanism is mandatory. Method(s): Healthy neutrophils (20 x 103/well) were stimulated with phorbol myristate acetate (PMA) or sera from severe COVID-19 patients (n = 16) in the presence or absence of dipyridamole (10 muM), aspirin (1 mM) and heparin (50 mug/mL). Neutrophils nuclei were stained with nuclear red and incubated with a medium containing the non-permeable cell membrane marker Sytox Green. Cell images were obtained using IncuCyte ZOOM and the number of cells that suffered netosis was monitored over time. NETs release was determined after 1 h of incubation and the percentage of NETs was calculated dividing the number of green cells by the total number of cells per well. Result(s): COVID-19 induced NETs was lower in neutrophils pretreated with heparin (median 2.6%, IQR 2.6-2.9) than in non-treated neutrophils (median 3.6%, IQR 3.2-4.0, p < 0.0001). Pretreatment with dipyridamole and aspirin did not change the effect of COVID-19 sera in inducing NETs. A similar pattern of inhibition was observed with PMA stimulation, in which heparin decreased NETs by 3 times (NETs after PMA 43.2% and NETs after PMA and heparin 14.8%) while dipyridamole and aspirin did not significantly affect the release of PMA-induced NETs (Figure 1). Figure 2 illustrates the identification of NETs. Conclusion(s): Heparin was capable of inhibiting in vitro NETs release induced by COVID-19, while dipyridamole and aspirin had no significant effect on this process. Such findings are in line with evidence that heparin use can improve COVID-19 prognosis. (Figure Presented).
ABSTRACT
Introduction During SARS-CoV-2 infection, a severe hypercoagulability state is observed due to the stimulus of multiple mechanisms of hemostasis, such as coagulation, activation of platelets, endothelial cells, monocytes and neutrophils and impaired fibrinolysis. As a consequence, thrombotic complications are common in the course of COVID-19. Microvesicles (MVs) are intracellular transmitters that participate in pathological conditions, such as inflammatory and infectious processes, and are capable of triggering prothrombotic mechanisms. Since MVs release is potentially associated with COVID-19-induced coagulopathy, our aim was to identify during the course of the disease when the stimulus for MVs release occurs and whether this was associated with adverse outcomes. Objective We evaluated changes in the levels of MVs markers during the first month of SARS-CoV-2 infection in patients (pts) with severe disease (hospitalized in an Intensive Care Unit ‒ ICU) as compared to outpatients. We also evaluated the association between MVs markers with: inflammatory biomarkers (C-reactive protein, CRP), hypercoagulability (D-dimer) and death. Methods Blood samples were collected on three occasions: before the 10th day of symptoms, in the 3rd week of symptoms and in the 4th week of symptoms for the quantification of the following MVs markers by flow cytometry: CD41A (platelet activation), CD162 (PSGL-1;leukocyte-platelet interaction), CD31 (endothelium-platelet interaction) and CD142 (tissue factor). Statistical tests of ANOVA with repeated measures, Mann-Whitney and regression methods were used. Results The population studied was 85 pts, being 25 from ICU. Mostly were men (51%), with a median age of 41 years. The concentration of MVs expressing CD31+, CD41+, CD162+ and CD142+ were persistently elevated in pts who required ICU compared to outpatients at the 3 moments studied, except for the levels of MVs-CD31+ and MVs-CD142+ that were similar between ICU and outpatients in the 4th week of symptoms. However, despite the differences between the groups, there were no significant changes in the levels of MVs during the course of the disease within the groups. In subgroup analysis, we observed that increases in the levels of MVs-CD162+ and MVs-CD142+ in the 3rd week of symptoms were associated with the risk of death (p=0.02 and p=0.06, respectively). We also observed that during the course of the disease an association between MVs, coagulability and inflammation was evident. In the 3rd week of symptoms, D-dimer levels were correlated with MV-CD31+ (r=0.52, p<0.0001), MV-CD162+ (r=0.35, p=0.001), MV-CD41A+ (r=0.44, p<0.0001) and MV-CD142+ (r=0.47, p<0.0001) and CRP values were correlated with MV-CD31+ (r=0.56, p=<0.0001), MV-CD162+ (r=0.48, p<0.0001), MV-CD41A+ (r= 0.41, p=0.0001), and MV-CD142+ (r=0.56, p<0.0001). By the 4th week of symptoms, both D-dimers and CRP correlations with the above MVs remained unchanged. Conclusion To conclude, MVs that express antigens related to platelet activation, leukocyte-platelet interaction and endothelium-platelet interaction, as well as those related to tissue factor are released during the course of COVID-19 in pts with severe disease. After the 4th week of symptoms, the release of these MVs was associated with signs of inflammation and hypercoagulability. Additionally, MVs that express tissue factor and leukocyte-platelet interaction antigens were particularly high among non-survivors, suggesting that these MVs may serve as markers of the risk of death. Finally, these findings suggest the participation of innate immunity and tissue factor pathways in the prognosis of COVID-19, and point towards a possible role of MVs as biomarkers of disease prognosis.
ABSTRACT
Objetivos: Evidências prévias sugerem que o risco trombótico é maior na COVID-19 do que em outros tipos de síndrome respiratória aguda grave (SRAG). Contudo, tal comparação se baseou principalmente em coortes históricas. O objetivo deste estudo foi avaliar a incidência de eventos tromboembólicos em pacientes com COVID-19 e outras SRAG internados em um mesmo período de tempo. Material e métodos: Foram selecionados pacientes internados entre março e junho de 2020 no Hospital de Clínicas - UNICAMP que atendiam aos critérios clínicos de SRAG segundo o Ministério da Saúde e a Definição de Berlim, e que apresentavam ao menos 2 resultados de RT-PCR ou ELISA confirmando ou excluindo o diagnóstico de COVID-19. Dos 253 indivíduos internados por SRAG nesse período, foram incluídos 101 pacientes COVID-19 e 102 pacientes não-COVID-19. Os demais foram excluídos por prontuário médico incompleto (n = 16) ou falta de exame laboratorial para COVID-19 (n = 34). Análise descritiva, testes de qui-quadrado, testes-t e regressão logística binária foram usados para comparar os pacientes COVID-19 e não COVID-19. Resultados: Pertenciam ao sexo masculino 62% e 48% dos pacientes COVID-19 e não-COVID-19, respectivamente (P = 0.07). A mediana de idade, em anos, foi 55.77 (IQR 42.31 a 66.68) no grupo COVID-19 e 59.04 (IQR 45.13 a 69.73, P = 0.39) no grupo não-COVID-19. Ambos os grupos apresentaram um escore de Pádua (COVID-19: 3 IQR 2 a 4;não-COVID-19: 3 IQR 2 a 5, P = 0.47) e uma saturação de oxigênio (COVID-19: 92% IQR 90% a 96%;não-COVID-19: 94% IQR 91% a 97%, P = 0.44) semelhantes à admissão. Contudo, a necessidade de suporte de oxigenação invasiva (37.6% vs. 14.7%, P = 0.0002), de drogas vasoativas (44.6% vs. 21.6%, P=0.0006) e de internação em UTI (55.4% vs. 40.2%, P = 0.04) foi maior entre aqueles infectados por SARS-CoV-2. Em conformidade, esses mesmos pacientes permaneceram internados por mais tempo (15 dias IQR 6 a 30.5 vs. 7 dias IQR 3 a 16.3, P < 0.0001) e vieram a óbito com mais frequência (27.7% vs. 14.7%, P = 0.03). Em relação a marcadores de coagulação, não houve diferença estatisticamente relevante entre os grupos quanto a tempo de protrombina, fibrinogênio e D-dímero (COVID-19: 1488 ng/mL IQR 726.5 a 3476;não COVID-19: 1773 ng/mL IQR 807.5 a 4153.8, P = 0.57). Apesar do uso de tromboprofilaxia ter sido mais comum entre pacientes COVID-19 (76.2% vs. 41.2%, P < 0.0001), a incidência de eventos tromboembólicos confirmados por exame de imagem se mostrou similar entre os grupos, mesmo após ajuste para múltiplos fatores (idade, sexo, tromboprofilaxia, hipertensão arterial, diabetes mellitus, escore de Pádua, internação em UTI, tempo de internação total): houve 7 eventos em 7 pacientes não COVID-19 e 13 eventos em 9 pacientes COVID-19 (OR ajustado 0.91, 95% IC 0.28-2.95, P = 0.87). Os eventos mais recorrentes no grupo COVID-19 foram embolismo pulmonar (53.8%) e trombose venosa profunda (23.1%), que representaram 57.1% (P = 0.37) e 14.3% (P = 0.37) dos eventos não-COVID-19, respectivamente. Discussão: Ao analisar pacientes internados em um mesmo período de tempo, constatamos que, embora elevado, o risco tromboembólico na COVID-19 é semelhante ao de outros tipos de SRAG, indicando que um estado de hipercoagulabilidade é inerente à SRAG em geral. Além disso, os resultados obtidos revelam que o uso de tromboprofilaxia foi significativamente maior no grupo COVID-19, e que não houve diferença estatisticamente relevante entre os níveis de D-dímero dos pacientes COVID-19 e não COVID-19. Conclusão: Tais achados fornecem uma melhor compreensão sobre o risco tromboembólico associado à infecção por SARS-CoV-2, e sugerem que evidências prévias de taxas de trombose mais elevadas na COVID-19 sofreram viés pelo uso de coortes históricas.
ABSTRACT
Previous evidence suggests that the thromboembolic risk is greater among patients with COVID-19 than among those affected by other types of acute respiratory distress syndrome (ARDS). However, such comparison has been primarily based on historical cohorts. In order to reduce the possible influence of such selection bias, the main goal of this study was to evaluate thromboembolic events in patients with COVID-19 and other ARDS hospitalized in the same time period. For this reason, we have selected patients admitted from March to June, 2020 at the UNICAMP Clinical Hospital who met the ARDS clinical criteria established by the Brazilian Ministry of Health and the Berlin Definition by presenting two or more flu-like symptoms and at least one ARDS-specific manifestation (dyspnea, persistent chest pressure, oxygen saturation lower than 95% at hospital admission, or lip/face cyanosis). Symptom onset or worsening occurred 30 days before hospital admission at the latest, and COVID-19 diagnosis was confirmed or excluded by at least 2 real time polymerase chain reactions or enzyme-linked immunosorbent assays. Descriptive analysis, chi-square and t-tests, as well as binary logistic regression, were used to compare COVID-19 and non-COVID-19 patients. Of the 253 patients hospitalized due to ARDS during this period, 101 COVID-19 and 102 non-COVID-19 patients were included in this study. The remaining patients were excluded due to incomplete medical records (n=16) or absence of COVID-19 testing results (n=34). Table 1 demonstrates the included patients' demographic and clinical baseline features. Both COVID-19 and non-COVID-19 groups showed similar baseline risk of hospital-associated thrombosis (assessed by reduced mobility within the past 3 days or more, previous thromboembolism event, recognized “thrombophilia”, and infarction, stroke, trauma or surgery within the past 4 weeks) and oxygen saturation at admission (COVID-19: 92% IQR 90% to 96%;non-COVID-19: 94% IQR 91% to 97%, P=0.44). However, the need for invasive oxygenation support (37.6% vs. 14.7%, P=0.0002) and vasoactive drugs (44.6% vs. 21.6%, P=0.0006) was greater in COVID-19 than in non-COVID-19 patients. Accordingly, those infected by SARS-CoV-2 were more frequently admitted in ICU (55.4% vs. 40.2%, P=0.04) and for a longer period of time (13 days IQR 6 to 22 vs. 3 days IQR 2 to 8.3, P=0.02) than those affected by other types of ARDS. In comparison to the non-COVID-19 group, the COVID-19 group's median total hospital stay was more lasting (15 days IQR 6 to 30.5 vs. 7 days IQR 3 to 16.3, P<0.0001), and its death rate, higher (27.7% vs. 14.7%, P=0.03), as shown in Table 2. With respect to coagulation markers (Table 3), activated partial thromboplastin time and C-reactive protein levels were greater in COVID-19 than in non-COVID-19 patients, while the latter presented higher median platelet counts. There was no statistically significant difference between both study groups in regards to prothrombin time, fibrinogen, and D-dimer levels (COVID-19: 1488 ng/mL IQR 726.5 to 3476;non-COVID-19: 1773 ng/mL IQR 807.5 to 4153.8, P=0.57). Although thromboprophylaxis was more commonly administered to COVID-19 (76.2%) than non-COVID-19 patients (41.2%, P<0.0001), the incidence of thromboembolic events confirmed by imaging examination was similar between groups even after adjusting for multiple factors (age, sex, thromboprophylaxis use, arterial hypertension, and cancer): there were 7 confirmed events in 7 non-COVID-19 patients, and 13 confirmed events in 9 COVID-19 patients (adjusted OR 0.74, 95% CI 0.24-2.25, P=0.59). Table 4 demonstrates the characteristics of such thrombotic manifestations. By analyzing patients hospitalized in the same time period, we have found that although high, the thromboembolic risk in COVID-19 is similar to that in other types of ARDS, indicating that a hypercoagulable state is inherent to ARDS in general. Additionally, the obtained results show that the use of thromboprophylaxis was significantly higher among COVID-19 patients, and that there was no tatistically relevant difference between COVID-19 and non-COVID-19 patients' D-dimer levels, a commonly used coagulation marker. Such findings provide a better understanding of the thromboembolic risk associated with SARS-CoV-2 infection, and suggest that previous evidence of higher thrombosis rates in COVID-19 suffered bias from the use of historical cohorts. [Formula presented] Disclosures: No relevant conflicts of interest to declare.
ABSTRACT
Background : Previous evidence suggest that the thromboembolic risk is greater among patients with COVID-19 than those affected by other types of severe acute respiratory syndrome (SARS). However, such comparison has been mainly evaluated in historical cohorts. Aims : To evaluate thromboembolic events in patients with COVID-19 and other SARS hospitalized in the same time period. Methods : Consecutive patients hospitalized for SARS from March to May, 2020 were selected. Descriptive statistics, chi-square and t -tests were used to compare COVID-19 and non-COVID-19 patients. Results : In all, 377 patients were admitted during this period. 100 COVID-19 and 100 non-COVID-19 patients were included. Table 1 demonstrates their baseline features. Both groups showed similar baseline risk of hospital-associated thrombosis, such as previous thromboembolism event, recognized 'thrombophilia', infarction or stroke within the past 4 weeks, and trauma or surgery within the past 4 weeks. Reduced mobility, however, was more frequent among non-COVID-19 than COVID-19 patients. Oxygen saturation was lower in COVID-19 (92% IQR 89% to 96%) than in non-COVID-19 patients (95% IQR 90% to 97%, P = 0.03);accordingly, the need for invasive oxygen support was greater (44% vs. 33%, P = 0.05) and longer (16.00 days IQR 8.50 to 22.50 vs. 12.50 days IQR 4.75 to 21.25, P = 0.002) in COVID-19 than in non-COVID-19 patients. Coagulation markers such as activated partial thromboplastin time, prothrombin time, platelet count, and D-dimer levels (1,700.00 ng/mL IQR 752.00 to 3,417.00 non-COVID-19;1,426.50 ng/mL IQR 744.25 to 3,461.00 COVID-19, P = 0.17) were similar between groups. Although thromboprophylaxis was more frequently administered to COVID-19 (76%) than non-COVID-19 patients (42%, P < 0.0001), thrombotic events were more recurrent in the former group (22% vs. 12%, P = 0.06). Table 2 demonstrates the thrombotic manifestations ' characteristics. Conclusions : In this study, we provide clinical evidence that the risk of thrombosis is more pronounced in COVID-19 than in other SARS. D-dimer analysis was not capable of differentiating the thrombotic risk between these conditions.