EXTRACELLULAR VESICLES AS MARKERS OF INFLAMMATION AND HYPERCOAGULABILITY DURING THE FIRST MONTH OF SARS-COV-2 INFECTION IN OUTPATIENTS AND HOSPITALIZED PATIENTS

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.

TIME COURSE OF THE DEVELOPMENT OF IMMUNOTHROMBOSIS DURING COVID-19 HOSPITALIZATION

Introduction: Hypercoagulability in COVID-19 has been attributed to immunothrombosis, a process that involves the formation of neutrophils extracellular traps (NETs). The moment of the COVID-19 evolution in which immunothrombosis mechanisms are triggered is not established. Aim: To describe the kinetics of NETs release during COVID-19 hospitalization associating with thrombosis and death. Methods: We quantified citrullinated H3 and inflammatory cytokines (TNF-α, IL-6), markers of NETs release, on 4 time points during COVID-19 hospitalization (admission, day 4, day 8 and last day) between May and July 2020. The association between changes in these markers levels and clinical outcomes was determined. Results: 101 patients were included, the median days in-hospital were 15, 62% were men, 27% were obese, 43% were diabetic, 54% were hypertensive, 59% were critically ill, 11% had a thrombotic event and 21% died. IL-6 levels were high on admission in survivors (median 25.32, IQR 24.19-28.15) and non-survivors (median 24.19, IQR 12.51-27.19), but gradually decreased on day 4 (median 12.07, IQR 6.32-17.81), day 8 (median 9.34, IQR 5.18-17.59) and last day (median 8.64, IQR 4.81-14.89) in survivors. TNF-α levels remained 2 times higher in non-survivors: admission (median 1.60, IQR 0.64-2.26), day 4 (median 1.78, IQR 1.02-2.60), day 8 (median 1.65, IQR 0.93-2.5), last day (median 2.41, IQR 1.31-4.06);than in survivors: admission (median 0.81, IQR 0.52-1.26), day 4 (median 0.84, IQR 0.44-1.16), day 8 (median 0.72, IQR 0.44-1.24), last day (median 0.69, IQR 0.4-1.14). CitH3 levels were similar between non-survivors at the beginning of hospitalization: admission (median 1.03, IQR 0.43-4.34), day 4 (median 1.1, IQR 0.65-3.45);as for survivors: admission (median 1.20, IQR 0.45-2.60), day 4 (median 1.27, IQR 0.64-3.29). On day 8, citH3 increased by 3-fold (median 3.80, IQR 1.98-10.15) in non-survivors and 2-fold (median 2.60, IQR 1.22-5.01) in survivors. While IL-6 and TNF-α levels were similar between patients with and without thrombosis, citH3 levels increased shortly on day 4, before the occurrence of a thrombotic event: admission (median 1.64, IQR 0.44-4.14), day 4 (median 3.21, IQR 2.57-9.31);but it didn't change on non-thrombotic event patients: admission (median 1.05, IQR 0.44-2.50), day 4 (median 1.06, IQR 0.58-2.95). Conclusion: Markers of inflammation and immunothrombosis were associated with poor outcomes in COVID-19;however, these disorders were detected in different moments during COVID-19 course. While an increased inflammatory response was observed since the beginning of hospitalization, markers of immunothrombosis arose latter during the course of the disease. Acknowledgment of the time-course of immunothrombosis development in COVID-19 is important for planning therapeutic strategies against this pathological process.

DIAGNÓSTICO DA COVID-19 E SEU IMPACTO SOBRE O RISCO TROMBÓTICO EM UMA COORTE DE PACIENTES SIMULTANEAMENTE HOSPITALIZADOS POR SÍNDROME RESPIRATÓRIA AGUDA GRAVE

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.

AVALIAÇÃO DOS NÍVEIS CIRCULANTES DE MEDIADORES DA INTEGRIDADE DA BARREIRA ENDOTELIAL NA COVID-19 E SUA RELAÇÃO COM A ATIVAÇÃO DA HEMOSTASIA

Objetivos: a imunotrombose consiste no processo que envolve a ativação concomitante da imunidade inata, hemostasia e endotélio como parte da resposta a patógenos, e vem sendo colocada no centro da fisiopatologia da Covid-19. Um elemento menos explorado da imunotrombose é a ruptura da barreira endotelial (BE), que permite o acesso dos leucócitos aos tecidos inflamados. Entre os reguladores da integridade da BE destacam-se as vias que envolvem a angiopoietina (Ang) 1 e 2 e seu receptor Tie2, e a via do VEGF-A/VE-caderina (VEC). Além deste papel, foi recentemente demonstrado que a ativação da via Ang/Tie2 inibe a ativação endotelial e a expressão de fator tecidual, estabilizando o endotélio no estado quiescente. Neste estudo determinamos os níveis circulantes de mediadores da integridade da BE na Covid-19, e exploramos sua associação com a gravidade da doença, assim como com a ativação da hemostasia através de um painel abrangente de biomarcadores. Materiais e métodos: as amostras foram obtidas de 30 pacientes internados por Covid-19 devido à hipoxemia e achados tomográficos típicos, e recrutados para um estudo clínico (REBEC: U1111-1250-1843). As amostras foram coletadas em até 24h do diagnóstico, antes de qualquer intervenção terapêutica. Os níveis de reguladores da BE foram medidos por métodos imunológicos (Elisa ou multiplex), e o de biomarcadores da hemostasia por kits comerciais específicos. Um grupo de 30 indivíduos saudáveis pareados por idade e sexo foram utilizados como controle. Dados clínicos e laboratoriais foram obtidos dos prontuários digitais. Resultados: o tempo médio de internação foi de 12,9 ± 9,8 dias, e 12 pacientes (40%) necessitaram de UTI. O dímero D médio foi de 3.609 ± 14.440 ng/mL. Os níveis circulantes de todos reguladores da integridade da BE encontraram-se aumentados em pacientes, quando comparado com controles (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p < 0.0001;Ang2: 1.926 (1.275-3.134) vs 1.215 (9-1.444) pg/mL, p < 0.0001;Tie2: 10.753 ± 2.377 vs 8.603 ± 1.851 pg/mL, p < 0.0001 e VEGF-A: 94.7 (73.4-116.0) vs 45.9 (39.7-57.0), p < 0.0001. Além disso, os níveis de alguns destes reguladores se associaram significativamente a desfechos de relevância clínica, a saber: (i) extensão da lesão pulmonar na tomografia: Ang2 e VEGF-A;(ii) tempo de internação em UTI: VEGF-A. Interessantemente, observamos correlações consistentes e significativas entre os níveis de reguladores da BE a proteínas envolvidas na ativação da hemostasia (fibrinogênio, VWF: Ag, uPAR, PAI-1 e P-selectina). Discussão: o interesse no estudo de reguladores da integridade da BE na Covid-19 já se justifica pelo fato de a doença envolver tanto o comprometimento da barreira alvéolo-capilar quanto a ativação da angiogênese, como demonstrado por outros autores. Nossos resultados reforçam a relevância destas vias através da associação observada com desfechos clínicos. Além disso, os resultados mostram pela primeira vez uma associação entre mediadores da integridade da BE e um painel amplo de biomarcadores da ativação da hemostasia, sugerindo um crosstalk entre estas vias na Covid-19, como demonstrado recentemente no contexto da sepse. Conclusões: nossos resultados apontam que a via Ang/Tie2 deve ser considerada um alvo terapêutico atrativo na Covid-19, por representar um elemento central da imunotrombose nestes pacientes.

AVALIAÇÃO DA INTEGRIDADE DA BARREIRA ENDOTELIAL E SUA RELAÇÃO COM AS MANIFESTAÇÕES CLÍNICAS E LABORATORIAIS NA COVID-19

Objetivos: Os mecanismos fisiopatológicos que determinam a gravidade da Covid-19 estão associados a ativação da hemostasia e da imunidade inata, em um processo coletivamente referido como imunotrombose, e que envolve ativação plaquetária, geração de NETs (do inglês, Nucleo extracelular traps), expressão de fator tecidual, ativação do complemento e ativação endotelial. Um elemento importante da ativação endotelial é a quebra da barreira endotelial (BE), que ocorre para facilitar o acesso de leucócitos aos tecidos, onde contribuem para erradicação dos patógenos. No entanto, a avaliação da integridade da BE é desafiadora, exigindo o uso de modelos celulares. O objetivo desse estudo foi avaliar o efeito do soro de pacientes com Covid-19 sobre a integridade da BE em monocamadas de células endoteliais, e sua correlação com características clínicas da doença. Materiais e métodos: A população do estudo consistiu em 30 pacientes com Covid-19 que apresentavam comprometimento pulmonar confirmado por tomografia de tórax, e necessidade de internação hospitalar por hipoxemia e 30 controles saudáveis pareados por sexo e idade. Os pacientes recrutados fizeram parte de um estudo clínico (REBEC: U1111-1250-1843), e as amostras utilizadas nesta avaliação foram obtidas no momento da internação, antes de qualquer intervenção. Monocamadas de células endoteliais de duas fontes (HUVECs: células de cordão umbilical;HULECs: células endoteliais pulmonares) foram estimuladas com soro de pacientes e indivíduos saudáveis (diluição 15% em meio de cultura) e a integridade da BE foi avaliada por um sensor de impedância celular (ECIS;Eletric Cell-substrate Impedance Sensing System) continuamente por 36 horas. Biomarcadores de gravidade e relacionados à ativação da hemostasia foram avaliados por kits comerciais. Dados clínicos foram obtidos a partir dos prontuários digitais. Resultados: O soro de pacientes com Covid-19 induziu quebra de BE significativamente mais acentuada que o de indivíduos saudáveis em HUVECs nos tempos 15 min (p < 0,01);30 min (p ≤ 0,001);1h (p ≤ 0,0001);2h (p ≤ 0,0001);3h (p ≤ 0,0001);4h (p ≤ 0,01) e 5h (p ≤ 0,05). Estes resultados foram confirmados no modelo de células endoteliais pulmonares (HULECs). A magnitude da quebra apresentou correlação significativa com desfechos clínicos relevantes como tempo de internação total (RS até 0.57) e tempo de UTI (RS = 0,47). Em relação a biomarcadores de interesse na Covid-19, a quebra da BE apresentou correlação significativa com neutrofilia, relação neutrófilo/linfócito, fator de Von Willebrand, fatores IX e XI, fibrinogênio, D-dímero e uPAR (Receptor de Uroquinase). Discussão: Através de um método considerado padrão-ouro para avaliação in vitro da integridade da BE nós demonstramos que componentes presentes no soro de pacientes com Covid-19 são capazes de promover a quebra da BE, e que a magnitude deste processo está relacionada à gravidade desta doença. A correlação com outros marcadores inflamatórios corrobora a conexão entre os mecanismos envolvidos na imunotrombose em pacientes com Covid-19. Conclusão: nossos resultados apontam a quebra da BE como um alvo terapêutico atrativo nestes pacientes.

COVID-19 Diagnosis and Its Impact on Thrombotic Risk in a Cohort of Patients Simultaneously Hospitalized Due to Acute Respiratory Distress Syndrome

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.

Circulating Levels of Ang/Tie2 and VEGF-a Pathway Mediators Are Associated with Clinical Severity, Endothelial Barrier Disruption and Coagulation Activation in COVID-19

Background: the pathogenesis of severe COVID-19 involves the deregulated activation of different compartments of immunothrombosis, which are otherwise important for pathogen eradication and tissue repair. Coagulation activation, angiogenesis and alterations of endothelial barrier (EB) are elements of immunothrombosis that have been shown to be involved in the pathogenesis of COVID-19. Angiopoietins (Ang) 1 and 2 and their receptor Tie2 and VEGF-A are well-known pro-angiogenic mediators that, during inflammation also mediate EB disruption. Recently, it has also been demonstrated that the Ang/Tie2 pathway is involved in coagulation activation. Here we explored whether increased levels of angiogenesis/EB regulators (which have been previously associated with disease severity in COVID-19) are also associated with both EB disruption and coagulation activation in this condition. Methods: the study population consisted of 30 patients with COVID-19 confirmed by RT-PCR and presenting typical CT findings admitted due to hypoxemia. Thirty sex- and age-matched healthy individuals were recruited at the same time, from the same geographic region. Patients were part of a clinical trial (REBEC: U1111-1250-1843) but samples were obtained before any study intervention, within 24 hours from diagnosis confirmation. Circulating levels of angiogenesis/EB regulation mediators and coagulation biomarkers were measured by commercial assays (immunological or functional). Monolayers of endothelial cells from umbilical veins (HUVECs) or lung (HULECs) were used for measurement of EB integrity using an impedance sensor system (ECIS, Electric Cell-substrate Impedance Sensing System). Cells were stimulated with serum from patients or healthy individuals and EB integrity was continuously monitored for 36 hours. Clinical outcomes were obtained from the digital medical records. Results: mean length of hospital stay (LOS) was 12.9 ± 9.8 days. Twelve patients (40%) required intensive care (ICU) and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Circulating levels of Ang1, Ang2, sTie2 and VEGF-A were all significantly increased in patients compared to healthy individuals (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p<0.0001;Ang2: 1,926 (1,275 - 3,134) vs 1,215 (9 - 1,440 pg/mL), p<0.0001;Tie2: 10,753 ± 2,377 vs 8,603 ± 1,851 pg/mL, p<0.0001 and VEGF-A: 94.7 (73.4 - 116.0) vs 45.9 (39.7 - 57.0 pg/mL), p<0.0001.). In contrast, soluble VE-cadherin levels were decreased in patients compared to healthy individuals (1,234 ± 318 vs 1,539 ± 363 ng/mL, P=0.001). Serum from COVID-19 patients induced decreases of EB integrity in monolayers of both HUVECs and HULECs as early as 15 minutes, lasting up to 5 hours after stimulation (figure 1). The magnitude of EB disruption was correlated with clinically relevant outcomes such as time of ICU stay and LOS (figure 1). Interestingly, levels of Ang1, Ang2 and soluble VE-cadherin levels were also significantly correlated with the magnitude of EB disruption, as well as with biomarkers of coagulation activation such as fibrinogen, Von Willebrand Factor antigen levels, PAI-1, P-selectin and urokinase receptor (uPAR). Conclusions: Ang-1/Ang-2 mediated Tie2 signaling has been shown to be important for the fine regulation of barrier integrity and coagulation activation at the endothelial level, which are two critical elements of immunothrombosis. Our results provide evidence supporting that the interplay between these processes can play a role in the mechanisms driving COVID-19 severity, and suggest that targeting the Ang/Tie2 and VEGF-A pathways could be attractive strategies to modulate not only changes of the alveolar-capillary barrier, but also of coagulation activation in COVID-19. Figure 1. In (a), endothelial barrier (EB) integrity of HUVEC monolayers upon stimulation by serum from COVID-19 patients and healthy individuals (n=27-30 per group). The lower the normalized resistance, the higher the magnitude of EB disruption. Significant differences (* to ****) are evident from 15 min to 5 hours (An va corrected for multiple comparisons). In the lower panels, the correlation of EB disruption with clinically relevant outcomes such as length of hospital stay (b) and days of intensive care (c) are shown. Negative correlations (Spearman test) indicate that the magnitude of EB disruption is associated with worse outcomes. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

Venous thromboembolism in critically-ill patients with pneumonia in the pre-COVID-19 era: Data from a large public database

Background: The high risk of venous thromboembolism (VTE) is a hallmark of COVID-19, particularly in intensive care units (ICU) patients. However, the magnitude of this risk is a matter of debate due to studies heterogeneity, significant changes on VTE management in COVID-19 era and scarce evidence of VTE risk in ICU patients with pneumonia in the pre-COVID-19 era. Aims: To evaluate the VTE risk in the pre-COVID-19 era in a large ICU database. Methods: Data of consecutive pneumonia patients admitted to ICU were retrieved from the Medical Information Mart for Intensive Care III (MIMIC-III) . VTE incidence during ICU stay was described. The association of thromboprophylaxis and VTE risk was determined by logistic regression, adjusted for age, sex, SOFA score, pneumonia diagnosis and type of ICU. Results: Among 6,842 pneumonia patients admitted to ICU, the median ICU stay was 11 (IQR 6-20) days. Tables 1 and 2 summarizes patients' characteristics and outcomes. 486 patients were diagnosed with VTE after a median of 3 (IQR 1-11) days in ICU. The overall cumulative incidence of VTE was 7% (95%CI 6.4-7.6), corresponding to a daily VTE incidence of 0.51% (95%CI 0.47-0.56). 1788 patients received thromboprophylaxis (out of 2958 for whom that data was available). The cumulative incidence of VTE was 10.7% (95%CI 8.9-12.6) among patients without thromboprophylaxis and 6.5% (95%CI 5.4-7.8) among those with thromboprophylaxis. Overall mortality was 19.3%, that was similar among patients with and without VTE (20.6% and 19.2%, respectively). (Table Presented) Conclusions: In pre-COVID-19 era, VTE rates in ICU patients with pneumonia was not substantially different from those reported in COVID-19 when VTE diagnosis is based on clinical suspicion. The risk of VTE was reduced by 46% with thromboprophylaxis. These findings can serve as comparator for future studies aiming at evaluating the impact of VTE on COVID-19.

Association of the angiopoietin/TIE2 and VEGF-a pathways with clinical and laboratory markers of disease severity incovid-19

Background : Endothelial barrier (EB) disruption is an important part of immunothrombosis, allowing the access of leukocytes to inflamed tissues. Pathways involving angiopoietin (Ang) 1 and 2 and their receptor Tie2, and VEGF-A/VE-cadherin (VEC) are key regulators of EB integrity. While the association of these mediators with sepsis severity have been known for more than 15 years, it was only recently that their role in coagulation activation was described. Moreover, these proteins also mediate angiogenesis, which has been shown to be upregulated in COVID-19. Aims : To measure circulating levels of key mediators of Ang/Tie2 and VEGF-A pathways in COVID-19 patients, and to explore their association with disease severity and hemostatic activation. Methods : Samples were obtained from patients admitted to a COVID-19 ward, within 24 h from COVID-19 confirmation. EB mediator levels were measured by immunological methods (Elisa or multiplex assays). The study was approved by the IRB and all participants provided written informed consent. Results : Data were obtained from 30 patients and 30 age and sexmatched healthy individuals. Mean length of hospital stay (LOS) was 12.9 ± 9.8 days respectively, twelve patients (40%) required intensive care (ICU), and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Levels of EB mediators are shown in Table 1. Associations between these parameters with relevant clinical and laboratory markers of disease severity are shown in Table 2. Conclusions : All mediators of EB disruption were significantly elevated in COVID-19 patients. In addition, these mediators were consistently associated with proteins involved in immunothrombosis, in particular with fibrinogen, VWF:Ag, uPAR, PAI-1 and P-selectin. Clinically significant associations were observed between Ang-2 and VEGF-A and extension of lung disease (for both) and ICU stay (for VEGF-A). Additional studies are warranted to explore the crosstalk between Ang/Tie2 and VEGF-A pathways with hemostasis in COVID-19.

Contact system and intrinsic pathway are activated and associated with clinical and laboratory outcomes in COVID-19patients

Background : Hemostasis activation is considered a key pathogenic element of COVID-19, as evidenced by the frequency of lung microvascular thrombosis and of venous thromboembolism. As shown in other conditions, pathways that mediate hemostasis activation during inflammation are not necessarily the same as those that drive hemostasis in non-inflammatory states. In particular, contact system and intrinsic pathway activation have been increasingly explored as potential mediators of hemostasis activation and prothrombotic states observed in both sterile and infectious inflammatory conditions. Aims : To assess the activation of contact system and intrinsic pathway activation in COVID-19. Methods : Protease:serpin complexes were measured in plasma from inpatients with COVID-19 and healthy individuals (HI). All samples were collected within 24 h of COVID-19 diagnosis. Associations with laboratory and clinical markers of hemostasis activation and disease severity were explored. The study was approved by the IRB and all participants provided written informed consent. Results : In total, 30 patients with COVID-19 and 30 age and sex-matched HI were enrolled. Main characteristics of the study population are shown in Table 1. As expected, higher levels of classical coagulation activation parameters were observed in COVID-19 patients. Contact system and intrinsic pathway activation in COVID-19 was demonstrated by increased levels of several protease:serpin complexes (Table 2). Interestingly, a consistent and significant association was observed between FIXa:antithrombin complexes with both clinical endpoints (need of ICU admission, length of ICU stay, total length of stay and extent of lung CT alterations), and with laboratory markers of immunothrombosis (neutrophil:lymphocyte ratio, C-reactive protein, D-dimer, PAP, VWF:Ag), as well as with several other activation markers of contact system and intrinsic pathway Conclusions : Contact system and intrinsic pathway activation contribute to hemostasis activation in COVID-19. The association of FIXa:antithrombin complexes with disease severity suggests that these pathways contribute to the pathogenesis of the prothrombotic state of COVID-19.