Evaluation of the protein C pathway in critically ill patients with severe COVID-19 as compared to bacterial sepsis

Introduction Endothelial dysfunction has been shown to play a role in severe COVID-19, the pathophysiology of which may be attributed to a myriad of factors including unmitigated immune and inflammatory response, viral-induced injury to the endothelium, end-stage organ failure, and coagulopathy. In addition, severe COVID-19 is most often seen in patients with multiple comorbidities, which themselves are often associated with endothelial dysfunction (such as myocardial and renal failure, as well as thrombotic disorders). However, the literature is still emerging on this topic and there appears to be no consensus on the extent to which endothelial dysfunction plays a role in severe COVID-19. Method The aim of this study was to assess the functionality of the endothelial protein C pathway in hospitalized patients > 18 years of age with severe COVID-19 as compared to those hospitalized with bacterial sepsis. COVID-19 (n = 31) and sepsis (n = 47) patients who were admitted to the ICU were assessed for rates of thrombin and activated protein C (APC) generation. Indirect markers of thrombin formation, including thrombin-antithrombin (TAT) complex, prothrombin fragment 1 + 2 (F1 + 2), as well as D-dimer, and protein C (PC) were measured additionally. Statistical analysis was performed via the Mann-Whitney test and a p value of < 0.05 was considered statistically significant. Results Plasma levels of free thrombin in COVID-19 and sepsis patients did not differ significantly, with (median, IQR) 0.59 (0.46-1.21) vs 0.57 (0.46-1.10) pmol/L, respectively. TAT was also increased at similar extent in both cohorts (192;111-325 pmol/L in COVID-19 patients, 148;73-213 pmol/L in sepsis patients), whereas F1 + 2 was higher in COVID-19 than in sepsis patients, with 850 (440-1940) vs 380 (130-620) pmol/L (p = 1.3 x 10-5). Interestingly, rates of APC formation did not significantly differ between the two groups, with 7.47 (1.99- 19.14) vs 9.87 (2.08-16.87) pmol/L ( Fig. 1). D-dimer and protein C were significantly higher in the COVID-19 patients than in those with sepsis (14.3 vs 8.1 mg/L, p = 0.01, and 92.9 % vs 58.5 %, p = 3x10-8, respectively). Conclusion We hypothesized that APC formation rates in response to thrombin formation would be significantly lower in patients with severe COVID-19 as compared to those with bacterial sepsis due to the well-known association between severe COVID-19 disease burden and endothelial dysfunction due to the downregulation of thrombomodulin expression. However, our results indicate that this may not be universally true in this patient population, as our observations suggest a largely intact functionality of the protein C pathway. Further studies are warranted to investigate the pathophysiology of severe COVID-19. (Figure Presented).

Effects of convalescent plasma infusion on the ADAMTS13-von Willebrand factor axis and endothelial integrity in patients with severe and critical COVID-19.

Background: Convalescent plasma infusion (CPI) was given to patients with COVID-19 during the early pandemic with mixed therapeutic efficacy. However, the impacts of CPI on the ADAMTS13-von Willebrand factor (VWF) axis and vascular endothelial functions are not known. Objectives: To determine the impacts of CPI on the ADAMTS13-VWF axis and vascular endothelial functions. Methods: Sixty hospitalized patients with COVID-19 were enrolled in the study; 46 received CPI and 14 received no CPI. Plasma ADAMTS13 activity, VWF antigen, endothelial syndecan-1, and soluble thrombomodulin (sTM) were assessed before and 24 hours after treatment. Results: Patients with severe and critical COVID-19 exhibited significantly lower plasma ADAMTS13 activity than the healthy controls. Conversely, these patients showed a significantly increased VWF antigen. This resulted in markedly reduced ratios of ADAMTS13 to VWF in these patients. The levels of plasma ADAMTS13 activity in each patient remained relatively constant throughout hospitalization. Twenty-four hours following CPI, plasma ADAMTS13 activity increased by ∼12% from the baseline in all patients and ∼21% in those who survived. In contrast, plasma levels of VWF antigen varied significantly over time. Patients who died exhibited a significant reduction of plasma VWF antigen from the baseline 24 hours following CPI, whereas those who survived did not. Furthermore, patients with severe and critical COVID-19 showed significantly elevated plasma levels of syndecan-1 and sTM, similar to those found in patients with immune thrombotic thrombocytopenic purpura. Both syndecan-1 and sTM levels were significantly reduced 24 hours following CPI. Conclusion: Our results demonstrate the relative deficiency of plasma ADAMTS13 activity and endothelial damage in patients with severe and critical COVID-19, which could be modestly improved following CPI therapy.

Prevention and treatment of atherothrombosis: Potential impact of nanotechnology.

Complications with atherosclerosis can often lead to fatal clot formation and blood vessel occlusion - also known as atherothrombosis. A key component to the development of atherosclerosis and atherothrombosis is the endothelium and its ability to regulate the balance between prothrombotic and antithrombotic activities. Endothelial surface glycocalyx has a critical role in maintenance of vascular integrity. The endothelial glycocalyx, nitric oxide, prostacyclins, heparan sulfate, thrombomodulin, and tissue factor pathway inhibitor all prevent thrombosis, while P-selectin, among many other factors, favors thrombosis. However, endothelial dysfunction gives rise to the acceleration of thrombotic development and eventually the requirement of antithrombotic therapy. Most FDA-approved anticoagulant and antiplatelet therapies today carry a side effect profile of major bleed. Within the past five years, several preclinical studies using different endothelial targets and nanotechnology as a drug delivery method have emerged to target the endothelium and to enhance current antithrombosis without increasing bleed risk. While clinical studies are required, this review illustrates the proof-of-concept of nanotechnology in promoting a greater safety and efficacy profile through multiple in vitro and in vivo studies.

Global Hemostasis Potential in COVID-19 Positive Patients Performed on St-Genesia Show Hypercoagulable State.

BACKGROUND: At the dawn of the pandemic, severe forms of COVID-19 were often complicated by thromboembolisms. However, routine laboratory tests cannot be used to predict thromboembolic events. The objective of this study was to investigate the potential value of the thrombin generation test (TGT) in predicting hypercoagulability and thrombotic risk in the aforementioned set of patients. METHODS: The study panel comprised 52 patients divided into two groups (26 COVID-19 positive and 26 COVID-19 negative); COVID-19-positive patients were further grouped in "severe" (n = 11) and "non-severe" (n = 15) categories based on clinical criteria. The routine blood tests and TGT of these patients were retrospectively analyzed. RESULTS: All 26 COVID-19-positive patients showed decreased lymphocyte, monocyte and basophil counts and increased lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine transaminase (ALT) compared with control patients. Conversely, we did not observe statistically significant differences between severe and non-severe patients despite anecdotal variations in the distribution patterns. TGT without thrombomodulin (TM) addition showed statistically significant differences in the thrombin peak heights between COVID-19-positive and negative patients. After addition of TM, peak height, Endogenous Thrombin Potential (ETP) and velocity index were increased in all COVID-19-positive patients while the percentage of inhibition of ETP was reduced. These trends correlated with the severity of disease, showing a greater increase in peak height, ETP, velocity index and a drastic reduction in the percentage of ETP inhibition in more severely affected patients. CONCLUSIONS: Our data suggest that all COVID-19 patients harbor a hypercoagulable TGT profile and that this is further pronounced in severely affected patients.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis.

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell-cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5'-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis.

Building a better NET: Neutrophil extracellular trap targeted therapeutics in the treatment of infectious and inflammatory disorders

Infectious and inflammatory stimuli induce the release of neutrophil extracellular traps (NETs), webs of cell-free (cf) DNA complexed with histones and antimicrobial proteins, that capture and kill pathogens. Despite their protective role in the initial stages of sepsis, excessive NET release accompanied by NET degradation, leads to the release of NET degradation products (NDPs), including cfDNA, histones, and myeloperoxidase that injure the microvasculature. Murine studies have shown that clearance or neutralization of NDPs improves outcomes, demonstrating that NETs have a causal link to disease and are not merely biomarkers. Recently, elevated NDPs have been associated with disease severity in sepsis and coronavirus disease 2019, raising further interest in targeting NETs. Many propose eliminating NETs, either by preventing their release, or by degrading them. However, NET inhibition may impede the innate immune response and is difficult to achieve in rapid-onset conditions such as sepsis. On the other hand, approaches that accelerate NET degradation have met with mixed results in murine studies, raising the concern that this strategy may liberate NET-captured pathogens while increasing circulating levels of harmful NDPs. Alternative NET-directed strategies include therapies that neutralize, sequester, or remove NDPs from the circulation. Others propose modifying released NETs to decrease their capacity to induce collateral tissue damage while enhancing their ability to capture microorganisms. Synthetic NETs have also been designed to combat antibiotic-resistant organisms. Although it is still in its infancy, the field of NET-targeted therapeutics is advancing rapidly and may soon find application in the treatment of sepsis and other inflammatory disorders.

The central role of neutrophil extracellular traps in SARS-CoV-2-induced thrombogenesis and vasculitis

Objective: Severe acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) seen in SARs-CoV-2 infection has been attributed to the disruption of the immune response in COVID-19 patients. Neutrophilia and marked lymphocyte reductions are associated with disease severity and seem predictive of disease outcome in moderate and severe COVID-19 patients. Herein, we aim to decipher possible mechanisms involved in extensive tissue injury observed in COVID-19 patients, accompanied by vasculopathy, coagulopathy, and a high incidence of thrombotic complications in severe patients. Method(s): We searched PubMED for keywords including COVID-19 pathogenesis, thrombosis, and vasculities. Result(s): Neutrophils can undergo a specialized form of apoptosis to yield thread-like extracellular structures termed neutrophil extracellular traps (NETs), termed NETosis, which form web-like scaffolds of DNA, histones, and toxic protein granules and enzymes, whose primary function is to trap and eliminate microbes. However, uncontrolled NET production can lead to ALI and ARDS, coagulopathy, multiple organ failure, and autoimmune disease. Dysregulation of NETs promotes production of anti-neutrophil cytoplasmic antibodies (ANCA) which affects small vessels through ANCA-associated vasculitis (AAV). Furthermore, NETs can also induce thrombosis via formation of scaffolds that trap platelets, RBCs, fibronectin, and other proteins, which can also induce coagulation. Conclusion(s): We suggest that NET production is central during SARS-CoV-2 infection and COVID-19 pathogenesis, associated with alveolar damage accumulation of edema, endothelial injury and coagulopathy, elevated platelet activation and thrombogenesis forming a NET production feed-forward loop, causing diffuse small vessel vasculitis in the lungs and other organs. Copyright © 2020 FSG Communications Ltd. All rights reserved.

Lung Ultrasound Findings and Endothelial Perturbation in a COVID-19 Low-Intensity Care Unit.

Hypercoagulability and endothelial dysfunction related to inflammation have been clearly demonstrated in COVID-19. However, their influence on thromboembolism, lung alterations and mortality in low-intensity-care patients with COVID-19 is not completely clarified. Our aims were to evaluate the prevalence of deep vein thrombosis (DVT) with compressive ultrasound (CUS); to describe lung ultrasound (LUS) features; and to study coagulation, inflammatory and endothelial perturbation biomarkers in COVID-19 patients at low-intensity care unit admission. The predictive value of these biomarkers on mortality, need for oxygen support and duration of hospitalization was also evaluated. Of the 65 patients included, 8 were non-survivors. CUS was negative for DVT in all patients. LUS Soldati and Vetrugno scores were strongly correlated (rho = 0.95) with each other, and both significantly differed in patients who needed oxygen therapy vs. those who did not (Soldati p = 0.017; Vetrugno p = 0.023), with coalescent B lines as the most prevalent pattern in patients with a worse prognosis. Mean (SD) levels of thrombomodulin and VCAM-1 were higher in non-survivors than in survivors (7283.9 pg/mL (3961.9 pg/mL) vs. 4800.7 pg/mL (1771.0 pg/mL), p = 0.004 and 2299 ng/mL (730.35 ng/mL) vs. 1451 ng/mL (456.2 ng/mL), p < 0.001, respectively). Finally, in a multivariate analysis model adjusted for age, sex and Charlson score, VCAM-1 level increase was independently associated with death [OR 1.31 (1.06, 1.81; p = 0.036)]. In conclusion, in a cohort of mild COVID-19 patients, we found no DVT events despite the highly abnormal inflammatory, endothelial and coagulation parameters. The presence of lung alterations at admission could not predict outcome. The endothelial perturbation biomarker VCAM-1 emerged as a promising prognostic tool for mortality in COVID-19.

HEMOSTATIC ACTIVATION MARKERS AND SEROLOGICAL RESPONSE IN SUBJECTS RECEIVING ANTICOVID- 19 VACCINATION

Background: SARS-COV2 infection is associated with inflammation, hypercoagulability and endothelial damage. Anti-SARS-COV2 vaccines have radically changed the course of the pandemic, however, reports on rare thrombotic events raise concern in the scientific community and the general population. Aims: In a prospectively enrolled cohort of adult subjects undergoing mRNA or adenovirus vector vaccination, we wanted to longitudinally evaluate the changes in levels of hemostatic biomarkers (i.e. activation of blood coagulation and perturbance of endothelium and fibrinolysis), together with the serological response, and occurrence of manifest thrombotic complications. Methods: Peripheral venous blood samples were collected at enrollment (day 0, D0) before the 1st vaccine dose, and on 15 (D15), 60 (D60), 90 (D90) and 180 (D180) days after the 1st dose. At each time point, hemostatic markers (i.e., fibrinogen, D-dimer, FVIII, von Willebrand Factor [vWF] antigen and activity, F1+2, thrombomodulin, protein C, protein S, FXIII, tPA, and PAI-1), and anti-Spike receptor-binding-domain protein (anti-S/RBD) IgG were measured. Follow up is currently continuing. Results: Fifty-three subjects (57% males) with a median age of 50 years (range 23-86) were enrolled into the study and followed-up for 6 months: 36 (68%) received BNT162b2, 6 (11%) mRNA-1273, and 8 (15%) ChAdOx1 nCoV-19 vaccines, in 2 doses over 21, 30 and 77 days, respectively;while 3 (6%) subjects received Ad26.COV2.S as single shot. Twenty individuals (38%) reported previous history of COVID-19, with a mean time from infection to vaccination of 10 months (4-18);only 1 required Hospitalization. Nine subjects presented cardiovascular risk factors and 4 a prior, non-active, cancer;3 were on anticoagulation for atrial fibrillation. The evaluation of the hemostatic biomarkers at the different time points showed variations in some of the parameters evaluated, with median values remaining within normal range levels. Specifically, compared to baseline, we observed a significant increase in thrombomodulin at D90 (p=0.001) and D180 (p=0.03), in parallel to a significant decrease in fibrinogen (D60), vWFAg (D60 and D180), FVIII (D60, D90 and D180), and TPA (D60 and D90) levels. The reduction of these biomarkers was particularly evident in individuals with a history of COVID-19. Of interest, this group of subjects was also characterized by significantly lower levels of PAI-1 both at baseline (7.18 ng/mL vs 17.53 ng/mL;p<0.0001), and at other time points (p<0.0001), and by an increase in F1+2 at D90 (p=0.02). The association between lower baseline PAI-1 levels with history of COVID-19 was confirmed by linear regression analysis (B= -10.351, p=0.013), and was independent by the time of infection resolution. Notably, no differences were observed in the hemostatic biomarkers according to vaccine types. All subjects positively responded to vaccination with a significant increase in anti-S/RBD IgG from baseline (D0) to each time point, especially COVID-19 subjects (D15, D60, and D90:p<0.0001;D180:p=0.031). No thrombotic or cardiovascular complications occurred during follow-up. Summary/Conclusion: No hypercoagulable state elicited by COVID-19 vaccination was observed, contrarily we detected an overall persistent reduction of coagulation activation over time. Subjects with previous SARS-COV2 infection had persistently low levels of PAI-1, supporting enhanced fibrinolysis activation. Compared with recent studies, our results provide a longer observation follow-up with all vaccine types and reassure on the safety of anti- SARS-COV2 vaccination.

Circulating Thrombomodulin: Release Mechanisms, Measurements, and Levels in Diseases and Medical Procedures.

Thrombomodulin (TM) is a type-I transmembrane protein that is mainly expressed on endothelial cells and plays important roles in many biological processes. Circulating TM of different forms are also present in biofluids, such as blood and urine. Soluble TM (sTM), comprised of several domains of TM, is the major circulating TM which is generated by either enzymatic or chemical cleavage of the intact protein under different conditions. Under normal conditions, sTM is present in low concentrations (<10 ng/mL) in the blood but is elevated in several pathological conditions associated with endothelial dysfunction such as cardiovascular, inflammatory, infection, and metabolic diseases. Therefore, sTM level has been examined for monitoring disease development, such as disseminated intravascular coagulation (DIC), sepsis and multiple organ dysfunction syndrome in patients with novel coronavirus disease 2019 (COVID-19) recently. In addition, microvesicles (MVs) that contain membrane TM (MV-TM) have been found to be released from activated cells which also contribute to levels of circulating TM in certain diseases. Several release mechanisms of sTM and MV-TM have been reported, including enzymatic, chemical, and TM mutation mechanisms. Measurements of sTM and MV-TM have been developed and explored as biomarkers in many diseases. In this review, we summarize all these advances in three categories as follows: (1) release mechanisms of circulating TM, (2) methods for measuring circulating TM in biological samples, and (3) correlation of circulating TM with diseases. Altogether, it provides a whole picture of recent advances on circulating TM in health and disease.

Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients.

Hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections, but the molecular mechanisms involved are unclear. Increased plasma levels of the inflammatory cytokine and TIE2 receptor antagonist Angiopoietin-2 were reported in severely ill COVID-19 patients. In vitro experiments suggest that Angiopoietin-2 bind and inhibits thrombomodulin. Thrombomodulin is expressed on the luminal surface of endothelial cells where it is an important member of the intrinsic anticoagulant pathway through activation of protein C. Using clinical data, mouse models, and in vitro assays, we tested if Angiopoietin-2 plays a causal role in COVID-19-associated hypercoagulation through direct inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Angiopoietin-2 was measured in 61 patients at admission, and after 10 days in the 40 patients remaining in the ICU. We found that Angiopoietin-2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. In support of a direct effect of Angiopoietin-2 on coagulation, we found that injected Angiopoietin-2 in mice associated to thrombomodulin and resulted in a shortened tail bleeding time, decreased circulating levels of activated protein C, and increased plasma thrombin/antithrombin complexes. Conversely, bleeding time was increased in endothelial-specific Angiopoietin-2 knockout mice, while knockout of Tie2 had no effect on tail bleeding. Using in vitro assays, we found that Angiopoietin-2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data suggest a novel in vivo mechanism for Angiopoietin-2 in COVID-19-associated hypercoagulation, implicating that Angiopoietin-2 inhibitors may be effective in the treatment of hypercoagulation in severe COVID-19 infection.

Automated measurement of coagulation and fibrinolytic activation markers: Outcomes in coronavirus disease 2019 (COVID-19) patients.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition. PATIENTS/METHODS: We present a retrospective observational study of 104 patients admitted to hospital with COVID-19. Plasma samples were collected within 72 h of admission. In addition to routine coagulation and haematology testing, soluble thrombomodulin (sTM), thrombin-antithrombin (TAT), tissue plasminogen activator-plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin-α2 antiplasmin complex (PIC) were performed by automated chemiluminescent enzyme immunoassays. RESULTS: Significantly higher levels of D-dimer, TAT, sTM and tPAI-C were observed in non-survivors compared to survivors. To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D-dimer, TAT. sTM, tPAI-C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for each 1.0 TU/mL increment (95% CI 1.025-1.115). CONCLUSIONS: Of the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID-19, and this suggests that endothelial dysfunction plays an important role in disease progression.

Association of COVID-19 with impaired endothelial glycocalyx, vascular function and myocardial efficiency four months after infection

Aims: SARS-CoV-2 infection may lead to endothelial and vascular dysfunction. We investigated alterations of arterial stiffness, endothelial coronary and myocardial function markers four months after COVID-19 infection. Methods: In a case-control prospective study, we included 100 patients four months after COVID-19 infection, 50 age- and sex-matched healthy individuals. We measured a) pulse wave velocity (PWV), b) flow-mediated dilation (FMD) of brachial artery, c) coronary Flow Reserve (CFR) by Doppler echocardiography d) left ventricular (LV) global longitudinal strain (GLS), e) left ventricular myocardial work index, constructive work, wasted work and work efficiency and e) von-Willenbrand factor and thrombomodulin as endothelial biomarkers. Results: COVID-19 patients had lower CFR and FMD values than controls (2.39 ± 0.39 vs 3.31 ± 0.59, p = 0.0122, 5.12 ± 2.95% vs 8.12 ± 2.23%, p = 0.006 respectively). Compared to controls, COVID-19 patients had higher PWV (PWVc-f 12.32 ± 2.44 vs 10.11 ± 1.85 m/sec, p = 0.033) and impaired LV GLS (-19.11 ± 2.14% vs -20.41 ± 1.61%, p = 0.001). Compared to controls, COVID-19 patients had higher myocardial work index, and wasted work (2067.7 ± 325.9 mmHg% vs 1929.4 ± 312.7 mmHg%, p = 0.026, 104.6 ± 58.9 mmHg% vs 75.1 ± 52.6 mmHg%, p = 0.008, respectively), while myocardial efficiency was lower (94.8 ± 2.5% vs 96.06 ± 2.3%, p = 0.008). and thrombomodulin were higher in COVID-19 patients than controls (3716.63 ± 188.36 vs 2590.02 ± 156.51pg/ml, p < 0.001). MDA was higher in COVID-19 patients than controls (10.55 ± 2.45 vs 1.01 ± 0.50 nmole/L, p = 0.001). Residual cardiovascular symptoms at 4 months were associated with oxidative stress markers. Myocardial work efficiency was related with PWV (F=-0.309, p = 0.016) and vWillenbrand (F=-0.541, p = 0.037). Myocardial wasted work was related with PWV (F = 0.255, p = 0.047) and vWillenbrand (F = 0.610, p = 0.016). Conclusions: SARS-CoV-2 may cause vascular dysfunction, followed by a waste of cardiac work, in order to compensate for increased arterial stiffness 4 months after infection.

Association of COVID-19 with impaired endothelial glycocalyx, coronary flow and longitudinal strain four months after infection

Aims: SARS-CoV-2 infection may lead to endothelial and vascular dysfunction. We investigated alterations of arterial stiffness, endothelial coronary and myocardial function markers four months after COVID-19 infection. Methods: In a case-control prospective study, we included 100 patients four months after COVID-19 infection, 50 age- and sex-matched healthy individuals. We measured a) perfused boundary region (PBR) of the sublingual arterial microvessels (increased PBR indicates reduced endothelial glycocalyx thickness), b) flow-mediated dilation (FMD), c) coronary Flow Reserve (CFR) by Doppler echocardiography d) pulse wave velocity (PWV) e) global left (LV) and right (RV) ventricular longitudinal strain (GLS), f) malondialdehyde (MDA), an oxidative stress marker, von-Willenbrand factor and thrombomodulin as endothelial biomarkers. Results: COVID-19 patients had lower CFR and FMD values than controls (2.39 ± 0.39 vs 3.31 ± 0.59, p = 0.0122, 5.12 ± 2.95% vs 8.12 ± 2.23%, p = 0.006 respectively). Compared to controls, COVID-19 had greater PBR5-25 (2.11 ± 0.14μm vs 1.87 ± 0.16μm, p = 0.002), higher PWV (PWVc-f 12.32 ± 2.44 vs 10.11 ± 1.85 m/sec, p = 0.033) and impaired LV and RV GLS (-19.11 ± 2.14% vs -20.41 ± 1.61%, p = 0.001 and -16.45 ± 3.33% vs -20.11 ± 2.48%, p < 0.001). MDA and thrombomodulin were higher in COVID-19 patients than controls (10.55 ± 2.45 vs 1.01 ± 0.50 nmole/L, p = 0.001 and 3716.63 ± 188.36 vs 2590.02 ± 156.51pg/ml, p < 0.001). Residual cardiovascular symptoms at 4 months were associated with oxidative stress and endothelial dysfunction markers. Conclusions: SARS-CoV-2 may cause endothelial and vascular dysfunction linked to.

Sars-Cov-2 Induced Endotheliopathy and the Impact on Clinical Outcomes in Critically Ill Pediatric Patients

Background: Coronavirus disease-19 (COVID-19) is an acute respiratory illness caused by the SARS-COV-2 virus. Patients with COVID-19 infection can present with thrombosis in the setting of inflammation (thromboinflammation), presumably from endothelial dysfunction, or “endotheliopathy”. Yu et al demonstrated in vitro that the spike protein subunit of SARS-COV2 acts as a potent activator of the alternative complement pathway (AP), one of three complement pathways within the innate immune system. Satyam et alreported the deposition of complement components on lung tissue of patients who succumbed to COVID-19, consistent with activation of classical and alternate pathways. These studies suggest complement dysregulation potentially causing endotheliopathy in COVID-19 patients. Thrombomodulin (TM) is an endothelial glycoprotein that plays two crucial roles in maintaining a healthy endothelium - as a natural anticoagulant and a negative regulator of complement. TM shed into the circulation due to endothelial injury can be measured in the plasma as soluble TM (sTM). Goshua et al showed elevated sTM in an adult cohort of patients with COVID-19. However, it is yet to be demonstrated if there is any correlation between endothelial injury and AP activation in COVID-19, or if either play a role in clinical outcome in the pediatric population. Objective: To 1) assess endothelial injury and AP activation in a cohort of critically ill pediatric patients with COVID-19 by measuring sTM and Ba (an AP activation product);2) determine the correlation between endothelial injury and AP activation;and 3) analyze the utility of sTM and Ba in predicting pediatric clinical outcomes. Methods: We collected plasma samples of patients admitted to the Pediatric Intensive Care Unit and found to be positive for SARS-CoV-2 between Dec 2, 2020 and Jan 22, 2021 at Texas Children's Hospital. For controls, we collected plasma samples from pediatric patients undergoing preoperative clearance, all at their baseline state of health. sTM levels and Ba levels were measured in plasma samples using commercially available TM and Ba ELISA kits. sTM greater than 7.6 ng/ml (based on the assay range in adults) and Ba greater than 1080 ng/ml (based on data from adult healthy controls) were considered elevated. Data regarding demographics, length of ICU stay, clinical indicators of end organ damage- mechanical ventilation, dialysis, use of vasopressors, ECMO, mortality were obtained retrospectively via chart review. Inclusion criteria included all patients with a positive SARS-COV2 PCR admitted to the ICU. Exclusion criteria was age greater than 21 years, pregnant female, patients with known inflammatory or complement-mediated disorders. Statistical analysis: For sTM and Ba levels between control and COVID-19 patients, mean +/- standard deviation was calculated and significance determined with an unpaired t-test. Fischer exact test, Wilcoxon rank sum and Pearson product-moment correlation tests were used for statistical analysis of clinical outcomes as appropriate. A p-value <0.05 was considered statistically significant. Results: A total of 38 control patients and 33 COVID-19 patients were enrolled. Ba and sTM levels were both significantly higher in the COVID-19 pediatric patients compared to the controls (Fig. 1). Within the COVID-19 patient cohort, 61% (n=20) had elevated sTM and 42% (n=14) had elevated Ba levels. There was a moderately positive correlation between sTM and plasma Ba levels in the COVID-19 cohort (Fig. 2). Within the COVID-19 patients' cohort, though higher Ba levels were not associated with an increased rate of intubation, they were associated with an increased duration of mechanical ventilation (p=.039) for those intubated (Table 1). Elevated sTM was associated with increased vasopressor use (p=.011). Although other clinical outcome variables did not reach statistical significance likely owing to small numbers, overall trend indicated worse outcomes in patients with elevated sTM. Conclusions: Our findings are consistent with the hypothesi that SARS-COV-2 activates AP and causes endothelial injury in children. The positive correlation between sTM and Ba suggest interplay between inflammation, coagulation and endotheliopathy supporting thromboinflammation in COVID-19. Higher sTM and Ba levels indicated worse clinical outcomes in children, but larger studies are needed to confirm our findings. [Formula presented] Disclosures: Sartain: Alexon Pharamaceuticals: Membership on an entity's Board of Directors or advisory committees.

Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the ‘cytokine storm’ resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even n patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures: Gligorov: Roche-Genentech: Research Funding;Novartis: Research Funding;Onxeo: Research Funding;Daichi: Research Funding;MSD: Research Funding;Eisai: Research Funding;Genomic Heatlh: Research Funding;Ipsen: Research Funding;Macrogenics: Research Funding;Pfizer: Research Funding. Terpos: Novartis: Honoraria;Janssen: Consultancy, Honoraria, Research Funding;Genesis: Consultancy, Honoraria, Research Funding;Celgene: Consultancy, Honoraria, Research Funding;BMS: Honoraria;Amgen: Consultancy, Honoraria, Research Funding;Takeda: Consultancy, Honoraria, Research Funding;Sanofi: Consultancy, Honoraria, Research Funding;GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria;BMS: Honoraria;Janssen: Honoraria;Beigene: Honoraria;Takeda: Honoraria.

The effect of prostacyclin infusion on markers of endothelial activation and damage in mechanically ventilated patients with SARS-CoV-2 infection.

BACKGROUND: In a pilot study, we found a significant reduction in mean daily sequential organ failure assessment score in mechanically ventilated patients with COVID-19 who received prostacyclin, compared to placebo. We here investigate the effect on biomarkers of endothelial activation and damage. METHODS: Post-hoc study of a randomized controlled trial in adult patients with confirmed SARS-CoV-2 infection, mechanically ventilated, with soluble thrombomodulin (sTM) plasma levels >4 ng/mL. Patients received prostacyclin infusion (1 ng/kg/min) or placebo. Blood samples were collected at baseline and 24 h. RESULTS: Eighty patients were randomized (41 prostacyclin, 39 placebo). The median changes in syndecan-1 plasma levels at 24 h were -3.95 (IQR: -21.1 to 2.71) ng/mL in the prostacyclin group vs. 3.06 (IQR: -8.73 to 20.5) ng/mL in the placebo group (difference of the medians: -7.01 [95% CI: -22.3 to -0.231] ng/mL, corresponding to -3% [95% CI: -11% to 0%], p = 0.04). Changes in plasma levels of sTM, PECAM-1, p-selectin, and CD40L did not differ significantly between groups. CONCLUSIONS: Prostacyclin infusion, compared to placebo, resulted in a measurable decrease in endothelial glycocalyx shedding (syndecan-1) at 24 h, suggesting a protective effect on the endothelium, which may be related to the observed reduction in organ failure.

Prostacyclin in Intubated Patients with COVID-19 and Severe Endotheliopathy: A Multicenter, Randomized Clinical Trial.

Rationale: The mortality in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who require mechanical ventilation remains high, and endotheliopathy has been implicated. Objectives: To determine the effect of prostacyclin infusion in mechanically ventilated patients infected with SARS-CoV-2 with severe endotheliopathy. Methods: We conducted a multicenter, randomized clinical trial in adults infected with coronavirus disease (COVID-19) who required mechanical ventilation and had a plasma level of thrombomodulin >4 ng/ml; patients were randomized to 72-hour infusion of prostacyclin 1 ng/kg/min or placebo. Measurements and Main Results: The main outcome was the number of days alive and without mechanical ventilation within 28 days. Key secondary outcomes were 28-day mortality and serious adverse events within 7 days. Eighty patients were randomized (41 prostacyclin and 39 placebo). The median number of days alive without mechanical ventilation at 28 days was 16.0 days (SD, 12) versus 5.0 days (SD, 10) (difference of the medians, 10.96 days; 95% confidence interval [CI], -5 to 21; P = 0.07) in the prostacyclin and the placebo groups, respectively. The 28-day mortality was 21.9% versus 43.6% in the prostacyclin and the placebo groups, respectively (risk ratio, 0.50; 95% CI, 0.24 to 0.96; P = 0.06). The incidence of serious adverse events within 7 days was 2.4% versus 12.8% (risk ratio, 0.19; 95% CI, 0.001 to 1.11; P = 0.10) in the prostacyclin and the placebo groups, respectively. Conclusions: Prostacyclin was not associated with a significant reduction in the number of days alive and without mechanical ventilation within 28 days. The point estimates, however, favored the prostacyclin group in all analyses, including 28-day mortality, warranting further investigation in larger trials. Clinical trial registered with www.clinicaltrials.gov (NCT04420741); EudraCT Identifier: 2020-001296-33.

Endothelial thrombomodulin downregulation caused by hypoxia contributes to severe infiltration and coagulopathy in COVID-19 patient lungs.

BACKGROUND: Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19. METHODS: We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy. FINDINGS: The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury. INTERPRETATION: Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2. FUNDING: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.

The Compass-COVID19-ICU Study: Identification of Factors to Predict the Risk of Intubation and Mortality in Patients with Severe COVID-19

Background: In some patients, SARS-CoV-2 infection induces cytokine storm, hypercoagulability and endothelial cell activation leading to worsening of COVID-19, intubation and death. Prompt identification of patients at risk of intubation or death is un unmet need. Objective: To derive a prognostic score for the risk of intubation or death in patients with critical COVID-19 by assessing biomarkers of hypercoagulability, endothelial cell activation and inflammation and a large panel of clinical analytes. Methods: We conducted a prospective, observational monocentric study enrolling 118 patients with COVID-19 admitted in the intensive care unit. At the 1st day of ICU admission all patients were assessed for the following biomarkers : protein C, protein S, antithrombin, D-Dimer, fibrin monomers, factors VIIa, V, XII, XII, VIII, von Willebrand antigen, fibrinogen, procoagulant phospholipid dependent clotting time, TFPI, thrombomodulin, P-selectin, heparinase, microparticles exposing tissue factor, IL-6, complement C3a, C5a, thrombin generation, prothrombin time, activated partial thromboplastin time, hemogram, platelet count) and clinical predictors. The clinical outcomes were intubation and mortality during hospitalization in ICU. Results: The intubation and mortality rate were 70 % and 18% respectively. Multivariate analysis led to the derivation of the COMPASS- COVID19-ICU score composed of P-Selectin, D-Dimer, free TFPI, TF activity, IL-6 and FXII, age and duration of hospitalization. The score predicted the risk of intubation or death with high sensitivity and specificity (0.90 and 0.92, respectively). Conclusions and Relevance: Critical COVID-19 is related with severe endothelial cell activation and hypercoagulability orchestrated in the context of inflammation. The COMPASS-COVID19-ICU score is an accurate predictive model for the evaluation of the risk of mechanical ventilation and death in patients with critical COVID-19. The assessment with the COMPASS- COVID-19-ICU score is feasible in tertiary hospitals. In this context it could be placed in the diagnostic procedure of personalized medical management and prompt therapeutic intervention. Disclosures: Terpos: Novartis: Honoraria;Janssen: Consultancy, Honoraria, Research Funding;Genesis: Consultancy, Honoraria, Research Funding;Celgene: Consultancy, Honoraria, Research Funding;BMS: Honoraria;Amgen: Consultancy, Honoraria, Research Funding;Takeda: Consultancy, Honoraria, Research Funding;Sanofi: Consultancy, Honoraria, Research Funding;GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria;BMS: Honoraria;Takeda: Honoraria;Beigene: Honoraria;Janssen: Honoraria.