key: cord-0824104-3xzgv370
authors: Khider, Lina; Gendron, Nicolas; Goudot, Guillaume; Chocron, Richard; Hauw‐Berlemont, Caroline; Cheng, Charles; Rivet, Nadia; Pere, Helene; Roffe, Ariel; Clerc, Sébastien; Lebeaux, David; Debuc, Benjamin; Veyer, David; Rance, Bastien; Gaussem, Pascale; Bertil, Sébastien; Badoual, Cécile; Juvin, Philippe; Planquette, Benjamin; Messas, Emmanuel; Sanchez, Olivier; Hulot, Jean‐Sébastien; Diehl, Jean‐Luc; Mirault, Tristan; Smadja, David M.
title: Curative anticoagulation prevents endothelial lesion in COVID‐19 patients
date: 2020-06-18
journal: J Thromb Haemost
DOI: 10.1111/jth.14968
sha: f55ac1232a262ed31f1827303491ac018fbd00e0
doc_id: 824104
cord_uid: 3xzgv370

BACKGROUND: Coronavirus disease‐2019 (COVID‐19) has been associated with cardiovascular complications and coagulation disorders. OBJECTIVES: To explore the coagulopathy and endothelial dysfunction in COVID‐19 patients. METHODS: Study analyzed clinical and biological profiles of patients with suspected COVID‐19 infection at admission, including hemostasis tests and quantification of circulating endothelial cells (CECs). RESULTS: Among 96 consecutive COVID‐19‐suspected patients fulfilling criteria for hospitalization, 66 were tested positive for SARS‐CoV‐2. COVID‐19 positive patients were more likely to present with fever (p=0.02), cough (p=0.03) and pneumonia at CT‐scan (p=0.002) at admission. Prevalence of D‐dimer >500 ng/mL was higher in COVID‐19 positive patients (74.2% vs. 43.3%; p=0.007). No sign of disseminated intravascular coagulation were identified. Adding D‐dimers >500 ng/mL to gender and pneumonia at CT scan in ROC curve analysis significantly increased AUC for COVID‐19 diagnosis. COVID‐19 positive patients had significantly more CECs at admission (p=0.008) than COVID‐19 negative ones. COVID‐19 positive patients treated with curative anticoagulant prior to admission had less CECs (p=0.02) than those without. Interestingly, patients treated with curative anticoagulation and ACEi or ARBs had even lesser CECs (p=0.007). CONCLUSION: Curative anticoagulation could prevent COVID‐19‐associated coagulopathy and endothelial lesion.

Authors contributions: 2 DMS, TM and JLD interpreted data, conceived and supervised the study. LK and NG interpreted 3 data and drafted the manuscript. RC analyzed the data and supervised statistical analysis. LK, NG, 4 GG, RC and BD analyzed the data and reviewed all patients' characteristics. All authors' 5 interpreted data, drafted and revised the manuscript, and approved the final version. concern. Compared to SARS-CoV which caused a SARS epidemic in 2003, SARS-CoV-2 has a 8 higher transmission capacity with a higher mortality (2). With a rapid increase in confirmed cases, 9 there is an unmet need of prevention and therapeutic strategies of COVID-19. Although COVID-10 19 clinical manifestations are dominated by respiratory symptoms, some patients have severe 11 cardiovascular damages (3, 4) and kidney disease participating to a multiple organ failure. Patients 12 with cardiovascular comorbidities may also be at higher risk of death (4). For example, COVID-19 13 patients with hypertension have an increased mortality and morbidity with hazard ratio ranging 14 from 1.7 to 3.05, depending of studies (3, 5). Based on this, raising concerns emerged regarding 15 angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARBs). 16 Indeed, angiotensin-converting enzyme 2 (ACE2) has been shown to be a co-receptor for viral 17 entry for SARS-CoV-2, and it has been demonstrated that ACEi and ARBs could enhance ACE2 18 expression (6) that may therefore jeopardize patient susceptibility to viral host cell entry and 19 dissemination.

During Chinese epidemic, coagulopathy has been reported in severe COVID-19 patients. D-21 dimer levels above 1000 ng/mL were an independent risk factor of in-hospital death (5) .

Coagulopathy was also found in fatal cases of COVID-19 patients, including a significant higher 23 proportion of patients with D-dimers above 500 ng/mL and prolonged prothrombin time (PT) in 24 non-survivors (7). This study was replicated in a second Chinese population where D-dimers 25 were still associated with in-hospital mortality (8). The hypothesis of microthrombi in kidney was 26 also suggested in COVID-19 patients since high creatinine level was correlated with D-dimers 27 above 500 ng/mL (9) . Finally, endothelial dysfunction might also play a role in the incidence of 28 severe respiratory symptoms and viral systemic dissemination. Indeed, SARS-CoV-2 receptor 29 ACE2 is strongly expressed on endothelial cells (10). We hypothesized that endothelial cell 30 infection may induce endothelium damage and dysfunction/activation that triggers coagulation 31 activation (11). Circulating endothelial cells (CECs) are considered as relevant markers of Accepted Article 1 endothelial lesion or dysfunction (12) and were used to explore the potential vascular dysfunction 2 in COVID-19 patients.

The aim of our study was to identify biological markers related to COVID-19 diagnosis and 4 severity. We also aimed at better characterizing subpopulations of patients at risk of coagulopathy 5 and/or endothelial dysfunction to target COVID-19 patients at risk for the worst outcomes. Table 1 . Suspicion of COVID-19 was defined by the presence of at least one of the followings: 9 fever, headache, myalgia, cough, dyspnea, rhinorrhea or digestive symptoms. All COVID-19-10 suspected patients had a clinical evaluation, blood test, CT-scan and were tested for SARS-CoV-2 11 infection by nasopharyngeal swabs before they were transfer to dedicated hospitalization units: Peripheral venous blood samples were collected on EDTA after having always discarded the first 7 milliliter of blood to avoid presence of endothelial cells dislodged by puncture. CECs were 8 isolated by immunomagnetic separation with mAb CD146-coated beads and staining with the 9 fluorescent probe acridin orange as previously described (12, 14-16). (p=0.03) and interstitial pneumonia at CT-scan (p=0.002). In terms of biological features (Table   10 3) COVID-19 positive patients had a significantly lower white blood cell count, including 11 neutrophil count (respectively p=0.008 and 0.02). Regarding hemostasis, the proportion of 12 COVID-19 positive patients with D-dimers above 500 ng/mL was significantly higher (74.2% vs. CECs were quantified as markers of endothelial lesion, using the reference method (14) as detailed 3 in the method section (12). Using this assay, the upper limit of normal range at 10 CECs per mL of 4 whole blood was previously determined and confirmed in several studies, including ours (12, 19-5 21). Among COVID-19 positive patients, 64% were above this threshold, suggesting a SARS- The originality of this study was to evidence an endothelial lesion during SARS-CoV-2 infection, 3 as witnessed by increased levels of CECs. Second, we show that this endothelial damage is 4 thwarted by curative anticoagulation. 5 Several Chinese studies found that increased D-dimer level correlated with in-hospital mortality 6 (5, 7, 8), suggesting a COVID-19-associated DIC (7, 8, 22) . However, in our population, no overt 7 DIC was diagnosed at admission. Indeed, patients had no significant thrombocytopenia, a normal 8 PT ratio and high fibrinogen levels. This was confirmed by a low level of fibrin monomers, which prognosis in severe COVID-19 patients meeting sepsis-induced coagulopathy criteria (28). 28 We further hypothesized that COVID-19-induced coagulopathy could be a consequence of 29 endothelial injury, based on the rationale that SARS-CoV-2 has an endothelial tropism linked to 30 ACE2 expression. Moreover, recently an endotheliitis has been described in SARS-CoV-2 31 infection and could be at the origin of impaired microcirculatory function affecting particularly the 32 lungs and kidneys (29). Thus, we explored CECs as a recognized non-invasive marker for Accepted Article 1 endothelial lesion, as demonstrated in acute cardiovascular conditions such as acute coronary 2 syndrome (30) and pulmonary arterial hypertension (12, 15, 19) . In agreement with the consensus 3 protocol from ISTH, we used the reference method for CECs quantification and the threshold of 4 10 CECs per mL as upper normal value (14). We found that over 60% of COVID-19 positive 5 patients were above this threshold. Interestingly, patients enrolled while they were treated with 6 curative anticoagulation had a significantly lower level of CECs, especially in the hypertensive 7 population treated with ACEi or ARBs. Increased mortality and/or morbidity of COVID-19 in 8 patients with hypertension has been described in China (3). One of the most important concerns is 9 the association between hypertension and treatment with ACEi or ARBs. Indeed, because ACE2 is 10 a receptor for viral entry of SARS-CoV-2, a link between ACEi or ARBs was considered. ACEi or 11 ARBs were described to increase ACE2 expression in the heart, brain, and even in urine after 12 treatment (6, 31). However, main scientific societies of cardiology and more specifically of Our study has several limitations. First, we are aware that false COVID-19 negative patients may 2 exist in our study population due to the imperfect sensitivity of the diagnostic test currently used 3 (35). Second, the study population size was necessarily small given the emergency of 4 understanding COVID-19; indeed we decided to investigate the endothelial dysfunction associated 5 to COVID-19 in order to consider new therapeutic alternatives. In addition, due to this small 6 population, our results concerning anticoagulant treatment and ACEi or ARBs therapy generated 7 hypothesis that need to be validated in larger cohorts. 

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This article is protected by copyright. All rights reserved CRP for C-reactive protein; Hs-TnI for high-sensitive troponin I. IQR for interquartile range. PT for thromboplastin time

CECs per mL, median

This article is protected by copyright. All rights reserved Accepted Article