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hypoxaemia (low oxygen levels in the blood) and dyspnoea (shortness of breath), Endothelial dysfunction and potentially evolving towards ARDS14. This stage of the disease is characterized by high immunothrombosis as key levels of pro-inflammatory cytokines, such as interleukin-6 (IL-6), IL-1β, IL-18 and pathogenic mechanisms in COVID-19 granulocyte–macrophage colony-stimulating factor (GM–CSF)2,15–17. This event has been 18,19 Aldo Bonaventura , Alessandra Vecchié, Lorenzo Dagna, Kimberly Martinod , referred to as a cytokine storm , although recent evidence suggests that cytokine Dave L. Dixon, Benjamin W. Van Tassell, Francesco Dentali, levels in patients with COVID-19 are Fabrizio Montecucco , Steffen Massberg , Marcel Levi and Antonio Abbate lower than those reported in patients with Abstract | Coronavirus disease 2019 (COVID-19) is a clinical syndrome caused by non-COVID-19 ARDS, sepsis, influenza virus infection and chimeric antigen infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). receptor (CAR) T cell-induced cytokine Patients with severe disease show hyperactivation of the immune system, which release syndrome20,21. SARS-CoV-2 has been can affect multiple organs besides the lungs. Here, we propose that SARS-CoV-2 shown to trigger the IL-1/IL-6 pathway to infection induces a process known as immunothrombosis, in which activated a larger extent than other coronaviruses22. neutrophils and monocytes interact with platelets and the coagulation cascade, Through the analysis of lungs of patients leading to intravascular clot formation in small and larger vessels. Microthrombotic who died from COVID-19-related ARDS, SARS-CoV-2 was found to activate the complications may contribute to acute respiratory distress syndrome (ARDS) and NLRP3 inflammasome23,24. In vitro studies also other organ dysfunctions. Therapeutic strategies aimed at reducing showed NLRP3 inflammasome activation immunothrombosis may therefore be useful. Several antithrombotic and by SARS-CoV-2 infection of primary immunomodulating drugs have been proposed as candidates to treat patients with human monocytes. In addition, NLRP3 SARS-CoV-2 infection. The growing understanding of SARS-CoV-2 infection inflammasome products, such as IL-1β and IL-18, are increased in patients with severe pathogenesis and how it contributes to critical illness and its complications may COVID-19 and positively correlated with help to improve risk stratification and develop targeted therapies to reduce the adverse clinical outcomes24. These findings acute and long-term consequences of this disease. support a potential relationship between SARS-CoV-2 infection and inflammatory Coronavirus disease 2019 (COVID-19) systems cooperate to block pathogens and features, such as the dysregulated cytokine is caused by the severe acute respiratory limit their spread10. In this Perspective, we response. syndrome coronavirus 2 (SARS-CoV-2) and describe how SARS-CoV-2 might induce Increased levels of ferritin have also can present with a range of manifestations, a prothrombotic state and discuss how been observed among patients with including pneumonia and acute respiratory currently available and developing therapies COVID-19, similar to what is observed distress syndrome (ARDS)1. Although may address COVID-19-associated in macrophage activation syndrome16,19,25. many individuals develop asymptomatic or immunothrombosis. Even though ferritin levels observed in paucisymptomatic SARS-CoV-2 infection, macrophage activation syndrome are those with severe-to-critical disease show Immunothrombosis in COVID-19 markedly higher than those currently a dysregulated hyperactivation of the COVID-19-associated hyperinflammation. observed in COVID-19 (≥10,000– immune system that can cause an abnormal The clinical spectrum of COVID-19 ranges 100,000 ng ml–1 versus 500–3,000 ng ml–1), cytokine immune response and is likely to from asymptomatic or paucisymptomatic ferritin levels remain predictive of poor be responsible for most of the organ damage (in up to 45% of individuals) to severe outcomes in COVID-19 (refs26,27). occurring in the late phase of the disease2. disease requiring admission of patients to With regard to inflammatory cells, large It is now apparent that SARS-CoV-2 the intensive care unit (ICU)11. Generally, numbers of CD68+ macrophages were infection induces a prothrombotic 3–10% of subjects requires hospitalization, observed in the alveoli of patients who died state manifesting especially with with up to 20% of them experiencing severe from COVID-19-associated pneumonia, microthrombosis3,4, as observed in patients disease and a high mortality rate12,13. The and SARS-CoV-2 proteins were detected in with COVID-19 and in post-mortem initial phase of COVID-19 is characterized CD68+ alveolar macrophages28,29. Analysis samples from individuals who died after by damage directly driven by the virus. of the bronchoalveolar fluid of patients SARS-CoV-2 infection5–9. This thrombotic After approximately 7–10 days of symptoms with severe or critical COVID-19 showed state is reminiscent of a process known (including fever, cough, fatigue, anorexia a higher percentage of macrophages and as immunothrombosis (Box 1; Fig. 1), and myalgias), a subgroup of patients neutrophils and a lower proportion of in which the immune and coagulation progresses to severe disease and develops myeloid dendritic cells, plasmacytoid

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dendritic cells and T cells than that of These features are more consistent with proposed the occurrence of two or more patients with moderate disease30. They a state of hypercoagulability coupled with a of the following four criteria in cases of also had higher levels of pro-inflammatory severe inflammatory state rather than with proven COVID-19: a decrease in platelet cytokines (such as IL-6, IL-8 and IL-1β) and classical disseminated intravascular coagulation count (<150 × 109 l–1); an increase in D-dimer chemokines (such as CCL2, CCL3, CCL4 (DIC)36. Indeed, DIC has been reported levels (more than two times the upper and CCL7) in the bronchoalveolar fluid30. in a few patients, usually in the advanced limit of normal); prothrombin time >1 s or These data suggest that lung macrophages stages of the disease33,37,38. Recently, low international normalized ratio >1.2; and the contribute to inflammatory responses in antithrombin levels were linked to poor presence of thrombosis (macrothrombosis SARS-CoV-2 infection, favouring excessive outcomes in COVID-19, especially and/or microthrombosis)45. The risk of inflammation in severe pneumonia. Similar among patients with obesity39. Hence, COVID-19-associated coagulopathy is to other respiratory infections, leukocyte it is possible that antithrombin activity defined by one of the aforementioned four recruitment to the lungs is finely tuned changes as SARS-CoV-2 infection worsens criteria and one of the following ones: by specific molecules31, the uncontrolled and a putative mechanism might be the increased fibrinogen levels; increased activity of which may result in complications cytokine-driven imbalance in endogenous VWF levels (more than two times the observed in patients with COVID-19 in both anticoagulant levels40. In macrophage upper normal limit); or the presence of the lungs and other organs5,32. activation syndrome, liver function lupus anticoagulant and/or high-titre impairment is a typical feature and may antiphospholipid antibodies45. Importantly, COVID-19-associated coagulopathy. contribute to coagulopathy, yet liver COVID-19-associated coagulopathy and An important pathophysiological feature impairment in COVID-19 is usually mild risk of COVID-19-associated coagulopathy of COVID-19 is the development of a and transient, without overt acute liver may further progress and/or predispose to prothrombotic state. The occurrence of injury41,42. DIC as COVID-19 severity increases. a COVID-19-specific coagulopathy is Apart from a state of hypercoagulability suggested by elevated levels of fibrinogen, in the lungs of patients with ARDS, a COVID-19-associated endothelial von Willebrand factor (VWF) and the fibrin hypofibrinolytic state in the alveolar space dysfunction. Although COVID-19 should degradation product D-dimer in the blood, was observed in COVID-19-related ARDS be regarded as a systemic disease, it acutely whereas patients generally show minor due to increased levels of fibrinolytic causes a lung-centred injury primarily or no changes in prothrombin time inhibitors43. Accordingly, high levels of affecting the vascular endothelium. COVID- (a measure of time to clot), activated plasminogen activator inhibitor 1 (PAI1) 19-associated pneumonia is characterized by partial thromboplastin time (coagulation were described in patients with SARS-CoV44. infiltration of the lungs with macrophages time), antithrombin levels, activated In order to properly identify COVID- and neutrophils that cause diffuse protein C levels and platelet count33–36. 19-associated coagulopathy, Iba et al. lung alveolar damage, the histological equivalent of ARDS46–48. Similar findings were previously reported in patients with Box 1 | Immunothrombosis: a conserved mechanism of host defence SARS-CoV49. However, the striking finding The term immunothrombosis was originally described by Engelmann and Massberg to refer to an in COVID-19 compared with SARS-CoV intrinsic effector pathway of innate immunity triggered by pathogens and injured cells to reduce is the more extensive viral infection within the spread and survival of the invading pathogens10,157. By contrast, the term ‘thromboinflammation’ the lungs, which results in more diffuse refers to a process in which inflammation and thrombosis coexist within microvessels in response to inflammation involving the pulmonary harmful stimuli (pathogens, injured cells or other irritants)158. Immunothrombosis is mainly triggered vessels50. This extensive infection reflects by neutrophils and monocytes and is supported by the formation of microthrombi in small vessels, in the particular tropism of SARS-CoV-2 for which endothelial cells exposed to microorganisms adopt a pro-adhesive phenotype159. angiotensin-converting enzyme 2 (ACE2) — During immunothrombosis, neutrophils and monocytes release tissue factor and extracellular nucleosomes and degrade endogenous anticoagulants, thereby facilitating activation of coagulation the cellular receptor for SARS-CoV-2 — 51 induced by inflammation160. Importantly, tissue factor decorates neutrophil extracellular traps expressed by type II pneumocytes , which (NETs), as found in sepsis161 and, more recently, in coronavirus disease 2019 (COVID-19)-related are anatomically close to the lung vascular immunothrombosis86. NETosis by neutrophils also enhances the activity of the coagulation system network and are typically described to be by increasing fibrin deposition162,163. Histones present in NETs, especially histones H3 and H4, can hyperplasic in samples from patients with dose-dependently enhance thrombin generation not only by reducing thrombomodulin-mediated COVID-19 (refs52–54). This hyperplasia of protein C activation164 but also by directly activating platelets141. Finally, NETs can bind plasma endothelial cells is likely to be caused by proteins needed for platelet adhesion and thrombus development, such as fibronectin and von lung tissue ischaemia due to congestion of 141 Willebrand factor (VWF) . In turn, platelets participate in this process in several ways: they increase small vessels by inflammatory cells (that the accumulation of immune cells that express tissue factor10,165; they bind directly to neutrophils 162,166,167 is, immunothrombosis) and thrombosis in and NETs ; they release damage-associated molecular patterns that promote tissue factor 55 expression within thrombi168; and they directly bind to microorganisms for presentation to innate large vessels . immune cells169,170. Endothelial dysfunction was suggested Microorganisms have evolved mechanisms to escape immunothrombosis, such as the expression to be an important pathophysiological event of streptokinase to dissolve fibrin171 or nucleases to degrade NETs172,173. Some bacteria use in infections by other coronaviruses, which immunothrombosis to avoid their killing, such as Staphylococcus aureus, whose staphylocoagulase and can directly infect endothelial cells56–59. VWF-binding protein trigger fibrin generation to form a biofilm and an abscess pseudocapsule174,175. SARS-CoV-2 directly infects vascular In sum, immunothrombosis can be considered a beneficial mechanism of intravascular immunity. endothelial cells and leads to cellular However, when immunothrombosis is uncontrolled, it causes dysregulated activation of the damage and apoptosis, thus decreasing coagulation cascade, leading to microthrombus formation and inflammation, which in turn the antithrombotic activity of the normal enhance each other and may, ultimately, develop into thrombosis (thromboinflammation) and endothelium7,55. Alveolar damage, vessel wall disseminated intravascular coagulation. oedema, hyaline thrombi, microhaemorrhage

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Pathogen P-selectin Endothelial cell

PRR Inactive TF Neutrophil PSGL1 a Activation releasing NETs of extrinsic coagulation pathway Inactivation Active TF of natural anticoagulants Pathogen e TFPA NLRP3 TM inflammasome Fibrin net C3a C5a C3a Microvesicles Active TF Blood Monocyte b VWF H3 NE f vessel Factor VWF c XII H3 MPO g H3 IL-1β, Activation of H4 IL-18 contact-dependent H4 coagulation pathway PEVs Platelets Factor VWF XIIa d DNA

Pathogen isolation and killing Microvascular thrombus formation

Fig. 1 | Immunothrombosis is important in promoting immune defence. coagulation through the release of polyphosphates and, along with Following recognition of a pathogen through pattern recognition receptors endothelial cells, may promote fibrin generation. Platelets can also be acti- (PRRs), monocytes and monocyte-derived microvesicles present activated vated by C3a and C5a (path f). Activated platelets release large amounts of tissue factor (TF) on their surfaces and release it at sites of pathogen locali- pro-inflammatory cytokines in platelet extracellular vesicles (PEVs) (path g). zation, thus activating the extrinsic pathway of coagulation (path a). Through this mechanism, pathogens such as severe acute respiratory syn- Pathogens also stimulate the NLRP3 inflammasome in monocytes and/or drome coronavirus 2 (SARS-CoV-2) become trapped within the fibrin-based macrophages, leading to the release of pro-inflammatory cytokines, such NETs and killed. The immunothrombotic process allows pathogen killing as interleukin-1β (IL-1β) and IL-18 (path b). Neutrophils are recruited and to be restricted to the intravascular compartment, thus limiting injury to contribute to this process through the release of neutrophil extracellular organs. Although it is clear that immunothrombosis participates in traps (NETs), which directly activate factor XII and, thus, the contact- SARS-CoV-2 pathogenesis, the exact mechanisms are still under investiga- dependent pathway of coagulation (path c). NETs also bind von Willebrand tion. These may include the following: direct injury of endothelial cells by factor (VWF) and help to recruit platelets (path d). Histones, in particular the virus and consequent activation of the coagulation cascade; infiltration H3 and H4, trigger activation of platelets. In addition, neutrophil elastase of neutrophils that lead to NET formation; induction of hypoxaemia causing (NE) and myeloperoxidase (MPO) in NETs cleave and inactivate natural anti- upregulation of TF expression by hypoxia-inducible transcription factors coagulants (tissue factor pathway inhibitor (TFPA) and thrombomodulin and formation of clots; activation of complement that promotes coagula- (TM)) (path e). Finally, NETs can externalize and bind TF, promoting activa- tion and recruits and activates platelets, monocytes and neutrophils, tion of the extrinsic pathway of coagulation. Platelets support the immuno- thus triggering TF expression; and an abnormal increase in the levels of thrombotic process by activating the contact-dependent pathway of pro-inflammatory cytokines causing direct cell damage. and diffuse thrombosis of peripheral small (ADAMTS13), a metalloproteinase that in patients admitted to the ICU than in vessels have emerged as key features of regulates the size of VWF multimers63. patients not in the ICU, whereas increased COVID-19 contributing to respiratory Endothelial cell perturbation along with levels of thrombomodulin (a specific failure6,8,46 (Fig. 2). the release of large VWF multimers and marker of endothelial activation generally Endothelial cells of lung blood vessels relative insufficient VWF cleavage owing to released during endothelial cell injury) can be activated by the high levels of ADAMTS13 consumption may account for were associated with increased mortality pro-inflammatory cytokines (IL-1, IL-6 increased platelet–vessel wall interactions risk35. Higher numbers of circulating and TNF) and ferritin in severe COVID-19 causing thrombotic microangiopathy. Indeed, endothelial cells were described in patients (ref.60). Moreover, increased levels of the a relative deficiency in ADAMTS13 activity with COVID-19, especially among those endothelial adhesion protein VWF were was previously described in inflammatory admitted to the ICU, and the presence reported both in patients admitted to the conditions with high levels of IL-6, as found of circulating endothelial cells positively ICU and in non-critically ill patients34,35,61,62, in patients with SARS-CoV-2 infection64. correlated with platelet and lymphocyte and this increase seems to be associated Other markers of endothelial cell injury have counts and with the classical endothelial with a mild reduction in the activity of a been recently investigated. Higher levels of marker soluble vascular cell adhesion disintegrin and metalloproteinase with a soluble P-selectin (a marker of endothelial molecule 1 (sVCAM1)65. This was recently thrombospondin type 1 motif, member 13 and platelet activation) were observed confirmed by another report from Italy,

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showing that patients with COVID-19 had of endothelial dysfunction in COVID-19 endothelial dysfunction in pulmonary increased circulating endothelial cells and (ref.66). microvessels acts as a trigger for higher plasma levels of soluble intercellular Considering the important role of the immunothrombosis, resulting in the adhesion molecule 1 (sICAM1) and endothelium in regulating haemostasis, coagulopathy observed in patients with sVCAM1, further supporting the concept fibrinolysis and vessel wall permeability, COVID-19 (refs27,47,67,68). In addition, endothelial dysfunction is recognized A as a risk factor of microvascular a b dysfunction through a shift towards vasoconstriction, by promoting ischaemia, inflammation and a procoagulant state69. In addition, the inflammatory environment triggers the expression of activated tissue factor on endothelial cells, macrophages and neutrophils, which amplifies activation of the coagulation cascade within the lungs60. These events

200 μm 100 μm fuel a positive immunothrombotic feedback loop in which hyperinflammation c d triggers the hypercoagulant state, with thrombosis occurring within pulmonary microvessels. Lastly, platelet activation within the microvasculature is increased in patients with severe COVID-19 and is associated with poor outcomes70,71. Platelet– vessel wall interactions, mediated by surface receptors (integrins and selectins) and adhesive proteins (VWF and fibrinogen), 80 μm 200 μm impair vessel wall integrity or increase endothelial injury, ultimately leading to B microvascular obstruction72. Moreover, a b platelets were found to be hyperactivated in patients with COVID-19 and to be a source of procoagulant extracellular vesicles73 and polyphosphates, which can exacerbate the thromboinflammatory cascade via neutrophils74. Of importance, activated platelets are critically involved in neutrophil extracellular trap (NET)

900 μm 600 μm formation71, which is an essential element of immunothrombosis. All of c d e these elements support the hypothesis that endothelial dysfunction, or endotheliopathy35,75, and platelet activation are key features of COVID-19-associated coagulopathy and may mediate the damage leading to severe disease. 60 μm COVID-19-associated ARDS. COVID- Fig. 2 | Seminal pathology features of patients with COVID-19. A | Hyaline membrane formation 19-related ARDS is considered atypical, is a typical finding in patients with alveolar damage irrespective of being ventilated or not (blue arrows with its main characteristic being a in panels a and b). Organizing fibrosis (blue arrowhead) and fibrin thrombi in small blood vessels (black dissociation between hypoxaemia arrows) with oedema, along with extensive haemorrhage (haematoxylin and eosin staining) and hya- severity and relatively good respiratory line membranes (blue arrows) (panel c). The bronchial respiratory mucosa is almost entirely intact and mechanics76,77. From a clinical standpoint, no squamous metaplasia is evident (panel d), different to that observed in patients with severe acute two phenotypes are described67,78. Patients | respiratory syndrome coronavirus 2 (SARS-CoV-2). B Two thrombi, one in a small pulmonary artery with ‘type L’ have high lung compliance, (blue arrow) and one in a pulmonary venule (blue arrowhead), in the haematoxylin and eosin staining lower lung weight as estimated by computed (panel a) and in the CD61 immunostaining for platelets within thrombi (panel b). Megakaryocytes within small vessels and alveolar capillaries (panel c, blue arrow). CD61 immunostaining of a fibrin- and tomography scan and poor response to platelet-rich thrombus in a small vessel (panel d), with a megakaryocyte stained below (blue arrow). positive end-expiratory pressure. Patients Small, perivascular aggregates of lymphocytes (panel e). COVID-19, coronavirus disease 2019. Images with ‘type H’ are those with a deteriorating reprinted from The Lancet, 8, Fox, S. E. et al., Pulmonary and cardiac pathology in African American disease or suboptimally treated disease patients with COVID-19: an autopsy series from New Orleans, 681–686, Copyright (2020), with that present with areas of accumulation permission from Elsevier (ref.6). of inflammatory exudate in the alveoli as

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Box 2 | The role of complement in sepsis strongly stimulate the production of NETs from healthy neutrophils71,80,85. In addition, The complement system is an important component of the innate immune system and activates neutrophils isolated from patients with following infections. After complement activation (through the classic, alternative or lectin COVID-19 showed elevated NET release pathways), the common pathway is responsible for the production of C3a and C5a, then stimulating at baseline similar to phorbol myristate formation of the C5b–9 membrane attack complex, which causes the lysis of pathogen cells176. In a mouse model of intranasal infection with severe acute respiratory syndrome coronavirus acetate (PMA)-stimulated neutrophils (SARS-CoV), early activation of the complement system, especially complement component C3, from healthy donors, indicating that the caused acute respiratory distress syndrome (ARDS)177. However, C3-deficient mice experienced COVID-19 plasma environment promotes reduced respiratory dysfunction, limited neutrophil and monocyte lung infiltration, and showed NET formation80. The virus itself is also lower levels of cytokines both in the lungs and the blood177. Excessive activation of complement in reported to directly activate neutrophils the advanced phases of sepsis may therefore cause tissue injury contributing to organ failure. towards NETosis, promoting epithelial cell The complement and coagulation pathways are closely linked178,179. In particular, complement end death89. NETs may provide a scaffold for the products can trigger the release of procoagulant products and reduce the production of natural deposition of C3, properdin and factor B, 178 anticoagulants . Additionally, the binding of C1q to its receptor on platelets triggers the expression thus directly participating in complement of integrins and P-selectin180, whereas C3 stimulates platelet activation and aggregation. The activation90. This mechanism may fuel activation of C5 during the inflammatory response is responsible for the expression of tissue factor on leukocytes and endothelial cells181,182. Complement may indirectly induce procoagulant changes; an amplifying feedback loop promoting for example, activated C3 and C5 contribute to cytokine release soon after the innate immune further NET formation and platelet and 91 response starts, particularly increasing the concentrations of tumour necrosis factor (TNF) and endothelial cell activation . The observation interleukin-6 (IL-6). of antiphospholipid antibodies, which can directly stimulate NETosis92, in patients with COVID-19, as well as in other infections93, observed through computed tomography SARS-CoV-2 infection80,85. Compared suggests that SARS-CoV-2 infection might scan, low lung compliance, greater lung with controls, patients with COVID-19 synergize with antiphospholipid antibodies weight and good response to positive have higher levels of serum85 or plasma80 to promote the immunothrombotic process. end-expiratory pressure. This clinical picture myeloperoxidase (MPO)–DNA complexes, Indeed, higher titres of antiphospholipid couples with endothelial damage, breaking which are biomarkers of circulating antibodies were associated with increased down pulmonary vasoregulation, stimulating NET fragments, and further increases activity of neutrophils and platelets and more ventilation–perfusion mismatch — which is were found in those patients requiring severe respiratory disease94. mainly responsible for hypoxaemia — and invasive mechanical ventilation. Whether NETs are direct contributors supporting immunothrombosis. In this MPO–DNA complexes and thrombin– to the dysregulated cytokine response is respect, the events leading to atypical antithrombin complexes were positively still unclear. Indeed, NETs participate in ARDS can be summarized by the term correlated in patients with COVID-19, and acute lung injury by inducing macrophage ‘microvascular COVID-19 lung vessels the anticoagulant dabigatran or complement release of IL-1β, which in turn can obstructive thromboinflammatory targeting inhibited NETosis86, further increase NET formation95,96. In addition, syndrome’ (MicroCLOTS)79. According to suggesting a central role for NETs in the most inflammatory mediators that are this proposed definition, ARDS is the result immunothrombotic milieu. MPO–DNA increased in patients with COVID-19 of a hyperimmune reaction of the host levels return to normal in convalescent are known to regulate neutrophil activity to SARS-CoV-2 that is both directly plasma, indicating that this is a transient through the expression of chemotactic mediated by the virus through cellular increase80. High-dimensional flow products16,97. These data suggest that injury and release of pro-inflammatory cytometric analysis of circulating neutrophils dysregulated cytokine release might be signals and indirectly mediated by revealed a highly activated phenotype in sustained by crosstalk between neutrophils complement activation (Box 2), leading patients with most severe COVID-19 (ref.71). and macrophages mediated by NETs, to leukocyte recruitment and increased Neutrophils can be found embedded in fibrin resulting in a deranged or exaggerated release of pro-inflammatory cytokines. clots and in close association with platelets immunothrombotic status. This results in a severe tissue injury with within microthrombi in the lungs80, heart vascular endothelial dysfunction and and kidneys of patients with COVID-19 COVID-19: a syndrome of deregulated alveolar cell damage, which may explain (ref.71). In severe disease, neutrophils adopted immunothrombosis? On the basis of the the progressive worsening of ventilation– the so-called low-density phenotype that aforementioned evidence, we propose that perfusion mismatch and pulmonary is more prone to spontaneously form exaggerated immunothrombosis, occurring microthrombosis79. NETs; this greater NETosis was linked for the most part within lung microvessels, to increased intravascular aggregation, drives the clinical manifestations of NETs in COVID-19-associated leading to microthrombosis and COVID-19 (ref.3), although systemic immunothrombosis. NETs are likely organ damage87. NETs have also been viraemia early in the disease course may to be important players in deregulated identified in the lung parenchyma and also be important98 (Fig. 1). The occurrence immunothrombosis in COVID-19 (ref.80). alveolar space in autopsy case reports9,88. of severe events, such as acute respiratory Indeed, ARDS can be considered as a Interestingly, neutrophils from healthy failure and ARDS, may be explained by a NETopathy 81; higher levels of NETs were donors showed increased adhesion to deregulated process of immunothrombosis described in the plasma and bronchoalveolar activated platelets in plasma from patients involving the pulmonary microcirculation. fluid of patients with transfusion-associated with COVID-19 compared with that from In support of this, there is increasing and pneumonia-related ARDS compared control patients, causing enhanced NET evidence that COVID-19 is a disease of with those without ARDS82–84. NETs are generation71. Indeed, both serum and the endothelium35 that results in elevated markers of disease severity in patients with plasma from patients with COVID-19 levels of PAI1 and VWF, increased platelet

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activation and a state of hypercoagulability, antithrombin-dependent factor Xa Multi-factorial Adaptive Platform Trial with consequent venous, arterial and inhibitor) in patients with COVID-19 was for Community-Acquired Pneumonia microvascular thrombosis. The exact factors reported in two papers, with contrasting (REMAP-CAP) (NCT02735707); the triggering this endotheliopathy have not results about its safety109,110. The same applies Accelerating COVID-19 Therapeutic yet been recognized, but potentially include to direct oral anticoagulants, the use of Interventions and Vaccines-4 (ACTIV-4) the virus itself (causing endothelial cell which in patients with COVID-19 has been trial (NCT04505774); and the injury that triggers the coagulation cascade little studied to date111,112. Several trials of Antithrombotic Therapy to Ameliorate involving fibrin and platelets47,99), immune heparin and other anticoagulant agents are Complications of COVID-19 (ATTACC) cell (neutrophil and/or macrophage and in progress: the Randomized, Embedded, trial (NCT04372589). These trials are platelet73,100,101) infiltration, hypoxaemia (that can induce hypoxia-inducible Glossary transcription factors that upregulate tissue factor expression102 and lead to Antithrombin Microthrombosis fibrin-based clot formation, thus supporting A glycoprotein produced by the liver that inactivates The formation of thrombi (clots) within small vessels, different enzymes of the coagulation cascade. which is typical of the immunothrombotic process. a thromboinflammatory feedback loop), Antithrombin activity is strongly increased by the Microthrombosis is often encountered in patients with complement-mediated damage or a surge in anticoagulant medication heparin, which enhances coronavirus disease 2019 (COVID-19). pro-inflammatory cytokine (such as IL-1β the binding of antithrombin to factor IIa (thrombin) and IL-6) release with direct cell damage. and factor Xa. NLRP3 inflammasome Macromolecular structure that is responsible for sensing Cytokine storm danger and triggering a local or systemic inflammatory Thrombosis management in COVID-19 A sudden surge in circulating levels of pro-inflammatory response. Following NLRP3 inflammasome activation, If we consider COVID-19 as a vascular cytokines, such as interleukin-1 (IL-1), IL-6, tumour large quantities of pro-inflammatory cytokines (in disease primarily involving the endothelium, necrosis factor (TNF) and interferon-γ (IFNγ). particular IL‑1β) are released for extracellular secretion. an ideal therapeutic approach would be both Clinically, this can result in hypotension, acute These cytokines are important in the inflammatory renal failure, impaired pulmonary function and response and responsible for symptoms such as fever. antithrombotic and anti-inflammatory. Here, even death. we describe some of the main approaches Neutrophil extracellular trap that are being explored to address these D-dimer (NET). Fibrillary structure consisting of DNA complexed with features of COVID-19 (Table 1). A small protein derived from fibrin degradation histones (H3 and H4) and proteins from neutrophil granules, that is found in the blood once a blood clot is especially neutrophil elastase (NE), myeloperoxidase degraded. The name comes from the presence (MPO) and cathepsin G. NETs act as a mesh that traps Antithrombotic therapies. As the of two D fragments of the fibrin protein united by microorganisms and exposes them to neutrophil-derived coagulation cascade is dysregulated in a cross-link. effector molecules. NETs also provide a scaffold for COVID-19, anticoagulation approaches thrombus formation and activation of coagulation. have been explored for the treatment, Disseminated intravascular coagulation (DIC). An acquired syndrome characterized by the Positive end-expiratory pressure particularly of patients with incident intravascular activation of coagulation without a Pressure within the lung alveoli above atmospheric venous thromboembolism (VTE) or precise localization in response to microvascular injury, pressure that is recorded at the end of expiration. Besides acute coronary syndrome due to plaque leading to organ dysfunction when sufficiently intrinsic positive end-expiratory pressure (caused by an rupture103. Indeed, a study reported that 40% severe. DIC involves large production of thrombin incomplete exhalation), extrinsic positive end-expiratory accompanied by platelet activation and consumption, pressure is provided by a ventilator during mechanical of patients hospitalized with COVID-19 altered fibrinolysis and microvascular thrombosis. ventilation and is meant to reduce end-expiratory alveolar 104 are at high risk of VTE and data from a It can occur in critically ill patients with overwhelming collapse, thus improving hypoxaemia or reducing large retrospective cohort of patients in the infection, fulminant sepsis, extensive tissue damage or ventilator-associated lung injury in patients with acute United States suggested that anticoagulation malignancy. respiratory distress syndrome (ARDS). may reduce mortality, especially among Haemostasis Thrombosis those with severe disease requiring invasive The combination of events that result in cessation of The formation of a thrombus (clot) inside blood vessels, 105 mechanical ventilation . Pharmacological bleeding. Haemostatic processes include constriction resulting in partial or complete vessel occlusion. VTE prophylaxis, including daily low of blood vessels, platelet aggregation and coagulation molecular weight heparin or twice-daily (clotting) of the blood. Thrombotic microangiopathy Pathology characterized by formation of thrombi in subcutaneous unfractionated heparin, may Hyaline thrombi capillaries owing to endothelial damage. In particular, also be of benefit to patients hospitalized Clots occurring in small vessels due to the formation decreased levels of the protease a disintegrin and with COVID-19 who are bedridden or need of hyaline membranes and surfactant dysfunction metalloproteinase with a thrombospondin type 1 motif, intensive care, or patients discharged from after an inflammatory injury. These formations are member 13 (ADAMTS13) lead to an accumulation of hospital but with relevant risk of VTE — the pathological equivalent of acute respiratory ultra-large von Willebrand factor (VWF) multimers, which distress syndrome (ARDS) occurring in patients promote platelet–vessel wall interactions, causing thrombus that is, individuals with reduced mobility, with severe-to-critical coronavirus disease 2019 formation. The clinical presentation comprises multiple pro-thrombotic comorbidities (active (COVID-19). organ dysfunctions, especially in the brain and kidneys. cancer or obesity), older age, a history of VTE and persistently elevated levels of Lung compliance Ventilation–perfusion mismatch 103,106,107 A measure of the ability of the lungs to stretch and Impairment of the ventilation to perfusion ratio, meaning D-dimer . expand. A low compliance refers to that of a stiff lung, that a part of the lung is adequately oxygenated but not Studies of heparin treatment of as occurs in fibrosis. A high compliance means a flexible perfused, or vice versa, caused by diseases such as COVID-19 have shown positive results103,106 lung, as seen in emphysema. pneumonia and pulmonary embolism. (Supplementary Table 1), and this is now one of a growing number of agents available to Macrophage activation syndrome Von Willebrand factor A syndrome in which a dysregulated activation (VWF). The main adhesive protein mediating interactions manage COVID-19 either as a prophylactic and proliferation of macrophages and T cells between platelets and the vessel wall, leading to platelet 108 or therapeutic regimen (Table 1). The leads to increased release of pro-inflammatory aggregation. Preformed multimers are released from use of fondaparinux (a selective indirect cytokines. Weibel–Palade bodies of activated endothelial cells.

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Table 1 | Putative therapeutic approaches for COVID-19 Therapeutic agent Target Outcome Clinical trial Refs Antithrombotic drugs Heparin Inactivation of thrombin and Controversial evidence on mortality – 105,152–154 activated factor X through an and need for mechanical ventilation antithrombin-dependent mechanism from observational studies Garadacimab Factor II blockade One study is ongoing NCT04409509 – Nafamostat Serine protease inhibitor of thrombin, Ongoing trials based on in vitro NCT04352400, NCT04418128 – mesylate plasmin and trypsin blockade of MERS-CoV infection Tissue-type Conversion of plasminogen into Benefit in a small study of patients NCT04357730 119 plasminogen plasmin, leading to breakdown of with severe COVID-19 activator cross-linked fibrin A phase IIa trial in patients with COVID-19-related ARDS is ongoing Dipyridamole Phosphodiesterase inhibitor In vitro suppression of SARS-CoV-2 NCT04391179 121,124,125 reversibly inhibiting platelet replication and improvement of aggregation; in addition, it potentiates lung pathology in a model of viral vascular-protective effects of pneumonia endothelium-derived nitric oxide In 14 patients, reported improvement Possible activity against in clinical status compared with positive-stranded RNA viruses and controls prevention of NET formation A trial is ongoing

Ticagrelor P2Y12-receptor antagonist and Ongoing trials NCT02735707 , NCT04518735 – attenuation of NET formation Anti-inflammatory drugs NE inhibitors NE inhibition blocks NET formation Sivelestat has been approved to treat – 131–133 and reduces SARS-CoV-2 spike protein ARDS in Japan and South Korea, but proteolytic activation findings are inconsistent No current indication is available for SARS-CoV-2-related ARDS DNase I Dissolution of NETs Dornase alpha has been tested in NCT04402944, NCT04355364, 138,139 two case studies in patients with NCT04432987 , NCT04359654, COVID-19-associated ARDS NCT04445285, NCT04402970 Several trials are ongoing Ruxolitinib JAK1/2 inhibitor In a small pilot study, clinical NCT04338958 142,155,156 improvement was observed after 1 week of treatment In a small RCT, ruxolitinib plus SOC did not significantly accelerate clinical improvement in patients with severe COVID-19 A trial is ongoing in Germany Eculizumab and C5 inhibitors blocking MAC formation Eculizumab showed good efficacy in NCT04288713, NCT04369469, 147,148 ravulizumab reducing inflammatory biomarkers NCT04390464 In ICU patients, eculizumab improved 15-day survival and hypoxia compared with SOC Trials with eculizumab and ravulizumab are ongoing AMY-101 Inhibition of C3 cleavage by direct Limited experience is now available, EudraCT number 2020-001550- 149 binding with encouraging results 22, EudraCT number 2020- 004408-32, NCT04395456 Three phase II clinical trials in patients with COVID-19-associated ARDS are ongoing ARDS, acute respiratory distress syndrome; COVID-19, coronavirus disease 2019; ICU, intensive care unit, JAK, Janus kinase; MAC, membrane attack complex; MERS-CoV, Middle East respiratory syndrome coronavirus; NE, neutrophil elastase; NET, neutrophil extracellular trap; RCT, randomized controlled trial; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SOC, standard of care. testing whether the use of full-dose versus However, enrolment of critically ill patients therapy may depend on the time of low-dose unfractionated heparin or low has been temporarily stopped owing to administration; if given too late, thrombi in molecular weight heparin benefits patients futility and safety concerns for this subgroup the microcirculation and large vessels may with COVID-19 requiring ICU admission. of patients. Response to this anticoagulant already be too extensive.

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An alternative approach to Anti-inflammatory therapies. released NETs include JAK1/2 inhibition pharmacologically disrupt the coagulation Immunomodulatory therapies, such as with ruxolitinib142, dipyridamole124, cascade involves blockade of factor XII inhibitors of IL-6, IL-1, GM-CSF and Janus ticagrelor126 and neonatal NET-inhibitory (FXII), which is responsible for activation kinases (JAKs), aim to blunt the cytokine factor, an endogenous peptide produced of the contact pathway (Table 1). FXII dysregulation associated with severe in newborns that restricts the ability of blockade has been shown to protect from COVID-19 (refs15,127,128) (Table 1), but little is neutrophils to produce NETs143. Neonatal occlusive thrombosis in animal models known about their impact on the thrombotic NET-inhibitory factor was shown to prevent without impairing haemostasis113,114. Thus, complications of the disease. COVID-19 plasma-induced NET formation a FXII-blocking antibody (garadacimab) is Given that NETs have a direct role by healthy donor neutrophils in vitro, but currently being explored in a multicentre, in the immunothrombotic process in the mechanism of action remains to be double-blind, placebo-controlled phase II COVID-19, blocking NETs may help elucidated80. trial in patients with respiratory distress to improve prognosis (Table 1). Some Finally, evidence is accumulating on and COVID-19 (NCT04409509). Similarly, drugs targeting NETs already exist or are the role of complement activation in the a serine protease inhibitor of thrombin, under development, such as inhibitors pathogenesis and severity of SARS-CoV-2 plasmin and trypsin known as nafamostat of neutrophil elastase (NE), PAD4 (ref.144) as well as on complement-mediated mesylate (used in Japan for the treatment of and gasdermin D129. For example, NE thrombotic microangiopathy in patients pancreatitis and DIC and the prevention inhibitors can block NET formation, limit with COVID-19 (ref.145). Thus, therapies of clots during dialysis115) is being the toxic activities of NE itself on lung for thrombotic microangiopathy — investigated in clinical trials for COVID-19 connective tissue and reduce spike protein complement inhibitors — may be used in (NCT04352400, NCT04418128). This drug proteolytic activation that is responsible this setting. C3 blockade can inhibit the showed potential to block Middle East for SARS-CoV-2 binding to host cells130. production of both C3a and C5a, but also of respiratory syndrome coronavirus infection Sivelestat, an NE inhibitor, has been intrapulmonary C3a and the release of IL-6 in vitro116 (Table 1). approved to treat ARDS in Japan and South from alveolar macrophages146. Currently, The hypofibrinolytic state observed Korea, but findings are inconsistent as the two FDA-approved complement inhibitors in ARDS has been targeted by the use severity of lung injury is likely to represent are available, eculizumab and ravulizumab, of tissue-type plasminogen activator, the most important predictor for patient both of which bind to C5 and sterically which is responsible for the conversion outcome131–133. However, no evidence is block the cleavage of C5 to C5a, and thus of plasminogen into plasmin, leading to currently available in favour of using NE membrane attack complex formation. the breakdown of cross-linked fibrin117,118. inhibitors for COVID-19-induced ARDS. Four patients with COVID-19 receiving Indeed, tissue-type plasminogen activator NETs can be degraded by recombinant eculizumab intravenously showed a prompt infusion showed benefit in three patients with DNase I (dornase alpha), which is reduction in C-reactive protein levels and severe COVID-19 (ref.119), and a phase IIa currently given by inhalation to patients an improvement of the lesions observed trial in patients with COVID-19-related with cystic fibrosis to target NETs in the through chest computed tomography scan147. ARDS is currently ongoing (NCT04357730). airways134. Dornase alpha may help to Among 35 patients admitted to the ICU with The antiplatelet agent dipyridamole may dissolve respiratory secretions in patients COVID-19, eculizumab treatment improved have therapeutic potential120 (Table 1). This with COVID-19 (ref.135), reducing the risk 15-day survival and hypoxia compared with agent, apart from its antiplatelet function, for secondary infections and improving patients treated with standard care alone148. was shown to provide broad-spectrum ventilation, and may also reduce the risk Trials with eculizumab (NCT04288713) antiviral activity (especially against of progression towards ARDS, as shown in and ravulizumab (NCT04369469, positive-stranded RNA viruses), suppress animal models136,137. Two case studies have NCT04390464) are currently ongoing. inflammation and favour mucosal healing, shown that administration of dornase alpha In a report from Italy, a patient with and prevent acute injury and fibrosis in the is well tolerated in patients with COVID-19- severe COVID-19-related ARDS was lungs, heart and kidney121–123. Dipyridamole associated ARDS138,139, and several trials are safely and successfully treated with the treatment in patients with COVID-19 may underway to determine the efficacy of this compstatin-based complement C3 inhibitor also have the added benefit of preventing approach (NCT04402944, NCT04355364, AMY-101 (ref.149). A recent study comparing NETosis by promoting 3′,5′-cyclic adenosine NCT04432987, NCT04359654, two different strategies targeting C3 and monophosphate (cAMP) generation in NCT04445285, NCT04402970). Nebulizer C5 has shown a general beneficial impact neutrophils, as was shown in the context administration of DNase provides direct in patients with COVID-19, although C3 of antiphospholipid syndrome124. Liu et al. access to NETs within the alveolar space, but inhibition led to a broader therapeutic demonstrated that dipyridamole can there may be a barrier to reaching systemic effect blocking cytokine dysregulation and suppress SARS-CoV-2 replication in vitro efficacy owing to the high reported levels of NET generation, thereby having a positive and improve lung pathology in an animal platelet factor 4 (PF4) in severe COVID-19 impact on immunothrombosis-driven model of viral pneumonia through a type I (ref.80). PF4 binding to NETs leads to their disease pathways150. The C3 inhibitor interferon response125. This drug was tested compaction and decreased susceptibility AMY-101 is currently being evaluated in in 14 patients and led to an improvement in to DNase degradation140. Therefore, DNase three phase II clinical trials in patients with clinical status compared with controls125. administration is unlikely to ameliorate COVID-19-associated ARDS (EudraCT A trial of dipyridamole is currently ongoing NET-mediated thrombosis, although the number 2020-001550-22, EudraCT number (NCT04391179). Another antiplatelet agent, combination with heparin (which also digests 2020-004408-32, NCT04395456). Another ticagrelor, showed the ability to attenuate NETs 141) may be able to overcome this DNase C3 inhibitor, the pegylated compound NET formation126. Accordingly, two trials of resistance. APL-9, is being tested in a phase I/II ticagrelor are ongoing or recently completed Other promising approaches to prevent randomized controlled trial for severe (NCT02735707, NCT04518735). NET formation rather than target already COVID-19 (NCT04402060). The C1

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