Thromboinflammation in coronavirus disease 2019: The clot thickens

Since the start of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic, a disease that has become one of the world's greatest global health challenges, the role of the immune system has been at the forefront of scientific studies. The pathophysiology of coronavirus disease 2019 (COVID‐19) is complex, which is evident in those at higher risk for poor outcome. Multiple systems contribute to thrombosis and inflammation seen in COVID‐19 patients, including neutrophil and platelet activation, and endothelial dysfunction. Understanding how the immune system functions in different patient cohorts (particularly given recent emerging events with the Oxford/AstraZeneca vaccine) is vital to understanding the pathophysiology of this devastating disease and for the subsequent development of novel therapeutic targets and to facilitate possible drug repurposing strategies that could benefit society on a global scale. LINKED ARTICLES This article is part of a themed issue on The second wave: are we any closer to efficacious pharmacotherapy for COVID 19? (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.10/issuetoc


| Coronaviruses
Coronaviruses (CoVs), of which there are four genera (alpha, beta, gamma and delta), are a family of enveloped, positive-sense, singlestranded and highly diverse RNA viruses . Three betacoronaviruses have been identified to cause severe respiratory illness in humans including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and SARS-CoV-2 (McFadyen et al., 2020). Although bats have been suggested to be the primary origin of SARS-CoV and SARS-CoV-2, the precise origin and mechanism by how SARS-CoV-2 manifested in humans is still unclear, with other possible hosts (such as the Malayan pangolins [Manis javanica]), under investigation (Lam et al., 2020).
SARS-CoV-2 is a single-stranded RNA virus, and entry into host cells initiates with their transmembrane spike (S) glycoproteins (GPs) encompassing S1 and S2 subunits. The S1 unit binds to host cell receptors, whereas the S2 subunit aids in viral and host cell fusion (Walls et al., 2020). Like SARS-CoV, SARS-CoV-2 binds via the human ACE2, a cell surface protein with peptidase activity, expressed on the endothelial surface (Davidson et al., 2020;Hoffmann et al., 2020). In comparison with SARS-CoV, SARS-CoV-2 has different amino acid residues within the receptor-binding domain (RBD) of the S protein and a polybasic furin cleavage site at the junction of S1 and S2. In addition, transmissibility and pathogenicity of SARS-CoV-2 is linked to cellular proteases such as furin and the transmembrane protease serine 2 (TMPRSS-2) (McFadyen et al., 2020). Although the functionality of these specific features of SARS-CoV-2 has yet to be fully elucidated, they could provide key information to the prothrombotic phenotype associated with SARS-CoV-2 and why patients with this specific CoV have higher thrombosis rates than SARS-CoV and MERS-CoV infections.

| Clinical manifestations and thromboinflammation
A number of clinical manifestations such as fever, dry cough, malaise, sore throat, chest pain, dyspnoea, nausea, diarrhoea and vomiting have been associated with infection with SARS-Cov-2 (Tian et al., 2020). The spectrum of symptomatic infection ranges from mild to critical, although there remains uncertainty around the proportion of asymptomatic infections. Among hospitalised patients, the proportion of critical or fatal disease is higher, with many progressing to acute respiratory distress syndrome (ARDS), respiratory failure and eventually death (Bost et al., 2021). Interestingly, the ARDS associated with COVID-19 patients differs from that caused by other infective or traumatic insults, with the 'cytokine storm' (i.e., increased cytokines released from the blood, which is associated with disease severity) being only partly involved in COVID-19 patients (Maier et al., 2020).
Furthermore, IL-6 levels are 60-fold to 90-fold higher in ARDS patients compared with COVID-19 patients, although the reasons for these differences are unknown. It is conceivable that SARS-CoV-2 takes over the host immune system, impairing antiviral immunity and triggering chronic inflammation by involving inflammatory cytokines (Bost et al., 2021;Sinha et al., 2020). Certainly, a hallmark of SARS-CoV-2 is the release of not only IL-6 but also other cytokines and chemokines including IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, IL-17, TNF-α, CCL2, CCL3 and CXCL10, all of which correlate with adverse clinical outcomes. The inflammatory effects of cytokines also activate vascular endothelial cells, disrupting endothelial function and integrity, which leads to increased platelet and leukocyte recruitment, resulting in a pro-inflammatory and prothrombotic state (Connors & Levy, 2020).
Autopsy reports from SARS-CoV-2 patients show multi-organ dysfunction, with highest viral titres in the lungs and immune cells, and the presence of endothelial inflammation and cell death (Gu et al., 2005;Mazzulli et al., 2004). It is likely that hypoxia also promotes a prothrombotic endothelial phenotype in SARS-CoV-2 via hypoxia-inducible transcription factor activation and endothelial tissue factor (TF) up-regulation.
The scale of COVID-19 severity also increases with comorbidities such as hypertension, chronic kidney disease, obstructive sleep apnoea, asthma diabetes and obesity (Tian et al., 2020). Patients with underlying cardiovascular disease are prone to increased severity of COVID-19 and a fivefold increase in mortality (Yang et al., 2020). Therefore, managing and controlling cardiovascular risk factors is a high priority.
COVID-19 is associated with a prothrombotic state, which can manifest as microvascular thrombosis, venous thromboembolism or arterial thrombosis (McFadyen et al., 2020). The cause of this prothrombotic state may relate to the appreciated link between thrombosis and inflammation (termed 'thromboinflammation') in which thrombosis can amplify inflammation and systemic inflammation can beget thrombosis. Although efforts to understand the complex immunological landscape in COVID-19 are evolving, both platelet activation and platelet-neutrophil interactions play a crucial role in thromboinflammation.

| ROLE OF NEUTROPHILS IN COVID-19
Neutrophils are the immune system's first responders having crucial functions in immunity and repair. Upon activation, they produce proinflammatory cytokines (including IL-6, TNF-α and IL-1β), generate ROS and release haematopoietic serine proteases (neutrophil elastase, proteinase 3 and cathepsin G), microparticles and neutrophil extracellular traps (NETs). Neutrophils possess antimicrobial properties capable of not only killing both Gram-positive and Gram-negative bacteria, but they can also act as a double-edged sword mediating tissue injury and perpetuating the inflammatory response (Kolaczkowska & Kubes, 2013). NETs, ROS and serine proteases can all independently, or collectively, up-regulate thromboinflammatory processes.
In the context of COVID-19, neutrophilia signifies worse outcomes, with autopsy studies showing neutrophil infiltration in pulmonary capillaries, acute capillaries with fibrin deposition, extravasation of neutrophils in the alveolar space and neutrophil mucositis of the trachea (Gu et al., 2005;Yao et al., 2020). Bost et al. (2021) revealed at least 10 different neutrophil states present in blood and bronchoalveolar lavage of COVID-19 patients, with resting state phenotype mainly associated with patients with mild disease, and activated or immature phenotype associated with patients with severe disease. These results suggest that COVID-19 is associated with a state of 'immune silence' (demonstrated by loss of neutrophil and monocyte immunosuppression and the replacement of lung memory CD8 + T cells by naïve T cells), correlating with severe clinical manifestation and outcome (Bost et al., 2021). Diao et al. (2020) showed that T cells are dysfunctional with increased expression of exhaustion molecules related to heightened systemic inflammation, including IL-6 levels. We have previously shown that IL-6 plays a major role in T cell-dependent thromboinflammatory responses to angiotensin II and in the activation and aggregation of platelets (Senchenkova, Russell, et al., 2019). These findings support the hypothesis that drug discovery programmes based on T cell-dependent, IL-6 signalling pathways may lead to protection against thromboinflammation in COVID-19 ( Figure 1).
As observed with other pathological conditions (e.g., sickle cell disease) (Ansari et al., 2021), NET levels are increased in COVID-19 patients, with sera from COVID-19 patients being shown to trigger NET release from control neutrophils and containing increased myeloperoxidase-DNA (MPO-DNA) complexes and citrullinated histone H3 levels, which correlated with disease severity (Veras et al., 2020;Zuo et al., 2020). These results suggest that NETs may contribute to COVID-19 pathology and NET biomarkers may help to predict clinical worsening and venous thromboembolism.
Neutrophil degranulation and NET formation lead to various intracellular danger-associated molecular patterns (DAMPs) activating pattern recognition receptors on nearby immune and non-immune (1) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds via the ACE2 expressed on the endothelial surface. The virus activates monocytes releasing a cytokine storm (including IL-6, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, IL-17, TNF-α, CCL2, CCL3, and CXCL10 , all of which correlate with adverse clinical outcomes). (2) The inflammatory effects of cytokines also activate vascular endothelial cells (ECs), disrupting endothelial function and integrity, which leads to increased platelet and neutrophil recruitment. (3) Increased neutrophil at inflammatory site results in increased expression of adhesion molecules such as L-selectin, Pselectin and intercellular adhesion molecule-1 on ECs. Activated neutrophils release their neutrophil extracellular traps (NETs, which contain a backbone of DNA and citrullinated histone [H3Cit], cathepsin G and neutrophil elastase [NE]), which trap platelets, increasing their activation and aggregation; this in turn further activates neutrophils to produce more NETs. (4) This cycle of events triggers coagulation cascade activation, which in turn increases fibrin formation and thrombosis. CAMs, cellular adhesion molecules; RBC, red blood cell; vWF, von Willebrand factorIFFAHANDGAVINS3 cells releasing pro-inflammatory mediators (Tomar et al., 2020).
DAMPs activate properdin, factor B and C3, all components of the alternative pathway necessary to induce the complement cascade.
These findings highlight the therapeutic strategy of complement targeted therapies for COVID-19-mediated thrombosis.
The initial neutrophil response also leads to interactions with platelets via a variety of different mechanisms including Mac-1 (CD11b/CD18)/glycoprotein Ib (CD42) and P-selectin/P-selectin

| ROLE OF PLATELETS AND IN COVID-19
Platelets are traditionally known for their role in haemostasis and formation of thrombi. However, they are increasingly being recognised as key effector cells in inflammation, influencing innate and adaptive immune responses (Senchenkova, Ansari, et al., 2019). Migration of single platelets acts as mechanosensors to collect and bundle bacteria for neutrophil phagocytosis (Gaertner et al., 2017), although their uncontrolled activation can result in pathogenic thrombosis. Platelets from COVID-19 patients show increased aggregation, adhesion and spreading on fibrinogen and collagen (via up-regulation of the MAPK pathway) and increased activity of thromboxane A 2 (marker of platelet activation) (Manne et al., 2020).
Thrombocytopenia in COVID-19 is common, with meta-analysis demonstrating a link with a fivefold increased risk of severe disease (Lippi et al., 2020). Thrombocytopenia has also been linked with issues of rare blood clotting events, which are now associated with the Oxford/AstraZeneca vaccine (ongoing pharmacovigilance will determine outcome), with seven deaths being reported in the 30 cases reported in the United Kingdom alone (at the time of writing this review, evidence is shifting towards a causal link between the vaccine and rare blood clots).

| THERAPEUTIC INTERVENTION THROMBOINFLAMMATION RESOLUTION
Understanding neutrophil and platelet responses in the context of thromboinflammation in COVID-19 has resulted in promising preclinical studies demonstrating that, for example, immune regulatory properties are being lost in patients with COVID-19, with the virus seemingly to 'suppress' or 'silence' the immune system (Bost et al., 2021). Drugs that can 'stimulate' or 'reawaken' the immune system may be the way forward, as seen with dexamethasone (Bost et al., 2021;The RECOVERY Collaborative Group et al., 2021). Other therapies may lie in targeting the cytokine storm with anti-inflammatory agents such as IL-6 and IL-1β antagonists.
Accumulating data linking inflammation and thrombosis supports the hypothesis that anti-inflammatory therapies may limit thrombosis and that antithrombotic therapies may reduce vascular inflammation (Vital et al., 2016). Due to the high incidence of thrombotic events seen in severe stages of COVID-19, most clinical guidelines agree to the administration of low-molecular-weight heparin (LMWH), as thrombo-prophylaxis, to hospitalised COVID-19 patients. However, the correct dose for hospitalised patients and thromboprophylaxis approach to ambulant patients remains controversial. A wide range of antithrombotic therapies are currently being assessed in ongoing and recent randomised controlled trials (RCTs) directed at outpatients, hospitalised patients and critically ill patients with COVID-19. These trials involve the use of antiplatelet agents, anticoagulants and fibrinolytic agents or a combination (Table 1). Current antithrombotic therapies consist of various interventions including thromboprophylaxis with unfractionated heparin (UFH), or LMWH; an intensive thromboprophylaxis protocol with LMWH, antithrombin, clopidogrel and salvage therapy with tissue plasminogen activator and heparin (Maldonado et al., 2020).
There is a growing evidence of the importance of resolution biology in vascular inflammation to develop innovative approaches for the treatment of diseases, which may include COVID-19. Inflammation resolution is an active process involving specific endogenous mediators (e.g., annexin A1 [AnxA1] and aspirin-triggered lipoxin A 4 ) T A B L E 1 Clinical trials examining the potential of antithrombotic therapies for the treatment of COVID-19-related side effects  (Senchenkova, Ansari, et al., 2019). FPR2 agonists are being developed and currently tested in man. In the context of thromobinflammation and the possible insights for therapeutic strategies for COVID-19 treatments, we recently discovered that targeting the AnxA1/Fpr2/ALX pathway promotes thromboinflammation resolution by altering both the platelet phenotype (from pro-pathogenic to regulatory) (Senchenkova, Ansari, et al., 2019) and the pathological neutrophil phenotype (from a pro-NETotic to pro-apoptotic) (Ansari et al., 2021).

| CONCLUSION
COVID-19 is a devastating disease that has affected the United Kingdom and our global community in unprecedented ways. The pathophysiology is complex with many systems likely to contribute to the prothrombotic state including inflammation, platelet and neutrophil activation, endothelial cell dysfunction, NETs and complement factors. However, the mechanism that drives COVID-19-associated thromboinflammation has not yet been fully elucidated and there is an urgent unmet clinical need to fully understand and characterise this disease. These discoveries will unearth ways to develop pharmacological strategies, which may also focus on resolution biology.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOL-OGY (http://www.guidetopharmacology.org) and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos et al., 2019;Alexander, Fabbro, et al., 2019a, b A placebo-controlled, double-blind, randomised Phase II dose-escalation study intending to study the safety and efficacy of tenecteplase for the treatment of COVID-19-associated respiratory failure. Hypothesis is that the administration of drug with heparin anticoagulation will improve patients' clinical outcome.