Endogenous anticoagulants and treatment of severe pneumonia and ARDS
Activation of the coagulation system is countered by endogenous anticoagulants, which act either alone or in concert to limit coagulation. A number of endogenous anticoagulants, including anti-thrombin (AT) and activated protein C (APC), have been trialled in severe pneumonia and ARDS, on the basis that they were shown to be protective against lung injury in experimental animal models by reducing fibrin generation and attenuating neutrophil recruitment [42–46].

Anti-thrombin
AT inactivates thrombin and factor Xa and has been shown to reduce systemic (after intravenous administration) and bronchoalveolar (after both intravenous and nebulised administration) thrombin and fibrin levels and reduce lung injury in preclinical models [42, 46]. However, the clinical data currently do not support using AT as a treatment to improve lung injury. The KyperSept trial of high-dose AT treatment in severe sepsis [47] showed no effect on 28-day mortality, and an increased risk of bleeding events in the AT-treated participants was observed, particularly in those who received concomitant heparin. Yet, a subgroup analysis revealed that the AT-treated group that had not received concomitant heparin had a 15% improvement in 90-day mortality compared to those treated with heparin. Nonetheless, a meta-analysis of 20 trials of a heterogeneous population of critically ill patients concluded that AT should be avoided owing to the risk of bleeding complications [45]. Natural or pharmacological thrombin inhibitors have not been tested in clinical trials in the setting of pneumonia and potential clinical translation has been inferred from studies of AT in sepsis, in which pneumonia is the leading cause in a large proportion of cases. In terms of extrapolation to COVID-19 pneumonia, we would propose that the current evidence would also argue against the use of AT, particularly if co-administered with heparin. Heparin alone, which exerts its anticoagulant effects via the activation of AT, is currently being evaluated in multiple trials in the context of COVID-19 pneumonia. This includes evaluation of a systemic prophylactic dose and a full therapeutic dose of low molecular weight heparin (LMWH). In H1N1 ARDS, anticoagulation with systemic heparin significantly reduced the high incidence of VTE [48]. Heparin also exerts anti-inflammatory and antiviral properties [49, 50]. However, the route of administration of heparin is likely to be critical. Nebulised heparin did not attenuate inflammation in a murine model of pneumonia [42], and, in clinical trials in patients with or at risk of ARDS, nebulised heparin had no major impact on physiological variables nor a beneficial effect on mortality, although it increased ventilator-free days [51–53].

Activated protein C
APC plays a critical role in terminating coagulation by proteolytically inactivating factors Va and VIIIa, and has been widely investigated in the context of sepsis [54–56]. Once the coagulation cascade is activated, thrombin binds to thrombomodulin and, facilitated by the endothelial protein C receptor (EPCR), protein C is activated. APC then binds to the EPCR and, together with its co-factor protein S, forms the EPCR-APC-protein S complex that binds and degrades factor Va and factor VIIIa [57], providing negative feedback for thrombin generation. APC also exerts pro-fibrinolytic and anti-inflammatory effects [57, 58]. Preclinical studies support a beneficial effect of APC administration, showing that rhAPC attenuates tissue injury and improves survival in models of sepsis and lung injury [59–61]. For example, in murine models of indirect lung injury (intravenous injection of lipopolysaccharide) and direct lung injury (Streptococcus pneumoniae infection), nebulised rhAPC specifically reduced local bronchoalveolar thrombin and fibrin generation without affecting intravascular thrombin generation or fibrinolytic activity [46] and without adversely affecting bacterial clearance [46]. This provides proof-of-principle that targeting the alveolar epithelium locally with nebulised rhAPC can attenuate coagulation activation without compromising host defence.
In clinical studies, systemic, rather than nebulised, delivery of APC has had initially promising results. In the original landmark PROWESS study, intravenous infusion of rhAPC (drotrecogin alpha (activated) (DrotAA)) significantly reduced 28-day all-cause mortality in patients with sepsis (secondary to CAP) by 28% and reduced the resolution time of respiratory failure [22]. This resulted in the Infectious Diseases Society of America/American Thoracic Society recommending APC for the treatment of refractory septic shock due to CAP [62]. Although APC reduced coagulation activation and lung injury scores in a study of 27 patients with ARDS [63, 64], in a subsequent randomised controlled trial APC did not improve the clinical outcomes of ARDS patients [65] or of patients with sepsis and low of risk of death [66]. Furthermore, a meta-analysis of five studies involving 5101 participants concluded that APC was associated with higher risk of bleeding and should not be used in patients with severe sepsis or septic shock [67]. Importantly, the PROWESS-Shock study [55] failed to demonstrate an improvement in survival in patients with septic shock and eventually led to the withdrawal of DrotAA (Xigris) from the market. However, the significant differences in patient characteristics between the PROWESS trials has led to the recommendation that a trial of DrotAA should be repeated using an optimised study design [68], and this could include a trial in a less heterogeneous high-risk population, such as COVID-19 pneumonia. The preclinical and clinical data suggest nebulised rhAPC may not carry the same bleeding liability as intravenous administration and may be an appropriate route of drug administration. Alternatively, the bleeding risk could be mitigated using an rhAPC variant with <10% anticoagulant activity [69], which in preclinical studies was as effective as wild-type APC in improving survival of mice in sepsis models [69].
In a small study of 11 COVID-19 patients, most had increased endogenous APC levels [70]; however, four patients had lower APC levels and in this subgroup of patients, particularly those with septic shock or at high risk of death, administration of rhAPC might warrant further investigation.

Endogenous fibrinolytics
Fibrin deposition and clearance is regulated by the control of plasmin activity, which in turn is regulated by the relative balance between plasminogen activators and plasminogen activator inhibitors (PAI)-1, -2 and -3. Plasminogen is mainly synthesised in the liver and converted into plasmin by the serine proteinases tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Several preclinical and clinical studies have demonstrated elevated levels of PAI-1 in pneumonia [30, 71, 72]. However, preclinical studies on the effects of PAI-1, tPA and uPA in murine models of pneumonia suggest the outcome is pathogen dependent. For example, in a Klebsiella pneumoniae pneumonia model, PAI-1 improved bacterial clearance and reduced mortality [73], but neither PAI-1 nor plasmin appeared to have a role in mouse models of S. pneumoniae [72] or Pseudomonas aeruginosa pneumonia [74]. Instead, the urokinase plasminogen activator receptor axis promoted host defence against S. pneumoniae by recruiting neutrophils to the alveoli and enhancing neutrophil-mediated bacterial killing [75]. Furthermore, in a model of sterile lung injury, tPA administration reduced alveolar leak but had no effect on pulmonary inflammation [76]. Data are limited regarding the role of this system in viral respiratory infection models but in a study of influenza A infection, the absence of plasminogen reduced inflammation [77]. A recent meta-analysis of preclinical studies of fibrinolytics in acute lung injury suggested increased fibrinolysis, attenuated inflammation and alveolar leak, and improved survival [78].
There are limited clinical data on the efficacy of targeting fibrinolysis in patients with severe infections or ARDS. However, patients with ARDS display evidence of a marked reduction in fibrinolysis, with evidence of reduced uPA activity and increased PAI-1 levels in bronchoalveolar lavage fluid [30]. Furthermore, one study demonstrated a shorter length of stay in the intensive care unit and improved survival in ARDS patients treated with nebulised streptokinase [79]; this pathway may represent an interesting target for the management of COVID-19-induced lung injury. Two trials targeting this pathway are currently underway, one in the UK (nebulised r-tPA) and another in the USA (alteplase) (NCT04356833, NCT04357730).