The suboptimal fibrinolytic response in COVID‐19 is dictated by high PAI‐1

Abstract Background Severe COVID‐19 disease is associated with thrombotic complications and extensive fibrin deposition. This study investigates whether the hemostatic complications in COVID‐19 disease arise due to dysregulation of the fibrinolytic system. Methods This prospective study analyzed fibrinolytic profiles of 113 patients hospitalized with COVID‐19 disease with 24 patients with non‐COVID‐19 respiratory infection and healthy controls. Antigens were quantified by Ella system or ELISA, clot lysis by turbidimetric assay, and plasminogen activator inhibitor‐1 (PAI‐1)/plasmin activity using chromogenic substrates. Clot structure was visualized by confocal microscopy. Results PAI‐1 and its cofactor, vitronectin, are significantly elevated in patients with COVID‐19 disease compared with those with non‐COVID‐19 respiratory infection and healthy control groups. Thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator were elevated in patients with COVID‐19 disease relative to healthy controls. PAI‐1 and tissue plasminogen activator (tPA) were associated with more severe COVID‐19 disease severity. Clots formed from COVID‐19 plasma demonstrate an altered fibrin network, with attenuated fiber length and increased branching. Functional studies reveal that plasmin generation and clot lysis were markedly attenuated in COVID‐19 disease, while PAI‐1 activity was elevated. Clot lysis time significantly correlated with PAI‐1 levels. Stratification of COVID‐19 samples according to PAI‐1 levels reveals significantly faster lysis when using the PAI‐1 resistant (tPA) variant, tenecteplase, over alteplase lysis. Conclusion This study shows that the suboptimal fibrinolytic response in COVID‐19 disease is directly attributable to elevated levels of PAI‐1, which attenuate plasmin generation. These data highlight the important prognostic potential of PAI‐1 and the possibility of using pre‐existing drugs, such as tenecteplase, to treat COVID‐19 disease and potentially other respiratory diseases.


| INTRODUC TI ON
Thrombosis is a common feature of severe COVID-19 disease arising from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1,2 Venous thromboembolism, particularly pulmonary embolism (PE), 3,4 but also arterial thromboembolic complications, are also prevalent. 5 Large vessel thrombi are present in almost half of patients who are critically ill with COVID-19 and focal platelet and fibrin microthrombi are described in over 80% of cases. 6 Importantly, these thromboembolic complications are observed despite prophylactic and full dose anticoagulation. 4 The coagulopathy seen in COVID-19 disease is characterized by elevated fibrinogen and D-dimer. 7 However, unlike disseminated intravascular coagulation resulting from bacterial sepsis, 8 there is minimal impact on the APTT/PT time and very mild thrombocytopenia. 7,9 Nonetheless, these individuals are predisposed to excessive fibrin deposition, particularly within the pulmonary vasculature.
Autopsy findings reveal evidence of thrombi in pulmonary arterioles and arteries with diffuse alveolar damage and hyaline membrane deposition, 10 even in patients with no clinical features of thromboembolism. 11 Dysregulation of hemostasis in patients with  is closely connected to the inflammatory state, consequential to the cytokine storm and endotheliopathy induced by SARS-CoV-2 infection. 12,13 The natural anticoagulant properties of the endothelium, such as the expression of thrombomodulin (TM) and glycocalyx, are reduced and release of nitric oxide is impaired, increasing the hypercoagulability of the blood and promoting platelet and leucocyte adhesion. 14,15 Platelets and neutrophils recruited to sites of endothelial damage become activated by multiple pathways. Neutrophils express ACE-2 and can be directly infected by SARS-CoV-2, resulting in chemokine release, complement system activation and platelet assisted neutrophil activation with formation of neutrophil extracellular traps (NETS). Prominent cytokines in the inflammatory 'storm' include interleukin-6 (IL-6), tumor necrosis factor α (TNFα) and C-reactive protein (CRP). IL-6 stimulates platelet production and activity, augments tissue factor expression on endothelial cells and monocytes, and promotes endothelial dysfunction. 16,17 Together, activated platelets, NETS, coagulation system dysregulation and endothelium disruption contribute to the prothrombotic state and resultant fibrin deposition.
D-dimer levels reflect both thrombin generation and proteolytic degradation of fibrin by plasmin. The high D-dimer levels in patients with COVID-19 indicate activation of the fibrinolytic system, but despite this thrombosis predominates, indicating that a suboptimal fibrinolytic response may underlie these prothrombotic changes in  Generation of plasmin is governed by the balance of the plasminogen activators, tissue plasminogen activator (tPA) and urokinase (uPA), and the main inhibitors of the system, plasminogen activator inhibitor-1 (PAI-1), α 2 antiplasmin (α 2 AP) and the activated form of thrombin activatable fibrinolysis inhibitor (TAFI). Circulating PAI-1 exists in complex with the plasma glycoprotein, vitronectin, which acts to stabilize the inhibitor 19,20 and localize it within fibrin. 21 Levels of PAI-1 are low in the circulation, but it is released from the endothelium, circulating platelets and adipocytes following stimulation. 22 Table 1). The maximum level of clinical support during hospital admission was grouped into mild-no oxygen support; moderate-oxygen delivered by face mask or normal nasal cannula; or severe-oxygen delivered through high flow nasal cannula (HFNC) or continuous positive airways pressure (CPAP) systems, invasive ventilation and above.

| Clinical data collection
Clinical notes and imaging results were reviewed for evidence of thromboembolism (venous or arterial) during admission, defined as high clinical suspicion (with or without bedside ultrasound evidence) or radiologically proven (consultant radiologist report).

| Laboratory sample preparation and handling
Blood samples were collected in 0.1 ml volume of 0.13 M trisodium citrate. Platelet poor plasma was obtained by centrifugation of whole blood samples at 2500 × g for 30 min at 4°C. Pooled normal plasma (PNP) was prepared as described 31 or kindly provided by National Institute for Biological Standards and Control (NIBSC).

| Statistical analysis
Statistical analysis was performed using GraphPad Prism Software (

| RE SULTS
Of the 137 patients presenting with suspected COVID-19 disease, 113 were positive by PCR. The remaining 24 tested negative and were managed for non-COVID-19 respiratory infection (Table S1).
The baseline characteristics of the two groups were largely similar, although BMI was higher in the COVID-19 cohort ( Table 2).
The median hospital stay of the COVID-19 cohort was significantly longer than those with non-COVID-19 respiratory infection were male and four (3.5%) experienced a thrombotic event (three PE and one ischemic stroke), of whom one required mechanical ventilation ( Table 2). Presentation full blood counts ( Table 3) showed significantly reduced leucocytes compared with patients with non-COVID-19 respiratory infections and reduced platelet counts, although these did not reach significance.
PAI-1 and its cofactor vitronectin were significantly elevated in patients with COVID-19 compared with healthy controls (p < .001) and non-COVID-19 respiratory infection (p < .01) (Figure 2A,B). We observed bands in COVID-19 plasma at a molecular mass consistent with the size of the reported PAI-1-Vn complex 19 ( Figure S1). Of note, tPA and TAFI levels were elevated in patients with COVID-19 disease compared with healthy controls (p < .001 and p < .05, respectively) but were not different compared with non-COVID-19 respiratory infection ( Figure 2C,F). Interestingly, plasminogen differed only between the two disease groups due to a reduced level in

TA B L E 2 (Continued)
non-COVID respiratory disease (p < .01), with uPA also attenuated in this disease group ( Figure 2D,E). There was no significant difference in sTM ( Figure 2G).
The relationship between inflammatory and fibrinolytic markers with COVID-19 disease progression was studied by grouping according to the level of maximum oxygen support required. CRP and D-dimer increased with disease severity, with CRP being a strong indicator of progression to more severe COVID-19 illness ( Figure S2A,B). IL-6 and IL-8 also increased with disease progression ( Figure S2D,E). PAI-1 and tPA were the only fibrinolytic proteins that stratified with escalating severity of COVID-19 ( Figure   S3A,D). Changes in PAI-1 activity did not reach statistical significance, potentially reflecting the difference in quantifying free versus total PAI-1. and D-dimer (r = .23, p < .05) but not with CRP ( Figure S4). PAI-1 levels did not correlate with BMI in COVID-19 disease (r = .039, p = .69), despite the documented relationship with obesity. Leptin levels, which are known to correlate with body fat, were not different between the patient groups and there was no relationship with PAI-1 ( Figure S5). Nonetheless, leptin significantly correlated with BMI in patients with COVID-19 ( Figure S5).

| Elevated PAI-1 drives the hypofibrinolytic state in COVID-19 disease
To determine the direct contribution of PAI-1 in the reduced fibrinolytic potential in COVID-19 disease we utilized tenecteplase, a variant of tPA that is resistant to inhibition by PAI-1. Plasma clots were  Figure S8B). In contrast, plasma clots with high PAI-1 lysed significantly slower with alteplase compared with tenecteplase ( Figure 5A & Figure S8B). A strong correlation between 50% lysis time and PAI-1 concentration existed with alteplase (r = .68, p < .001), which was reduced with tenecteplase (r = .45, p < .05, Figure 5B). No relationship was observed between 50% lysis time and endogenous tPA antigen ( Figure 5C). These data suggest that there is a shift in the balance of fibrinolysis in COVID-19 disease towards inhibition, arising from high PAI-1.

| DISCUSS ION
We examined the hemostatic dysregulation, specifically the contri-   disease, 63 reduced PAI-1 in this group of patients providing direct evidence of the link between the proinflammatory state and PAI-1 levels. 64 The endotheliopathy and prothrombotic state are known to persist in patients with COVID-19 4-12 months post-discharge, 18,65 with elevated PAI-1 a component of this syndrome. It remains to be seen whether therapeutic agents, like tocilizumab, that dampen the inflammatory response, could be beneficial in reducing the prothrombotic tendency of these individuals.

| Limitations
The

CO N S ENT S TATEM ENT
All authors provide consent to publication of this data.

ACK N OWLED G M ENTS
The authors would like to thank all the patients who consented to be part of this study, and all the staff at the Emergency Department

CO N FLI C T O F I NTE R E S T
The authors have no relevant conflict of interests to declare.