Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients

Patients with coronavirus disease-19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions. However, bleeding complications have also been observed in some patients. Understanding the balance between coagulation and fibrinolysis will help inform optimal approaches to thrombosis prophylaxis and potential utility of fibrinolytic-targeted therapies. 118 hospitalized COVID-19 patients and 30 healthy controls were included in the study. We measured plasma antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) and performed spontaneous clot-lysis assays. We found markedly elevated tPA and PAI-1 levels in patients hospitalized with COVID-19. Both factors demonstrated strong correlations with neutrophil counts and markers of neutrophil activation. High levels of tPA and PAI-1 were associated with worse respiratory status. High levels of tPA, in particular, were strongly correlated with mortality and a significant enhancement in spontaneous ex vivo clot-lysis. While both tPA and PAI-1 are elevated among COVID-19 patients, extremely high levels of tPA enhance spontaneous fibrinolysis and are significantly associated with mortality in some patients. These data indicate that fibrinolytic homeostasis in COVID-19 is complex with a subset of patients expressing a balance of factors that may favor fibrinolysis. Further study of tPA as a biomarker is warranted.

The close relationship between COVID-19 and thrombosis is of significant clinical importance. There are increasing reports of venous thromboembolism in COVID-19 patients 1,2 , and arterial thrombosis including strokes and myocardial infarctions have been described 2,3 . Histopathology of lung specimens from patients with severe disease demonstrate fibrin-based occlusion of small vessels [4][5][6] .
COVID-19 is characterized in most patients by minimum prolongation of activated partial thromboplastin time (aPTT) and/or prothrombin time (PT), and mild, if any, thrombocytopenia 7,8 suggesting that it is distinct from traditional descriptions of sepsis-induced coagulopathy 9,10 . There are several (possibly synergistic) mechanisms by which SARS-CoV-2 infection may result in macrovascular and microvascular occlusions including cytokine-mediated activation of leukocytes, endothelium, and platelets; hypoxic vasoconstriction; direct activation of endothelial cells by viral transduction 11 ; and potentiation of thrombosis by neutrophil extracellular traps (NETs) [12][13][14][15] . At the same time, bleeding has been described in some patients with COVID-19. For example, a recent multicenter observation of 400 patients hospitalized with COVID-19 demonstrated an overall bleeding rate of 4.8% and a severe bleeding event (World Health Organization grade 3 or 4) rate of 2.3% 16 .
Fibrinolysis is a tightly controlled process whereby a fibrin-rich thrombus is degraded and remodeled by the protease plasmin 17 . This process is regulated by plasminogen activators and inhibitors with the conversion of plasminogen to plasmin being the end result that supports fibrinolysis 17 . The interplay of plasminogen activators-both tissue-type (tPA) and urokinase-type (uPA)-and their principal inhibitor, plasminogen activator inhibitor-1 (PAI-1), plays a key role in regulating fibrinolytic activity 17 . Impaired fibrinolysis has been suggested Scientific Reports | (2021) 11:1580 | https://doi.org/10.1038/s41598-020-80010-z www.nature.com/scientificreports/ among COVID-19 patients, which could further heighten thrombotic risk. This has been evidenced by markedly reduced clot lysis at 30 min via thromboelastography (TEG) in critically-ill patients with COVID-19 18 . Ex vivo evaluation of COVID-19 plasma also noted a prolonged clot lysis time, which was more pronounced among critically-ill COVID-19 patients 19 . Furthermore, a case series demonstrated that 11 of 21 COVID-19 patients who underwent rotational thromboelastometry in an intensive care unit met the criteria for fibrinolytic shutdown; 9 of those 11 patients developed thrombosis during their hospitalization 20 . Elevated PAI-1 levels observed in COVID-19 patients has further suggested impaired fibrinolytic ability 21 . The cause of this fibrinolytic shutdown has yet to be elucidated. Here, we aimed to evaluate the potential roles of tPA and PAI-1 in regulating fibrinolytic homeostasis among COVID-19 patients. Given that both bleeding and clotting have been described in COVID-19, we hypothesized that plasma of some patients would demonstrate fibrinolytic shutdown while plasma of others might present a hyper-fibrinolytic state.

Measurement of PAI-1 and tPA antigen.
Total PAI-1 and tPA protein was measured as described 23 .
Briefly, 25 μg of either rabbit anti-human PAI-1 (Molecular Innovations) or mouse anti-human tPA clone 2A153 (Molecular Innovations) was coupled to color-coded superparamagnetic beads. 25 μL of standard or diluted sample and 25 μL coupled beads (4000) were incubated for 2 h in the dark. 25 μL of 2 μg/mL biotinylated rabbit anti-hPAI-1 or biotinylated rabbit anti-htPA antibody (Molecular Innovations) was added to the plate, followed by incubating with phycoerythrin-conjugated streptavidin. The plate was read with a Luminex 100 System; the setting was 100 μL sample size and 100 events per well. Levels of PAI-1 and tPA were presented as mean ± standard deviations in the text. Active PAI-1 was detected by the same method but using the human uPA protease coupled to the beads as the capture 23 .

Spontaneous lysis assay.
To determine the rate of spontaneous lysis, 40 µL of diluted plasma (1:1 in TBS) was added to a microtiter plate and pre-read at 405 nm. Then, 40 µL of 25 nM alpha human thrombin (Haemtech) and 15 mM CaCl 2 was added and incubated at 37 °C for 30 min, and the absorbance was read at 405 nm. Twenty µL of TBS was then added to the plate to prevent clot drying during the extended incubation and the plate was read again after 30 min and then at 60-min intervals up to 8 h.

Statistical analysis.
When two groups were present, normally-distributed data were analyzed by two-sided t test and skewed data were analyzed by Mann-Whitney test or Wilcoxon test. For three or more groups, analysis was by one-way ANOVA or Kruskal-Wallis test with correction for multiple comparisons. Normality was assessed by Shapiro-Wilk test. Correlations were tested by Spearman's method. Data analysis was with Graph-Pad Prism software version 8. Statistical significance was defined as p < 0.05.

Results
Tissue-type plasminogen activator and plasminogen activator inhibitor-1 in COVID-19. Utilizing established Luminex platforms, we measured total PAI-1 and tPA levels (detecting both free and complexed PAI-1 and tPA, respectively) in the plasma of 118 patients hospitalized with COVID-19. We similarly assessed 30 healthy controls whose samples had been banked prior to December 2019. Of the 118 COVID-19 patients, the mean age was 61 with a standard deviation of 17 (range 25-95); 54 were female (46%) ( Table 1). In our cohort, 42% of patients were supported by mechanical ventilation, 8% were receiving high-flow oxygen, 27% were supported by standard nasal cannula, and 24% were breathing ambient air at the time of sample collection. Markedly elevated levels of both PAI-1 and tPA were detected in patients with COVID-19 as compared with healthy controls (mean ± standard deviation 75 ± 46 vs. 40 ± 42 ng/mL, p < 0.0001; and 78 ± 68 vs 2.4 ± 2.6 ng/mL, p < 0.0001, respectively Fig. 1a,b). There was a significant correlation between levels of PAI-1 and tPA among COVID-19 patients (r = 0.52, p < 0.0001) (Fig. 1c). In summary, both PAI-1 and tPA are markedly elevated in the plasma of patients hospitalized with COVID-19.

Plasma level of tPA and PAI-1 and their association with clinical biomarkers.
We assessed potential correlations with D-dimer and platelet count. We limited the analysis of clinical laboratory measurements to those performed on the same day as plasma used for the PAI-1 and tPA assays. No significant correlation was found between D-dimer and either PAI-1 (r = 0.23, p = 0.11) or tPA (r = -0.01, p = 0.94) (Fig. 2a,b). We did observe a strong correlation between PAI-1 and platelet count (r = 0.33, p = 0.0003) (Fig. 2c); the same was not true for tPA (r = 0.06, p = 0.5) (Fig. 2d). Given that activated neutrophils and their products can exert anti-fibrinolytic effects 24 , we next asked how absolute neutrophil count and calprotectin (a marker of neutrophil activation) compared to tPA and PAI-1. Both PAI-1 and tPA demonstrated strong positive correlations with same-day absolute neutrophil count (r = 0.32, p = 0.03 and r = 0.23, p = 0.03) (Fig. 2e,f), as well as levels of calprotectin (r = 0.42, p < 0.0001 and r = 0.23, p = 0.01) (Fig. 2g,h). In summary, levels of PAI-1 and tPA demonstrated strong correlations with neutrophil numbers and activation.  (Fig. 3a), but not tPA or D-dimer (Fig. 3b,c). Beyond mode of respiratory support, oxygenation efficiency can also be measured by comparing pulse oximetry (SpO 2 ) to the fraction of inspired oxygen (FiO 2 ). We tested the correlation between PAI-1, D-dimer and SpO 2 /FiO 2 ratio and found a strong negative association (r = -0.35, p = 0.0002 for PAI-1; r = -0.37, p = 0.009 for D-dimer;) (Fig. 3d,f). A negative association was also appreciated between oxygenation efficiency and tPA (r = -0.19, p = 0.04), albeit less robust than for PAI-1 and D-dimer (Fig. 3e). Among the 118 patients, 24 died, 92 were discharged, and two remained hospitalized at the time of this analysis. Significantly higher levels of both PAI-1 (p = 0.04) and tPA (p = 0.0003) were observed among patients who died as compared with those who were discharged, with this difference being especially robust for tPA (Fig. 3g,h). Surprisingly, we did not see a significant difference in D-dimer levels between those two groups (Fig. 3i). In summary, high levels of tPA and PAI-1 were associated with worse respiratory status and poor clinical outcomes; in particular, high levels of tPA were strongly associated with death.
High tPA COVID-19 samples have enhanced spontaneous fibrinolysis. Finally, we asked whether COVID-19 plasma with the highest tPA levels might demonstrate enhanced spontaneous fibrinolysis as compared with low-tPA COVID-19 plasma or control plasma. A spontaneous fibrinolysis assay was performed on 10 COVID-19 plasma samples with high tPA (> 100 ng/mL), 10 COVID-19 samples with low tPA (< 20 ng/mL), and 10 healthy control plasma samples (mean value 2.4 ng/mL). Notably, the high-tPA COVID-19 samples significantly enhanced spontaneous fibrinolysis as compared with low-tPA and healthy control plasma samples (Fig. 4a,b). Consistent with this observation, we found that tPA levels were on average 2.2-fold higher than PAI-1 in the high tPA patients ( Supplementary Fig. 1A,B). This was in contrast to the ratio in control plasma samples (where PAI-1 levels averaged more than tenfold greater than tPA) or in COVID-19 patients with tPA less than 20 ng/mL (where PAI-1 levels were > twofold greater than tPA). No significant difference in age or oxygenation efficiency were observed in a subset of ten high tPA and ten low tPA patients ( Supplementary Fig. 2). Detailed demographic and clinical characteristics of those COVID-19 patients with high and low tPA are presented in Supplementary Table 1.

Discussion
Fibrinolytic homeostasis in COVID-19 is likely complex and influenced by various factors. Normal lung physiology has a pro-fibrinolytic tendency 25 . However, during acute respiratory distress syndrome (ARDS) impaired fibrinolysis results in accumulation of fibrin that promotes hyaline membrane formation and alveolar injury 26 . Fibrin is removed by plasmin. It is believed that depressed fibrinolysis in ARDS is at least partially driven by increased circulating PAI-1 that exerts a negative effect on the plasminogen activation system 25 . Indeed, elevated PAI-1 is an independent risk factor for poor ARDS outcomes 27 . Elevated PAI-1 and its associated hypo-fibrinolytic state were observed in the 2002 SARS-CoV epidemic 28 , while recent characterizations of COVID-19 patients have suggested impaired global fibrinolysis 18,21 . Interestingly, in our large cohort of hospitalized COVID-19 patients, we observed elevated levels of not only PAI-1, but also tPA. While high PAI-1 and D-dimer tracked most closely with impaired oxygenation efficiency, tPA was the best predictor of death. A recent study of 78 hospitalized COVID-19 patients also detected elevations of both PAI-1 and tPA, particularly among critically-ill COVID-19 patients 21 ; however, the mechanistic role of the elevated tPA among COVID-19 patients was not specifically investigated. Furthermore, the level of tPA we detected in the COVID-19 patients, 78 ± 68 ng/mL, is striking and www.nature.com/scientificreports/ much higher than the 23.9 ± 14.5 ng/mL described in this prior report in 48 patients admitted to ICU 21 . Notably these levels are even higher than is observed in trauma patients who have exaggerated fibrinolytic activity and in patients with hantavirus cardiopulmonary syndrome, which carries high alveolar hemorrhage risk [29][30][31] . The major source of these high levels of tPA among COVID-19 patients is likely endothelial cells. The source of PAI-1 could also be the endothelium or perhaps release from activated platelets (as we found a strong correlation between PAI-1 and platelet counts). High PAI-1 expression in other cell types such as macrophages has also been reported during hantavirus infections 29 . One hallmark of COVID-19 ARDS is the sequestration of leukocytes, particularly neutrophils, in the microvasculature of the lung-contributing to alveolar injury and unrestricted inflammation 27 . This local proinflammatory environment is further exaggerated by the formation of NETs and results in massive release of proinflammatory cytokines 6 . Those cytokines likely trigger endothelial cell activation and thereby promote local release of tPA and PAI-1 6,32 . Notably, we observed a strong correlation www.nature.com/scientificreports/ between tPA/PAI-1 and both absolute neutrophil counts and circulating calprotectin, a neutrophil activation marker. In addition to endothelial activation, it is possible that direct infection and destruction of endothelial cells by SARS-CoV-2 may also potentiate the release of tPA and PAI-1 11 . While the prothrombotic risk associated with COVID-19 is well recognized, the risk of bleeding should not be ignored. One recent large multicenter study observed an overall bleeding risk of 4.8% among hospitalized COVID-19 patients and this risk increased to 7.6% among critically-ill patients 16 . Elevated D-dimer was associated with both thrombotic and bleeding complications 16 . It has been suggested that high PAI-1 levels overcome the effects of local tPA and produce a net prothrombotic hypofibrinolytic state in COVID-19 patients 21 . However, we here found a subset of COVID-19 patients with extremely high levels of tPA (> 100 ng/mL) in which fibrinolysis seems to dominate. This may at least partially explain the enhanced bleeding risk observed in some groups of patients with COVID-19.
Our study has some limitations. We did not have access to fresh plasma samples each day of a patient's hospitalization. PAI-1 and tPA levels were therefore not tested on a defined day of hospitalization, but rather when a plasma sample became available to the research laboratory. It should however be noted that when assessing correlations of PAI-1 and tPA with clinical variables, same-day laboratory and clinical status data were used. Due to research restrictions during the pandemic we were not allowed to recruit new healthy controls. Healthy controls were recruited during the pre-COVID-19 era and we were not able to match gender and age to COVID-19 patients. Future studies should endeavor to systematically track PAI-1 and tPA levels over the full course of hospitalization of COVID-19 patients and to compare with gender-and age-matched controls. We also recognize that tPA is not the sole activator of plasminogen, as uPA also plays a role in the fibrinolysis regulation and PAI-1 can also inhibit uPA 17 . Dysregulation of uPA and its receptor system have been implicated in the pathogenesis of pulmonary fibrosis and ARDS 33,34 . The role of uPA and its receptor in COVID-19 warrants further investigation.
Because the COVID-19 associated prothrombotic risk is known, prophylactic anticoagulation has become part of standard COVID-19 treatment. High rates of thromboembolic events from early studies prompted some experts to recommend a more intensive dose of anticoagulation among COVID-19 patients 2 . We would urge caution regarding this recommendation (pending randomized studies) as the coagulopathy of COVID-19 is complex and potentially dynamic. Therapies aimed at promoting fibrinolysis, such as administration of aerosolized or intravenous tPA, have been trialed in ARDS models where there have been some promising preclinical results 35,36 . Profibrinolytic therapy has been suggested as a potential beneficial therapy in COVID-19 patients suffering from ARDS 27 and is currently being tested in multiple clinical trials (https ://clini caltr ials.gov/ct2/resul ts?cond=Covid 19&term=tpa). We have now found that a hyperfibrinolytic state exists in some COVID-19 patients. Targeted therapies that promote fibrinolysis therefore need to be selective and cautious to minimize bleeding risk. Finally, our data suggests that high systemic tPA may be a biomarker for poor clinical outcomes and supports further studies of tPA levels during the course of disease progression. patients' plasma with high (> 100 ng/mL) and low tPA (< 20 ng/mL) to promote spontaneous lysis of an ex vivo plasma clot formed by the addition of alpha human thrombin was evaluated. (a) Lysis over time was recorded for 10 high-tPA plasma samples. (b) The rate of lysis determined from the slope of the absorbance from t 0min to t 480min was compared between COVID-19 patients with high tPA, low tPA, and healthy controls by one-way ANOVA; *p < 0.05. Statistics were calculated and the figure was produced in GraphPad Prism https ://www.graph pad.com/scien tific -softw are/prism /, using version 8.3.