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

Abstract

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.

Full text

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
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Plasma tissue plasminogen
activator and plasminogen
activator inhibitor‑1 in hospitalized
COVID‑19 patients
Yu Zuo1, Mark Warnock2, Alyssa Harbaugh1, Srilakshmi Yalavarthi1, Kelsey Gockman1,
Melanie Zuo3, Jacqueline A. Madison1, Jason S. Knight1, Yogendra Kanthi2,4 &
Daniel A. Lawrence2*
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 brinolysis will help inform optimal approaches
to thrombosis prophylaxis and potential utility of brinolytic‑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
signicant 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 brinolysis and
are signicantly associated with mortality in some patients. These data indicate that brinolytic
homeostasis in COVID‑19 is complex with a subset of patients expressing a balance of factors that may
favor brinolysis. Further study of tPA as a biomarker is warranted.
e close relationship between COVID-19 and thrombosis is of signicant clinical importance. ere are increas-
ing reports of venous thromboembolism in COVID-19 patients1,2, and arterial thrombosis including strokes
and myocardial infarctions have been described2,3. Histopathology of lung specimens from patients with severe
disease demonstrate brin-based occlusion of small vessels4–6.
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, thrombocytopenia7,8 suggesting that it is distinct
from traditional descriptions of sepsis-induced coagulopathy9,10. ere are several (possibly synergistic) mecha-
nisms 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 activa-
tion of endothelial cells by viral transduction11; and potentiation of thrombosis by neutrophil extracellular traps
(NETs)12–15. 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 brin-rich thrombus is degraded and remodeled by the
protease plasmin17. is process is regulated by plasminogen activators and inhibitors with the conversion of
plasminogen to plasmin being the end result that supports brinolysis17. e interplay of plasminogen activa-
tors—both tissue-type (tPA) and urokinase-type (uPA)—and their principal inhibitor, plasminogen activator
inhibitor-1 (PAI-1), plays a key role in regulating brinolytic activity17. Impaired brinolysis has been suggested
OPEN
Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI,
USA. Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor,
MI, USA. Division of Geriatric and Palliative Medicine, Department of Internal Medicine, University of Michigan,
Ann Arbor, MI, USA.           
 *email: dlawrenc@umich.edu
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among COVID-19 patients, which could further heighten thrombotic risk. is has been evidenced by markedly
reduced clot lysis at 30min via thromboelastography (TEG) in critically-ill patients with COVID-1918. Exvivo
evaluation of COVID-19 plasma also noted a prolonged clot lysis time, which was more pronounced among
critically-ill COVID-19 patients19. 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 brinolytic shutdown;
9 of those 11 patients developed thrombosis during their hospitalization20. Elevated PAI-1 levels observed in
COVID-19 patients has further suggested impaired brinolytic ability21. e cause of this brinolytic shutdown
has yet to be elucidated. Here, we aimed to evaluate the potential roles of tPA and PAI-1 in regulating brinolytic
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 brinolytic shutdown while plasma of
others might present a hyper-brinolytic state.
Methods
Human samples. Plasma from 118 patients hospitalized with COVID-19 were used in this study. Blood
was collected into EDTA by a trained phlebotomist. Aer completion of hematological testing ordered by the
clinician, the remaining plasma was stored at 4°C for up to 48h before it was released to the research labora-
tory. Samples were immediately divided into aliquots and stored at -80°C until testing. All 118 patients had a
conrmed COVID-19 diagnosis based on FDA-approved RNA testing. is study complied with all relevant
ethical regulations and was approved by the University of Michigan IRB (HUM00179409). Healthy volunteers
were recruited through a posted yer; exclusion criteria for controls included history of a systemic autoimmune
disease, active infection, and pregnancy. For the COVID-19 samples the University of Michigan Institutional
Review Board waived the requirement for informed consent given the discarded nature of the patient samples.
All healthy controls provided signed informed consent before blood donation (HUM00044257). e 30 controls
included 20 females and 10 males, mean age of 41.7 ± 14.4. All COVID-19 plasma samples were treated with sol-
vent/detergent (0.3% v/v tri-(n-butyl) phosphate and 1% Triton X-100) to inactivate the virus22. Control plasma
samples were similarly treated with the same solvent/detergent.
Measurement of PAI‑1 and tPA antigen. Total PAI-1 and tPA protein was measured as described23.
Briey, 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. e 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 ± stand-
ard 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 capture23.
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. en, 40 µL of 25nM alpha human thrombin (Hae-
mtech) and 15mM CaCl2 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 aer 30min and then at 60-min intervals up to 8h.
Quantication of S100A8/A9 (calprotectin). Calprotectin levels were measured with the Human
S100A8/S100A9 Heterodimer DuoSet ELISA (DY8226-05, R&D Systems) according to the manufacturer’s
instructions.
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, analy-
sis 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 soware version 8. Statistical signicance was dened as p < 0.05.
Results
Tissue‑type plasminogen activator and plasminogen activator inhibitor‑1 in COVID‑19. Uti-
lizing established Luminex platforms, we measured total PAI-1 and tPA levels (detecting both free and com-
plexed 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-ow 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 (me an ± 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). ere was a signicant 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.
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Plasma level of tPA and PAI‑1 and their association with clinical biomarkers. We assessed poten-
tial 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 signicant 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-brinolytic
eects24, we next asked how absolute neutrophil count and calprotectin (a marker of neutrophil activation) com-
pared 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 correla-
tions with neutrophil numbers and activation.
Table 1. Demographic and clinical characteristics of COVID-19 patients. *Mean ± standard deviation (range).
a At time of sample collection. b 22 patients died as a result of acute respiratory distress syndrome from COVID-
19, 1 died as a result of septic shock from a superinfection, and 1 died of necrotizing pancreatitis.
Demographics
Number 118
Age (years)* 61 ± 17 (25–95)
Female 54 (46%)
White/Caucasian 50 (42%)
Black/African-American 51 (43%)
Comorbidities
Diabetes 51 (43%)
Heart disease 37 (31%)
Renal disease 37 (31%)
Lung disease 27 (23%)
Autoimmune 7 (6%)
Cancer 14 (12%)
Obesity 64 (54%)
Hypertension 76 (64%)
Immune deciency 7 (6%)
History of smoking 28 (24%)
Medicationsa
Hydroxychloroquine 19 (16%)
Anti-IL6 receptor 16 (14%)
ACE inhibitor 6 (5%)
Angiotensin receptor blocker 1 (0.8%)
Antibiotic 40 (34%)
Remdesivir 7 (6%)
Dexamethasone 3 (3%)
IV heparin 55 (47%)
Subcutaneous heparin 24 (20%)
Subcutaneous enoxaparin 30 (25%)
Alteplase 0 (0%)
In-hospital thrombosis
Arterial thrombosis 3 (3%)
Venous thrombosis 9 (8%)
Both 1 (0.8%)
Respiratory statusa
Room air 28 (24%)
Nasal Cannula 32 (27%)
High ow oxygen 9 (8%)
Mechanical ventilation 49 (42%)
Awake-prone positioning strategy 35 (30%)
Final outcomes
Discharged 92 (78%)
Deathb24 (20%)
Remains hospitalized 2 (2%)
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Levels of PAI‑1 and tPA associate with severe disease and worse outcomes. Compared with
patients breathing room air, patients requiring oxygen had signicantly higher levels of PAI-1 (p = 0.02) (Fig.3a),
but not tPA or D-dimer (Fig.3b,c). Beyond mode of respiratory support, oxygenation eciency can also be
measured by comparing pulse oximetry (SpO2) to the fraction of inspired oxygen (FiO2). We tested the correla-
tion between PAI-1, D-dimer and SpO2/FiO2 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 oxy-
genation eciency 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. Sig-
nicantly 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 dierence being especially robust for tPA (Fig.3g,h).
Surprisingly, we did not see a signicant dierence in D-dimer levels between those two groups (Fig.3i). In sum-
mary, 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 brinolysis. Finally, we asked whether
COVID-19 plasma with the highest tPA levels might demonstrate enhanced spontaneous brinolysis as com-
pared with low-tPA COVID-19 plasma or control plasma. A spontaneous brinolysis 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
signicantly enhanced spontaneous brinolysis 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). is 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 signicant dierence in age or oxygenation
eciency 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 inuenced by various factors. Normal lung physiol-
ogy has a pro-brinolytic tendency25. However, during acute respiratory distress syndrome (ARDS) impaired
brinolysis results in accumulation of brin that promotes hyaline membrane formation and alveolar injury26.
Fibrin is removed by plasmin. It is believed that depressed brinolysis in ARDS is at least partially driven by
increased circulating PAI-1 that exerts a negative eect on the plasminogen activation system25. Indeed, elevated
PAI-1 is an independent risk factor for poor ARDS outcomes27. Elevated PAI-1 and its associated hypo-brino-
lytic state were observed in the 2002 SARS-CoV epidemic28, while recent characterizations of COVID-19 patients
have suggested impaired global brinolysis18,21. 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 eciency, 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
patients21; however, the mechanistic role of the elevated tPA among COVID-19 patients was not specically
investigated. Furthermore, the level of tPA we detected in the COVID-19 patients, 78 ± 68ng/mL, is striking and
Figure1. High levels of tPA and PAI-1 among patients with COVID-19. (a,b) PAI-1 and tPA were measured
in individuals with COVID-19, or healthy controls. Levels of PAI-1 and tPA were compared by Mann–Whitney
test as samples were not normally distributed when assessed by Shapiro–Wilk test; ****p < 0.0001 as compared
with the control group. Dotted line indicates high tPA cut-o. c, e relationship between tPA and PAI-1 was
assessed by Spearman’s correlation test. Statistics were calculated and the gure was produced in GraphPad
Prism https ://www.graph pad.com/scien tic -sow are/prism /, using version 8.3.
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much higher than the 23.9 ± 14.5ng/mL described in this prior report in 48 patients admitted to ICU21. Notably
these levels are even higher than is observed in trauma patients who have exaggerated brinolytic activity and
in patients with hantavirus cardiopulmonary syndrome, which carries high alveolar hemorrhage risk29–31.
e major source of these high levels of tPA among COVID-19 patients is likely endothelial cells. e source
of PAI-1 could also be the endothelium or perhaps release from activated platelets (as we found a strong cor-
relation between PAI-1 and platelet counts). High PAI-1 expression in other cell types such as macrophages has
also been reported during hantavirus infections29. 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 inammation27. is local proinammatory environment is further exaggerated by the formation
of NETs and results in massive release of proinammatory cytokines6. ose cytokines likely trigger endothelial
cell activation and thereby promote local release of tPA and PAI-16,32. Notably, we observed a strong correlation
Figure2. Association between PAI-1 and tPA and clinical biomarkers in plasma. Levels of PAI-1 and tPA
were compared to D-dimer (a,b), platelet counts (c,d), absolute neutrophil counts (e,f), and calprotectin (g,h).
Spearman’s correlation coecients were calculated. Statistics were calculated and the gure was produced in
GraphPad Prism https ://www.graph pad.com/scien tic -sow are/prism /, using version 8.3.
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Figure3. Association between PAI-1, tPA, D-dimer and respiratory status as well as nal outcomes. (a–c)
COVID-19 patients were grouped by clinical status (room air vs. supplemental oxygen) and analyzed for PAI-1,
tPA, and D-dimer. Level of PAI-1, tPA, and D-dimer were not normally distributed based on Shapiro–Wilk
test. Groups were compared by Mann–Whitney test; *p < 0.05. (d–f) PA1-1, tPA, and D-dimer were compared
to SpO2/FiO2 ratio for each patient, and correlations were determined by Spearman’s test. (g–i) COVID-19
patients were also grouped by nal outcomes (death vs. discharge). Level of PAI-1, tPA and D-dimer were not
normally distributed based on Shapiro–Wilk test. us groups were compared by Mann–Whitney test; *p < 0.05,
***p < 0.001. Statistics were calculated and the gure was produced in GraphPad Prism https ://www.graph pad.
com/scien tic -sow are/prism /, using version 8.3.
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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-111.
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 patients16. Elevated D-dimer was associ-
ated with both thrombotic and bleeding complications16. It has been suggested that high PAI-1 levels overcome
the eects of local tPA and produce a net prothrombotic hypobrinolytic state in COVID-19 patients21. How-
ever, we here found a subset of COVID-19 patients with extremely high levels of tPA (> 100ng/mL) in which
brinolysis seems to dominate. is 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 hos-
pitalization. PAI-1 and tPA levels were therefore not tested on a dened 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 brinolysis regulation and PAI-1
can also inhibit uPA17. Dysregulation of uPA and its receptor system have been implicated in the pathogenesis of
pulmonary brosis and ARDS33,34. e 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 patients2. We would urge cau-
tion regarding this recommendation (pending randomized studies) as the coagulopathy of COVID-19 is complex
and potentially dynamic. erapies aimed at promoting brinolysis, such as administration of aerosolized or
intravenous tPA, have been trialed in ARDS models where there have been some promising preclinical results35,36.
Probrinolytic therapy has been suggested as a potential benecial therapy in COVID-19 patients suering from
ARDS27 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 hyperbrinolytic state exists in some COVID-19 patients. Targeted
therapies that promote brinolysis 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.
Figure4. Spontaneous lysis rate among COVID-19 patients with high and low tPA. e ability of COVID-19
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) e rate of lysis determined from the slope of the absorbance from t0min
to t480min 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 gure was produced in GraphPad Prism https ://www.graph
pad.com/scien tic -sow are/prism /, using version 8.3.
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Received: 23 October 2020; Accepted: 14 December 2020
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Acknowledgements
e work was supported by a COVID-19 Cardiovascular Impact Research Ignitor Grant from the Michigan Medi-
cine Frankel Cardiovascular Center (to JSK and YK), the A. Alfred Taubman Medical Research Institute (to JSK
and YK), and the National Institutes of Health (HL055374 to DAL). YZ was supported by a career development
grant from the Rheumatology Research Foundation. JSK was supported by grants from the NIH (R01HL115138),
Lupus Research Alliance, and Burroughs Wellcome Fund. YK was supported by the NHLBI Intramural Research
Program, and grants from the NIH (ITAC Award, K08HL131993, R01HL150392), .
Author contributions
Y.Z., M.W., A.H., S.Y., K.G., M.Z., and J.A.M. conducted experiments and analyzed data. Y.Z., J.S.K., Y.K., and
D.A.L. conceived the study and analyzed data. All authors participated in writing the manuscript and gave
approval before submission.
Completing interests
Daniel A. Lawrence is a member of the board and holds equity in MDI erapeutics which is developing thera-
peutic inhibitors of PAI-1. All other authors report that they have no conicts of interest.
Additional information
Supplementary Information e online version contains supplementary material available at https ://doi.
org/10.1038/s4159 8-020-80010 -z.
Correspondence and requests for materials should be addressed to D.A.L.
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