Circulating Levels of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 Are Independent Predictors of Coronavirus Disease 2019 Severity: A Prospective, Observational Study

Abstract

A substantial pool of evidence suggests that the pathophysiology of the severe form of coronavirus disease 2019 (COVID-19) is mediated, in part, by a hypercoagulable state termed COVID-19-associated coagulopathy (CAC),[1] [2] [3] and characterized by micro- and macrovascular thrombosis.[4] [5] Approximately 27% of critically ill intensive care unit (ICU)-admitted COVID-19 patients develop venous thromboembolism during admission,[6] while 4.4% experience arterial thrombotic complications.[7] Autopsy studies on COVID-19 patients have demonstrated extensive presence of microthrombi within pulmonary capillaries, associated with diffuse alveolar damage and neoangiogenesis.[8] [9] Other organs may also be affected by this form of thrombotic microangiopathy, resulting in multiple organ dysfunction syndrome and eventually death.[10] Laboratory profiles seen in patients with severe COVID-19 (elevated D-dimers and fibrin degradation products [FDPs], and prolonged prothrombin time) are also consistent with a hypercoagulable state.

The role of the fibrinolytic system in CAC is not yet well established. It has been hypothesized that patients with severe COVID-19 have elevated levels of antifibrinolytic proteins, which create a hypofibrinolytic state, with subsequent failure of clot lysis. This hypothesis is supported by a recent thromboelastographic study by Wright and colleagues where a complete lack of lysis of clot at 30 minutes was reported in approximately 57% of ICU-admitted COVID-19 patients.[11] Henry and colleagues recently demonstrated that ICU-admitted COVID-19 patients had lower levels of circulating plasminogen than non-ICU patients, suggesting an exhaustion of fibrinolysis.[12] Recent small clinical studies have also demonstrated potential therapeutic effects of supplementing the fibrinolytic system, where exogenous administration of tissue plasminogen activator (tPA) resulted in improvement of respiratory function and reduced mortality in critically ill COVID-19 patients with acute respiratory distress syndrome (ARDS).[13] [14]

Herein, we performed a prospective observational study to determine whether circulating plasma levels of plasminogen activator inhibitor-1 (PAI-1) and tPA may be significantly elevated in patients with severe COVID-19. We additionally determined the diagnostic performance of baseline PAI-1 and tPA for predicting progression to severe disease.

Adults (>18 years old) presenting to the University of Cincinnati Medical Center Emergency Department (ED) with symptoms of COVID-19 with a positive reverse transcription-polymerase chain reaction test for COVID-19 on nasopharyngeal swab were included. This study was approved by the Institutional Review Board of the University of Cincinnati. Blood samples were collected via routine draws for clinical indications in the ED. Circulating levels of tPA and PAI-1 were determined using enzyme-linked immunosorbent assay (Technozym, Diapharma, West Chester, OH). The clinical course of these patients was then monitored for 30 days. The severity of COVID-19 at the time of presentation to the ED was the primary outcome of this study, while peak severity of COVID-19 within 30 days of index visit to the ED or hospital discharge was the secondary outcome. Severe disease was defined as a composite of (1) partial pressure of oxygen in arterial blood/fraction of inspired oxygen ≤300 mm Hg or (2) patients requiring noninvasive ventilation using high flow nasal devices/mechanical ventilation/vital life support/ICU admission, or admission to the ICU.

Analysis of the data was performed using Prism 7 (GraphPad Software, San Diego, CA) and SPSS (IBM Statistics Software Version 25, Armonk, NY). Categorical data were reported as frequencies (%) while continuous data were reported as median and interquartile range (IQR). Comparison of baseline tPA and PAI-1 levels between patients with and without severe COVID-19 at the time of index ED visit was performed using the Mann–Whitney U-test. The diagnostic performance of baseline tPA and PAI-1 for predicting peak 30-day severity of COVID-19 was assessed using receiver operating characteristic (ROC) curve, with calculation of the area under the curve (AUC) and its 95% confidence interval (95% CI). Logistic regression was performed to estimate the effect of elevated tPA and PAI-1 (≥4 and >52.3 ng/mL respectively, based on ROC curve analysis) on the secondary outcome of peak 30-day severity of COVID-19, adjusting for age, sex, and comorbidities, and to calculate adjusted odds ratios (ORs) with the corresponding 95% Wald CI. Additionally, correlation between circulating levels of tPA/PAI-1 and inflammatory biomarkers (interleukin [IL]-6, IL-10, tumor necrosis factor α [TNFα], ferritin and C-reactive protein [CRP]) as well as platelet counts was performed using Spearman's correlation.

A total of 52 patients were included in this study. Of these, 46 had nonsevere disease at the time of index ED presentation, while six had severe disease. The median age in the severe group was 57 (IQR: 47–67) versus 50.5 (IQR: 37.8–66) years in the nonsevere group (p = 0.445). Males made up 83% of the severe group and 54.3% of the nonsevere group (p = 0.181). The comorbidities of these patients are presented in [Fig. 1A]. Patients with severe disease had significantly higher levels of PAI-1 than those with the nonsevere disease (115.6 [IQR: 49.5–357] vs. 47.6 [IQR: 27.9–88.3] ng/mL; p = 0.045) ([Fig. 1B]). Circulating tPA levels were also elevated in patients with severe disease compared with those with nonsevere disease, although this did not achieve statistical significance (4.7 [IQR: 2.4–6.3] vs. 2.3 [IQR: 1.2–4.0] ng/mL; p = 0.055) ([Fig. 1C]). A total of 16 patients developed severe disease within 30 days of index ED presentation. ROC curves were generated for tPA and PAI-1 at the time of initial ED evaluation for predicting peak disease severity within 30 days of index ED visit. The AUC of tPA was 0.73 (95% CI: 0.58–0.89; p = 0.008). Analysis of ROC data determined that a tPA cut-off of ≥4 ng/mL was associated with 0.56 sensitivity and 0.83 specificity, respectively ([Fig. 1D]). The AUC for PAI-1 was 0.63 (95% CI: 0.47–0.79; p = 0.142), with ≥52.3ng/mL cut-off displaying 0.69 sensitivity and 0.58 specificity ([Fig. 1E]). Multivariable logistic regression (adjusted for age, sex, and comorbidities) for tPA and PAI-1 levels above the ROC cut-offs (i.e., ≥4 and ≥52.3 ng/mL, respectively) as independent predictors of peak 30-day severity revealed that elevated tPA and PAI-1 levels were associated with increased odds of developing severe disease (OR = 6.67, 95% CI: 1.42–31.21, p = 0.016 and OR = 7.79, 95% CI: 1.34–45.21, p = 0.022, respectively) ([Table 1]). Circulating tPA positively correlated with IL-10 (r = 0.431, 95% CI: 0.168–0.637; p = 0.002), TNFα (r = 0.424, 95% CI: 0.159–0.631; p = 0.002), serum ferritin (r = 0.471, 95% CI: 0.219–0.664; p < 0.001), and lactate dehydrogenase (r = 0.443, 95% CI: 0.183–0.641; p < 0.001), while PAI-1 positively correlated with IL-10 (r = 0.318, 95% CI: 0.038–0.551; p = 0.023), TNFα (r = 0.310, 95% CI: 0.029–0.546; p = 0.027), and platelet counts (r = 0.347, 95% CI: 0.061–0.058; p = 0.016) ([Table 2]).