Covid-19 severity predictors

Abstract

To provide a COVID-19 severity prediction method and COVID-19 severity prediction reagent by discovering novel COVID-19 severity predictors.SOLUTION: A measurement method involves measuring blood concentration of one or more substances selected from uPA, uPA / PAI-1 complex, and non-complex PAI-1, and using the measurements as severity predictors of a COVID-19 patient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for predicting the severity of COVID-19 and a reagent for predicting the severity of COVID-19. [Background technology]

[0002] Many patients with COVID-19 have abnormalities in the coagulation and fibrinolysis system, and the Japanese Society on Thrombosis and Hemostasis published an article on its website on May 13, 2020, entitled "Warning about the increased risk of thrombosis due to COVID-19 infection" (Non-Patent Document 1). According to this article, the onset of thrombosis and DIC due to coagulation abnormalities are prognostic factors for COVID-19, and it is recommended that anticoagulant therapy be administered, taking into account clinical symptoms, D-dimer values, fibrinogen values, and platelet counts, especially in moderate and severe patients. In addition, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) have been proposed as biomarkers in COVID-19 patients (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Japanese Society on Thrombosis and Hemostasis website (May 13, 2020) [Non-Patent Document 2] SciRep 2021,11,1580 Summary of the Invention [Problem to be solved by the invention]

[0004] However, predicting the severity of COVID-19 in many patients is difficult, and new severity predictors are needed. Therefore, an object of the present invention is to find a new predictor of COVID-19 severity and to provide a method for predicting the severity of COVID-19 and a reagent for predicting the severity of COVID-19. [Means for solving the problem]

[0005] Therefore, the inventors conducted a study on many COVID-19 patients in Japan to find factors that predict the severity of the fibrinolytic system. They unexpectedly found that in patients who become seriously ill, the blood concentrations of urokinase-type plasminogen activator (uPA) and uPA / PAI-1 complexes decrease, while the concentration of uncomplexed PAI-1 increases, and that measuring these concentrations makes it possible to predict the severity of COVID-19 patients, thus completing the present invention.

[0006] That is, the present invention provides the following [1] to [4]. [1] A method for measuring the blood concentration of one or more selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1, wherein the obtained blood concentration is used as an indicator for predicting the severity of COVID-19 patients. [2] The measurement method according to [1], wherein the severity prediction index is an index indicating progression to severe disease when the blood concentration of uPA or the uPA / PAI-1 complex is lower than a predetermined standard value, and an index indicating progression to severe disease when the blood concentration of uncomplexed PAI-1 is higher than a predetermined standard value. [3] A reagent for predicting the severity of COVID-19 patients, comprising one or more blood concentration measurement reagents selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1. [4] A severity prediction reagent according to [3], comprising a protocol for predicting the progression of disease when the blood concentration of uPA or uPA / PAI-1 complex is lower than a predetermined standard value, and predicting the progression of disease when the blood concentration of uncomplexed PAI-1 is higher than a predetermined standard value. Effect of the Invention

[0007] According to the present invention, it is possible to predict whether a COVID-19 patient will develop severe symptoms, and therefore, by treating these factors in a favorable manner, the risk of developing severe symptoms can be reduced. [Brief description of the drawings]

[0008] [Figure 1] Figure 1. Hyperfibrinolysis with increased plasmin / α2-antiplasmin in Japanese COVID-19 patients in the complex phase. (A) Levels of D-dimer in COVID-19 patients in the adaptive clinical phase using ELISA, (B) Western blot analysis of plasminogen, (C) plasmin / α2-antiplasmin complex, and (D) α2-antiplasmin by immunoblot after loading equal amounts of protein in serum COVID-19 patient samples. All Western blots were performed at least twice with similar results. (B-D) Band intensities were quantified using signal intensity and normalized to one sample of a healthy donor. *p<0.1; **p<0.05; ***p<0.01; n / s-not significant difference. [Diagram 2] Circulating levels of free uPA, uncomplexed PAI-1 and uPA / PAI-1 complexes are highest in COVID-19 patients with severe COVID-19. (A) PAI-1 activity was measured in patient and healthy donor serum using a functional ELISA. Western blot analysis was used to assess serum levels of uncomplexed PAI-1 antigen with a molecular weight of 45 kDa (B), tPA with a molecular weight of 70 kDa (C) and uPA with a molecular weight of 50 kDa (E). Quantification of band intensities normalized to samples from healthy donors was performed for tPA / PAI-1 (D) and uPA / PAI-1 (F) complexes detectable as 120 kDa and 100 kDa bands (Figure 3). *p<0.1; **p<0.05; ***p<0.01; n / s; not significant. [Diagram 3] The results of the Western blot used in the test in FIG. 2 are shown. [Figure 4] Correlation analysis of PAs / PAI-1 versus plasmin / α2-antiplasmin (n=36) was performed using Spearman's coefficient r. [Diagram 5]Changes in serum markers of endothelial dysfunction in COVID-19 patients in relation to plasmin activity. Serum soluble VCAM-1 (sVACM) (A) and uPAR (B) were measured using Western blot analysis with quantification of band intensity normalized to samples from healthy donors. n indicates the number of patients tested in each group. *p<0.1; **p<0.05; ***p<0.01; n / s; not significant. [Figure 6] Correlation analysis of serum sVCAM1 and suPAR against PAI-1 activity (n=45) and uncomplexed PAI-1 antigen (n=36) is shown. Spearman coefficient r was used for correlation analysis (C,D). Separate Spearman correlations were also performed for sVCAM1 and suPAR against plasmin / α2 antiplasmin (Pm / α2AP) (n=36 / group) (E,F). n indicates the number of patients tested in each group. *p<0.1; **p<0.05; ***p<0.01; n / s; not significant. [Figure 7] Antigen levels of inflammatory markers and proinflammatory cytokines CRP (A) and IL6 (B) in COVID-19 were measured by ELISA. Serum IL1β (C), TGFβ (D), and TNFα (E) were detected by Western blot analysis, with quantification of band intensity normalized to samples from healthy donors. (F) Fold change expression of TNFα transcript levels measured by qPCR was normalized using β-actin expression in the same samples. Each sample was performed. n indicates the number of patients tested in each group. *p<0.1; **p<0.05; ***p<0.01; n / s; no significant difference. [Figure 8] Correlation of fibrinolytic and inflammatory markers in COVID-19 patients. (A) Heatmap of Spearman correlation coefficients. Pairs with positive coefficients are in brown, and pairs with negative coefficients are in blue. (B) P-values ​​associated with the Spearman correlation for each parameter. [Figure 9]Pearson correlation coefficients for biomarkers in patients with COVID-19. Heatmap correlation coefficients are represented by the change in intensity of orange / blue shown in the color bar, whereby variables shown in green were positively correlated and variables in orange were negatively correlated. Correlations were statistically significant (p<0.05) with 95% CI for each correlation coefficient. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to a method for predicting the severity of COVID-19 and a reagent for predicting said severity. One aspect of the present invention is a method for measuring the blood concentration of one or more selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1, and the obtained blood concentration is used as an indicator for predicting the severity of COVID-19 patients. Another embodiment is a reagent for predicting the severity of COVID-19 patients, comprising one or more blood concentration measurement reagents selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1.

[0010] COVID-19 refers to a novel coronavirus (SARS-CoV2) infection, and COVID-19 patients are those in whom SARS-CoV2 has been detected by PCR or antigen testing. The symptoms of COVID-19 patients are classified into four stages in the SARS-CoV2 treatment guide published by the Ministry of Health, Labor and Welfare: mild (no signs of pneumonia, oxygen saturation 96% or higher), moderate I (dyspnea, no signs of pneumonia, oxygen saturation 93-96%), moderate II (oxygen administration required, oxygen saturation 93% or lower), and severe (intensive care unit, artificial respirator required). The prediction of severity in the present invention refers to the prediction of the stage of these symptoms, for example, predicting that there is a high possibility of developing moderate I, moderate II, or severe, which is different from the severe classification.

[0011] The measurement targets of the present invention, uPA and PAI-1, are both factors constituting the fibrinolytic system. Plasminogen in plasma is activated by tPA or uPA to become plasmin. Plasmin breaks down clotted fibrin and converts it into D-dimer and other breakdown products. On the other hand, PAI-1 is a factor that forms a complex with plasminogen and inactivates it. The present inventors found that when the symptoms of COVID-19 patients become severe, the concentrations of uPA and uPA / PAI-1 complexes in the blood decrease, while the concentration of uncomplexed PAI-1 increases. Changes in the concentrations of these fibrinolytic factors in COVID-19 patients have not been reported to date.

[0012] In the present invention, the concentration of the factor in blood refers to the concentration in whole blood, plasma, or serum, preferably the concentration in plasma or serum, and more preferably the concentration in serum.

[0013] The uPA concentration may be measured by any known means, for example, Western blot, ELISA, immunoassays using magnetic beads, or other immunoassays such as non-enzymatic electrochemical, fluorescent, or colorimetric assays using fluorescent or other detectable probes as detection antibodies.

[0014] Here, Western blotting can be used to detect a 1.50 kDa protein band by (1) non-reducing protein 6-12% polyacrylamide gel electrophoresis in either Tris-glycine, Bis-Tris, Tris-borate, Tricine, or Tris-acetate buffer systems, (2) semi-dry or wet transfer to a PVDF or nitrocellulose membrane, and (3) subsequent blocking with neutral proteins (2-5% skim milk, 0.5-2% serum albumin, diluted serum, or a commercially available blocking solution). Detection is performed using primary unconjugated or conjugated polyclonal or monoclonal antibodies against human uPA (urokinase-type plasminogen activator, also known as urokinase or PLAU) or against other species of uPA with cross-reactivity to human uPA. Enzyme labels (horseradish peroxidase, alkaline phosphatase or glucose oxidase), radioactive or fluorescent labels can be used for antibody conjugation. Secondary conjugated polyclonal or monoclonal antibodies against the host species of the primary antibody coupled with a detection label (horseradish peroxidase, radioactive or fluorescent label) can be used. Visualization of specific protein bands can also be performed depending on the labeling of the primary or secondary antibodies. Enhanced chemiluminescence reagents (luminol-coumaric acid system), enzyme-labeled fluorescent or chromogenic substrates with automated (using a reader) or manual (using X-ray film) signal detection; automated or manual radioactive and fluorescent label signal detection can also be employed. The amount of uPA and / or uPA / PAI-1 complex can be measured using densitometry, calculated as relative optical density normalized to total sample protein concentration, and compared to a standard, where pooled healthy donor plasma or serum with normalized protein concentration can be used as the standard.

[0015] Total uPA protein by ELISA can be detected using polystyrene microtiter plates with preadsorbed coating antibodies with high or medium protein binding capacity blocked with neutral protein solution, detergent or commercial blocking solutions, with a pair of monoclonal or monospecific polyclonal antibodies against different epitopes in the human uPA molecule (urokinase-type plasminogen activator, also known as urokinase or PLAU), or other species of uPA with cross-reactivity to different epitopes in the human uPA molecule, one of which is used as a coating for the unconjugated antibody and the other as a detection antibody conjugated with an enzyme label (horseradish peroxidase, alkaline phosphatase or glucose oxidase). Enzyme-label specific chromogenic or fluorogenic substrates can be used. The reaction can be stopped with a stopping solution (acidic, alkaline, high salt, or inhibitor solution) and can be performed with automated detection by colorimetric or fluorometric instruments. ELISA can also be performed using microfluidic, microchip, or electrochemical devices.

[0016] The concentration of the uPA / PAI-1 complex may be measured by any means known per se, but may also be measured by other immunoassays such as Western blot, ELISA, immunoassays using magnetic beads, non-enzymatic electrochemical, fluorescent, or colorimetric assays using fluorescent or other detectable probes as detection antibodies.

[0017] Western blotting can be used to detect protein bands between 1.90 and 100 kDa by (1) nonreducing protein 6-12% polyacrylamide gel electrophoresis in either Tris-glycine, Bis-Tris, Tris-borate, Tricine, or Tris-acetate buffer systems, (2) semi-dry or wet transfer to a PVDF or nitrocellulose membrane, and (3) subsequent blocking with neutral proteins (2-5% nonfat milk, 0.5-2% serum albumin, diluted serum, or a commercially available blocking solution). Detection is performed with primary unconjugated or conjugated polyclonal or monoclonal antibodies against human uPA (urokinase-type plasminogen activator, also known as urokinase or PLAU) or against other species uPA with cross-reactivity to human uPA. Enzyme labels (horseradish peroxidase, alkaline phosphatase or glucose oxidase), radioactive or fluorescent labels can be used for antibody conjugation. Secondary conjugated polyclonal or monoclonal antibodies against the host species of the primary antibody coupled with a detection label (horseradish peroxidase, radioactive or fluorescent label) can be used. Visualization of specific protein bands is performed depending on the label of the primary or secondary antibody: with enhanced chemiluminescence reagents (luminol-coumaric acid system), with fluorescent or chromogenic substrates of enzyme labels with automatic (using a reader) or manual (using X-ray film) signal detection, and with automatic or manual radioactive and fluorescent label signal detection. The amount of uPA / PAI-1 complex can be measured using densitometry, calculated as the relative optical density normalized to the total protein concentration of the sample, and compared to the uPA / PAI-1 complex of a standard sample, or pooled healthy donor plasma or serum with a normalized protein concentration can be used as a standard sample.

[0018] The ELISA method uses commercially available polystyrene microtiter plates with high or medium protein binding capacity, with preadsorbed coating antibodies and blocked with neutral protein solutions, detergents, or commercial blocking solutions, and is performed with a pair of monoclonal or specific polyclonal antibodies against human uPA (urokinase-type plasminogen activator, also known as urokinase or PLAU) and human PAI-1 (plasminogen activator inhibitor-1, also known as serpin-1); a pair of monoclonal or monoclonal antibodies against other species uPA and PAI-1 with cross-reactivity to human uPA and PAI-1; one of which is used as a non-conjugated coating antibody and the other as a detection antibody conjugated with an enzyme label (horseradish peroxidase, alkaline phosphatase, or glucose oxidase). Enzyme-label specific chromogenic or fluorogenic substrates can be used. The reaction can be optionally stopped with a stopping solution (acidic, alkaline, high salt, or inhibitor solution). Automated detection by colorimetric or fluorometric instruments is also available. ELISAs can also be performed using microfluidic, microchip, or electrochemical devices.

[0019] The concentration of uncomplexed PAI-1 may be measured by any means known per se, for example, by Western blot, ELISA, immunoassays using magnetic beads, or other immunoassays such as non-enzymatic electrochemical, fluorescent, or colorimetric assays using fluorescent or other detectable probes as detection antibodies.

[0020] Western blotting can be performed for detection of a 45 kDa protein band by (1) nonreducing protein 6-12% polyacrylamide gel electrophoresis in either glycine, Bis-Tris, Tris-borate, Tricine, or Tris-acetate buffer systems, (2) semidry or wet transfer to a PVDF or nitrocellulose membrane, and (3) subsequent blocking with neutral proteins (2-5% nonfat milk, 0.5-2% serum albumin, diluted serum, or a commercially available blocking solution). Detection is performed using primary unconjugated or conjugated polyclonal or monoclonal antibodies against human PAI-1 (also known as plasminogen activator inhibitor-1, serpin-1) or other species PAI-1 with cross-reactivity to human PAI-1, enzyme labels (horseradish peroxidase, alkaline phosphatase or glucose oxidase), radioactive or fluorescent labels can be used for antibody binding. Secondary conjugated polyclonal or monoclonal antibodies against the host species of the primary antibody conjugated with a detection label (horseradish peroxidase, radioactive or fluorescent label) can be used. Visualization of specific protein bands is performed depending on the label of the primary or secondary antibody: enhanced chemiluminescence reagents (luminol-coumaric acid system), enzyme-labeled fluorescent or chromogenic substrates with automatic (using a reader) or manual (using X-ray film) signal detection, automatic or manual radioactive and fluorescent label signal detection can be used. Alternatively, the amount of uncomplexed PAI-1 can be measured using densitometry, calculated as the relative optical density normalized to the total sample protein concentration, and compared to the PAI-1 bands of standard samples. Pooled healthy donor plasma or serum with normalized protein concentrations can be used as standards.

[0021] ELISA can be performed using total PAI-1 protein as the detection means, polystyrene microtiter plates with high or medium protein binding capacity, with pre-adsorbed coating antibodies, blocked with neutral protein solutions, surfactants, or commercial blocking solutions, with different monoclonals or pairs of monoclonal antibodies against different epitopes on the human PAI-1 molecule or other species of PAI-1, or PAI-1 with cross-reactivity against different epitopes on the human PAI-1 molecule, with one used as the coating of unconjugated antibodies, and the other used as the detection antibody conjugated with an enzyme label (horseradish peroxidase, alkaline phosphatase, or glucose oxidase). The reaction can be stopped with a stopping solution (acidic, alkaline, high salt, or inhibitor solution) using an enzyme-indicated chromogenic or fluorogenic substrate. Detection can be performed using automated detection with colorimetric or fluorometric instruments. ELISA can also be performed using microfluidic, microchip, or electrochemical devices.

[0022] To predict the severity of the disease using the blood concentrations of the above-mentioned factors as an indicator, it is possible to predict that the disease will become severe when the blood concentration of uPA or the uPA / PAI-1 complex is lower than a specified standard value, and that the disease will become severe when the blood concentration of uncomplexed PAI-1 is higher than a specified standard value. Here, the predetermined reference values ​​can be, for example, the reference values ​​of healthy individuals or the blood concentrations of these factors in patients with mild COVID-19.

[0023] In the reagent for predicting the severity of COVID-19 patients of the present invention, the above-mentioned measurement reagents can be used as the blood concentration measurement reagent for one or more types selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1. The reagent for predicting the severity of COVID-19 patients of the present invention preferably includes a protocol for predicting the progression of the disease when the blood concentration of uPA or the uPA / PAI-1 complex is lower than a predetermined standard value, and predicting the progression of the disease when the blood concentration of uncomplexed PAI-1 is higher than a predetermined standard value.

[0024] According to the present invention, it is possible to predict whether a COVID-19 patient will develop severe symptoms, and therefore, by treating these factors in a favorable manner, the risk of developing severe symptoms can be reduced. EXAMPLES

[0025] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0026] Example 1 (1) Patients and methods (1.1) Study Design, Participants, and Data Collection Patients were Japanese adults hospitalized at Juntendo University Hospital. All study participants gave informed consent for the de-identified use of their clinical data. Inclusion criteria for study participants were adult patients aged 18 years or older, inpatients and outpatients, patients with a positive PCR or rapid antigen test for SARS-CoV2, and patients with basic medical information available, including ethnicity, patient history, initial blood test data, and outcome data. Eight Japanese volunteers (aged 35-57 years) and 12 European volunteers (aged 22-57 years) were recruited as control groups. The clinical stage of COVID-19 at the time of diagnosis was defined as 1) uncomplicated (asymptomatic or with symptoms of upper respiratory tract infection, fever, nausea, or diarrhea), 2) complicated (clinically significant increase in need of supplemental oxygen or history of home oxygen therapy, room oxygen tension (PaO2) <70 mmHg, SO2 <90% on room air, new cardiac arrhythmias, new pericardial effusion >1 cm, pulmonary edema, congestive liver injury, or new heart failure with peripheral edema), or 3) critical (need for life-sustaining therapy such as mechanical ventilation, catecholamine dependency, life-threatening arrhythmias, liver failure with INR >3.5, qSOFA score >+2, or acute renal failure requiring dialysis). Criteria for discharge were absence of fever for ≥3 days, substantial improvement in both lungs, clinical remission of respiratory symptoms, or negative PCR test results for SARS-CoV2 RNA. The patient's blood test results included coagulation / fibrinolysis factors D-dimer (≦1μg / ml), INR (<1.25), fibrinogen (≦400mg / dl), platelet count (120,000-450,000 / μl), as well as inflammatory factors CRP (≦0.3mg / dl), IL6 (≦1.8pg / ml), WBC (4000-8000 / ul), and neutrophil count (2000-8900 / ul). Normal values ​​are listed in parentheses.

[0027] (1.2) Blood collection, serum preparation After whole blood collection, clotting, and centrifugation at 400 g for 10 min, serum samples were collected at room temperature, and undiluted serum was then aliquoted and stored in polypropylene tubes at −80°C for subsequent analysis.

[0028] (1.3) Quantification of PAI-1 activity Activity in freshly thawed serum was measured using a PAI-1 Serpin E1 / PAI-1 Active ELISA kit (Cell Sciences, Newbridge, USA) according to the manufacturer's protocol.

[0029] (1.4) Western blotting Serum levels of plasminogen, tPA, uPA, PAI-1, suPAR, α2-antiplasmin, TGFβ1, TNFα, IL1β, and sVCAM1 were measured by Western blot analysis. Blood serum was mixed with 2xLaemmli Sample Buffer (#1610737, Bio-Rad, Hercules, USA) and boiled for 1 min. Serum samples (20–70 μg protein) were applied to 10–12% acrylamide gels, transferred to PVDF membranes (#IPVH00010, Millipore, Burlington, USA), blocked, and incubated with primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Protein band detection was performed with ECL Prime Western blotting reagent (#RPN2236, Amersham, Amersham, UK) using an image analyzer (Image-Quant LAS4000, GE Healthcare Life Sciences, Chicago, USA). List of antibodies used in the analysis (using working dilutions): PAI-1 rabbit polyclonal antibody (1:1000, #ab66705, Cambridge, UK); tPA rabbit polyclonal antibody (1:sc-15346, Dallas, USA); uPA rabbit polyclonal antibody (1:1000, #1768-1-AP, San Diego, USA); uPAR rabbit polyclonal antibody (1 ug / ml) 10.1182 / blood-2005-3176 {Fietz, 2006 #18707}; Plg rabbit polyclonal antibody (0,5 ug / ml) 10.15407 / Biotechnology 13.050; TGFβ1 rabbit polyclonal antibody (1:1000, #sc-146, Santa Cruz, Dallas, USA), TNFα rabbit polyclonal antibody (Cell 3707S, 1:1000), VCAM1 rabbit polyclonal antibody (1:1000, #sc8304, Santa Cruz, Dallas, USA), IL1β hamster polyclonal FACS Core Facility of Juntendo University (1 ug / ml); secondary rabbit anti-hamster HRP-conjugated (1:2000, #ab5745, Abcam, Cambridge, UK); secondary goat anti-rabbit polyclonal HRP-conjugated (1:8000, #sc2004, Santa Cruz, Dallas, USA). Quantification of protein bands was performed using ImageJ software.

[0030] (1.5) Quantitative reverse transcription polymerase chain reaction (qPCR) Total RNA was extracted from peripheral mononuclear cells of patients using Trizol reagent and reverse transcribed into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit (#4368813, Applied Biosystems, Pleasanton, USA) according to the manufacturer's instructions. cDNA was stored at -30°C. mRNA expression levels were measured by qPCR using QuantStudio 3 real-time PCR system (Applied Biosystems, Pleasanton, USA) with TB Green Premix Ex Taq II (#RR820A, TaKaRa, Shiga, Japan). Relative mRNA expression was calculated using the 2-ΔΔCt method.

[0031] (1.6) Statistical analysis Statistical analysis was performed using Prizm GraphPad 8 for Windows. The Shapiro-Wilk test was performed to verify the distribution of certain variables. Differences between two independent groups were evaluated by Student's t test (for normally distributed variables) or Mann-Whitney test (for variables without normal distribution). Linear correlation tests and Spearman coefficient measures were applied to estimate the association between the tested variables. Regarding heatmap generation: All comparisons were performed using Pearson correlations, except for comparisons using ARDS calculated using Point Biserial Correlation, as its values ​​are categorical (yes / no ARDS occurred). For survival analyses of biomarkers or comorbidities, patients were divided into two groups: those with normal biomarker values ​​(yes), those with pathological biomarker values ​​(no) or with the above comorbidities (yes), or those with the above comorbidities (no).

[0032] (2) Results (2.1) Phenotype and clinical profile of COVID-19 patients Of 69 Japanese SARS-CoV2-positive patients, 43 were defined as uncomplicated and 26 as complicated. No differences were found between the two patient groups in terms of body mass index (BMI), age, sex, and COVID-19-related comorbidities such as heart disease, diabetes, and hypertension. No thrombotic events occurred in any of the 69 patients throughout their hospital stay. Circulating COVID-19 severity markers related to inflammation and coagulation were examined in univariate models at admission. C-reactive protein (CRP), procalcitonin, ferritin, and interleukin 6 (IL6) were higher in the complicated stage (Table 1). Troponin T levels were higher than normal levels, regardless of disease stage. White blood cell counts were in the normal range, whereas lymphocyte counts were almost two-fold lower in the complicated group, but still in the normal range. The coagulation markers D-dimer and fibrinogen were elevated in the complicated cases compared with the uncomplicated phase, but just above the upper limit of normal, confirming previously published results. Other coagulation-related factors, such as platelet counts or prothrombin time (PT INR) in both patient groups, were similar and within the normal range. Overall, moderate coagulation activation with normal extrinsic and common coagulation pathways and mild hypofibrinolysis characterized the Japanese COVID-19 cohort.

[0033] [Table 1]

[0034] Data are n (%). P values ​​were calculated by Fischer's exact test by comparing biomarker values ​​within each country separately, taking into account the clinical phase at diagnosis.

[0035] (2.2) Higher PAI-1 activity in healthy Europeans compared with Japanese subjects BMI-effective PAI-1 levels were higher in Europeans than in Japanese healthy donors (26.28±11.68IU / ml and 20.2±5.2IU / ml, respectively). Serum samples from healthy Japanese subjects were used as a control group for further investigations.

[0036] (2.3) Mild fibrinolytic activation in severe Japanese COVID-19 patients D-dimer levels were increased approximately two-fold in uncomplicated COVID-19 patients compared to healthy controls (1.2–1.9 ng / ml and 1.8–3.8 ng / ml compared to <1 ng / ml) (Figure 1A). No differences were observed in the plasmin precursor molecule plasminogen in blood samples independent of clinical phase, as measured by Western blot (Figure 1B). We found that COVID-19 patients had higher levels of plasmin / α2AP complexes and free α2AP compared with healthy controls (Figure 1C-D), indicating enhanced fibrinolytic activation with disease progression.

[0037] (2.4) PAI-1, uPA, and uPA / PAI-1 concentrations in severe Japanese COVID-19 patients Active PAI-1 concentrations were similar in the serum of COVID-19 patients and healthy donors (Figure 2A). Surprisingly, circulating uncomplexed PAI-1 protein (inactive and active protein) was highest in COVID-19 patients compared to the uncomplicated strata and healthy donors (Figure 2B). Circulating free uncomplexed active PAI-1 can be reversibly converted to an inactive latent form, a proteolytically inactivated free form, or an inactive form after PA binding with the formation of tPA / PAI-1 or uPA / PAI-1 complexes. Serum concentrations of tPA were similar in COVID-19 patients and healthy controls (Figure 2C). Serum concentrations of tPA / PAI-1 complexes were higher in COVID-19 patients compared to healthy controls regardless of clinical phase (Figure 2D). Serum concentrations of uPA were lower in COVID-19 patients than in healthy controls, regardless of clinical phase (Fig. 2E). Strikingly, circulating uPA / PAI-1 protein was downregulated in COVID-19 patients compared to healthy controls, with the lowest uPA / PAI-1 complex levels found in patients with complex disease strata (Fig. 2C, D). These data suggested that circulating PAI-1 was upregulated, whereas uPA and uPA / PAI-1 complexes were downregulated during disease progression. To understand whether these PA changes reflect fibrinolytic impairment, we correlated the plasmin / α2AP complex, a good measure of actual plasmin production and fibrinolysis in blood, with uPA / PAI-1 and tPA / PAI-1. The plasmin / α2AP complex negatively correlated with uPA / PAI-1 but not with tPA / PAI-1 complex levels (Spearman correlation [efficient r s ]=-0.488, vs. [r s ]=0.0068 (Figure 4H, I). This suggests that uPA and hence cellular plasminogen activation contributes to plasmin activity.

[0038] (2.5) Endothelial dysfunction markers in COVID-19 are associated with fibrinolytic activation SARS-CoV2 induces endothelial dysfunction in human pulmonary microvascular endothelial cells (HPMECs). The uPA receptor (uPAR) and the endothelium-associated adhesion molecule VCAM-1 (vascular cell adhesion protein 1) are cleaved from the activated endothelium by pericellular proteases, as well as plasmin, elastase, and matrix metalloproteinases. Soluble uPAR (suPAR) and sVCAM1 released in the serum of COVID-19 patients indicate endothelial dysfunction during COVID-19. Serum concentrations of suPAR and sVCAM1 at admission were higher in COVID-19 patients in the uncomplicated phase compared with healthy controls and were highest in COVID-19 patients in the complicated phase (Figure 5A and B). Increased PAI-1 has been recognized as an early marker of endothelial dysfunction. We next analyzed the correlation between uncomplexed PAI-1 protein, PAI-1 activity, and suPAR and sVCAM. Uncomplexed PAI-1 protein and plasmin / α2AP complex, but not PAI-1 activity, correlated with suPAR and sVCAM (Fig. 6C-F). These data suggest that uncomplexed PAI-1 protein and plasmin / α2AP complexes are good indicators of endothelial dysfunction and that pericellular plasmin contributes to suPAR and sVCAM shedding.

[0039] (2.6) Inflammatory markers (IL1β / 6, CRP, TGFβ) are elevated in critically ill patients, but TNFα is not elevated. Proinflammatory factors such as cytokines (IL1β / 6, TGFβ, and TNFα) can regulate the expression of PAI-1. Similar to other reports, CRP, IL6, and IL1β levels increased with the severity of COVID-19 (Figure 7A-C). Increased TGFβ levels were only observed in patients in the complicated phase compared to the uncomplicated phase and healthy controls (Figure 7D). TNFα and IL1β upregulate PAI-1 synthesis in endothelial cells and lipopolysaccharide, and induce tissue-type plasminogen activator inhibitor in vivo and in cultured endothelial cells. Strikingly, lower TNFα serum concentrations and transcripts in peripheral blood mononuclear cells (PBMCs) were observed in severely ill patients compared to those in the uncomplicated stage (Figures 7E, F). These data showed that PAI-1-associated inflammatory cytokines, except for TNFα, were elevated in critically ill patients.

[0040] To elucidate the correlation of total PAI-1 and uPA / PAI-1 with inflammatory cytokines, Spearman analysis was performed. Total PAI-1 protein showed a similar pattern as D-dimer, but stronger correlations with CRP, IL6, and IL1β (Spearman coefficient r: 0.59, 0.61, 0.69; p<0.001), but not with TGFβ (r=-0.035, p=0.2). The latter showed a moderate correlation with uPA (r=0.42, p=0.02). In contrast, uPA / PAI-1 was negatively correlated with CRP, IL6, and IL1β. The negative correlations of PAI-1 antigen and uPA / PAI-1 with IL6 / 1β and CRP were significant. Our data suggest that PAI-1 in Japanese COVID-19 patients reflects the inflammatory response and is responsible for the low risk of thrombosis in these patients, but showed a weak correlation with IL6 and CRP (r: 0.42 and 0.36; p < 0,01), whereas uPA / PAI-1 complex showed a strong correlation with inflammatory cytokines (Figure 8).

[0041] (2.7) uPA / PAI-1 and uncomplexed PAI-1 are associated with ARDS ARDS in COVID-19 is accompanied by lymphopenia. Our data suggested that uPA and / or uPA-PAI-1 complexes may be novel biomarkers indicative of worsening clinical parameters during COVID-19, such as the development of ARDS or lymphopenia, even in patient cohorts with low D-dimer levels. Point Biserial Correlation analysis was performed to estimate correlations with ARDS, and Pearson correlation analysis was used for numerical parameters (Figure 9). Uncomplexed PAI-1 protein correlated strongly with ARDS and inversely correlated with lymphocytes or TNFα. uPA / PAI-1 complexes, but not tPA-PAI-1 complexes, showed the exact inverse correlation pattern. uPA / PAI-1 complexes correlated negatively with ARDS and positively with lymphocytes. Interestingly, TNFα correlated positively with uPA / PAI-1 complexes and negatively with uncomplexed PAI-1 protein.

[0042] Our data suggest that analysis of uPA, uPA / PAI-1 complex, and uncomplexed PAI-1 antigen should be included in the fibrinolysis status of COVID-19 patients, as it can identify “fibrinolysis-sleep / arrested” patients with low D-dimer levels who rapidly progress to the complicated phase. We speculate that uPA administration may be beneficial for patients in the complicated phase with low uPA / PAI-1 levels. Dysregulation of uPA and its receptor system is involved in the pathogenesis of pulmonary fibrosis and ARDS. It is intriguing that uPA and uPA / PAI-1 levels can predict post-COVID-19 complications such as pulmonary fibrosis. Our data are in line with the notion of reduced cell-associated capacity for plasmin generation by uPA, moderately elevated uncomplexed PAI-1, and increased suPAR shedding promoted by endothelial dysfunction.The significant reduction in circulating uPA and uPA / PAI-1 complex levels, markers of attenuated pericellular fibrinolysis, may represent novel biomarkers of COVID-19 severity. These findings suggest that PAI-1 may play a role in the development of complications in COVID-19, such as blood clots and lung injury, and suggest that measurement of uncomplexed and active PAI-1 levels, as well as PAI-1 complexes, may serve as biomarkers for disease severity and prognosis.

Claims

1. A method for measuring the blood concentration of one or more selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1, wherein the obtained blood concentration is used as an indicator for predicting the severity of a COVID-19 patient.

2. The method according to claim 1, wherein the severity prediction index is an index indicating progression to severe disease when the blood concentration of uPA or the uPA / PAI-1 complex is lower than a predetermined reference value, and an index indicating progression to severe disease when the blood concentration of uncomplexed PAI-1 is higher than a predetermined reference value.

3. A reagent for predicting the severity of COVID-19 patients, comprising a reagent for measuring the blood concentration of one or more types selected from uPA, uPA / PAI-1 complex, and uncomplexed PAI-1.

4. The severity prediction reagent according to claim 3, comprising a protocol for predicting aggravation when the blood concentration of uPA or the uPA / PAI-1 complex is lower than a predetermined reference value, and predicting aggravation when the blood concentration of uncomplexed PAI-1 is higher than a predetermined reference value.

Images