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SCUBE1 is associated with thrombotic complications, disease severity, and in-hospital mortality in COVID-19 patients

      Abstract

      Introduction

      COVID-19 disease, which has recently become an important cause of mortality and morbidity all over the world, is remarkably associated with thrombotic complications. Although many factors are responsible for these increased thrombotic complications in COVID-19 disease, its relationship with a marker that increases the risk of thrombosis such as Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1) has not yet been clarified. This is the first study to examine the potential diagnostic and prognostic value of SCUBE1 levels in patients with COVID-19. In this study, we aimed to clarify the relationship between the increased risk of thrombosis and SCUBE1 in the course of COVID-19 disease.

      Materials and methods

      553 patients with COVID-19 and 553 healthy controls were compared in terms of SCUBE1 levels. Additionally, patients with COVID-19 were divided into two groups according to their SCUBE1 levels and compared in terms of severity of disease, thrombotic complications and ın-hospital mortality.

      Results

      SCUBE1 levels were significantly higher in patients with COVID-19 compared to the control group (p < 0.001). Plasma SCUBE1 levels were significantly higher in patients with severe disease and thrombotic complications, those with mild to moderate disease, and those without thrombotic complications (p < 0.001, for both). In addition, SCUBE1 was found to be an independent predictor of in-hospital mortality (p < 0.001).

      Conclusions

      SCUBE1 may be one of the major determinants of thrombotic complications, which is an increased cause of mortality and morbidity in COVID-19 patients so inhibition of this peptide may be among the therapeutic targets in patients with COVID-19.

      Keywords

      1. Introduction

      COVID-19 disease is a systemic disease that has recently caused major mortality and morbidity all over the world [
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      ]. Among these, thrombotic complications can be observed at an increased rate even during the course of the disease or even after the disease has survived [
      • Gómez-Mesa J.E.
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      ]. Although many pathophysiological mechanisms have been blamed for this predisposition, the exact cause has not been fully elucidated [
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      ]. Recent studies have shown that vasculopathy accompanied by platelet and endothelial dysfunctions plays a key role in susceptibility to thrombosis during the course of COVID-19 disease [
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      • Zhang S.
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      • Dong J.
      • Yuan Z.
      • Ding Z.
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      ]. However, the relationship between this pro-thrombotic state in COVID-19 patients and SCUBE1 (signal peptide-CUB-epidermal growth factor [EGF] domain-containing protein 1) levels, which have been closely associated with pro-thrombotic states in previous studies, has not been studied so far [
      • Liao W.J.
      • Wu M.Y.
      • Peng C.C.
      • Tung Y.C.
      • Yang R.B.
      Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation.
      ,
      • Dai D.F.
      • Thajeb P.
      • Tu C.F.
      • Chiang F.T.
      • Chen C.H.
      • Yang R.B.
      • Chen J.J.
      Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.
      ].
      SCUBE1 is a cell surface glycoprotein expressed and secreted from platelet and endothelial cells during early embryogenesis [
      • Tu C.F.
      • Yan Y.T.
      • Wu S.Y.
      • Djoko B.
      • Tsai M.T.
      • Cheng C.J.
      • Yang R.B.
      Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.
      ]. When SCUBE1 is overexpressed, it can be secreted into the conditioned medium as a soluble protein or bound to the cell surface as a peripherally associated membrane protein [
      • Yang R.B.
      • Ng C.K.
      • Wasserman S.M.
      • Colman S.D.
      • Shenoy S.
      • Mehraban F.
      • Komuves L.G.
      • Tomlinson J.E.
      • Topper J.N.
      Identification of a novel family of cell-surface proteins expressed in human vascular endothelium.
      ]. SCUBE1 binds to the plasma membrane by electrostatic and lectin-glycan interactions and is responsible for different functions depending on soluble or membrane protein distribution [
      • Tu C.F.
      • Yan Y.T.
      • Wu S.Y.
      • Djoko B.
      • Tsai M.T.
      • Cheng C.J.
      • Yang R.B.
      Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.
      ,
      • Yang R.B.
      • Ng C.K.
      • Wasserman S.M.
      • Colman S.D.
      • Shenoy S.
      • Mehraban F.
      • Komuves L.G.
      • Tomlinson J.E.
      • Topper J.N.
      Identification of a novel family of cell-surface proteins expressed in human vascular endothelium.
      ,
      • Tu C.F.
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      • Li L.T.
      • Chen Y.L.
      • Cheng C.J.
      • Dai D.F.
      • Yang R.B.
      Localization and characterization of a novel secreted protein SCUBE1 in human platelets.
      ,
      • Liao W.J.
      • Tsao K.C.
      • Yang R.B.
      Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.
      ]. Besides endothelial cells, SCUBE1 is also produced and stored in thrombocyte, is released on the cell surface, undergoes proteolysis upon activation by adenosine diphosphate (ADP), thrombin or collagen stimulation, and is incorporated into the thrombus [
      • Wu M.Y.
      • Lin Y.C.
      • Liao W.J.
      • Tu C.F.
      • Chen M.H.
      • Roffler S.R.
      • Yang R.B.
      Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.
      ,
      • Dai D.F.
      • Thajeb P.
      • Tu C.F.
      • Chiang F.T.
      • Chen C.H.
      • Yang R.B.
      • Chen J.J.
      Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.
      ,
      • Tu C.F.
      • Yan Y.T.
      • Wu S.Y.
      • Djoko B.
      • Tsai M.T.
      • Cheng C.J.
      • Yang R.B.
      Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.
      ,
      • Yang R.B.
      • Ng C.K.
      • Wasserman S.M.
      • Colman S.D.
      • Shenoy S.
      • Mehraban F.
      • Komuves L.G.
      • Tomlinson J.E.
      • Topper J.N.
      Identification of a novel family of cell-surface proteins expressed in human vascular endothelium.
      ,
      • Tu C.F.
      • Su Y.H.
      • Huang Y.N.
      • Tsai M.T.
      • Li L.T.
      • Chen Y.L.
      • Cheng C.J.
      • Dai D.F.
      • Yang R.B.
      Localization and characterization of a novel secreted protein SCUBE1 in human platelets.
      ]. It is thought that plasma SCUBE1 actively participates in platelet aggregation by bridging activated platelets that come together and aggregate during thrombus formation [
      • Wu M.Y.
      • Lin Y.C.
      • Liao W.J.
      • Tu C.F.
      • Chen M.H.
      • Roffler S.R.
      • Yang R.B.
      Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.
      ]. It has been shown in experimental studies that soluble SCUBE1 is a biomarker for platelet activation and also an active participant in thrombosis, and plays a critical role in arterial thrombosis through its in vivo adhesive EGF-like repeats of platelet-derived SCUBE1 [
      • Liao W.J.
      • Wu M.Y.
      • Peng C.C.
      • Tung Y.C.
      • Yang R.B.
      Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation.
      ,
      • Wu M.Y.
      • Lin Y.C.
      • Liao W.J.
      • Tu C.F.
      • Chen M.H.
      • Roffler S.R.
      • Yang R.B.
      Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.
      ]. In a clinical study, it was shown that the plasma SCUBE1 level released from activated platelets is significantly increased in acute coronary syndrome and acute ischemic stroke, and therefore SCUBE1 may be a biomarker of platelet activation [
      • Dai D.F.
      • Thajeb P.
      • Tu C.F.
      • Chiang F.T.
      • Chen C.H.
      • Yang R.B.
      • Chen J.J.
      Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.
      ].
      Both the increased prothrombogenic environment in COVID-19 patients and the active role of SCUBE1 in platelet activation made us think that there may be a relationship between COVID-19 and SCUBE1. Since the relationship between COVID-19 and SCUBE1 has not been examined before, we aimed to reveal the possible relationship between them in this study.

      2. Materials and methods

      2.1 Study design and subjects

      In this observational, prospective study, 553 patients hospitalized for COVID-19 disease between May 2020 and December 2021, and 553 healthy volunteers matched for age and sex who did not have COVID-19 disease, who applied to the outpatient clinic for routine check-ups as the control group, were included. In-hospital mortality and thrombotic complications of patients were recorded.
      During the course of the disease, 76 patients requiring intensive care unit (ICU) follow-up were evaluated in the severe disease category, and 477 patients requiring hospitalization and clinical service follow-up were evaluated in the mild to moderate disease category. Patients who use antiaggregant or anticoagulant for any reason, have any previous history of thrombotic complications, have had COVID-19 vaccine, history of active cancer or take chemotherapeutic drugs, genetically known coagulation disorders, hematologic and endocrine disorders, autoimmune diseases and chronic inflammatory diseases, advanced kidney or liver failure, and patients who could not obtain consent were excluded from the study. Baseline demographic and clinical characteristics were recorded for all patients and the control group. The study was approved by the local ethics committee and was performed in accordance with the Declaration of Helsinki (date: 05.04.2020, no: 20/05/02).

      2.2 Biochemical measurements

      For SCUBE1 measurement, venous blood samples were taken from the patients and control group at the time of admission, before any treatment was started. Blood samples were collected into tubes containing K2EDTA or SST™ II as an anticoagulant and serum separator. Plasma and serum specimens were obtained after centrifugation at 2500 ×g for 10 min. Routine biochemistry analyses, complete blood counts and coagulation tests were carried out on blood samples. Complete blood counts were assessed by an automated blood cell counter (Coulter LH 780 Hematology Analyzer, Beckman Coulter Corp, Hileh, Florida). Serum creatinine and blood urea nitrogen levels were measured using the Architect Plusci 4100 m (Abbott Laboratories, Abbott Park, Illinois, USA). D-dimer serum levels were measured using an STA Compact analyzer (Houston, USA). The reference range of D-dimer is 0–0.55 mg/L and results are expressed in mg/L. Serum samples for SCUBE1 analysis were frozen, and kept at −80 °C until testing. SCUBE1 levels were measured by using commercial SCUBE1 Enzyme-Linked Immunosorbent Assay (ELISA) kit (Molecular Devices, Sunnyvale, CA, USA) according to the manufacturer's instructions. The absorbance of samples was measured at 450 nm on a VERSA max tunable microplate reader. Results are presented in ng/mL for SCUBE1; intra-assay and inter-assay coefficients of variation were < 10 %.

      2.3 Diagnostic and treatment modalities

      The diagnosis was made in accordance with current guidelines according to clinical signs, reverse transcription-polymerase chain reaction (RT-PCR), chest computed tomography findings, IgM and IgG antibody tests [
      • Jin Y.H.
      • Zhan Q.Y.
      • Peng Z.Y.
      • Ren X.Q.
      • Yin X.T.
      • Cai L.
      • et al.
      Evidence-Based Medicine Chapter of China International Exchange and Promotive Association for Medical and Health Care (CPAM); Chinese Research Hospital Association (CRHA). Chemoprophylaxis, diagnosis, treatments, and discharge management of COVID-19: An evidence-based clinical practice guideline (updated version).
      ]. Patients were categorized as mild, moderate and severe illness according to their present symptoms as follows; Mild illness: Individuals who have any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell) but who do not have shortness of breath, dyspnea, or abnormal chest imaging; Moderate illness: Individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have an oxygen saturation measured by pulse oximetry (SpO2) ≥94 % on room air at sea level; Severe illness: Individuals who have SpO2 < 94 % on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2 /FiO2) <300 mmHg, a respiratory rate > 30 breaths/min, or lung infiltrates >50 %. Those with mild and moderate illness were followed up in the inpatient service, and those with severe illness were followed up in the intensive care unit [
      • Disease Coronavirus
      (COVID-19) Treatment Guidelines [Internet].
      ].
      We diagnosed disseminated intravascular coagulation (DIC) based on low platelet count, high D-dimer concentration, decreased fibrinogen concentration, and prolongation of clotting times such as prothrombin time (PT) [
      • Levi M.
      Diagnosis and treatment of disseminated intravascular coagulation.
      ]. ST elevation myocardial infarction (STEMI) is diagnosed if there is >1–2 mm of ST elevation in two contiguous leads on the electrocardiography (ECG) or new left bundle branch block (LBBB) with a clinical picture consistent with ischemic chest pain [
      • Ibanez B.
      • James S.
      • Agewall S.
      • Antunes M.J.
      • Bucciarelli-Ducci C.
      • Bueno H.
      • et al.
      ESC Scientific Document Group. 2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC).
      ]. Non-ST elevation myocardial infarction (NSTEMI) is diagnosed through elevated levels of creatine kinase-myocardial band (CK-MB), troponin I, and troponin T and an ECG [
      • Roffi M.
      • Patrono C.
      • Collet J.P.
      • Mueller C.
      • Valgimigli M.
      • Andreotti F.
      • et al.
      ESC scientific document group. 2015 ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC).
      ]. The diagnosis of pulmonary embolism was diagnosed by detecting a filling defect in the pulmonary arteries on computed tomography (CT) pulmonary angiography [
      • Konstantinides S.V.
      • Meyer G.
      • Becattini C.
      • Bueno H.
      • Geersing G.J.
      • Harjola V.P.
      • et al.
      The task force for the diagnosis and management of acute pulmonary embolism of the european Society of Cardiology (ESC). 2019 ESC guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the European Respiratory Society (ERS): the task force for the diagnosis and management of acute pulmonary embolism of the european Society of Cardiology (ESC).
      ]. Duplex ultrasonography was used as the standard imaging test to diagnose deep vein thrombosis (DVT) [
      • Streiff M.B.
      • Agnelli G.
      • Connors J.M.
      • Crowther M.
      • Eichinger S.
      • Lopes R.
      • et al.
      Guidance for the treatment of deep vein thrombosis and pulmonary embolism.
      ]. CT venography (CT-V) protocol was used in the diagnosis of cerebral vein thrombosis [
      • Rodallec M.H.
      • Krainik A.
      • Feydy A.
      • Hélias A.
      • Colombani J.M.
      • Jullès M.C.
      • Marteau V.
      • Zins M.
      Cerebral venous thrombosis and multidetector CT angiography: tips and tricks.
      ]. The treatment of the patients was arranged according to the current guidelines [
      • Disease Coronavirus
      (COVID-19) Treatment Guidelines [Internet].
      ]. Antithrombotic medication was started in all patients during hospitalization in accordance with current guidelines [
      • Disease Coronavirus
      (COVID-19) Treatment Guidelines [Internet].
      ].

      2.4 Prospective follow-up and primary end point

      In-hospital mortality and thrombotic complications of patients were recorded. All-cause mortality was considered the primary end point.

      2.5 Statistical analysis

      Statistical Program for Social Sciences 26 (IBM SPSS, Chicago, IL, USA) was used for all statistical calculations. Kolmogorov-Smirnov test was used to determine whether the data fit the normal distribution. Continuous variables that fit the normal distribution were expressed as means ± standard deviation (SD), and those that did not fit the normal distribution were expressed as median with interquartile range (IQR). Comparisons between groups were analyzed using the Mann-Whitney U test, independent t-test and one-way analysis of variance (ANOVA),where appropriate. Chi-square test was applied to categorical variables. Pearson correlation coefficient was used to determine the relationship between SCUBE1 levels and other variables. Multivariate regression analyses were performed to determine the independent predictors of presence of thrombotic complications. Baseline variables with significant significance (p < 0.05) by univariate analysis were included in the multivariate logistic regression analysis.
      To determine the predictive value of SCUBE1 in predicting thrombotic complications and in-hospital mortality, a pairwise comparison with D-dimer and fibronogen was performed by performing Receiver operating characteristic (ROC) analysis. The predictive validities were quantified as the area under the ROC curve (c statistics), and these comparisons were done using MedCalc 16 statistic software (MedCalc Software Ltd., Ostend, Belgium). The optimal cut-off value was calculated from the point of maximum sensitivity and specificity (Youden's index). Also, patients' baseline characteristics were classified as lower SCUBE1 and higher SCUBE1 based on their admission SCUBE1 level. The median value of 2.30 ng/mL of SCUBE1 levels of patients with COVID-19 was taken as the cut-off value to categorize patients according to SCUBE1 levels.
      The cumulative events of these two groups were evaluated using Kaplan-Meier curves and compared using the log-rank test.Overall survival time was calculated to the in-hospital day of death or the date of last discharge. In addition, univariate and multivariate Cox proportional hazards model analysis was used to evaluate the relationship between admission SCUBE1 levels and all-cause in-hospital deaths. Five models were created to show the effects of possible confounders on the relationship between SCUBE1 level and in-hospital mortality. The model 1 was unadjusted. Model 2 was adjusted for sex and age. Based on model 2, model 3 was further adjusted for status of smoking, history of diabetes mellitus, hypertension, dyslipidemia. Based on model 3, model 4 was further adjusted for white blood cell, lymphocyte, neutrophil, phosphorus and platelet. Based on model 4, model 5 was further adjusted for D-dimer and fibrinogen. Results of Cox regression analysis was presented as hazard ratio (HR) and 95 % confidence interval (CI). Two-tailed p-values of <0.05 were considered to be statistically significant.

      3. Results

      553 patients who had COVID-19 and were hospitalized and 553 healthy subjects were included in the study as the control group (mean age 62.0 ± 12.7, 71.0 % male; 61.7 ± 12.3 years, 66.5 % male; respectively). These two groups were compared in terms of baseline demographic, clinical, biochemical characteristics and SCUBE1 levels (Table 1). Cardiovascular comorbidities, dyslipidemia, and smoking were more common in the COVID-19 group compared to the control group (p = 0.004, p = 0.001; respectively). In the group with COVID-19, fasting plasma glucose (FPG), lactate dehydrogenase (LDH), C-protein (CRP), white blood cell (WBC), neutrophil, platelet counts, fibrinogen, D-dimer and SCUBE1 levels were higher than in the control group, in addition, the estimated glomerular filtration rate (e-GFR), lymphocyte, phosphorus, calcium and hemoglobin levels were lower (p < 0.05, for all). In the patient group, hypertension was more common in the higher SCUBE1 group than in the lower SCUBE1 group (p = 0.026) and WBC, neutrophil counts and fibrinogen, D-dimer levels were higher, lymphocyte, platelet counts and phosphorus levels were lower (p < 0.05, for all).
      Table 1Demographic, clinical and biochemical characteristics of COVID-19 patients and control group.
      VariablesControl Group

      (n = 553)
      COVID -19 Group

      (n = 553)
      p value
      Demographics and medical history
      Age, years61.7 ± 12.362.0 ± 12.70.662
      Gender,male,n (%)368 (66.5)393 (71.0)0.174
      BMI, kg/m224.34 ± 3.0424.45 ± 3.910.290
      Diabetes mellitus, n (%)73 (13.2)93 (16.8)0.102
      Hypertension, n (%)317 (57.3)344 (62.2)0.098
      Dyslipidemia, n (%)401 (72.5)442 (79.9)0.004
      Smoking, n (%)261 (47.1)315 (56.9)0.001
      Laboratory results
      FPG, (mg/dL)122.6 (118.4–127.5)132.3 (127.1–138.4)<0.001
      Creatinine, (mg/dL)0.85 ± 0.300.88 ± 0.370.166
      Uric acid, (mg/dL)5.48 ± 2.685.22 ± 1.530.054
      Albumin, (mg/dL)4.49 ± 3.754.23 ± 1.670.143
      LDH, (mg/dL)250.0 (202.0–330.0)262 (225.0–351.0)0.003
      Triglycerides, (mg/dL)151.0 (108.0–216.0)139.0 (86.5–180.0)0.078
      TC, (mg/dL)183.0 (149.0–217.0)175.0 (141.0–201.0)0.609
      HDL-C, (mg/dL)35.0 (30.0–41.0)35.0 (29.0–41.0)0.609
      LDL-C, (mg/dL)111.5 (84.0–137.0)107.0 (83.0–130.0)0.176
      CRP, (mg/dL)0.57 (0.46–0.76)1.55 (1.30–1.83)<0.001
      e-GFR, (ml/min)91.12 ± 18.0986.77 ± 19.88<0.001
      WBC, (×1000/mm3)10.1 (8.2–13.5)10.2 (7.6–14.1)0.028
      Lymphocyte, (×1000/mm3)2.50 (2.20–2.87)1.40 (1.13–1.70)<0.001
      Monocytes, (×1000/mm3)0.57(0.53–0.60)0.57 (0.40–0.72)0.323
      Neutrophil, (×1000/mm3)5.90 (4.44–7.80)6.75 (4.41–10.07)<0.001
      Phosphorus, (mg/dL)3.20 (2.80–3.70)3.10 (2.60–3.60)0.001
      Calcium, (mg/dL)9.20 (8.90–9.60)9.0 (8.61–9.38)<0.001
      Hemoglobin, (mg/dL)14.2 ± 2.7813.80 ± 1.900.001
      Hematocrit, (%)44.01 ± 7.9943.09 ± 9.150.076
      Platelet count, (×1000/mm3)261.0 (224.0–308.0)288.0 (217.5–363.0)<0.001
      Fibrinogen, (mg/dL)163.0 (154.0–228.8)288.0 (241.0–350.0)<0.001
      D-dimer, mg/L1.10 (0.21–1.55)2.10 (1.50–2.80)<0.001
      SCUBE1, ng/mL0.70 (0.18–1.47)2.30 (1.56–2.84)<0.001
      Values are mean ± SD, n (%), or median (interquartile range) unless otherwise stated.
      Abbreviations: BMI: body mass index; CRP: C- reactive protein; FPG: Fasting plasma glucose; eGFR: estimated glomerular filtration rate; TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; WBC: White blood cell.
      In addition, the higher SCUBE1 group had more severe cases than the lower SCUBE1 group (52 [18.7 %] vs. 24 [8.6 %]; p = 0.001) (Table 2).
      Table 2Demographic, clinical and biochemical characteristics of patients with COVID-19 according to SCUBE1 levels.
      VariablesGroups of SCUBE1p-Value
      Lower SCUBE1

      (≤2.30 ng/mL)

      (n:276)
      Higher SCUBE1

      (>2.30 ng/mL)

      (n:277)
      Demographics and medical history
      Age, years61.50 ± 12.6662.67 ± 12.780.196
      Gender,male,n (%)194 (70)199 (72)0.687
      BMI, kg/m224.76 ± 2.9825.59 ± 4.110.158
      Diabetes mellitus, n (%)39 (14.1)54 (19.4)0.092
      Hypertension, n (%)159 (57.6)185 (66.7)0.026
      Dyslipidemia, n (%)226 (81.8)216 (77.9)0.252
      Smoking, n (%)139 (50.3)176 (63.5)
      Laboratory results
      FPG, (mg/dL)114.0 (94.0–141.0)117.0 (100.0–143.0)0.210
      Creatinine, (mg/dL)0.90 ± 0.460.86 ± 0.240.204
      Uric acid, (mg/dL)5.27 ± 1.585.17 ± 1.480.262
      Albumin, (mg/dL)4.33 ± 2.334.13 ± 0.430.085
      LDH, (mg/dL)261.0 (220.5–344.5)262.0 (225.0–378.0)0.249
      Triglycerides, (mg/dL)126.0 (90.25–183.0)115.0 (84.0–176.0)0.097
      TC, (mg/dL)174.0 (140.0–199.0)176.0 (144.0–203.0)0.549
      HDL-C, (mg/dL)35.0 (30.0–40.7)35.0 (28.5–41.0)0.909
      LDL-C, (mg/dL)105.4 (81.0–131.7)109.0 (86.0–129.0)0.653
      CRP, (mg/dL)0.49 (0.15–1.06)0.54 (0.16–2.28)0.013
      e-GFR, (ml/min)86.41 ± 20.5687.13 ± 19.210.862
      WBC, (×1000/mm3)10.0 (7.4–14.2)10.7 (8.40–14.4)0.016
      Lymphocyte, (×1000/mm3)1.65 (1.25–1.80)1.30 (0.98–1.57)<0.001
      Monocytes, (×1000/mm3)0.55 (0.40–0.72)0.58 (0.40–0.73)0.697
      Neutrophil, (×1000/mm3)5.90 (4.09–8.97)7.80 (5.15–11.0)<0.001
      Phosphorus, (mg/dL)3.10 (2.70–3.70)3.00 (2.50–3.50)0.007
      Calcium, (mg/dL)9.10 (8.70–9.40)9.00 (8.50–9.36)0.166
      Hemoglobin, (mg/dL)13.69 ± 1.8713.92 ± 1.920.246
      Hematocrit, (%)42.41 ± 5.4243.478 ± 11.720.173
      Platelet count, (×1000/mm3)308.0 (237.2–369.0)260.0 (204.5–350.5)<0.001
      Fibrinogen, (mg/dL)258.0 (211.7–308.0)322.0 (258.0363.5)<0.001
      D-dimer, mg/L1.59 (1.21–2.11)2.59 (1.66–3.11)<0.001
      Severity of disease, severe cases, n (%)24 (8.6)52 (18.7)0.001
      Values are mean ± SD, n (%), or median (interquartile range) unless otherwise stated.
      Abbreviations: BMI: body mass index; CRP: C- reactive protein; FPG: Fasting plasma glucose; eGFR: estimated glomerular filtration rate; TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; WBC: White blood cell.
      Thrombotic complications were seen in 30 patients during admission and hospitalization. Of these, 4 had disseminated intravascular coagulation (DIC), 12 had Non-ST-leveled myocardial infarction (NSTEMI), 2 had ST-elevation myocardial infarction (STEMI), 3 had pulmonary embolism (without evidence of DVT), 8 had deep vein thrombus (DVT), central vein thrombus was detected in 1 of them (Table 3). SCUBE1 levels were significantly higher in patients with COVID-19 than in patients without (p < 0.001), and SCUBE1 levels were significantly higher in patients with severe disease than those with mild to moderate disease (p < 0.001) (Fig. 1A ). In addition, SCUBE1 levels were significantly higher in patients with thrombotic complications than in patients without (p < 0.001) (Fig. 1B).
      Table 3Thrombotic complications seen in patients.
      Disseminated intravascular coagulation, n4
      Non-ST elevation myocardial infarction, n12
      ST elevation myocardial infarction, n2
      Pulmonary embolism (without DVT), n3
      Deep vein thrombosis, n8
      Central vein thrombosis, n1
      Fig. 1
      Fig. 1In patients with COVID-19, SCUBE1 levels were significantly higher in patients with both mild to moderate and severe disease than in the control group, and SCUBE1 levels were significantly higher in patients with severe disease than in those with mild to moderate disease (A), Patients with COVID-19 also had significantly higher SCUBE1 levels in the group with thrombotic complications compared to the group with no thrombotic complications (B).
      A positive correlation was found between SCUBE1 and D-dimer (Fig. 2A ) and fibrinogen (Fig. 2B) in the correlation analysis (r = 604, p < 0.001; r = 458, p < 0.001; respectively). D-dimer, Severity of Disease, SCUBE1 were determined as independent predictors for thrombotic complications in multivariate analysis (OR 2.499, 95 % CI 1.240–5.038, p = 0.010; OR 5.708, 95 % CI 1.746–18.660, p = 0.004; OR 4.343, 95 % CI 1.818–10.373, p = 0.001; respectively) (Table 4). When the predictive value of thrombotic complications of SCUBE1, D-dimer and fibrinogen were compared pairwise with ROC analysis, there was no statistically significant difference between SCUBE1 and D-dimer (p = 0.3601), while the difference between SCUBE1 and fibrinogen was significant (p = 0.0044) (Fig. 3A ). At the same time, when the in-hospital mortality predictive values of SCUBE1, D-dimer and fibrinogen were compared pairwise with ROC analysis, there was no statistically significant difference between SCUBE1 and D-dimer (p = 0.0867), but the difference between SCUBE1 and fibrinogen was significant (0.0033) (Fig. 3B).
      Fig. 2
      Fig. 2Scatter/dot plots showing positive correlation of SCUBE1 with D-dimer (A) and Fibrinogen (B).
      Table 4Univariate and Multivariate Logistic Regression Analysis for Identifying Independent Predictors of Thrombotic Complications.
      VariablesUnivariate analysesMultivariate analyses
      Nagelkerke R2 in final step = 0.607
      OR (95 % CI)p-ValueOR (95 % CI)p-Value
      Fibrinogen1.011 (1.007–1.015)<0.0011.003 (0.998–1.008)0.193
      Platelet0.989 (0.984–0.994)<0.0010.997 (0.991–1.002)0.271
      D-dimer6.184 (3.531–10.830)<0.0012.499(1.240–5.038)0.010
      Severity of disease29.137 (11.936–71.127)<0.0015.708 (1.746–18.660)0.004
      SCUBE113.886 (6.828–28.243)<0.0014.343 (1.818–10.373)0.001
      Abbreviations: OR: Odds ratio; CI: Confidence Interval.
      Fig. 3
      Fig. 3Pairwise comparison of ROC curves of SCUBE1, D-dimer and Fibrinogen to predict thrombotic complications (A), Pairwise comparison of ROC curves of SCUBE1, D-dimer and Fibrinogen to predict in-hospital mortality (B). Abbreviations: AUC: Area under the curve; ΔAUC: Difference between areas under the curve; ROC: Receiver operating characteristics; CI: Confidence Interval.
      The Kaplan-Meier cumulative survival curve shows that in-hospital mortality was significantly higher in the higher SCUBE1 group than in the lower SCUBE1 group (Log-rank test: p < 0.001) (Fig. 4). Severe cases, thrombotic complications, and in-hospital mortality rates were significantly higher in the higher SCUBE1 group than in the lower SCUBE1 group (p = 0.001, p < 0.001, p < 0.001; respectively) (Table 5). The Cox regression analysis for in-hospital mortality related with the plasma SCUBE1 levels are presented in Table 6. Even after adjustment for multiple confounding factors (model 5), SCUBE1 was found to be an independent predictor of in-hospital mortality (HR = 2.692, 95 % CI: 1.879–3.858; p < 0.001) (Table 6).
      Fig. 4
      Fig. 4Kaplan-Meier survival curve of in-hospital all-cause mortality according to SCUBE1 level.
      Table 5Severity of disease, thrombotic complications and in-hospital mortality rates according to plasma SCUBE 1 levels.
      In-hospital follow-upLower SCUBE1

      (≤2.30 ng/mL)

      (n: 276)
      Higher SCUBE1

      (>2.30 ng/mL)

      (n: 277)
      p value
      Severity of disease, severe cases, n (%)24 (8.6)52 (18.7)0.001
      Thrombotic complications, n (%)2 (0.7)28 (10.1)<0.001
      In-hospital mortality, n (%)7 (2.5)40 (14.4)<0.001
      Table 6Cox Regression Models to Estimate the Effects of SCUBE 1 Levels on in-hospital mortality.
      In-hospital mortality
      Hazard ratio (95 % CI)p-Value
      Model 15.296 (3.731–7.517)<0.001
      Model 25.296 (3.731–7.517)<0.001
      Model 35.296 (3.731–7.517)<0.001
      Model 44.226 (2.623–5.807)<0.001
      Model 52.692 (1.879–3.858)<0.001
      Model 1: Unadjusted. Model 2: adjusted for age and sex. Model 3: further adjusted for smoking, history of diabetes mellitus, hypertension, dyslipidemia. Model 4: further adjusted for white blood cell, lymphocyte, neutrophil, phosphorus and platelet. Model 5: further adjusted for D-dimer and fibrinogen. CI: confidence interval.

      4. Discussion

      First, the major finding of this study is that SCUBE1 levels were found to be higher in patients who had COVID-19 compared to those who did not. In addition, patients with severe disease (patients requiring intensive care follow-up) had higher levels of SCUBE1 in plasma than those with mild to moderate disease. Second, thrombotic complications and in-hospital mortality during the disease course were higher at higher SCUBE1 levels and elevated SCUBE1 levels were found to be an independent predictor for thrombotic complications and in-hospital mortality in patients with COVID-19. To the best of our knowledge, this is the first study to investigate the relationship between SCUBE1 and thrombotic complications and in-hospital mortality during the course of COVID-19.
      The COVID-19 pandemic has heavily affected the world in recent years as a major cause of mortality and morbidity [
      • Ochani R.
      • Asad A.
      • Yasmin F.
      • Shaikh S.
      • Khalid H.
      • Batra S.
      • et al.
      COVID-19 pandemic: from origins to outcomes. A comprehensive review of viral pathogenesis, clinical manifestations, diagnostic evaluation, and management.
      ]. Thrombotic complications are prominent among these increased mortality and morbidities [
      • Levi M.
      Diagnosis and treatment of disseminated intravascular coagulation.
      ]. These complications may be the manifestation of a systemic involvement, or they may accompany an isolated pulmonary involvement alone [
      • Ibanez B.
      • James S.
      • Agewall S.
      • Antunes M.J.
      • Bucciarelli-Ducci C.
      • Bueno H.
      • et al.
      ESC Scientific Document Group. 2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC).
      ]. Although many mechanisms have been suggested for this prethrombogenic environment seen during the course of COVID-19 disease, the main cause has not been understood and it has been mentioned that vasculopathy, which is the result of the interaction of many thrombogenic parameters, may play an important role in this process [
      • Roffi M.
      • Patrono C.
      • Collet J.P.
      • Mueller C.
      • Valgimigli M.
      • Andreotti F.
      • et al.
      ESC scientific document group. 2015 ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC).
      ].
      The primary targets of the COVID-19 are endothelial cells, pneumocytes, immune cells [
      • Konstantinides S.V.
      • Meyer G.
      • Becattini C.
      • Bueno H.
      • Geersing G.J.
      • Harjola V.P.
      • et al.
      The task force for the diagnosis and management of acute pulmonary embolism of the european Society of Cardiology (ESC). 2019 ESC guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the European Respiratory Society (ERS): the task force for the diagnosis and management of acute pulmonary embolism of the european Society of Cardiology (ESC).
      ]. The main causes of acute lung failure in COVID-19 are virus-induced alveolar damage and thrombosis in the pulmonary microvascular bed [
      • Streiff M.B.
      • Agnelli G.
      • Connors J.M.
      • Crowther M.
      • Eichinger S.
      • Lopes R.
      • et al.
      Guidance for the treatment of deep vein thrombosis and pulmonary embolism.
      ]. Vasculopathy occurring during the course of the disease results from the activation of a pathological process directly caused by the COVID-19 infection and involving the immune system, including thrombo-inflammatory responses [
      • Streiff M.B.
      • Agnelli G.
      • Connors J.M.
      • Crowther M.
      • Eichinger S.
      • Lopes R.
      • et al.
      Guidance for the treatment of deep vein thrombosis and pulmonary embolism.
      ]. As a result of this process, both microvascular and macrovascular thrombotic complications occur in the arterial, venous and capillary vascular bed [
      • Rodallec M.H.
      • Krainik A.
      • Feydy A.
      • Hélias A.
      • Colombani J.M.
      • Jullès M.C.
      • Marteau V.
      • Zins M.
      Cerebral venous thrombosis and multidetector CT angiography: tips and tricks.
      ]. In studies, it has been shown that besides endothelial damage in vasculopathy caused by COVID-19 virus, vasculitis with direct effect and disruption of coagulation hemostasis between endothelium and platelet play a role in the precoagulative state in the disease process [
      • Roffi M.
      • Patrono C.
      • Collet J.P.
      • Mueller C.
      • Valgimigli M.
      • Andreotti F.
      • et al.
      ESC scientific document group. 2015 ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC).
      ]. COVID-19 disease causes hypercoagulation, platelet activation and endothelial dysfunction with acutely increased inflammatory process [
      • Gómez-Mesa J.E.
      • Galindo-Coral S.
      • Montes M.C.
      • Muñoz Martin A.J.
      Thrombosis and coagulopathy in COVID-19.
      ]. In COVID-19 patients presenting with thrombotic complications, increased fibrinogen and D-dimer levels are usually observed initially, whereas minor changes in prothrombin time and platelet count are detected [
      • Ibanez B.
      • James S.
      • Agewall S.
      • Antunes M.J.
      • Bucciarelli-Ducci C.
      • Bueno H.
      • et al.
      ESC Scientific Document Group. 2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC).
      ,
      • Wool G.D.
      • Miller J.L.
      The impact of COVID-19 disease on platelets and coagulation.
      ]. In critically severe patients, decreased fibrinogen and thrombocytopenia can be observed in the late stages of the disease [
      • Iba T.
      • Connors J.M.
      • Levy J.H.
      The coagulopathy, endotheliopathy, and vasculitis of COVID-19.
      ].
      SCUBE1 is highly expressed in both vascular endothelial cells and platelets [
      • Becker R.C.
      COVID-19-associated vasculitis and vasculopathy.
      ,
      • Ackermann M.
      • Verleden S.E.
      • Kuehnel M.
      • Haverich A.
      • Welte T.
      • Laenger F.
      • Vanstapel A.
      • Werlein C.
      • Stark H.
      • Tzankov A.
      • Li W.W.
      • Li V.W.
      • Mentzer S.J.
      • Jonigk D.
      Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19.
      ]. This glycoprotein is secreted and surface-exposed on activated platelets, promotes platelet-platelet interaction and promotes platelet-matrix adhesion and agglutination and therefore, increased plasma level of SCUBE1 is used as a biomarker of platelet activation in acute thrombotic diseases [
      • Ackermann M.
      • Verleden S.E.
      • Kuehnel M.
      • Haverich A.
      • Welte T.
      • Laenger F.
      • Vanstapel A.
      • Werlein C.
      • Stark H.
      • Tzankov A.
      • Li W.W.
      • Li V.W.
      • Mentzer S.J.
      • Jonigk D.
      Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19.
      ,
      • Flaumenhaft R.
      • Enjyoji K.
      • Schmaier A.A.
      Vasculopathy in COVID-19.
      ]. Clinical studies have shown significantly higher plasma SCUBE1 levels in acute thrombotic complication processes such as acute coronary syndrome, acute ischemic stroke, high thrombus load in patients presenting with STEMI, and the no-reflow phenomenon after stent implantation [
      • Levi M.
      • Thachil J.
      • Iba T.
      • Levy J.H.
      Coagulation abnormalities and thrombosis in patients with COVID-19.
      ,
      • Eljilany I.
      • Elzouki A.N.
      D-dimer, fibrinogen, and IL-6 in COVID-19 patients with suspected venous thromboembolism: a narrative review.
      ,
      • Barale C.
      • Melchionda E.
      • Morotti A.
      • Russo I.
      Prothrombotic phenotype in COVID-19: focus on platelets.
      ]. In the experimental study by Wu et al., they showed that plasma SCUBE1 is not only a biomarker in acute thrombotic processes, but also plays an active role in the thrombosis process [
      • Wu M.Y.
      • Lin Y.C.
      • Liao W.J.
      • Tu C.F.
      • Chen M.H.
      • Roffler S.R.
      • Yang R.B.
      Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.
      ]. In addition, in a study by Heit et al., SCUBE1 gene polymorphism was found to be associated with venous thromboembolism [
      • Yang R.B.
      • Ng C.K.
      • Wasserman S.M.
      • Colman S.D.
      • Shenoy S.
      • Mehraban F.
      • Komuves L.G.
      • Tomlinson J.E.
      • Topper J.N.
      Identification of a novel family of cell-surface proteins expressed in human vascular endothelium.
      ]. In a study by Topcu et al., higher serum SCUBE1 levels were found in breast cancer patients known to cause a procoagulant state compared to the control group [
      • Tu C.F.
      • Su Y.H.
      • Huang Y.N.
      • Tsai M.T.
      • Li L.T.
      • Chen Y.L.
      • Cheng C.J.
      • Dai D.F.
      • Yang R.B.
      Localization and characterization of a novel secreted protein SCUBE1 in human platelets.
      ]. Various circulating inflammatory coagulation biomarkers directly involved in coagulation such as fibrinogen, D-dimer, P-selectin and von Willebrand Factor (VWF) have been shown to take an active role in the procoagulant process in the COVID-19 disease [
      • Tu C.F.
      • Yan Y.T.
      • Wu S.Y.
      • Djoko B.
      • Tsai M.T.
      • Cheng C.J.
      • Yang R.B.
      Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1.
      ]. An imbalance between procoagulant and anticoagulant factors in the disease process causes thrombotic complications during the disease [
      • Dai D.F.
      • Thajeb P.
      • Tu C.F.
      • Chiang F.T.
      • Chen C.H.
      • Yang R.B.
      • Chen J.J.
      Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.
      ]. Our findings suggest that SCUBE1 also plays an active role in the prothrombotic process during the course of COVID-19 disease.
      In COVID 19 patients, in accordance with our study, thrombotic complications are more common in severe cases, and mortality is higher in patients with thrombotic complications [
      • Dai D.F.
      • Thajeb P.
      • Tu C.F.
      • Chiang F.T.
      • Chen C.H.
      • Yang R.B.
      • Chen J.J.
      Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke.
      ]. In our study, high SCUBE1 levels in patients with severe course requiring ICU follow-up and those with thrombotic complications make us think that SCUBE1 may play an active role especially in severe cases with thrombotic complications. In this context, SCUBE1 may be a therapeutic target in patients with COVID-19.

      4.1 Limitations

      Our study had some limitations. First of all, this study was an observational study and the number of patients was relatively small. Secondly, outpatient follow-up patients with COVID were not included in the study. Third, SCUBE1 measurements were based on a single plasma sample only. Serial follow-ups of SCUBE1 in the course of the disease were not performed.

      5. Conclusions

      SCUBE 1 levels were higher than in the control group, and higher SCUBE1 levels were associated with disease severity, in-hospital mortality, and thrombotic complications. Based on these findings, it can be said that SCUBE1 can indicate disease severity, in-hospital mortality and hypercoagulability. Therefore, high SCUBE1 levels measured in the early stages of the disease may predict more intensive medical therapy to prevent disease progression and more intensive antithrombotic therapy to prevent thrombotic complications. In this respect, it may be a useful biomarker and therapeutic target in patients with COVID-19. Further studies are needed to confirm these findings.

      Declaration of competing interest

      The authors report no conflict of interest or financial support.

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