key: cord-0997137-2msvg4nm authors: Al‐Tamimi, Abaher O.; Yusuf, Ayesha M.; Jayakumar, Manju N.; Ansari, Abdul W.; Elhassan, Mona; AbdulKarim, Fatema; Kannan, Meganathan; Halwani, Rabih; Ahmad, Firdos title: SARS‐CoV‐2 infection induces soluble platelet activation markers and PAI‐1 in the early moderate stage of COVID‐19 date: 2022-03-09 journal: Int J Lab Hematol DOI: 10.1111/ijlh.13829 sha: 22affc5667d2ace2540eb0cf64e8249cf110420d doc_id: 997137 cord_uid: 2msvg4nm INTRODUCTION: Coagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID‐19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID‐19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID‐19 to understand the pathogenesis. METHODS: To understand this, hospitalized COVID‐19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including prothrombin time (PT), activated partial thromboplastin time (APTT), D‐Dimer, and tissue factor pathway inhibitor (TFPI). The flow cytometry‐based multiplex assays were performed to assess FXI, anti‐thrombin, prothrombin, fibrinogen, FXIII, P‐selectin, sCD40L, plasminogen, tissue plasminogen activator (tPA), plasminogen activator inhibitor‐1 (PAI‐1), and D‐Dimer. RESULTS: The investigations revealed induction of plasma P‐selectin and CD40 ligand (sCD40L) in moderate COVID‐19 cases, which were significantly abolished with the progression of COVID‐19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID‐19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI‐1 more prominently in moderate, and tPA particularly in severe COVID‐19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease. CONCLUSIONS: In summary, induction of soluble P‐selectin, sCD40L, fibrinogen, and PAI‐1 suggests the activation of platelets and coagulation system at the moderate stage before COVID‐19 patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID‐19 severity. Severe acute respiratory syndrome coronavirus-2 (SARS- infection often leads to respiratory conditions like acute respiratory distress syndrome (ARDS) and pneumonia in severe cases. 1, 2 Preliminary cohort studies show a higher incidence of venous thromboembolic events in COVID-19 patients who particularly required intensive care. 3, 4 Acute respiratory disease progression includes an early infection phase in which the virus attacks epithelial cells using angiotensin-converting enzyme 2 (ACE2) receptors, which leads to pneumonia and induction of severe systemic inflammation. 5 Coagulopathy is one of the most concerning features in severe COVID-19 patients which is characterized by prolonged prothrombin and activated partial thromboplastin times, elevated Ddimer, and fibrinogen levels. 6, 7 The elevated coagulopathy markers are found to be associated with a higher mortality rate. 8, 9 SARS-CoV-2-induced endothelial damage releases a variety of soluble markers of endothelial dysfunction including von Willebrand factor (VWF), soluble E-selectin, P-selectin, thrombomodulin. 10, 11 Such biologically active molecules eventually promote platelet activation. Platelet receptors regulate not only thrombosis but also initiate delayed cellular activities underlying inflammatory response against diverse pathogens, including viruses. 12 Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of TF and plays a critical role in balancing the initiation phase of coagulation. TFPI is a serine protease inhibitor that inhibits TF-FVIIa and prothrombinase complex (FXa/FVa) and regulates thrombin generation. 13, 14 In a variety of infections, endothelial cells release proinflammatory cytokines such as TNFα, IL-1, and IL-6 which in turn not only increases the expression of TF on the endothelial lining but also increases the plasminogen activator inhibitor-1 (PAI-1). Endotoxininduced expression of TF and PAI-1 by endothelium thus may provide a stimulus that promotes thrombotic complication. 15, 16 Several viral infections including dengue and influenza were found to be associated with platelet activation and inflammation through different molecular pathways. 17 The inflammatory reaction can also lead to a release of reactive oxygen species (ROS) and proteases which contribute to endothelial damage and further pathological remodeling of the venous wall. 18 Studies have revealed that patients infected with SARS-CoV-1 or MERS virus develop fibrin/thrombi within the pulmonary vasculature. 19 Studies with SARS-CoV-1-infected mice further suggest the abnormal expression of procoagulant genes such as thrombin, VII, XI, XII, and plasminogen activators especially in those who had fatal consequences. 20, 21 Sepsis-induced coagulopathy (SIC) and disseminated intravascular coagulopathy (DIC) have been documented with severe disease, especially in non-survivors COVID-19 cases. 6 Though the dysregulation of plasma CD40 ligand (sCD40L), 22 TFPI, 23 tissue plasminogen activator (tPA), PAI-1, 24 and FXIII 25 have already been reported, most of the studies were focused particularly on severe COVID-19. However, it is still unclear particularly when these key platelet activation markers and components of the coagulation system are dysregulated during the COVID-19 pathogenesis and how they correlate with the progression of disease severity. Therefore, to better understand the pathogenesis of COVID-19, here, we sought to identify the levels of plasma platelet activation markers P-selectin and sCD40L at the early moderate and severe stages of COVID-19. To identify the role of coagulation and fibrinolysis dysfunction in COVID-19 pathogenesis, we assessed levels of coagulation factors including FIX, TFPI, prothrombin, anti-thrombin, fibrinogen, FXIII, tPA, and PAI-1 in the moderate and severe COVID-19 patients. The study was conducted on COVID- 19 The hospitalized moderate (without pneumonia or the requirement of intensive care) and severe COVID-19 patients (presented pneumonia and/or acute respiratory distress syndrome confirmed by Chest X-ray or CT scan and required intensive care) were included in the study. Patients with a preexisting history of cardiovascular diseases including those with myocardial infarction, stroke, or deep vein thrombosis (DVT) were excluded from the study. Patients were also excluded if received thromboprophylaxis like low molecular weight heparin or other anti-coagulant. The healthy controls were recruited after taking the medical history and informed consent. Individuals on any type of medication or with a recent history of any medical conditions were excluded. Individuals without any current medications and/or a recent history of any disease were only included as healthy control. Blood samples from COVID-19 patients and healthy controls were collected in sodium citrate or EDTA-coated vials and processed quickly. The whole blood sample was centrifuged at 700 g for 10 min, and platelet-poor plasma was separated quickly and transferred to a fresh tube and stored at −80°C until further use. Upon hospitalization, basic laboratory investigations including complete blood count (CBC), prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, and C-reactive protein (CRP) were performed. PT, APTT, and D-dimer assays were performed using semi-automated coagulometer (STAGO-STAR, Seine Cedex, France) and complete blood count (CBC) was measured under Beckman Coulter (Brea, CA, USA). The level of CRP was measured using Cobas b101 system, and plasma ferritin level was measured using an immunoturbidimetric assay kit Cobas 6000. (Roche Diagnostics, Gerrmany). To detect the plasma levels of coagulation, thrombosis, and fibrinolysis markers including FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue plasminogen activator (tPA), plasminogen activator inhibitor-I (PAI-I), and D-Dimer, we performed multiplex bead-based assays using LegendPlex Human thrombosis (BioLegend #740906) and LegendPlex Human Fibrinolysis Panels (BioLegend #740761) following the manufacturer's instruction. Briefly, diluted plasma samples from COVID-19 patients and healthy controls were used to measure the levels of soluble markers. Data were acquired under BD FACS Aria III using FACS Diva software, and data analysis was done using LegendpLex software (Biolegend, CA, USA). All the samples were assessed in duplicate, and the average was taken as a final reading. To quantify the tissue factor pathway inhibitor (TFPI) levels in the plasma samples of the COVID-19 patients and healthy controls, we performed a sandwich enzyme-linked immunosorbent assay (ELISA) using Human TFPI ELISA kit (Abcam# ab274392) according to the manufacturer's instruction. All the samples were assessed in duplicate, and the average was taken as a final reading. Data group differences were evaluated for significance using unpaired t-test or one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (Graph Pad Prism Software Inc., San Diego, CA). Data are expressed as mean ± SEM. For all tests, a p-value <.05 was considered for statistical significance. The age range of recruited patients was between 32 and 69 years (Table 1 ). Similarly, the level of sCD40L was found significantly elevated in the moderate COVID-19 cases and the level profoundly diminished with the advancement of severity ( Figure 1C ). These findings strongly suggest that SARS-CoV-2 triggers the release of P-selectin and sCD40L at the early moderate stage of COVID-19 potentially from activated endothelial. Accumulating data sets suggest that severe SARS-CoV-2 infection leads to coagulation dysfunction characterized by increased thrombin generation and elevated levels of D-Dimer. 2, 6, 29 Therefore, next, we assessed the potential dysregulation of intrinsic, extrinsic, and/or common coagulation pathways that might TA B L E 1 Table shows Figure 3D ). Importantly, the plasminogen level was comparable in moderate and severe patients vs. healthy controls ( Figure 3E ). Overall, these findings revealed the aberrant fibrinolysis in the COVID-19 patients. Our findings show profoundly elevated levels of endothelial cell and platelet activation markers P-selectin and sCD40L in moderate cases and the levels were significantly abolished in critically ill patients. Moreover, attenuated levels of TFPI and FXIII, and a higher level of fibrinogen were observed particularly in the severe COVID-19. The level of PAI-1 was elevated in both the patient groups and increased tPA level was seen only in severe COVID-19 cases. Prothrombin time and APTT are the laboratory tools that predict the defective extrinsic and intrinsic coagulation pathways. 30 In contrast to our observations, a study has reported prolonged PT and APTT, 31 elevated level of tPA was significantly associated with higher mortality. 24 The higher level of tPA is consistent with our findings where we have observed a higher level of tPA in the cohort of critically ill COVID-19 patients. In conclusion, here, we report the induction of platelet activation markers in the moderate stage of COVID-19, and the levels mark- The work was supported by COVID-19 (CoV-0302), Targeted (1801090144-P), and Competitive (1901090162-P) research grants from the University of Sharjah to Firdos Ahmad. This work was also supported by the Cardiovascular Research Group at the University of Sharjah. Declared none. AOA, AMY, and MNJ performed experiments and collected data. AWA, ME, and FA helped with sample and clinical data collection. MK and RH performed data interpretation and helped in manuscript writing. FA designed study, acquired funding, supervised the project, performed data analysis and interpretation, wrote and revised the manuscript. All the supporting data are included within the main article and additional information is available from the corresponding author on reasonable request. 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