The quantified data were shown in a bar graph
The quantified data were shown in a bar graph. plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2CSP-PVCinduced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2CSP-PVCinduced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug AdministrationCapproved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19. Introduction Although respiratory dysfunction is the primary feature of the COVID-19, many studies showed that a hypercoagulability state is frequently present in patients with COVID-19.1,2 Thromboembolic complications play a crucial role in the pathogenesis and mortality of COVID-19.3,4 Aberrant expression of tissue factor (TF) is associated with (S)-(-)-5-Fluorowillardiine many forms of thrombosis.5 Recent reviews suggest that TF may play a central role in thrombosis in patients with COVID-19.6,7 Increased expression of TF was found in platelet-monocyte aggregates from critically ill patients with COVID-19.8 Skendros et al9 (S)-(-)-5-Fluorowillardiine showed the expression of TF in neutrophils of patients with severe COVID-19. However, in the above studies, TF expression analyses were limited to microscopy, flow cytometry, or mRNA levels. Recently, Rosell et al10 reported that levels of extracellular vesicle (EV) TF activity were significantly higher in patients with COVID-19 than control subjects, and the levels of EV TF (S)-(-)-5-Fluorowillardiine activity were associated with disease severity and mortality. Interestingly, in contrast to the above studies, Mast et al11 reported no increase in TF mRNA transcript in bronchoalveolar lavage fluids of patients with COVID-19. TF expressed on cells, either constitutively or induced, possess very low or no procoagulant activity.12 TF has to undergo posttranslational modifications, commonly referred to as decryption or activation, to exhibit optimal TF procoagulant activity.12 Our recent studies showed that high sphingomyelin (SM) (S)-(-)-5-Fluorowillardiine content in the outer leaflet of plasma membrane maintains TF in an encrypted state, and the hydrolysis of SM induced by pathophysiologic stimuli leads to TF activation.13,14 The breakdown of SM in the plasma membrane also prospects to the generation of EVs.13,14 Many stimuli, including viral (S)-(-)-5-Fluorowillardiine infections, can release sphingomyelinases, such as acidity sphingomyelinase (ASMase), that can breakdown SM in the plasma membrane.15 Recent studies suggest that ASMase plays a role in the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).16 Here, we investigated the role of ASMase in SARS-CoV-2 infectionCinduced TF-mediated coagulation. Study design Generation and titration of SARS-CoV-2 spike protein enveloped pseudovirus (SARS-CoV-2CSP-PV) Two hundred ninety-three T cells (1??107 cells) were transfected with 20 g vector plasmid pLVX-IRES-ZsGreen (Takara Bio), 16 g packaging plasmid psPAX2 (Addgene), and 10 Btg1 g envelope plasmid pCAGGSCSARS-CoV-2 spike (from BEI Resources) using polyethyleneimine (Polysciences, Warrington, PA). After 30 minutes of transfection, Dulbeccos revised Eagle medium supplemented with 10% fetal bovine serum and antibiotics was added to the cells, and the cells were cultured at 37C under 5% CO2. After 48 to 72 hours, the supernatants were collected, filtered, and centrifuged at 25?000for 2.5 hours at 4C to sediment the pseudovirus particles. The pseudovirus pellet was resuspended in phosphate-buffered saline, and the viral titers were determined by infecting a human being lung cell collection, H441, with serially diluted pseudovirus preparations. Generation of human being monocyteCderived macrophages and isolation of extracellular vesicles.