Results: In the mHam test, addition of S1α 20 µg/mL to NHS led to significantly more complement-mediated cell killing after 2 hours as compared to S1O ( P<.05) ( Figure 1). The Student t test was used to assess the difference between unpaired groups with a value of P<.05 considered statistically significant. Data is presented as mean ± standard error. Experiments were performed in triplicate. Blood 2002), a tick salivary protein that inhibits FVIIa/TF-induced FX activation, was incubated with cells and plasma at 37☌ for 10 minutes prior to plasma recalcification in select samples. Clotting activity was measured by the recalcification time of the plasma using a BioTeK ELx808 plate reader at 405 nm. After 6 hours incubation, cells (150,000-200,000 cells/well) were washed with PBS then resuspended in pooled human plasma. Tissue factor (TF) relative mRNA expression was quantified using RT-PCR at various time points and normalized to GADPH expression. For coagulation assays, the spike proteins were incubated with THP-1 cells, a monocytic cell line, at 37☌ in RPMI with 10% fetal bovine serum. Complement assays including the modified Ham (mHam) test and flow cytometry for cell surface deposition of complement C5b-9 were performed as previously described using the spike proteins added to normal human serum (NHS) to activate complement on TF1 PIGAnull cells (Yu et. Methods: The SARS-CoV-2 α variant S1 subunit (S1α) and Omicron variant BA.1.1.529 S1 (S1O) recombinant proteins were obtained from Raybiotech and human coronavirus OC43 recombinant spike protein (OC43 S) from Sino Biological. Using ex vivo assays of complement and coagulation, we sought to characterize the effects of the S1 subunit from the α and O SARS-CoV-2 spike protein variants as compared to the OC43 spike protein, a common human coronavirus associated with mild upper respiratory tract illness. Further, complement amplification is associated with disease severity in hospitalized patients. We previously showed that the S1 subunit of the α spike protein containing the receptor binding domain critical for viral host cell entry leads to dysregulation of the alternative pathway of complement through binding to heparan sulfate and competing for binding of factor H. Signal Transduction and Targeted Therapy 2022). Clinically, the first alpha (α) variant was marked by elevated incidence of thromboembolism, intensive care unit (ICU) admissions and mortality, whereas the omicron BA.1 (O) variant has been associated with lower rates of hospitalization, ICU admissions, and need for mechanical ventilation (Fan et. Notwithstanding individual patient risk factors and more widespread immunity, mutations in SARS-CoV-2, commonly involving the spike protein, have yielded fluctuations in disease manifestations and severity. Introduction: Infection caused by the SARS-CoV-2 virus is marked by both a localized and systemic inflammatory response and coagulopathy leading to end organ damage and mortality.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |