SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation

Abstract

Microvascular thrombosis is associated with multiorgan failure and mortality in coronavirus disease 2019 (COVID-19). Although thrombotic complications may be ascribed to the ability of SARS-CoV-2 to infect and replicate in endothelial cells, it has been poorly investigated whether, in the complexity of viral infection in the human host, specific viral elements alone can induce endothelial damage. Detection of circulating spike protein in the sera of severe COVID-19 patients was evaluated by ELISA. In vitro experiments were performed on human microvascular endothelial cells from the derma and lung exposed to SARS-CoV-2-derived spike protein 1 (S1). The expression of adhesive molecules was studied by immunofluorescence and leukocyte adhesion and platelet aggregation were assessed under flow conditions. Angiotensin converting enzyme 2 (ACE2) and AMPK expression were investigated by Western Blot analysis. In addition, S1-treated endothelial cells were incubated with anti-ACE2 blocking antibody, AMPK agonist, or complement inhibitors. Our results show that significant levels of spike protein were found in the 30.4% of severe COVID-19 patients. In vitro, the activation of endothelial cells with S1 protein, via ACE2, impaired AMPK signalling, leading to robust leukocyte recruitment due to increased adhesive molecule expression and thrombomodulin loss. This S1-induced pro-inflammatory phenotype led to exuberant C3 and C5b-9 deposition on endothelial cells, along with C3a and C5a generation that further amplified S1-induced complement activation. Functional blockade of ACE2 or complement inhibition halted S1-induced platelet aggregates by limiting von Willebrand factor and P-selectin exocytosis and expression on endothelial cells. Overall, we demonstrate that SARS-CoV-2-derived S1 is sufficient in itself to propagate inflammatory and thrombogenic processes in the microvasculature, amplified by the complement system, recapitulating the thromboembolic complications of COVID-19.

Complement activation in COVID-19 and targeted therapeutic options: A scoping review

“… Kovacs-Kasa [ 69 ], 2022 Experimental cell HLMVEC Endothelial permeability measurement of HLMVEC exposed to plasma from SARS-CoV-2 patients ECIS C3aR and C5aR antagonists Non-severe, severe SARS-CoV-2 induced permeability is not affected by C3a or C5a inhibitors. Perico [ 68 ], 2022 Experimental HMEC-1 Endothelial cells exposed to SARS-CoV-2 derived spike protein 1 Immunofluorescence C3aR and C5aR antagonists Severe Endothelial dysfunction induced by SARS-CoV-2-derived S1 protein triggers exuberant complement deposition on activated microvascular endothelial cells C3a and, to a lesser extent, C5a, further amplify complement activation that fuels inflammation in response to S1. Zhang [ 66 ], 2021 Experimental cell Neutrophils and HUVECs Neutrophils exposed to plasma from HCs or COVID-19 patients and HUVECs exposed to supernatant of neutrophils cultured with COVID-19 plasma MPO-DNA, cell viability assay Anti-C3a and anti-C5a antibodies Mild or severe Anaphylatoxins C3a and C5a in the plasma of COVID-19 patients strongly induced NET formation, which could be relieved by CPB.…”

“…Inhibition of C3aR (SB 290157) and C5aR (mix of W-54011 and DF2593) in human airway epithelial cells led to mitigation of viral infection and a decrease of the inflammatory response in airways, whereas inhibition of C5aR also maintained the epithelial integrity of human airway epithelia infected with SARS-CoV-2 [ 64 ]. One experimental study in human microvascular endothelial cells (HMEC-1) showed that endothelial dysfunction induced by SARS-CoV-2 derived S1 protein triggered complement deposition on activated microvascular endothelial cells [ 68 ]. In particular C5a further amplified activation of the complement system, which contributed to inflammation in response to S1 [ 68 ].…”

“…One experimental study in human microvascular endothelial cells (HMEC-1) showed that endothelial dysfunction induced by SARS-CoV-2 derived S1 protein triggered complement deposition on activated microvascular endothelial cells [ 68 ]. In particular C5a further amplified activation of the complement system, which contributed to inflammation in response to S1 [ 68 ]. However, in one experimental cell study, pulmonary microvascular endothelial cell permeability induced by SARS-CoV-2 was not affected by C3aR (SB SB290157) and C5aR (W54011) antagonists [ 69 ].…”

“…C3 and C4 were most often measured and were generally either decreased in severe patients or non-survivors [ [100] , [101] , [102] , [103] , [104] , [105] ], or did not differ compared with non-severe patients or survivors [ [106] , [107] , [108] , [109] , [110] , [111] , [112] , [113] , [114] , [115] , [116] , [117] , [118] ]. Anaphylatoxins C3a and notably C5a were elevated and correlated with disease severity, ICU admission and mortality [ 12 , 14 , 65 , 68 , 85 , [119] , [120] , [121] , [122] , [123] , [124] , [125] , [126] , [127] , [128] ]. Soluble C5b-9 levels were also increased and correlated with C5a, markers of inflammation and coagulation [ 12 , 33 , 36 , 68 , 70 , 71 , 78 , 120 , 125 , [129] , [130] , [131] , [132] , [133] , [134] ].…”

Weighted Gene Co-Expression Network Analysis to Identify Potential Biological Processes and Key Genes in COVID-19-Related Stroke

“…The production of C3a and C5a further amplifies S1-induced complement activation. This eventually leads to increased platelet aggregation on the endothelial cells of microvessels [ 30 ]. Platelets, in addition to their conventional involvement in thrombosis and hemostasis, also play important roles in inflammatory and immunological processes [ 31 – 33 ], which indirectly influence immune responses by the release of antimicrobial peptides and cytokines and directly amplify immune responses by interacting with lymphocytes, monocytes, and neutrophils once pathogens have been detected [ 34 ].…”