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

Background: 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 specific viral elements can induce endothelial damage. Methods: We performed an enzyme-linked immunosorbent assay (ELISA) to detect circulating spike protein in the sera of 23 severe COVID-19 patients, 9 mild disease patients, and 9 infection-naive subjects. In vitro experiments were performed on human microvascular endothelial cells (HMEC-1) exposed to SARS-CoV-2-derived spike protein 1 (S1) protein at the subtoxic concentration of 10 nM for 24 hours. The expression of adhesive molecules was studied by immunofluorescence on S1-primed HMEC-1 and, in parallel, leukocyte adhesion and thrombus formation were assessed under flow conditions. In selected experiments, S1-treated HMEC-1 were incubated anti-angiotensin converting enzyme 2 (ACE2) functional blocking antibody or different complement inhibitors. Findings: We show that the activation of HMEC-1 with S1 protein, via ACE2, induced 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 HMEC-1, along with C3a and C5a generation that further amplified S1-induced complement activation. Functional blockade of ACE2 or complement inhibition halted S1-induced platelet-thrombi formation by limiting von Willebrand factor exocytosis and deposition. Interpretation: Collectively, we demonstrate that SARS-CoV-2-derived S1 is sufficient per se to propagate inflammatory and thrombogenic processes in the microvasculature, amplified by the complement system, recapitulating the thromboembolic complications of COVID-19. Funding Information: The authors declare no conflicts of interest. Declaration of Interests: The present study was supported by generous contributions from: Associazione Calcio Monza, Monza; Barabino Immobiliare, Milano; Brembomatic Pedrali, Pontirolo Nuovo, Bergamo; Fondazione Aiutiamoli a vivere, Ranica, Bergamo; GF-ELTI, Sovere, Bergamo; Mrs. Mariella Guzzoni, Bergamo; Mrs. Hazzazi Malika El, Milano; Mr. Alberto Paccanelli, Bergamo; SMILAB Srl, San Pellegrino Terme, Bergamo; Mrs. Paola Suardi, Bergamo. Ethics Approval Statement: The study was approved by the Ethical Committee of the Azienda Sanitaria Locale Bergamo, Italy. Written informed consent was obtained from all enrolled patients.