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. 2022 Mar 7:13:827146.
doi: 10.3389/fimmu.2022.827146. eCollection 2022.

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

Luca Perico  1 , Marina Morigi  1 , Miriam Galbusera  1 , Anna Pezzotta  1 , Sara Gastoldi  1 , Barbara Imberti  1 , Annalisa Perna  1 , Piero Ruggenenti  1   2 , Roberta Donadelli  1 , Ariela Benigni  1 , Giuseppe Remuzzi  1
Affiliations

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

Luca Perico et al. Front Immunol. .

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.

Keywords: COVID-19; SARS-CoV-2 spike protein 1; complement system; endothelial dysfunction; inflammation; thrombosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
SARS-CoV-2-derived Spike 1 (S1) protein affects microvascular endothelial viability and phenotype in vitro. (A) Quantification of cell viability in HMEC-1 exposed for 24 h to medium alone (CTR) or S1 at the concentration of 0.5 nM, 10 nM, or 50 nM. (B) Quantification and representative images of the binding of the S1 protein (red) to HMEC-1 treated with medium alone (CTR) or S1 at the concentration of 0.5 nM and 10 nM for 24 h. (C, D) Quantification and representative images of ICAM-1 expression [(C), red] and vWF deposition [(D), red] on HMEC-1 treated with medium alone (CTR) or S1 at the concentration of 0.5 nM and 10 nM for 24 h. All experiments were repeated 3 times. Data represent mean ± SEM and were analysed with Tukey’s multiple comparison test. **p-value<0.01, and ***p-value<0.001 vs CTR; $$$ p-value<0.001 vs 0.5 nM S1; °°p-value<0.01, and °°°p-value<0.001 vs 10 nM S1. All the slides were counterstained with DAPI (blue). Scale bar 20 μm.
Figure 2
Figure 2
S1, through ACE2, upregulates adhesive molecules on HMEC-1 by impairing AMPK signalling. (A) Quantification and representative Western Blots of ACE2 protein expression in HMEC-1 exposed for 24h to medium alone (CTR) or S1 (10 nM). GAPDH was used as a sample loading control. (B–D) Quantification and representative images of ICAM-1 expression [(B), red], P-selectin expression [(C), red], and vWF deposition [(D), red] on HMEC-1 incubated with medium alone (CTR) or with S1 (10 nM) in the presence of anti-ACE2 Ab (2 μg/ml) or Irr Ab (2 μg/ml). (E) Quantification and representative Western Blots of AMPK activation, evaluated as the ratio between the expression of pAMPKThr172 and total AMPK in HMEC-1 exposed for 24h to medium alone (CTR) or S1 (10 nM). (F, G) Quantification of ICAM-1 expression (F) and vWF deposition (G) on HMEC-1 incubated with medium alone (CTR) or with S1 (10 nM) in the presence or absence of AMPK agonist AICAR (2 mM). All experiments were repeated at least 3 times. Data represent mean ± SEM and were analysed with unpaired t-test or Tukey’s multiple comparison test, as appropriate. **p-value<0.01, and ***p-value<0.001 vs CTR; °°°p-value<0.001 vs 10 nM S1; ###p-value<0.001 vs 10 nM S1+Irr. All slides were counterstained with DAPI (blue). Scale bar 20 μm for (B, C) and 50 μm for (D).
Figure 3
Figure 3
S1 induces C3 and C5b-9 deposition on HMEC-1 by interacting with ACE2. (A) Quantification and representative images of C3 deposition (green) on HMEC-1 pre-exposed for 24h to medium alone (CTR) or to S1 (10 nM) in the presence of anti-ACE2 Ab (2 μg/ml) or the corresponding Irr Ab (2 μg/ml) and then incubated with human serum (HS, 50%) for 2h in the presence or in the absence of complement inhibitors (Compstatin, Comp; C1 inhibitor, C1inh; C3a receptor antagonist, C3aRa; C5a receptor antagonist, C5aRa). (B) Quantification and representative images of C5b-9 formation (green) on HMEC-1 pre-exposed for 24h to medium alone (CTR) or S1 (10 nM) in the presence of anti-ACE2 Ab (2 μg/ml) or Irr Ab (2 μg/ml) and then incubated with HS 50% in the presence or in the absence of complement inhibitors (Comp, C1inh, C3aRa, and C5aRa). All experiments were repeated at least 3 times. Data represent mean ± SEM and were analysed with Tukey’s multiple comparison test. *p-value<0.05, and ***p-value<0.001vs CTR; °p-value<0.05, °°p-value<0.01, and °°°p-value<0.001 vs 10 nM S1; ###p-value<0.001 vs 10 nM S1+Irr. All slides were counterstained with DAPI (blue). Scale bars 50 μm.
Figure 4
Figure 4
S1 promotes leukocyte adhesion and NET formation on HMEC-1 under flow. (A) Quantification of leukocyte adhesion under flow conditions on HMEC-1 exposed for 24h to medium alone (CTR) or to subtoxic concentration of S1 (10 nM) in the presence of anti-ACE2 functional blocking Ab (ACE2, 2 μg/ml) or the corresponding Irr Ab (2 μg/ml). (B) Adhesion of leukocytes, incubated for one hour with control medium or with S1 (S1*, 10 nM) and perfused under flow conditions (1.5 dynes/cm2) on HMEC-1 exposed to medium alone (CTR) or with S1 (10 nM). (C) Representative images of leukocytes treated with medium alone (CTR) or S1 (S1*, 10 nM), which adhered to HMEC exposed for 24h to medium alone or to S1. In this setting, neutrophils were co-stained with histone H3 citrullinated (citHH3, green) and neutrophil elastase (NE, red). The release of neutrophil extracellular traps (NETs) was observed only when leukocytes were activated with S1 (10 nM S1+ 10 nM S1*, inset). All experiments were repeated at least 3 times. Data represent mean ± SEM and were analysed with Tukey’s multiple comparison test. ***p-value<0.001 vs CTR; °p-value<0.05, and °°°p-value<0.001 vs 10 nM S1; ###p-value<0.001 vs 10 nM S1+Irr. Slides were counterstained with DAPI (blue). Scale bar 20 μm.
Figure 5
Figure 5
S1 activates the complement system amplifying platelet aggregate formation on HMEC-1 through ACE2. (A) Quantification of platelet aggregate formation on HMEC-1 pre-exposed for 24h to medium alone (CTR) or S1 (10 nM). Platelets aggregates on HMEC-1 perfused with heparinised blood under flow conditions (60 dynes/cm2) were evaluated and expressed as pixels2/field analysed. (B, C) Quantification (B) and representative images (C) of platelet aggregate formation on HMEC-1 pre-exposed for 24h to medium alone (CTR), S1 (10 nM), or 10 nM S1 in the presence of anti-ACE2 Ab (2 μg/ml) or the corresponding Irr Ab (2 μg/ml) and then incubated with 50% human serum (HS) for 2h. In selected samples, S1-treated HMEC-1 were incubated with 50% HS in the presence of complement inhibitors (Compstatin, Comp; C3a receptor antagonist, C3aRa; C5a receptor antagonist, C5aRa). Platelet aggregate formation on HMEC-1 under flow conditions (60 dynes/cm2) was quantified and expressed as pixel2/field analysed. (D, E) Quantification (D) and representative images (E) of vWF deposition on HMEC-1 pre-exposed for 24h to medium alone (CTR) or S1 (10 nM) and then incubated with 50% HS in the presence or absence of complement inhibitors (Comp, C3aRa, and C5aRa). All experiments were repeated at least 3 times. Data represent mean ± SEM and were analysed with unpaired t-test or Tukey’s multiple comparison test, as appropriate. **p-value<0.01, and ***p-value<0.001 vs CTR; °p-value<0.05, °°p-value<0.01, and °°°p-value<0.001 vs 10 nM S1; ##p-value<0.01 and ###p-value<0.001 vs 10 nM S1+Irr. Scale bars 50 μm.
Figure 6
Figure 6
S1, through ACE2, upregulates adhesive molecules on HPMEC by impairing AMPK signalling. (A) Quantification and representative images of the binding of the S1 protein (red) to HPMEC treated with medium alone (CTR) or S1 (10 nM) for 24 h. (B) Quantification and representative Western Blots of ACE2 protein expression in HPMEC exposed for 24h to medium alone (CTR) or S1 (10 nM). GAPDH was used as a sample loading control. (C, D) Quantification and representative images of ICAM-1 expression [(C), red], and vWF deposition [(D), red] on HPMEC incubated with medium alone (CTR) or with S1 (10 nM) in the presence or absence of anti-ACE2 Ab (2 μg/ml) or AICAR (2 mM). (E) Quantification and representative Western Blots of AMPK activation, evaluated as the ratio between the expression of pAMPKThr172 and total AMPK in HPMEC exposed for 24h to medium alone (CTR) or 10 nM S1. All experiments were repeated at least 3 times. Data represent mean ± SEM and were analysed with unpaired t-test or Tukey’s multiple comparison test, as appropriate. *p-value<0.05, and ***p-value<0.001 vs CTR; °°p-value<0.01, and °°°p-value<0.001 vs 10 nM S1. All slides were counterstained with DAPI (blue). Scale bar 50 μm.
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