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. 2022 Sep 9;23(18):10436.
doi: 10.3390/ijms231810436.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis

Biju Bhargavan  1 , Georgette D Kanmogne  1
Affiliations

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis

Biju Bhargavan et al. Int J Mol Sci. .

Abstract

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell-cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5'-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis.

Keywords: DTNB; Factor-V; SARS-CoV-2 spike proteins; TFPI; fibrinogen; human lung endothelial cells; neutrophils; thrombin; thrombomodulin; tissue factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cytotoxicity of S-proteins, rTFPI, and DTNB on HLMEC. HLMEC were treated with S-proteins (A), rTFPI (B), and DTNB (C) and cytotoxicity at 24 to 48 h assessed by AlamarBlue assay. ** p < 0.004; *** p < 0.0007; # p < 0.0001, compared to untreated controls.
Figure 2
Figure 2
S-proteins and endothelial–neutrophil interactions induce Factor-III transcriptional upregulation in HLMEC and neutrophils. HLMEC were treated with 1 nM S-protein Wuhan (SW) or Delta (SD) variants for 6–24 h (A). In separate experiments, HLMEC were treated with S-proteins for 6 h, washed, and co-cultured with neutrophils for 6–24 h (B,C); neutrophils were treated with S-proteins for 6 h, washed, and co-cultured with HLMEC for 6–24 h (D,E). Each cell type was harvested separately and Factor-III mRNA levels in endothelial cells (A,B,D) and neutrophils (C,E) were quantified by real-time PCR. Data presented as mean ± standard deviation. Control: untreated cells; ACE2: cells treated with recombinant human ACE2 (1 µg/mL). Hi: cells treated with heat-inactivated S-proteins. * p = 0.01; ** p = 0.002; *** p < 0.0008; # p < 0.0001.
Figure 3
Figure 3
S-proteins and endothelial–neutrophil interactions increase Factor-V expression and release in HLMEC. (A,B) HLMEC treated with S-proteins (1 nM SW or SD) for 6–24 h; (C) HLMEC treated 6 h with S-proteins, washed, and co-cultured with neutrophils for 6–24 h; (D) neutrophils treated 6 h with S-proteins, washed, and co-cultured with HLMEC for 6–24 h. Following treatments, Factor-V levels in culture supernatants (A,C,D) and endothelial cell lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi-SW: cells treated with 1 nM heat-inactivated SW; Hi-SD: cells treated with 1nM heat-inactivated SD; ACE2: cells treated with rhACE2 (1 µg/mL). ** p = 0.002; *** p < 0.0002; # p < 0.0001.
Figure 4
Figure 4
S-proteins and endothelial–neutrophil interactions induce thrombin expression and release in HLMEC. (A,B) HLMEC treated with S-proteins (1 nM SW or SD) for 6–24 h; (C) HLMEC treated 6 h with S-proteins, washed, and co-cultured with neutrophils for 6–24 h; (D) neutrophils treated 6 h with S-proteins, washed, and co-cultured with HLMEC for 6–24 h. Following treatments, thrombin levels in culture supernatants (A,C,D) and endothelial cell lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi-SW: cells treated with 1 nM heat-inactivated SW; Hi-SD: cells treated with 1nM heat-inactivated SD; ACE2: cells treated with rhACE2 (1 µg/mL). * p < 0.05; ** p < 0.008; *** p < 0.0006; # p < 0.0001.
Figure 5
Figure 5
S-proteins and endothelial–neutrophil interactions induce fibrinogen expression and release in HLMEC. (A,B) HLMEC treated with S-proteins (1 nM SW or SD) for 6–24 h; (C) HLMEC treated 6 h with S-proteins, washed, and co-cultured with neutrophils for 6–24 h; (D) neutrophils treated 6 h with S-proteins, washed, and co-cultured with HLMEC for 6–24 h. Following treatments, fibrinogen levels in culture supernatants (A,C,D) and endothelial cell lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi-SW: cells treated with 1 nM heat-inactivated SW; Hi-SD: cells treated with 1nM heat-inactivated SD; ACE2: cells treated with rhACE2 (1 µg/mL). *** p = 0.0006; # p < 0.0001.
Figure 6
Figure 6
S-proteins and endothelial–neutrophil interactions inhibit TFPI transcription in HLMEC and neutrophils. (A) HLMEC treated with S-proteins (1 nM SW or SD) for 6–24 h; (B,C) HLMEC treated 6 h with S-proteins, washed, and co-cultured with neutrophils for 6–24 h; (D,E) Human neutrophils treated 6 h with S-proteins, washed, and co-cultured with HLMEC for 6–24 h. Following treatment, each cell type was harvested separately and TFPI mRNA levels in endothelial cells (A,B,D) and neutrophils (C,E) were quantified by real-time PCR. Data presented as mean ± standard deviation. Control: untreated cells; ACE2: cells treated with rhACE2 (1 µg/mL); Hi: cells treated with heat-inactivated S-proteins. * p = 0.025; *** p = 0.0002; # p < 0.0001.
Figure 7
Figure 7
rTFPI and thiol blockers prevent S-protein-induced Factor-V expression and release in HLMEC, whereas TM had no effect: (A,B) HLMEC treated (24 h) with S-proteins (1 nM SW or SD), with or without rTFPI, DTNB, and BDCA3 (200 ng/mL); (C) HLMEC were treated 6 h with S-proteins, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured with neutrophils for 24 h; (D) human neutrophils were treated 6 h with S-proteins, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured with HLMEC for 24 h. Following treatments, Factor-V levels in culture supernatants (A,C,D) and endothelial cells lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: heat-inactivated (SW, SD, rTFPI, DTNB, BDCA3). # p < 0.0001.
Figure 8
Figure 8
rTFPI and thiol blockers prevent S-proteins-induced thrombin expression and release in HLMEC, whereas TM had no effect: (A,B) HLMEC treated (24 h) with S-proteins (1 nM SW or SD), with or without rTFPI, DTNB, and BDCA3 (200 ng/mL); (C) HLMEC were treated 6 h with S-proteins, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured with neutrophils for 24 h; (D) human neutrophils were treated 6 h with S-proteins, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured with HLMEC for 24 h. Following treatments, thrombin levels in culture supernatants (A,C,D) and endothelial cells lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: heat-inactivated (SW, SD, rTFPI, DTNB, BDCA3). # p < 0.0001.
Figure 9
Figure 9
rTFPI, TM, and thiol blockers prevent S-proteins-induced fibrinogen expression and release in HLMEC: (A,B) HLMEC treated (24 h) with S-proteins (1 nM SW or SD), with or without rTFPI, DTNB, and BDCA3 (200 ng/mL); (C) HLMEC were treated 6 h with S-proteins, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured with neutrophils for 24 h; (D) human neutrophils were treated 6 h with S-proteins, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured with HLMEC for 24 h. Following treatments, fibrinogen levels in culture supernatants (A,C,D) and endothelial cell lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: heat-inactivated (SW, SD, rTFPI, DTNB, BDCA3). # p < 0.0001.
Figure 10
Figure 10
Model illustrating the coagulation cascade pathway in S-protein-induced TF, Factor-V, thrombin, and fibrinogen. Arrows indicate direct activation. The red ⊥ symbol indicate pharmacological inhibitors. Yellow boxes indicate factors associated with the extrinsic pathway of blood coagulation. Blue boxes indicate factors associated with the common pathway of blood coagulation.
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