Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis

Abstract

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.

Keywords: COVID-19; Complement; Immunology; Neutrophils; Thrombosis.

Figure 1. NETs in the coagulopathy of COVID-19.

(A) Myeloperoxidase (MPO)/DNA complex levels representing NET release in plasma from healthy individuals (controls, n = 10) and patients with COVID-19 (n = 25). (B) Thrombin-antithrombin (TAT) complex levels in plasma from controls and patients with COVID-19. In A and B, red squares indicate severe patients; red triangles, critical patients. (C) Correlation between MPO-DNA levels and TAT levels in patients with COVID-19. (D) Confocal fluorescence microscopy showing TF/neutrophil elastase (NE) staining in control and COVID-19 neutrophils. A representative example of 4 independent experiments is shown. Original magnification: ×600; scale bar: 5 μm. Blue: DAPI; green: TF; red: NE. (E) MPO-DNA levels in NETs isolated from controls and patients with COVID-19. Data are from 4 independent experiments (mean ± SD). (F) TAT levels in plasma from controls treated with NETs isolated from controls and patients with COVID-19. Data are from 4 independent experiments (mean ± SD). (G) Fold expression of TF mRNA in neutrophils isolated from controls and patients with COVID-19. Data are from 4 independent experiments (mean ± SD). D–G were performed in the same 4 patients (identified by an asterisk in Supplemental Table 1). All conditions were compared with controls. *Statistical significance is P < 0.05. A, B: Student’s t test; C: Spearman’s correlation test; E–G: Friedman’s test.

Figure 2. Inhibition of PRP-neutrophil interactions in COVID-19.

(A) Relative fold expression of TF mRNA in control neutrophils treated with COVID-19–derived PRP (COV PRP) and inhibited with dabigatran (thrombin inhibitor), HCQ (autophagy inhibitor), or C5aR1 antagonist (C5aRa/PMX-53). Data are from 4 independent experiments (mean ± SD). (B) MPO-DNA complex levels in NETs isolated from control neutrophils treated with COV PRP and inhibited with dabigatran, HCQ, or C5aRa/PMX-53. Data are from 4 independent experiments (mean ± SD). (C) TAT complex levels in control plasma stimulated with NET structures isolated from control neutrophils treated with COV PRP and inhibited with dabigatran, HCQ, or C5aR. Data are from 4 independent experiments (mean ± SD). (D and E) Confocal fluorescence microscopy showing TF/neutrophil elastase (NE) staining in control neutrophils treated with COV PRP and inhibited with (F) dabigatran, (G) FLLRN (PAR1 receptor inhibitor), (H) HCQ, or (I) C5aRa/PMX-53. A representative example of 4 independent experiments is shown. Original magnification: ×600; scale bar: 5 μm. Blue: DAPI, green: TF, red: NE. All conditions were compared with control/untreated. *Statistical significance at P < 0.05. A–C: Friedman’s test. The in vitro experiments mentioned above are also summarized in Supplemental Table 2. Conditions of real-time RT-PCR are described in Supplemental Table 3.

Figure 3. C3 inhibition disrupts neutrophil-driven thromboinflammation in COVID-19.

(A) Soluble terminal complement complex (sTCC) levels in plasma from controls (n = 10) and patients with COVID-19 (n = 25). Red squares: severe patients; red triangles: critical patients. (B) Relative fold expression of TF mRNA in control neutrophils stimulated with serum from healthy individuals (HS), HS incubated with COVID-19 serum (COV serum), or HS treated with compstatin analog Cp40 and then COV serum, COV serum alone, or COV serum treated with Cp40. Data are from 4 independent experiments (mean ± SD). (C) Fluorescence microscopy showing TF/NE staining in control neutrophils stimulated with HS, HS incubated with COV serum, or HS treated with Cp40 and then COV serum. A representative example of 4 independent experiments is shown. Original magnification: ×1000; scale bar: 5 μm. Blue: DAPI, green: TF, red: NE. All conditions were compared with HS alone (control). *Statistical significance at P < 0.05. A: Student’s t test, B: Friedman’s test. These in vitro experiments are also listed in Supplemental Table 2. Conditions of real-time RT-PCR are described in Supplemental Table 3.

Figure 4. Proposed mechanism of COVID-19 immunothrombosis.

During COVID-19, SARS–CoV-2 triggers complement activation by interacting with mannan-binding lectin (MBL) serine proteases (MASPs) or possibly through (auto)antibodies or/and immunocomplexes. C3 activation, as a point of convergence of all complement pathways, leads to C3a, C5a, and sC5b-9 (TCC) generation. Subsequently, C3a might activate platelets (PLTs), while C5a and PLT-derived thrombin induce both neutrophil TF expression and NETs carrying active TF. These thrombogenic NETs may induce endothelial cell activation toward TF expression, thus increasing their procoagulant activity. This may further amplify (e.g., via PAR1), inflammation and PLT activation, thereby fueling a complement/NET-driven vicious cycle of immunothrombosis. Complement, thrombin, and NETosis represent promising therapeutic targets. The central pink box includes components of the COVID-19 thromboinflammatory environment. Question marks and dotted lines indicate provisional pathways/connections that have not yet been investigated in COVID-19. FP, properdin; PMX-53, specific C5a receptor antagonist.