Protease-Activated Receptor-1 IgG Autoantibodies in Patients with COVID-19

COVID-19 Patients Putting on the Brakes with PAR1 Autoantibodies

Dysregulated humoral immunity and autoimmune responses in severe coronavirus disease 2019 (COVID-19) remain incompletely understood. Growing evidence suggests autoantibody (AAb) production upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[1] However, the full range of AAb in COVID-19 and their distinct pathophysiological role remain unclear. Recently, increased levels of protease-activated receptor-1 (PAR-1) AAbs were linked to severe COVID-19.[2]

Here, we investigated the plasma levels of PAR-1 AAb using solid-phase sandwich ELISA Kits detecting only AAbs of the immunoglobulin G (IgG) isotype (CellTrend GmbH, Luckenwalde, Germany)[3] in healthy control subjects (n = 10), hospitalized COVID-19 patients with mild-to-moderate symptoms (n = 35),[4] and COVID-19 patients with a severe course who developed clinical deterioration with respiratory failure and acute respiratory distress syndrome requiring mechanical ventilation (n = 47) ([Fig. 1A, B]) (local ethics committee approval identifier EA2/066/20, EA4/147/15). Additionally, PAR-1 AAbs were measured in patients with non-COVID-19 pneumonia with mild symptoms (n = 9) or with severe disease course (n = 13) due to bacterial infections ([Fig. 1B]).

Fig. 1 Increased PAR-1 autoantibodies (AAb) deregulate thromboinflammatory response in severe COVID-19 patients. (A) Overview of the number of plasma samples from healthy donors and COVID-19 patients according to the WHO clinical progression scale. (B) Plasma level of PAR-1 AAb in healthy control subjects (n = 10), hospitalized mild-to-moderate COVID-19 patients (n = 35), severe COVID-19 patients (n = 47), and in patients with mild (n = 9) or severe (n = 13) non-COVID-19 pneumonia. Data were analyzed with one-way ANOVA followed by the Dunn post-hoc test for multiple comparisons. (C) Effect of IgG from healthy (SARS-CoV-2 negative) donors versus severe COVID-19 patients on PAR-1 GPCR activation by TRAP6 stimulation as assessed by PathHunter β-Arrestin eXpress GPCR Assay. Results are shown as the percentage of PAR-1 activation detected by β-Arrestin recruitment relative to healthy donors (100%). (D) Effect of IgG from healthy (SARS-CoV-2 negative) donors versus severe COVID-19 patients on p38y phosphorylation levels (relative to total p38y expression) and total p38y protein levels (relative to GAPDH expression) in platelets stimulated by 1 µM TRAP6. Representative western blots of phospho-p38y(Thr180/Tyr182), p38y, and GAPDH expression are shown below. Results were analyzed by a two-tailed unpaired t-test. (E) Multiplate (Roche) platelet impedance aggregometry analysis in whole blood of control subjects pretreated with IgG from healthy donors versus severe COVID-19 patients shows platelet aggregation in response to TRAP6 as the area under the curve over time (AU*min). Data in (C) and (E) were analyzed by two-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons. (F) Comparison of PAR-1 AAb levels between patients who experienced thrombotic events and patients without thrombotic events. Results were analyzed by a two-tailed unpaired t-test. ANOVA, analysis of variance; GPCR, G protein-coupled receptor; IgG, immunoglobulin G.

The patients were predominantly recruited during the spread of the α variant and were unvaccinated at the time of blood sampling. The mean age in the mild-to-moderate COVID-19 group was 69 years, with 66% male patients. In the severe COVID-19 group, the mean age was 66 years, with 80% male patients. In the mild non-COVID-19 group, the mean age was 76 years, with 67% male patients, and in the severe non-COVID-19 group, the mean age was 77 years, with 54% male patients.

Our findings confirmed significantly higher levels of PAR-1 AAb in patients with severe COVID-19 as compared with healthy controls (mean level [U*mL⁻¹]: 2.71 vs. 17.70, p = 0.015) ([Fig. 1B]). Increased PAR-1 AAb levels appear to be specific for COVID-19, as patients with non-COVID-19 pneumonia did not show elevated PAR-1 AAb levels (mild non-COVID-19: 3.478 U*mL⁻¹, p = 0.999, severe non-COVID-19: 3.185 U*mL⁻¹, p = 0.999). PAR-1 is a G protein-coupled receptor (GPCR) activated by distinct plasma proteases, such as thrombin, thereby promoting platelet activation.[5] Importantly, PAR-1 mediates the interplay between coagulation and immune responses, e.g., in viral infections,[6] and thus, is considered to play a crucial role in associated pathobiological processes, particularly in thrombotic complications of COVID-19.[7] To assess the functional properties of PAR-1 AAb, we tested their potential effects on PAR-1 activity in vitro using a cell-based reporter assay (Eurofins DiscoverX, France), which detects intracellular β-Arrestin recruitment in response to GPCR activation.[8] Surprisingly, pretreatment of platelets from healthy donors with IgG from patients with severe COVID-19 who had the highest levels of anti-PAR1 AAb attenuated the GPCR signaling response to TRAP6 (thrombin receptor activator peptide 6) stimulation as compared with IgG pretreatment from healthy controls, pointing toward an inhibitory effect of these AAbs ([Fig. 1C]). Unlike TRAP6, the inhibitory effects of IgG treatment from patients with high PAR-1 AAb were absent upon stimulation with collagen, supporting a PAR-1-specific effect (data not shown). Analogously, TRAP6-induced activation of PAR-1 downstream signaling pathways was decreased, as indicated by reduced phosphorylation of p38y(Thr180/Tyr182) in platelets of healthy donors upon IgG treatment from severe COVID-19 ([Fig. 1D]). Finally, treatment of control subjects' platelets with IgG from severe COVID-19 patients attenuated platelet aggregation in response to TRAP6 in vitro compared with IgG from healthy donors ([Fig. 1E]). Notably, higher PAR-1 AAb levels were detected in patients who experienced thrombotic events than those without ([Fig. 1F]).

Cumulatively, our data support previous findings of increased levels of PAR-1 AAb in patients with severe COVID-19 and provide evidence of the potential inhibitory effects of these AAbs on PAR-1 signaling and platelet aggregation. This may reflect a compensatory circuit where humoral immune processes counteract deregulated thrombocyte activation in COVID-19 with an antibody-mediated response targeting PAR-1 signaling.

^* These authors contributed equally as first authors.

^** These authors contributed equally as senior authors.

Publikationsverlauf

Eingereicht: 17. Oktober 2023

Angenommen: 05. November 2023

Accepted Manuscript online:
06. November 2023

Artikel online veröffentlicht:
29. Dezember 2023

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