Study finds patients with long COVID exhibit complement dysregulation with signs of thromboinflammation

By Pooja Toshniwal PahariaJan 23 2024Reviewed by Benedette Cuffari, M.Sc.

In a recent study published in Science, researchers explore inflammatory signatures restricted to patients with active post-acute coronavirus disease 2019 (COVID-19) sequelae (PASC).

Study: Persistent complement dysregulation with signs of thromboinflammation in active Long Covid. Image Credit: ninjapoy / Shutterstock.com

What is PASC?

PASC, which is otherwise known as long COVID, arises following the recovery from acute infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19.  Tissue injury, autoimmunity, SARS-CoV-2 reservoirs, and sustained inflammation influence PASC, which is associated with a wide range of symptoms including fatigue, post-exertional malaise, and cognitive impairment.

To date, there is a lack of diagnostic tests and therapeutics available for individuals affected by long COVID. Moreover, the factors underlying PASC remain poorly understood.

About the study

In the present multicenter and longitudinal study 113 SARS-CoV-2-infected individuals and 39 healthy controls were monitored for one year after acute COVID-19. The study also included healthy controls, PASC patients, and individuals fully recovered from mild or severe COVID-19.

Over 6,500 proteins were measured in the sera of all study participants, with the top candidate biomarkers identified using computational tools and further evaluated experimentally. Serum proteins were analyzed using synthetic aptamers, which are short single-stranded nucleic acids with unique binding specificities to proteins.

To confirm complement dysregulation in PASC, serological proteomic parameters were measured in acute COVID-19 samples of an independent external cohort, which included 198 patients hospitalized due to acute COVID-19, 73% of whom developed six-month PASC.

C7 aptamer specificity was determined using an in-house enzyme-linked immunosorbent assay (ELISA) using C7 aptamers to capture different complement components. Statistical modeling was applied to determine which terminal complement complex (TCC) components were associated with increased sC5b-9 formation upon complement activation.

Mass spectrometry was performed at six months on COVID-19 patient sera. Thromboinflammatory signatures at the cellular level were explored by spectral flow cytometry on peripheral blood mononuclear cells (PBMCs) from seven healthy controls, five individuals with COVID-19 without six-month PASC, and 10 six-month PASC patients, six of whom progressed to 12-month PASC.

Antibodies potentially contributing to classical complement activation in six-month PASC patients were measured by phage immunoprecipitation sequencing for 87,890 epitopes in 57 patients during acute COVID-19. To translate observed differences into a predictive model for six-month PASC, the team included up to 61 uncorrelated protein epitope measurements of involved biological pathways, two protein ratios, and information on medical history.

Study findings

PASC symptoms were observed in 40 patients at six months, with changes to blood serum proteins indicating activation of the complement cascade, altered coagulation, and tissue injury. Complement signaling is a component of the intrinsic immunological system and improves immune and homeostatic functions by targeting damaged cells and pathogens.

PASC patients exhibited imbalanced TCC formations with increased C5b-C6 protein complexes and reduced C7-comprising TCC formations inserted into cellular membranes. Increased membrane incorporation of TCCs was also observed among individuals with PACS, thereby leading to tissue injury.

PASC patients also exhibited increased levels of tissue damage biomarkers in the blood and thromboinflammation, which was characterized by erythrocyte lysis and endothelial activation markers like von Willebrand factor (vWF). Signs of enhanced cleavage induced by thrombin proteins that drive TCC were accompanied by low levels of antithrombin III in PASC patients.

PASC patients also exhibited increased platelet activation biomarkers and platelet-monocyte aggregations at six months, especially in the case of PASC persistence for over one year. These patients also presented with features of antibody-regulated classical complement signaling activation related to elevated anti-cytomegalovirus (anti-CMV) and anti-Epstein-Barr virus (anti-EBV) immunoglobulin G (IgG) titers.

In PASC patients, protein analysis showed dysregulated stimulation of complement signaling that altered innate immunity and homeostasis. Terminal complement dysregulation and the activation of classical complement and alternative pathways, which was related to elevated titers of antibodies targeting herpesviruses that activate the pathway, characterized PASC.

The six-month PASC group was associated with a higher prevalence of severe acute COVID-19 as compared with the 73 other COVID-19 patients.

Conclusions

Increased thromboinflammation and complement activation was observed in the blood proteins of PASC patients, including platelets and erythrocyte lysis markers. The tissue damage might be complement-regulated and stimulate complement pathways induced by antigen-antibody protein complexes, autoantibodies, antibodies targeting herpesviruses, and cross-talks with dysregulated coagulation systems.

Taken together, the current study identified common patterns in the serum proteome of PASC patients who had not recovered six months after acute infection, thus revealing an inflammatory signature restricted to active PASC patients.