Links between the complement and coagulation systems could lead to Long Covid therapies

WOLFRAM RUF Authors Info & Affiliations, SCIENCE, 18 Jan 2024, Vol 383, Issue 6680, pp. 262-263, DOI: 10.1126/science.adn1077, BY CARLO CERVIA-HASLER, SARAH C. BRÜNINGK, TOBIAS HOCH, ET AL.

Acute infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause a respiratory illness that can be associated with systemic immune cell activation and inflammation, widespread multiorgan dysfunction, and thrombosis. Not everyone fully recovers from COVID-19, leading to Long Covid, the treatment of which is a major unmet clinical need (1). Long Covid can affect people of all ages, follows severe as well as mild disease, and involves multiple organs. The persistence of lingering symptoms after acute disease creates a considerable challenge for understanding the specific pathophysiology and risk factors underlying Long Covid. On page 273 of this issue, Cervia-Hasler et al. (2) report a multicenter, longitudinal study of 113 patients who either fully recovered from COVID-19 or developed Long Covid, identifying localized activation of the innate immune defense complement system as a likely culprit that induces thromboinflammation and prevents the restoration of fitness after acute COVID-19.

Patients with Long Covid display signs of immune dysfunction and exhaustion (1), persistent immune cell activation (3), and autoimmune antibody production (1), which are also pathological features of acute COVID-19. Cervia-Hasler et al. undertook a proteomic screen measuring serum levels of 6596 human proteins that are recognized by 7289 epitope-specific DNA oligonucleotide aptamer probes. The patients with severe or mild acute COVID-19 were analyzed during the acute infection and 6 months later. Comparison of the 40 Long Covid patients, 73 recovered patients, and 39 healthy controls revealed that most serum biomarkers that were elevated in patients with Long Covid at 6 months overlapped with those that were altered in the subgroup of the cohort with severe acute COVID-19.

In particular, the blood antimicrobial defense systems of complement and pentraxin 3 stood out as being significantly associated with Long Covid development. Complement components and pentraxins, which include serum amyloid proteins, serve humoral immunity by destroying and opsonizing pathogens for rapid clearance by innate immune cells. These proteins are up-regulated in hepatocytes as part of the inflammationinduced systemic acute phase response (4). However, the up-regulated markers of this response in Long Covid—pentraxin 3 and certain complement factors—are produced mainly by immune and other tissue-resident cells and not the liver (5), indicating that persistent inflammation in Long Covid patients is local rather than systemic.

The complement system is crucial for innate immune defense by effecting lytic destruction of invading microorganisms, but when uncontrolled, it causes cell and vascular damage. The complement cascade is activated by antigen–antibody complexes in the classical pathways or in the lectin pathway by multimeric proteins (lectins) that recognize specific carbohydrate structures, which are also found on the SARS-CoV-2 spike protein that facilitates host cell entry. Both pathways may contribute to the pronounced complement activation in acute COVID-19 (6). Independent of these pathogen recognition–triggered mechanisms, the so-called alternative pathway, in which C3b recruits complement factor B, leading to its proteolysis, can amplify complement activation on cell surfaces. The consecutive proteolytic cleavages in the complement cascade ultimately generate C5b that binds C6 and C7. C7 serves as the cell membrane anchor for the C5b-C7 complex and enables subsequent recruitment of C8 and C9 in the terminal complement complex (TCC), which mediates lysis of pathogens and host cells.

Focusing on Long Covid–specific proteome changes and taking into consideration age, sex, and hospitalization, Cervia-Hasler et al. detected increased C5b-C6 levels, supporting excessive complement activation. However, an aptamer probe measuring C7 showed surprisingly decreased levels. Careful validation of the aptamer target specificity revealed recognition of C7 in complex with other TCC components, but not of free C7. The reduced levels of circulating C7-containing complexes indicated that assembly of C7 with C5b-C6 resulted in increased membrane insertion and consequently cell damage in Long Covid. Consistently, a decreased ratio of complexed C7/C5b-C6 was a strong predictor for developing Long Covid (see the figure).

Cervia-Hasler et al. found that Long Covid patients also showed a marked up-regulation of serum von Willebrand factor (vWF) and thrombospondin 1, which are both released from damaged or activated endothelial cells as well as from platelets. Conversely, levels of a disintegrin and metalloproteinase with thrombospondin motif 13 (ADAMTS13), which cleaves vWF, were reduced in Long Covid patients, indicating an imbalanced regulation of vWF in the circulation. ADAMTS13 plays a crucial role in controlling ultralarge vWF multimers that are released from activated endothelial cells and are highly prothrombotic by facilitating platelet adhesion. Moreover, vWF multimers bind C3b and thereby promote alternative complement pathway activation, which was also evident in the Long Covid patients based on measurement of activated factor B fragments. By contrast, processing of ultralarge vWF by ADAMTS13 increases the susceptibility of C3b to degradation and thereby attenuates local complement amplification (7). In addition to inefficient vWF degradation, Cervia-Hasler et al. found evidence for red blood cell lysis and release of heme, which may further contribute to the persistent local complement activation in Long Covid patients.

Coagulation activation leads to blood clots consisting of platelets and fibrin, which is lysed by fibrinolytic proteases. Acute, severe COVID-19 is associated with elevated levels of the fibrin degradation product D-dimer and by multiorgan thrombosis typically without reduction in platelet counts. Increased D-dimer and fibrin levels in acute COVID-19 predict the development of cognitive dysfunction (“brain fog”) in Long Covid patients (8). D-dimer and fibrin elevations did not persist in Long Covid patients, but—apart from vWF—Cervia-Hasler et al. found that levels of coagulation factor 11 (F11) were elevated as a potential additional procoagulant mechanism. F11 serves in a coagulation amplification loop that generates thrombin on platelets that are recruited to endothelial vWF, and F11 can promote vascular inflammation without causing overt thrombosis (9). The lingering thromboinflammatory dysregulation in Long Covid occurs with apparently otherwise normal endothelial function. In particular, the biomarker profiles in Long Covid patients provided no evidence for an impaired endothelium-protective thrombomodulin pathway that not only regulates fibrinolysis and complement activation but also supports the degradation of inflammation- inducing anaphylatoxins C3a and C5a (10), which were unchanged in Long Covid patients. Furthermore, ongoing limited coagulation in Long Covid is indicated by the detection of circulating fibrinolysis-resistant microclots that are enriched in vWF, C7, acute-phase serum amyloid proteins, and fibrin (11), proteins identified in the proteomics screen by Cervia-Hasler et al. as contributors to Long Covid.

The formation of neutrophil extracellular traps (NETs), prothrombotic DNA “webs” that are ejected from neutrophils to immobilize pathogens, and other signs of immunothrombosis are frequent in acute COVID-19 but absent in Long Covid. Instead, platelets were found by Cervia-Hasler et al. to be associated with classical monocytes in the blood of patients with persistent Long Covid symptoms, in line with the demonstrated increased levels of thrombospondin, which stimulates platelet interactions with monocytes in addition to other platelet-derived proteins. Although single-cell sequencing of a small subset of Long Covid patients did not detect procoagulant or proinflammatory gene induction in classical monocytes, these showed down-regulation of nuclear receptor subfamily 4 group A member 1 (NR4A1) expression, the key transcription factor required for monocyte conversion into endothelial-protective, patrolling monocytes (12). In addition, the transcriptome of CD16+ patrolling monocytes showed an upregulation in the expression of interferonregulated genes, consistent with previous studies implicating persistent interferon signaling in Long Covid (3). Because monocytes can rapidly change their procoagulant properties in the context of complement activation without transcriptional induction of the coagulation initiator tissue factor, contributions of altered monocyte surveillance to endothelial dysfunction and local generation of thrombin, which also activates C3, should be considered in future studies as additional mechanisms for Long Covid–associated thromboinflammation.

Long Covid symptoms include a postexertional exhaustion reminiscent of other postviral illnesses, such as myalgic encephalomyelitis– chronic fatigue syndrome (MECFS) with suspected latent viral reactivation (1). Antibody titer changes in Long Covid patients indicate an association of fatigue with reactivation of latent Epstein-Barr virus (EBV) infections (13), and Cervia-Hasler et al. found that the severity of Long Covid symptoms is associated with cytomegalovirus (CMV) reactivation. In addition, various autoantibody alterations have been observed in Long Covid with unclear contributions to pathophysiology (1). Cervia-Hasler et al. found that autoantibodies to the B cell–stimulating C-X-C motif chemokine 13 (CXCL13) were decreased in patients with persistent Long Covid symptoms, which together with increased interferon signaling may contribute to persistent autoimmune pathologies. Intriguingly, complement-dependent prothrombotic antiphospholipid antibodies, which are observed in acute COVID- 19, intersect with coagulation signaling to induce interferon responses in monocytes and promote autoimmunity by type I interferon production in dendritic cells (14). A better understanding of the connections between viral reactivation, persistent interferon signaling, and autoimmune pathologies promises to yield new insights into the thromboinflammation associated with Long Covid.

Although therapeutic interventions with coagulation and complement inhibitors in acute COVID-19 produced mixed results, the pathological features specific for Long Covid suggest potential interventions for clinical testing. Microclots are also observed in ME-CFS patients (1), indicating crucial interactions between complement, vWF, and coagulation-mediated fibrin formation in postviral syndromes. Target-specific coagulation inhibitors can reprogram innate immune phenotypes and interrupt the microangiopathic roles of vWF (15). Rebalancing complement activation can be achieved by blocking distinct amplification points with monoclonal antibodies that are clinically approved or in development. Complement and coagulation systems are not only an integral part of the innate immune response but also are connected at several levels in feedforward amplification loops. A better definition of these interactions in preclinical and clinical settings will be crucial for the translation of new therapeutic concepts in chronic thromboinflammatory diseases.