Long COVID Is Caused By The Increased Levels Of Six Inflammatory Molecules Triggering Thrombotic Endotheliitis

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A new study conducted by researchers from Stellenbosch University-South Africa has found that the increased levels of six different inflammatory molecules in most post-COVID individuals known to be key drivers of endothelial and clotting pathology, causes thrombotic endotheliitis, a contributing factor to Long COVID.

The study findings were published on a preprint server and are currently being peer reviewed.
https://www.medrxiv.org/content/10.1101/2022.10.13.22281055v1

Post-Acute Sequelae of COVID-19 (PASC) or Long COVID is a major global health burden with its symptoms significantly impacting physical and cognitive function, health-related quality of life, and participation in society (1-9). The mechanisms for the persistent symptoms can be narrowed down to

  • 1) viral persistence,
  • 2) microclotting and platelet hyperactivation,
  • 3) autoantibodies,
  • 4) immune dysregulation,
  • 5) widespread organ damage,
  • 6) reactivation of dormant viruses (9-12).

SARS-CoV-2 (COVID-19) viral remnants including RNA and spike protein have been identified in patients with Long COVID. Increases in antibody responses directed against non-SARS- CoV-2 viral pathogens, particularly Epstein-Barr virus, have been noted and are likely to be related to reactivation of dormant viruses (13, 14).

There is also increasing evidence that autoantibodies may be involved in the lingering symptoms of individuals with Long COVID (15, 16). We have demonstrated that individuals with Long COVID have a significant fibrin amyloid microclot load in their circulation (2, 10, 17).

These clots can be induced in normal plasma by (recombinant) SARS-CoV-2 spike protein (18), and are resistant to fibrinolysis (10, 19). Microclots are microscopic (fibrin-amyloid-containing) clots present in the plasma of recruited individuals (10, 19). Entrapped in these fibrinolysis-resistant microclots are numerous inflammatory molecules, including alpha 2-antiplasmin (α2AP), various fibrinogen chains, Von Willebrand factor (VWF), platelet factor 4 (PF4), Serum Amyloid A (SAA), as well as numerous antibodies (10, 19).

If the coagulopathy present in the acute phase of the disease, is not adequately treated, it is likely that both tissue hypoxia and impaired oxygen exchange may linger for months (10, 11, 17, 19, 20). Recently it was noted that the incidence of vascular events remains elevated up to 49 weeks after COVID-19 diagnosis (21).

The published evidence so far points to widespread endothelial inflammation (22-24), and the underlying causes are a failed (i.e. inadequate) fibrinolytic system and persistent coagulopathy (9).

Although characterizing the inflammatory molecule content of the fibrinolytic-resistant microclots is of considerable interest and value (2, 10, 17, 20), patients desperately need more readily accessible diagnostic markers at general pathology laboratories. In this paper we focus on identifying biomarkers in the soluble fraction of blood that could be used as a suite to confirm the widespread endothelial damage and coagulation pathologies see in Long Covid patient.

The soluble molecules that were chosen for this study, were chosen from previously published research that found these molecules entrapped inside solubilized microclots, as well as two additional molecules. The previously identified molecules include VWF, PF4, SAA and a-2AP, with the two new markers , that are well known endothelial damage markers, include endothelial-leukocyte adhesion molecule 1 (E-selectin), and the platelet endothelial cell adhesion molecule1 (PECAM-1).

Von Willebrand Factor

Following damage to the endothelial layers, VWF is released by endothelial cells (25) and megakaryocytes; it mediates platelet adhesion to the damaged vascular surface, and the aggregation of platelets (26, 27). We included VWF as it is stored in endothelial Weibel-Palade bodies (28) and platelet a-granules (29), and is also secreted during platelet activation and endothelial damage (30, 31). Back in 2020, we and other researchers discussed the importance of VWF as a disease marker in acute COVID-19 (22, 32-36). It has also been noted that VWF/ADAMTS-13 imbalance persists in endothelial cell activation and angiogenic disturbance in Long COVID (37).

Platelet Factor 4

Another important inflammatory molecule driving coagulation and platelet pathology, is PF4 (38-40). Platelets produce and store PF4 in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin (41). Heparin can bind to PF4 and thereby promotes PF4 aggregation, resulting in the formation of PF4/heparin complexes, which have antigenic properties (38). PF4 and VWF may also form complexes that are involved in thrombus propagation (39).

α-2-antiplasmin

Central to the persistent nature of microclots in Long COVID (in addition to the simple resistance to proteolysis of amyloid-like proteins (42) entrapping inflammatory molecules), is a plasmin and antiplasmin imbalance in individuals with Long COVID. We have found that α- 2AP is trapped inside microclots (10, 19).

This molecule prevents the fibrinolytic system from functioning optimally and impedes the digestion of pathological thrombi that cause microvascular thrombosis, thereby increasing the risk of thromboembolic events (10, 19). High blood levels of α-2AP, an ultrafast, covalent inhibitor of plasmin- increase the risk of a poor outcome in cardiovascular diseases (43).

Serum amyloid A

We also included, as part of our suite of soluble inflammatory markers, the more general inflammatory marker, SAA. The human SAA protein family comprises the acute phase SAA1 and SAA2, involved in innate and adaptive immunity and the constitutive SAA4 (44, 45). SAA activates the complement system and the nucleotide-binding domain, leucine-rich repeat (NLR) family pyrin domain containing 3 (NLRP3) inflammasome, alters vascular function, affects HDL function, and increases thrombosis (46).

Endothelial factors

As endothelial dysfunction is central in persistent symptoms of Long COVID, we also chose to include two well-known endothelial damage markers, namely, CD62 antigen-like family member E (CD62E) a commonly known as E-selectin (sometimes also called endothelial- leukocyte adhesion molecule 1 (ELAM-1), and the platelet endothelial cell adhesion molecule-

1 (PECAM-1). E-selectin is a glycoprotein cell adhesion receptor that is expressed on endothelial cells activated by cytokines (47), and can be used as a marker to evaluate endothelial dysfunction (48, 49). Platelet endothelial cell adhesion molecule-1 (PECAM-1), also referred to as CD31, functions as an inhibitory receptor in circulating platelets and is highly expressed at endothelial cell-cell junctions, where it functions as an adhesive stress- response protein to both maintain endothelial cell junctional integrity (50). As PECAM-1 is also expressed on platelets and leukocytes, it is at the nexus of thrombosis and inflammation (51).

In addition to determining if E-selectin and PECAM-1 are upregulated in the soluble part of the blood, we also wished to determine if these they may indeed be involved in protein-protein interactions with plasma proteins to cause the formation of (amyloid) protein misfolding (resulting in microclot formation).

Furthermore, we investigated if these two molecules can interact with and bind to platelet receptors to cause platelet hyperactivation. This part of the investigation was planned as a laboratory model investigation and the final exposure concentration was chosen to mimic concentrations found in Long COVID.

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