Stellenbosch University Study Finds That Long COVID Is Caused By The Increased Levels Of Six Inflammatory Molecules Triggering Thrombotic Endotheliitis

Long COVID News: 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.


These six inflammatory molecules were:
 
-1) Von Willebrand Factor,
-2) platelet factor 4 (PF4),
-3) serum amyloid A (SAA),
-4) alpha-2-antiplasmin (2AP),
-5) E-selectin and
-6) platelet endothelial cell adhesion molecule-1 (PECAM-1)
 
The presence of fibrinaloid microclots and multiple inflammatory molecules in the soluble part of blood points to thrombotic endotheliitis as one of the key pathological processes in Long COVID.
 
At present, the prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation.
 
The study team demonstrate the significantly increased concentrations of Von Willebrand Factor, platelet factor 4, serum amyloid A, alpha-2-antiplasmin E-selectin, and platelet endothelial cell adhesion molecule-1, in the soluble part of the blood of Long COVID patients.
 
The study findings significantly found that the mean level of alpha-2-antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls.
 
The Long COVID study team warned that this is alarming if it was taken into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules).
 
The study team also determined that by individually adding E-selectin and PECAM-1 to healthy blood, these molecules may indeed be involved in protein-protein interactions with plasma proteins (contributing to microclot formation) and platelet hyperactivation.
 
The study was performed as a laboratory model investigation and the final exposure concentration of these molecules was chosen to mimic concentrations found in Long COVID.
 
The study findings conclude that presence of microclotting, together with relatively high levels of six inflammatory molecules known to be key drivers of endothelial and clotting pathology, points to thrombotic endotheliitis as a key pathological process in Long COVID. This has implications for the choice of appropriate therapeutic options in 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
 
Despite official figures showing that only about 629.7 million individuals around the world have already contracted the SARS-CoV-2 virus so far, the actual figures could be as high a 5 to 6-fold! In the coming catastrophic surges in the next 4 months driven by a multit ude of new SARS-CoV-2 variants and sub-lineages, it is anticipated that as at least 80 percent of the world population would be exposed to the SARS-CoV-2 virus. Re-infections and co-infections will be also be common occurrences.
 
Recent Long COVID News coverages show that based on various studies, anything between 20 to 70 percent of all those exposed to the SARS-CoV-2 virus irrespective of whether they were asymptomatic or symptomatic will end up developing Long COVID-19 issues. We at Thailand Medical News refutes parts of all these studies as we strong believe that as long as anyone has contracted the SARS-CoV-2 virus, they ultimately develop Long COVID complications. The only issue is that many are not even aware of the effects taking place as a result of the virus and its viral proteins on their bodies just yet! It some cases these effects can be slow and not detectable or manifesting symptoms yet.
 
The study team had previously determined that various inflammatory molecules are entrapped inside fibrinolysis-resistant microclots in individuals with Long COVID.
https://pubmed.ncbi.nlm.nih.gov/36131342/
 
https://pubmed.ncbi.nlm.nih.gov/34425843/
 
The study team undertook detailed proteomics analysis and with a double digestion method to liberate these molecules.
 
The study team identified, amongst others, VWF, -2AP, SAA and PF4 entrapped inside these microclots.
 
These molecules and similar such molecules are also well-known to activate platelets, by binding to their receptors. Both microclots and hyperactivated platelets, may interact with damaged endothelial cells and further perpetuate widespread vascular damage.
 
Furthermore, E-selectin and PECAM-1 are well-known markers of endothelial damage, that was also analyzed in this study.
 
The study team chose to determine if the concentrations of the above 6 inflammatory markers are also upregulated in the soluble part of blood (serum and plasma).
 
The study team have also shown that both E-selectin and PECAM-1 can trigger considerable microclot formation as well as platelet hyperactivation similar to that previously noted in samples from individuals suffering from Long COVID.
 
In order to comprehend how these six molecules may be central in perpetuating widespread endothelial damage and persistent coagulation pathology noted in Long COVID, the researchers needed to turn also turn their attention to the physiological pathways that are affected by them.
 
The study team focused on receptor activation pathways in endothelial cells and platelets.
 
Von Willebrand Factor
 
Von Willebrand Factor or VWF is well-known inflammatory molecule that is widely used as a marker of inflammation and vascular damage, and is commonly available in pathology laboratories.
 
The study team had found VWF to be trapped inside microclots and also upregulated in the soluble part of the plasma. When in circulation, it binds to two main receptors on the platelet membrane, including, GPIbα (in the GPIb-IX-V complex) and integrin αIIbβ3 (in the GPIIb-IIIa complex). When VWF binds to GPIbα in the GPIb-IX-V complex, tyrosine protein kinases LYN and FYN are activated. When these kinases are activated, they promote tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) on the FcRγ or FcγRIIα. Once the GPIb-IX-V complex is engaged, it activates intracellular signalling events that instigate platelet activation and aggregation through the αIIbβ3 integrin. This also allows for more VWF to bind to αIIbβ3 as binding of VWF to GPIb-IX-V further upregulates αIIbβ3 integrin affinity. When additional VWF molecules bind to αIIbβ3, platelet adhesion and platelet aggregation are upregulated. This ultimately contributes to thrombus formation by binding to fibrinogen, which is mediated via different pathways.
 
Although the fundamental role of VWF is to activate platelets, VWF can also bind to endothelial cells via the integrin αvβ3 receptor. An important function of VWF is to ensure endothelial cell adhesion, migration, proliferation, differentiation, and survival.
 
Furthermore, when VWF binds to the αvβ3 integrin on endothelial cells, it encourages Weibel-Palade bodies to secrete additional VWF into the circulation. The αvβ3 receptor on endothelial cells also binds fibrinogen and fibronectin.
 
Platelet Factor 4 (PF4)
 
The study team also noted a significant upregulation of PF4 in their Long COVID samples. PF4 is released from the α-granules of activated platelets as a complex with a chondroitin sulfate proteoglyan carrier. After secretion from platelets, PF4 quickly transfers to higher affinity heparan sulfate on endothelial cells where it inhibits local antithrombin activity and promotes coagulation. When heparin-induced thrombocytopenia antibodies bind to PF4/heparin complexes, these immune complexes can activate platelets via the FcγIIα receptor, further promoting thrombin generation and increased coagulation. Interestingly, PF4 does not directly bind to endothelial cells, but rather enhances platelet–neutrophil interactions that activate the vascular endothelium.
 
Serum Amyloid A (SAA)
 
Serum Amyloid A was also significantly upregulated in the Long COVID samples. SAA contains binding sites for the extracellular matrix components, laminin and heparin/heparan sulfate, as well as the RGD-like (arginine-glycine aspartic acid) adhesion motif. Immobilized SAA also binds to the αIIbβ3 receptor on platelets and may also directly bind to toll-like receptors (TLR) on endothelial cells.
 
Dysregulated SAA is associated with endothelial dysfunction and early-stage atherogenesis. SAA binding to on endothelial cells results these cells express ICAM-1, VCAM-1, E-selectin, TNF-α, IL-1, and IL-6, together with pro-thrombotic factors.
 
E-Selectin
 
The study team also found that E-selectin is significantly upregulated in serum from individuals with Long COVID. This is a notable finding as E-selectin is typically undetected in endothelial cells that have not yet been activated. Upon inflammatory stimulation, E-selectin acts as an adhesion molecule and tethers leukocytes to endothelial cells through supporting the initial rolling of leukocytes on activated endothelium. Several adhesion molecules including E-selectin, are induced and upregulated on endothelial cells during inflammation. The rolling of leukocytes on E-selectin engages P-selectin glycoprotein ligand-1 (PSGL-1) and cluster of differentiation 44 (CD44) to initiate signaling through a common pathway that requires lipid rafts, the cytoplasmic domain of PSGL-1, all 3 Src family kinases (SFKs), the immunoreceptor tyrosine-based activation motif (ITAM) adaptors, DNAX activating protein of 12 kDa (DAP12) and Fc receptor (FcR) y, the Tec family kinase Btk, and lastly, p38.
 
This signaling cascade subsequently activates integrin leukocyte function associated antigen 1 (LFA-1) to a conformation that enables slow rolling to occur. Rolling and adhesion of leukocytes due to E-selectin signals, result in an increase of CD11b/CD18 (Mac-1), ultimately indirectly facilitating binding of leucocyte s to platelets.
 
PECAM-1
 
The study team also found a significant upregulation of PECAM-1 in serum from individuals with Long COVID. PECAM-1 has been found to be expressed on endothelial cell membranes, platelets, and leukocytes. In endothelial cells, PECAM-1 signals through the Syk/PECAM1 signaling pathway as a response to shear stress, causing tension changes across the junctional proteins VE-cadherin and PECAM-1. Onset of flow results in decreased force across VE-cadherin, and a simultaneous increase in force across PECAM-1. The changes in tension across VE-cadherin and PECAM-1, activate vascular endothelial growth factor receptor 2 (VEGFR2), followed by PI3K activation and the transcription of proinflammatory genes through the NF-κB pathway. Even though PECAM-1 is known to be expressed on platelets, its role in platelets is still unclear. Unlike the other inflammatory markers, PECAM-1 is thought to function as a selective inhibitor for platelet aggregation and signaling. Mice models with decreased platelet derived PECAM-1 showed increased responsiveness to collagen stimulation and led to larger thrombus formation in vitro, further implicating PECAM-1 in the inhibition of platelet function.
 
In addition, PECAM-1 has shown to inhibit platelet protein tyrosine phosphorylation stimulated by thrombin, leading to inhibition of calcium mobilization from intracellular stores. Apart from showing that both E-selectin and PECAM-1 are upregulated in blood from individuals with Long COVID, the study team was also able to induce significant microclot formation and platelet hyperactivation when adding these two purified molecules to healthy samples.
 
Importantly, these results suggest that when both E-selectin and PECAM-1 are in circulation, they may interact with soluble plasma proteins by engaging in direct protein-protein interactions. This protein-protein interaction results in misfolding of the plasma proteins and microclot formation. Both the molecules were also found to trigger platelet activation, suggesting direct platelet receptor-molecule interactions. These results further provide evidence of significant interactions between circulating inflammatory molecules in individuals with Long COVID.
 
α-2-Antiplasmin
 
The study team had previously found that α-2AP is significantly upregulated inside microclots found in samples from individuals suffering from Long COVID.
 
In this current study, they also found a significant upregulation of α-2AP in the soluble part of the blood. This antifibrinolytic protein covalently binds to plasmin, and in so doing inhibits its function to solubilize fibrin polymers. When α-2AP is increased in circulation and comes in contact with plasmin, the C-terminal lysine residue of α-2AP binds non-covalently to the kringle domains of plasmin and forms a 1:1 stoichiometric complex. Increased levels of α-2AP therefore prevent the fibrinolytic system from dissolving pathologic thrombi resulting in venous thrombosis, pulmonary embolism, arterial thrombosis, and ischemic strokes.
 
Study Conclusions
 
The study team concluded that dysregulation of coagulation, and in particular the formation of fibrin amyloid microclots that are resistant to proteolysis and can then cause ischaemia-reperfusion injury, and are a key part of the pathogenesis of Long COVID.
 
The study team further added that whilst there may be many mechanisms at play in the pathogenesis of Long COVID, the common pathological process is thrombotic endotheliitis. This may be characterized by the formation of anomalous fibrinaloid microclots, hyperactivated platelets, endotheliitis and elevated levels of prothrombotic inflammatory molecules which interact with each other as well as with platelets and the endothelium.
 
The study team demonstrated significantly increased concentrations of VWF, PF4, SAA, α-2AP, E-selectin, and PECAM-1 in the soluble fraction of blood from individuals with Long COVID when compared to those without Long COVID. Each of the individual molecules can cause significant endothelial dysfunction and platelet activation, ultimately resulting in severe clotting imbalance and endothelial pathology.
 
To date, the same study team has demonstrated significant microclot formation and platelet activation in all Long COVID patients using fluorescence microscopy, as well as detailed proteomics analysis revealing several-fold increased concentrations of inflammatory molecules entrapped within the microclots.
 
https://pubmed.ncbi.nlm.nih.gov/34425843/
 
https://pubmed.ncbi.nlm.nih.gov/36131342/
 
https://cardiab.biomedcentral.com/articles/10.1186/s12933-022-01579-5
 
However, both fluorescence microscopy and proteomics are complex techniques usually only available in research facilities and are not currently available in clinical laboratories.
 
The study team noted that in the current findings whilst only the mean level of alpha-2-antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, it was noteworthy that the average levels of the other 5 molecules measured were significantly higher in Long COVID patients as compared to the controls- even though technically they were within the ‘normal’ range.
 
The study team warned that this is alarming if it was taken into consideration that a significant proportion of the total burden of these inflammatory molecules is entrapped inside fibrinolysis-resistant microclots.
 
Considering the significant increase in the rates of thrombotic events following even a mild COVID19 infection, it is imperative that the thrombotic endotheliitis is treated urgently.
https://pubmed.ncbi.nlm.nih.gov/35614233/
 
The study team stress that there is an immediate need for a microclot detection method that can be rolled out for clinical use.
 
The study team says that such a microclot detection method together with the demonstration of relatively high levels of the six molecules described in this paper, would be good evidence for an ongoing thrombotic endotheliitis. The optimal therapeutic regimen is yet to be defined; however, given the complex pathophysiology, such therapy is likely to incorporate antiplatelet drugs, as well as agents acting on the enzymatic pathway of coagulation.
 
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