Inflammation and Endothelial Biomarkers for Long Covid
Pointing the way towards endothelitis???
Recently I’ve been trying to move beyond focusing on the Coagulation Cascade and Platelet Activation pathways to understand how inflammation is caused in Long Covid.
Specifically, I’ve been curious about inflammation of the endothelium (the lining of the blood vessels), which results in endothelitis. And how microclots and platelet hyperactivation are either a cause or an effect of that inflammation.
As I shared with you last week, when I plunged head-on and somewhat naively into that topic, I was overwhelmed with a ton of disjointed data that was difficult to fit into one overall, unified picture.
Now I want to back up and start with one paper that may help point the way to piecing together the inflammation story in Long Covid endothelitis.
This paper (see below and Ref 1) reports measuring six biomarkers for inflammation and coagulation in blood samples from Long Covid patients and comparing them to blood samples from “healthy” controls who do not have Long Covid.
The goal of this work is to try to develop biomarkers that could be easily measured in a clinical setting to more quickly and accurately determine whether a patient has Long Covid and whether they could benefit from therapeutic treatments like Triple Anticoagulant Therapy.
The six biomarkers that were chosen and measured all relate in some way to inflammation, coagulation, or damage to the endothelium. Here are the biomarkers and their known functions:
VWF (von Willebrand factor) - Promotes Coagulation & Clotting
VWF is non-enzymatic protein that is contained in 𝛼-granules of the platelets. When platelets are activated, VWF is released into circulation. VWF then binds to a multitude of cell types (including platelets themselves and collagen at the site of wounds) and proteins (including coagulation factors like Factor VIII and platelet cell surface receptors like gpIb/IX/V) in order to promote clotting and wound healing.
PF4 (Platelet Factor 4) - Promotes Coagulation & Clotting
PF4 is small signaling protein that is also contained in 𝛼-granules of the platelets and released when platelets are activated. PF4 promotes coagulation and clot formation by inhibiting the action of human heparin (a natural anticoagulant) and heparin-like molecules. PF4 is also a chemoattractant for neutrophils (a type of white blood cells) and fibroblasts (which make collagen) during the inflammation process.
SAA (Serum Amyloid A) - Links Inflammation with Immune Response
SAA is a protein produced during the acute phase of inflammation. SAA localizes to the site of inflammation and recruits immune cells. The activation profile of SAA is similar to CRP (C-Reactive Protein) and SAA levels increase quickly in the acute phase of inflammation.
𝛼-2AP (𝛼-2-antiplasmin) - Inhibits Fibrinolysis and Breaking Down Clots
𝛼-2AP is a protein that inhibits and inactivates the plasmin protein. Plasmin is the enzyme responsible for breaking down clots as part of the natural process called fibrinolysis. Elevated 𝛼-2AP levels inhibit plasmin and inhibit fibrinolysis, leading clots to persist and not break down.
E-Selectin - Biomarker for Endothelial Damage, Links Inflammation with Immune Response
E-selectin is a cell adhesion protein that is expressed only on the surface of endothelial cells that have been activated by cytokines such as PF4 or P-selectin at a site of injury, thereby making it a biomarker for endothelial damage or trauma. Following activation, E-selectin binds leukocytes (white blood cells) and recruits the immune response to the site of injury.
PECAM-1 (Platelet endothelial cell adhesion molecule) - Biomarker for Endothelial Damage
PECAM-1 is a cell adhesion protein found on the surface of endothelial cells and makes up a large portion of the endothelial cell intercellular junctions. At this location, PECAM-1 is responsible for the maintenance of cell junction integrity and regulates leukocyte (white blood cell) transmigration across the endothelium, which is a hallmark of inflammation. Damage to the endothelium leads to PECAM-1 activation, increased leukocyte migration, and inflammation.
These biomarkers were detected circulating in their soluble form in the blood and were measured using either an ELISA laboratory assay (for PF4, SAA, 𝛼-2AP, E-selectin, and PECAM-1) or clinical services laboratory test (for VWF). The Long Covid patients were also tested for microclots and platelet activation, which were detected in all the Long Covid patients using fluorescence microscopy.
So what did the study find?
Once measured, “All six [biomarkers] were significantly upregulated in the soluble part of the blood (serum and plasma) in individuals with Long Covid when compared to healthy participants.”
Here’s the data:
When we look at the data in the table, we see that for 5 of the 6 biomarkers (SAA, PF4, VWF, E-selectin, and PECAM-1), the mean values in the Long Covid patients are higher by a statistically significant amount (p<0.05 or lower). BUT the mean values for each of these biomarkers in the Long Covid patients are all still inside the reference range. Which means the “high” reading on a blood test might not be treated as high by a physician because it’s inside the reference range.
However, for one of the biomarkers - 𝛼-2AP (highlighted in yellow in the table) - the mean value is not only higher in the Long Covid patients than in the control group, but it’s also so high that it’s outside the top end of the reference range. Which means it would actually be flagged on blood work as an abnormal result.
So what does this mean?
From a biomarker point of view, that means that 𝛼-2AP might actually be a good biomarker to test for clinically to detect Long Covid. To prove that and have 𝛼-2AP adopted as a Long Covid clinical biomarker will require a much larger experiment (this one had 25 patients with Long Covid and 15 healthy volunteers) and a lot more testing and validation.
And what can we learn about inflammation, coagulation, and endothelitis from these results?
Overall, we see that the five biomarkers that are elevated but within the reference range all deal with coagulation, inflammation, endothelial damage, and recruiting the immune response.
The fact that the biomarkers dealing with coagulation (VWF & PF4) are elevated isn’t a big surprise, given what we know about microclots and platelet hyperactivation.
The biomarkers that deal with inflammation (SAA, E-selectin, and PECAM-1) give us a starting point to look at the molecular mechanisms that underlie their activation, in order to start really investigating the inflammation and endothelitis in Long Covid. That’s one of the things that I’ll be working on next.
And the one biomarker that is both high and above the reference range - 𝛼-2AP - is tied to microclots, but in a way that we have not talked about yet. The high 𝛼-2AP means that clots are not being broken down by the fibrinolysis process. So it’s worth asking whether there are any ways to promote fibrinolysis or somehow inhibit 𝛼-2AP in order to restore the function of plasmin and break down clots. That’s also something that I’m going to start taking a look at.
So in summary, looking at these biomarkers have give us a few things:
A potential biomarker (𝛼-2AP) to use clinically to identify Long Covid patients.
Three inflammation biomarkers to start examining for the link between inflammation and coagulation.
A new molecular pathway (fibrinolysis) and target (𝛼-2AP) to examine for additional ways to treat microclots.
Not a bad week’s work. Stay tuned for more on those topics
I hope you enjoyed this piece.
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Reference
Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis. Seminars in Thrombosis & Hemostasis. 2023. https://doi.org/10.1055/s-0043-1769014.