Spike Protein, novel binding sites and coagulopathy

Endless bag of tricks

As I wrote in my Twitter feed. I prefer sticking to what I said, but I spent most of the day helping a family member (leukemia, 4 mRNA doses), so no way I can publish my mimicry post today. I will write something else, but, as important.

First a throwback to one of my most pertinent observations to this day made half as a joke, half serious.

John Paul @ThingsHiddenn

I am amazed how many receptors SARS 2 can use. I will just assume it can use all receptors know to man 🤷🏻‍♂️

From this paper.

von Willebrand factor (vWF) is primarily secreted by endothelial cells (ECs) and functions as a transporter of pro-coagulant factor VIII (FVIII). It is also an initiator of platelet adhesion and aggregation, leading to thrombus formation. A clinical study reported that ICU patients with COVID-19 had a significantly higher level of vWF in blood than non-ICU patients,2 suggesting a role of vWF in COVID-19-associated coagulopathy. To examine the effects of vWF regulation in COVID-19-associated thrombosis, we used the recombinant purified Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to mimic SARS-CoV-2 invasion. Spike protein had no effects on vWF expression but induced vWF secretion in a dose- and time-dependent manner in human umbilical vein ECs. Simultaneously, FVIII-vWF binding and platelet adhesion to ECs were significantly elevated after Spike protein stimulation. These data suggest a potential role of vWF secretion in COVID-19-associated coagulopathy.

Not highlighted but the authors bring to attention that despite many concluding that ACE2 had a direct role in the induction of vWF secretion, here they bring up the fact that it doesn’t, and if ACE2 does have a role, it is indirect.

But when they tested the Spike Protein and Receptor-binding domain of the same protein, they found that CKAP4 (otherwise known as P63) is a novel receptor to the Spike/RBD, and you don’t need damage to endothelial cells to activate the coagulopathy, the Spike protein binding to CKAP4 itself will induce set off this cascade of events. As you can read in the following paragraph.

In addition, EC-specific overexpression of CKAP4 did not affect EC structure in mice with or without Spike protein-stimulation. Therefore, Spike protein-caused coagulopathy were primarily attributed to its binding with CKAP4 and downstream vWF secretion, rather than impairing EC structure.

Now we have evidence that the Spike Protein alone can bind to a fairly important protein and set off a cascade that ends in coagulation and endothelial dysfunction, another contributor to the endothelial dysfunction aspect of the virus/spike. And it is a rather important find because it fits into the puzzle (sent by a friend).

Neurobiochemical Cross-talk Between COVID-19 and Alzheimer’s Disease

Third time within days I mention this class of protein, the Galectin, in this case, Galectin 9, a sister protein of Galectin 3, and both play a big role in immune regulation in the brain (microglia), a dysregulation or expression of one above the other shifts the immune system out of balance. Galectin 3 will be of extreme importance when I publish the Molecular Mimicry piece.

And then we have CKAP4, which you can clearly read that ablation (fancy word for elimination) of it results in quite the severe cognitive impairment, which is another angle now the Spike Protein can cause cognitive disruption besides the Kynurenine Pathway.

You can gauge how troublesome the Spike binding to this protein is from this short text alone. Now we go into more novelty. The hidden aspects of the Spike Protein.

Identification and characterization of a novel cell binding and cross-reactive region on spike protein of SARS-CoV-2

Given that COVID-19 continues to wreak havoc around the world, it is imperative to search for a conserved region involved in viral infection so that effective vaccines can be developed to prevent the virus from rapid mutations. We have established a twelve-fragment library of recombinant proteins covering the entire region of spike protein of both SARS-CoV-2 and SARS-CoV from Escherichia coli. IgGs from murine antisera specifically against 6 spike protein fragments of SARS-CoV-2 were produced, purified, and characterized. We found that one specific IgG against the fusion process region, named COVID19-SF5, serologically cross-reacted with all twelve S-protein fragments. COVID19-SF5, with amino acid sequences from 880 to 1084, specifically bound to VERO-E6 and BEAS-2B cells, with Kd values of 449.1 ± 21.41 and 381.9 ± 31.53 nM, and IC50 values of 761.2 ± 28.2 nM and 862.4 ± 32.1 nM, respectively. In addition, COVID19-SF5 greatly enhanced binding of the full-length CHO cell-derived spike protein to the host cells in a concentration-dependent manner. Furthermore, COVID19-SF5 and its IgGs inhibited the infection of the host cells by pseudovirus. The combined data from our studies reveal that COVID19-SF5, a novel cell-binding fragment, may contain a common region(s) for mediating viral binding during infection. Our studies also provide valuable insights into how virus variants may evade host immune recognition. Significantly, the observation that the IgGs against COVID19-SF5 possesses cross reactivity to all other fragments of S protein, suggesting that it is possible to develop universal neutralizing monoclonal antibodies to curb rapid mutations of COVID-19.

Here the authors “cut” the Spike Protein into 12 fragments and created a library, an archive of said proteins, and a way to recombine them. 6 fragments from SARS-CoV (the first SARS), and 6 from SARS-CoV-2.

You can visualize it below.

From their testing, the strongest possible protein sequence was the SF5 (Spike Fragment 5) which bears a sizable portion of the S2 part of the Spike Protein. In their testing, the SF5 was able to bind to a cell and facilitate the binding of the entire Spike Protein. Worth noting IgG (type of antibody) that reacts to Fragment 5, was also able to cross-react to the whole Spike.

This cross-reaction enables the antibodies to be able to inhibit infection at a variable rate of 20 to 50%, where 50% was achieved by SF5. This is an argument made by the authors that you can develop new potent monoclonals from SF5, and further research would enable the development of new vaccines.

My argument will soon follow.

At present, the highly transmissible Omicron variant is spreading rapidly across the world, causing new infections, reinfections, and breakthrough infections among fully vaccinated people31,32. There may be an alternative infection pathway for mutant RBD-ACE-2 virus variants to infect host cells and some other functional or active regions on the SARS-CoV-2 spike protein to be further identified.

In summary, a new cell-binding region (COVID19-SF5) on spike protein of SARS-CoV-2 has been identified and characterized. The binding of COVID19-SF5 to the cell can greatly enhance the association of glycosylated full-length S protein with the host.

If there is one thing I could agree with the authors is the highlighted paragraph, there are other hidden, functional regions in the Spike Protein that need to be further studied, and some are using semantics to do the research, or not doing it at all. And glycosylation is extremely important in the context of the Spike protein and its effects.

That free e-book will sure come in handy. I will now leave the reader with the same question I left on Twitter not long ago.

What is hidden inside that Spike Fragment 5, in the S2 region?

In summary, we now have 2 novel mechanisms for the Spike. One is for binding to a cell and activating coagulopathy on its own, without the need for infection or anything else, and the other is a big piece of the Spike enabling easier infection. Here is the kicker, these regions, and others I will cover in my Molecular Mimicry piece are all in the vaccines. You can now understand pretty easily why the vaccinated immune system is shifting heavily towards a non-inflammatory state… one that will be counterproductive shortly.

I will do my best to publish the Molecular Mimicry one tomorrow !

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