A Mechanism for Spike Protein Damage

In which I present a hypothesis.

First, a note on the importance of this article. When discussing COVID-19 associated damage or potential vaccine adverse events it is common to encounter the question, “what’s the mechanism for that?” Though the question is also asked by well meaning people, the most common form is in the snide, self-aggrandizing voice of the pseudo-skeptic. Detailed mechanistic knowledge of how diseases function usually comes about long after the general acknowledgment of cause and effect has been accepted and a pseudo-skeptic knows that their interlocutor is unlikely to be able to explain the mechanism even if sufficient studies have been done.

Yet working towards a mechanistic understanding of COVID-19 and COVID-19 vaccine adverse events is an absolute necessity. In developing a series of testable frameworks we are able to make and test predictions and prioritize future research directions.

Herein I lay out a mechanism by which SARS-CoV-2’s Spike Protein can cause damage. Nothing is unique to me, except perhaps some of the synthesis as I haven’t seen everything all together before. Much of what I will discuss is so well known and well documented as to risk boring experienced readers, yet somehow I haven’t seen any major voices mention the connection. The question yet to be answered is to what extent this mechanism is responsible for damage in COVID-19 and COVID-19 vaccine adverse events. Although the topic gets technical at times I’ve tried to write this article in a plainer language than what is found in scientific journals. This article is meant to be an introduction to the ideas and not necessarily exhaustive. People familiar with RAGE and AT1R may choose to skip Part 1, which is background on these systems.

At the end I will discuss predictions, limitations, and shortcomings of the hypothesis.

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PART 1. Background

COVID-19 and Spike Protein Damage
Initially, one of the defining aspects of COVID-19 was the level of fibrosis or scarring in the lungs of patients. Figure 1 shows an example of this from an early patient. As more people were infected, an interesting picture of disease began to take shape. Poorer outcomes were mostly limited to very specific groups of people and the disease seemed to affect a wide variety of systems. In order to make informed choices, to understand the past and plan for the future, we must work to understand this disease.

Figure 1: From Liu et al. Sci China Life Sci., 2020. CT Scan of a 66yo Male taken at 8, 13, and 16 days after COVID-19 onset. Images show increased signs of inflammation, edema, and fibrosis over the time course.

How much of COVID-19 as a disease is due to active viral replication? How much is due to other aspects, such as the presence of viral proteins, and, specifically relevant to the COVID-19 vaccines, what is the outcome after exposure to the Spike Protein?

Let’s look at an example research paper which tried to answer this question. Figure 2 shows pictures of the lung tissue from two hamsters. Both hamsters were infected with a pseudo-virus, a virus that has been genetically modified so that it cannot replicate on its own, however the virus which infected the hamster whose tissue is on the right had been further modified to express the SCV-2 Spike Protein. Five days after infection, the researchers killed the hamsters and preserved the lung tissue for staining and imaging. Examine the image below.

Figure 2: From Lei et al. Circ. Research, 2021. Select photos compare hamster lung tissue after infection with pseudo-virus which was (pseudo-spike) or wasn’t (mock) modified to express Spike Protein.

The authors use arrows to bring special attention to the signs of immune response and inflammation: the thickening of the air sac walls, the reduction of space for oxygen exchange, and the movement of the immune cells into the air sacs. Both animals were infected with a virus, but there is a visible difference in the two images. This implies that the difference between these two animals is whether or not the Spike Protein was present, therefore the Spike Protein is the cause of the observed changes. 

Is there a plausible mechanism by which the Spike Protein might cause this?

Background on RAS

To answer this question, let’s start with background information on the Renin Angiotensin System (RAS). The main role of the RAS is in maintaining normal levels of blood pressure. Signals are sent to the blood vessels which cause them to either constrict or relax depending on the circumstances. Below is a brief description of the RAS highlighting the parts necessary to understand the main idea of this article.

The major molecule of RAS action is Angiotensin. Initially found in its inactive form, Angiotensinogen, it is activated by a molecule called renin and turned into Angiotensin I (Ang I). Angiotensin I is cut by Angiotensin Converting Enzyme (ACE) to form Angiotensin II (Ang II). Ang II is the constriction and inflammation signal. It can either bind to one of its receptors or be further cut by Angiotensin Converting Enzyme 2 (ACE 2) to form Angiotensin 1,7 (Ang 1,7) which is the relaxation and dilation signal.

In other words, Ang II and Ang 1,7 work in balance with each other with Ang II constricting blood vessels and Ang 1,7 dilating them. This is the normal function of the body, yet it can turn pathological in the proper circumstances. Associated with stress, poor diet and lifestyle, and advanced age, Ang II can enter a positive feedback loop in which Ang II’s inflammatory signaling through its receptor, Angiotensin II Receptor Type 1 (AT1R), leads to increased levels of Ang II and AT1R. This occurs slowly and over the course of years in most cases, but can be acute if the delicate signaling balance is overridden by disease.

Spike Protein and the RAS

Figure 3: Modified from Bosso et al. Molecular Therapy, 2020. A cartoon model of the interaction of SCV-2 Spike protein on the ACE 2 receptor. The red, blunted arrow indicates inhibition (stopping the creation or function of the target).

How does exposure to the Spike Protein impact this system? As seen in Figure 3, ACE 2 inhibition by the Spike Protein blocks one of the body’s main paths to counter Ang II. The response to too much Ang II is to create more Ang I which is also then converted into Ang II. Obviously this is a dangerous spiral, but the level of damage is dependent on several factors. One factor is the quantity of AT1R is being made by the body. This depends on how much Ang II signaling has been going on in the person. Someone who has hypertension, kidney disease, is diabetic or obese is likely to have higher AT1R expression than someone who is not. The second factor will be explored further in the article.

Below, Figure 4 and Figure 5 show evidence of ACE 2 reduction and increased serum Ang II respectively.

Figure 4: Lei et al. Circ. Research 2021. Top: Select Western Blot Images. Bottom: aggregate western blot data showing an increase in endothelial damage markers and a decrease in ACE-2 Expression.

Figure 5: Liu et al. Sci China Life Sci., 2020. Graphs showing increase serum Angiotensin II in 12 COVID-19 patients. Increase was proportional to viral load and was associated with poorer gas exchange.

Before we continue, there is one more section of background which is necessary.

Background: AGE and RAGE

It’s time to introduce more necessary background. First, the idea of Advanced Glycation End-products (AGEs). AGEs are proteins, fats, or nucleic acids that have been so modified by the presence of sugars that they are permanently modified. They’re the end products after advanced levels of sugar interaction, or glycation. The rate of AGE formation in our bodies is dependent on the amount of sugar present, for example it is increased while blood sugar is high after eating. People with poorer diets which lead to increased levels of blood sugar more often or for longer have increased AGE formation. Perhaps the most extreme example of this is people who have uncontrolled Type 2 Diabetes as their blood sugar is nearly constantly elevated. AGEs are thought to be difficult to breakdown and therefore may build up over time.

AGEs are one group among several which activate the Receptor for Advanced Glycation End-products (RAGE). When activated, RAGE induces NFkB mediated oxidative stress, inflammation, and fibrosis. See references at the end for further reading. RAGE signaling is also a positive feedback loop; RAGE binding to its ligands results in more RAGE being produced.

Part 2: The Connection

It has long been surmised that there was a connection between AT1R and RAGE. Two papers were recently published by Pickering et al. and Kawai et al. in 2019 and 2021 respectively that show evidence that AT1R and RAGE form a dimer on the cell surface and trans-activate each other. This is the single point which is most often overlooked. I will try to explain the implications.

Figure 6: A cartoon of a dimerized RAGE and AT1R and their ligands.

Dimerization is a process by which two receptors, in this case, bind to each other as seen in cartoon form in Figure 6. Due to this dimerization, these receptors are able to trans-activate. Please, examine Figure 7.

Figure 7: A cartoon showing transactivation in which the binding of one receptor’s ligand activates both receptors. A) Ang II binding to AT1R activates AT1R and RAGE. B) AGEs binding to RAGE results in RAGE and AT1R activation.

Binding of the ligand of one receptor causes the signaling of both. This mechanism implies a much more intimate interaction between RAGE and AT1R than previously thought. More than that, Pickering et al.’s 2019 paper suggest that RAGE may actually be necessary for the inflammatory outcome of AT1R signaling. If this turns out to be the case, the amount of damage from AT1R signaling may be directly related to the quantity of RAGE expression. Because of this, any analysis on the impact of the Spike Protein on the RAS which doesn’t look at RAGE signaling is incomplete.

What we have are two positive feedback loop systems which seem to be able to stimulate each other. Each has been studied for decades as a path of disease. The quantity of each is dependent on factors such as lifestyle, diet, and age. The degree to which AT1R / RAGE transactivation is responsible for Spike Protein related damage is not yet known.

PART 3. Predictions and Limitations

Comorbidity

Figure 8: Kompaniyets et al. Preventing Chronic Disease, 2021. Those comorbidities that were found to have increased risk of poor C19 outcome. Numbers > 1 show increased risk of poor outcome.

One of clearest instances in which this hypothesis predicts outcomes has to do with the comorbidities, diseases that one has at the time of infection, which predict poorer outcomes from COVID-19 infection as seen in the table above. There is a connection between every disease shown in the example, with the possible connection of aplastic anemia which I am still connecting, and increased signaling through RAGE / AT1R.

This study reported that poor outcomes were strongly correlated not only with comorbidities, but with the number of comorbidities one had. An elderly person without comorbidities had a similar risk of poor outcomes from COVID-19 to that of a younger person.

Interestingly, this study showed normal or decreased risk of poor outcome in people who were hypertensive and those who had ‘controlled’ diabetes. This finding may be evidence against the hypothesis I’ve presented, but I believe it more likely to be due to the medications these patients are taking. The AT1R / RAGE transactivation hypothesis of Spike Protein damage would predict that medications like ACE inhibitors and Angiotensin II Receptor Type 1 Blockers (ACEi and ARB respectively) may be protective against Spike Protein damage. Currently the evidence here is conflicting, likely because those people who would be taking the medications are the same people who would have the comorbidities such that they need these medications. We’ll have to wait for more results.

Vitamin D deficiency also plays a role in the Spike Protein damage hypothesis. vitamin D is a negative regulator of renin. Recall that the action of renin is to start the process by turning inactive Angiotensinogen into active Ang I. Having normal levels of vitamin D keeps renin in check. In people who are vitamin D deficient, this regulator is not properly maintained. This lack of a control mechanism may bias the body towards runaway Angiotensin II signaling. See Figure 9.

Figure 9: Yan Chun Li. Vitamin D (3rd Edition). Cartoon of the effect of inhibitors on the RAS. Blue text indicates inhibitor, ie ACEi inhibits ACE, ARB inhibits AT1R, vitamin D inhibits renin gene expression.

Outcomes of Spike Exposure
The majority of the outcomes predicted by the mechanism I’ve detailed would be vascular or related to the blood vessels. However, this can take many, seemingly disconnected, forms. AT1R and RAGE signaling have well studied detrimental impacts on the vasculature and the heart causing scarring, inflammation, and heart failure. In other areas of the body this can lead to new or worsening kidney disease and pulmonary fibrosis, and increased permeability of the blood brain barrier. Ang II signaling leads to the creation of new blood vessels. Aberrant levels of new vessel creation can supply new cancers with the nutrients necessary to overcome the body’s immune system or to reinvigorate cancers in remission. Increased Ang II levels would result in a pro-clotting state through aberrant platelet activity, coagulation, and fibrinogen breakdown.

To recap, one might expect spike protein across a population exposure to result in: increases in certain types of cancer, especially fast growing and soft tissue cancers, unusual clotting, vascular and heart inflammation, brain inflammation, intestinal inflammation, worsening of kidney function, diabetes and associated diseases including neuropathy. One might expect these things to appear most in people already predisposed to these states because of preexisting conditions and lifestyles and those with vitamin D deficiency. Different people would be expected to have different reactions depending on which systems were most vulnerable at the time of exposure. If the dosing amount of Spike Protein was inconsistent, one might expect that these outcomes would occur inconsistently but with a general overarching pattern.

Limitations
However, a hypothesis that predicts any outcome is useless. So, what would this not look like? When I ask myself this question, the first thing that comes to mind is it would not look like an acute toxin. I would not expect to see this mechanism cause things like biliary track damage associated with different metabolic genotypes, this would likely be evidence for LNP toxicity.

The amount of Spike Protein which is created by any one shot of the COVID-19 vaccine is stochastic, that is to say unpredictable. The mechanism I’ve laid out assumes that the dose of Spike Protein is large enough to cause an increase in serum Angiotensin II levels. In the same vein, hypotheses which do not include the production of Spike Protein are mutually exclusive with the AT1R / RAGE transactivation proposal, such as in strongest versions of the mis-folding Spike leads to prion disorder hypotheses. If little or no Spike Protein is created, then little or none of what I’ve discussed here is likely to occur. This distinction could end up being a general case or dependent on individual propensity to mis-folding and prion disease.

Many of the other candidate mechanisms for Spike Protein damage from COVID-19 and vaccine adverse events are compatible with what I have laid out here. Examples include anti-Spike antibody damage, direct Spike-induced clotting, and others. This mechanism is also agnostic to other hypotheses regarding vaccine related damage including vaccine LNP toxicity, bolus formation from adenovirus or LNP, batch contaminants, etc. That being said, excepting in cases where those hypotheses provide increased explanatory power, this transactivation mechanism should likely take priority until further evidence is acquired.

It is yet to be determined to what extent Spike Protein remains active once bound with antibodies. It may be that the formation of the spike-immunocomplex stops the mechanism described here. If that is the case, then this mechanism is likely to play its biggest role in the first week post exposure.

I do not yet know whether this mechanism has any extra explanatory power when it comes to COVID-19 or vaccine injury in young or healthy people. My personal interests have been in older populations and those with chronic illness. Further exploration of this question is still to come.

To me, the argument I’ve laid out here is the most parsimonious. It does not assume any maliciousness or conspiracy, nor does it rule those things out. This mechanism is sure to be happening to some extent in both COVID-19 and COVID-19 vaccine adverse events and is very likely happening along some of the other hypotheses of Spike Protein and transfection vector related damage. While this of course does not instill it with any sacred quality, it does mean that this is a hypothesis which must be seriously considered.

Citations and References
For those of you looking for more information, consider them my recommendations for where to start.

Ramasamy et al. Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiology, 2005.

Sellegounder et al. Advanced glycation end products (AGEs) and its receptor, RAGE, modulate age-dependent COVID-19 morbidity and mortality. A review
and hypothesis International Immunopharmacology. 2021.

Lei et al. SARS-CoV-2 Spike Impairs Endothelial Function Via Downregulation of ACE-2. Circulation Research, 2021.

Forrester et al. Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology. Physiol Rev., 2018.

Yan Chun Li. Chapter 40: Vitamin D and the Renin-Angiotensin System. Vitamin D (3rd Edition) 2011.

Kompaniyets et al. Underlying Medical Conditions and Severe Illness Among 540,667 Adults Hospitalized With COVID-19, March 2020–March 2021. Preventing Chronic Disease, 2021.

Chiappalupi et al. Targeting RAGE to prevent SARS-CoV-2-mediated multiple organ failure: Hypotheses and perspectives. Life Sciences, 2020.

Delpino and Quarleri. SARS-CoV-2 Pathogenesis: Imbalance in the Renin-Angiotensin System Favors Lung Fibrosis. Front. Cell. Infect. Microbiol., 2020.

Crackower et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature, 2002.

Li et al. Advanced glycation end products and neurodegenerative diseases: Mechanisms and perspective. Journal of Neurological Sciences, 2012.

Liu et al. NF-κB signaling in inflammation. Sig Transduct Target Ther, 2017.

Jangde et al. RAGE and its ligands: from pathogenesis to therapeutics. Critical Reviews in Biochemistry and Molecular Biology, 2020.

Senchenkova et al. Angiotensin II Mediated Microvascular Thrombosis Circulation Research, 2010.

Kawai et al. AGE ligands stimulate angiotensin II type I receptor (AT1) via RAGE/AT1 complex on the cell membrane Sci Rep., 2021.

Pickering et al. Transactivation of RAGE mediates angiotensin-induced inflammation and atherogenesis. J. Clin. Invest. 2019

Trougakos et al. Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis. Trends in Molecular Medicine 2022