[Preprint] SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy, Ryu et al, 2021

[SNT Gatchaman]

SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy (Non peer-reviewed)
Jae Kyu Ryu, Elif G Sozmen, Karuna Dixit, Mauricio Montano, Yusuke Matsui, Yixin Liu, Ekram Helmy, Thomas J Deerinck, Zhaoqi Yan, Renaud Schuck, Rosa Meza Acevedo, Collin M Spencer, Reuben Thomas, Alexander R Pico, Scott S Zamvil, Kara L Lynch, Mark H Ellisman, Warner C Greene, Katerina Akassoglou

Abstract
Blood clots are a central feature of coronavirus disease-2019 (COVID-19) and can culminate in pulmonary embolism, stroke, and sudden death. However, it is not known how abnormal blood clots form in COVID-19 or why they occur even in asymptomatic and convalescent patients. Here we report that the Spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the blood coagulation factor fibrinogen and induces structurally abnormal blood clots with heightened proinflammatory activity. SARS-CoV-2 Spike virions enhanced fibrin-mediated microglia activation and induced fibrinogen-dependent lung pathology. COVID-19 patients had fibrin autoantibodies that persisted long after acute infection. Monoclonal antibody 5B8, targeting the cryptic inflammatory fibrin epitope, inhibited thromboinflammation. Our results reveal a procoagulant role for the SARS-CoV-2 Spike and propose fibrin-targeting interventions as a treatment for thromboinflammation in COVID-19.

One-sentence summary
SARS-CoV-2 spike induces structurally abnormal blood clots and thromboinflammation neutralized by a fibrin-targeting antibody.

Link | PDF

 

[SNT Gatchaman]

This paper does not relate to the amyloid fibrin(-ogen) / microclot findings, and in fact does not reference those papers.

Selected quotes —

Background

The central structural component of blood clots, and a key regulator of inflammation in disease, is insoluble fibrin, which is derived from the blood coagulation factor fibrinogen

Hypercoagulability in COVID-19 is associated with inflammation and the formation of fibrin clots resistant to degradation despite adequate anticoagulation

The high prevalence of thrombotic events with these unique hypercoagulability features suggests an as yet unknown mechanism of abnormal blood clot formation in COVID-19.

Fibrinogen is a 340 kDa protein consisting of three pairs of polypeptide chains Aα, Bβ, and γ.

Fibrinogen is causally linked to the activation of macrophages and microglia in autoimmune and inflammatory diseases in the brain and periphery. Fibrin is a driver of microglia-induced cognitive dysfunction and is associated with perivascular-activated microglia and macrophages in brains of COVID-19 patients even without signs of infection.

Findings

Incubation of SARS-CoV-2 recombinant trimeric spike protein with healthy donor plasma increased fibrin polymerization. Spike strikingly altered the fibrin clot structure resulting in thinner fibers with a rough appearance and increased clot density as shown by scanning electron microscopy (SEM), identifying direct effects of SARS-CoV-2 Spike on fibrin clot architecture.

Spike bound to the γ364-395 peptide, which encompasses the γ377-395 cryptic fibrinogen binding site to complement receptor 3 that activates innate immune responses. Spike also bound to the γ163-181 peptide, whose function is unknown.

Mapping of the Spike binding peptides onto the crystal structure of fibrinogen revealed proximity of the γ163-181 and γ377-395 peptides, suggesting that a 3D conformational epitope in the carboxy-terminal γ-chain of fibrinogen (γC domain) is involved in fibrinogen binding to Spike.

Since Spike binds to fibrinogen sites that regulate plasmin cleavage and binding to complement receptor 3 [...] Incubation of Spike with fibrin delayed plasmin degradation of both the β-chain and the γ-γ dimer, suggesting that Spike delays fibrinolysis.

Spike increased fibrin-induced release of reactive oxygen species (ROS) in a concentration-dependent manner in bone marrow- derived macrophages (BMDMs), while Spike alone did not have an effect.

Conversion of fibrinogen to fibrin exposes the cryptic inflammatory γ377–395 epitope in the fibrinogen γ-chain. [...] These findings reveal a previously unknown interaction between SARS- CoV-2 Spike protein and fibrin γ377–395 epitope that promotes innate immune activation.

We tested longitudinally collected serum samples ranging from acute to convalescent disease stages from 54 COVID-19 asymptomatic, mild, and severe disease patients requiring admission to the intensive care units. Fibrin autoantibodies were abundant in all three groups of COVID-19 patients and persisted during the convalescent stage, but were scarce in healthy donor controls or in subjects with non-COVID respiratory illnesses.

Conclusions

Our data shed new light on the enigmatic coagulopathy found in COVID-19 revealing a causal role for fibrinogen in thromboinflammation – even independent of active viral replication.

Our findings now show that coagulopathy is not merely a consequence of inflammation. Rather, the interaction of SARS-CoV-2 Spike with fibrinogen and fibrin results in abnormal blood clot formation that in turn drives inflammation.

This mechanism might be in play at sites of local fibrin deposition and microvascular injury perpetuating a hypercoagulable and inflammatory state.

Overall, these results reveal an unanticipated role for SARS-CoV-2 Spike as a fibrinogen binding protein that alone accelerates the formation of abnormal clots with altered structure and increased inflammatory activity.

 

[Trish]

Assuming this research is accurate, this seems like a pretty significant finding.
What I don't understand is why it causes major, even deadly, clotting in some individuals, and seemingly has no ongoing ill effect in others.

 

[SNT Gatchaman]

What I don't understand is why it causes major, even deadly, clotting in some individuals, and seemingly has no ongoing ill effect in others.

It could be a matter of degree of fibrinolytic imbalance. Alternatively or additionally other factors such as the degree of endothelitis and platelet activation may come to bear.

From my personal experience, I've observed that I have been clearly hypercoagulable: either by clotting blood tubing during phlebotomy or looking at thromboelastography (or in vitro fluorescing microclots). However, routine coagulation tests have remained steadfastly normal. <touches wood, non-scientifically> Despite this I haven't had any observable macrothrombotic issues.

Related to "all tests are normal", one thing I've been considering is the possibility that clinical fibrinogen assays are reading incorrectly in me and other LC (±ME) patients. The assay indirectly quantifies fibrinogen by seeing how long it takes for fibrinogen to form a fibrin clot (the Clauss method).

To determine plasma fibrinogen concentration, several assays have been developed. The most frequently used in a diagnostic routine is the Clauss assay: a method based on clotting time that measures the ability of fibrinogen to be enzymatically converted to a fibrin clot. In principle, diluted citrated plasma is activated with a high concentration of thrombin, and the clotting time is inversely proportional to the functional fibrinogen concentration.

We know that glycated fibrinogen makes poorer clots and hyperglycaemic patients will read erroneously low on this test.

... fibrinogen concentration measured by the Clauss method significantly decreased with increasing glucose contamination (0–5–10–20%)

If fibrinogen in LC is similarly defective and/or abnormally glycated due to associated metabolic derangements, then it might too read artificially low.

the functional intact fibrinogen test (FiF), a monoclonal antibody-based assay that measures the amount of fibrinogen antigen, may be a better overall predictor of the overall risk of cardiovascular disease.

This might be a more accurate indicator of hyperfibrinoginaemia and would complement (pun intended) this paper's and others' findings.

See:

1. Fibrinogen Glycation and Presence of Glucose Impair Fibrin Polymerization—An In Vitro Study of Isolated Fibrinogen and Plasma from Patients with Diabetes Mellitus (2020)
2. Glucose Concentration Affects Fibrin Clot Structure and Morphology as Evidenced by Fluorescence Imaging and Molecular Simulations (2018)
3. Determinants of Increased Fibrinogen in COVID-19 Patients With and Without Diabetes and Impaired Fasting Glucose (2021)
   

 

[SNT Gatchaman]

Just to note that despite this paper being in pre-print, the same authors published in Nature in 2018 where they covered some of the same (non spike-related) ground:
Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration

Abstract
Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, the mechanisms that link disruption of the blood–brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions.

Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377–395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration.

Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

 

[Boba]

Thanks for posting. Looking at the study in Austria where they found viral remnants in all IBS patients experiencing Long Covid symptoms this could be an ongoing pathology right?

https://www.s4me.info/threads/post-...tory-bowel-diseases-2022-zollner-et-al.27446/

 

[SNT Gatchaman]

Looking at the study in Austria where they found viral remnants in all IBS patients experiencing Long Covid symptoms this could be an ongoing pathology right?

I'm with you on this question. The IBS biopsy findings certainly found remnant viral elements that included spike protein.

We detected viral RNA in 31% of biopsies, with expression of the RNA dependent RNA polymerase (RdRP) in 13.6% of biopsies, the surface glycoprotein (Spike) in 11.4% of biopsies, the nucleocapsid phosphoprotein (Nucleocapsid) in 10.6% of biopsies, and the envelope protein in 6.1% of biopsies

These aren't replication-competent whole virions that they have found. But the cells contain spike protein - which could be exocytosed. More importantly if there is RNA and RNA polymerase, the cell could make more RNA and more spike protein (cf mRNA vaccines which - in theory - make a limited amount of spike).

SARS-CoV-2 is a positive-sense, single-stranded RNA virus. Being positive-sense, its RNA acts like mRNA and can directly interact with a cell's ribosomes to make proteins. So I'm assuming it could make spike protein which could be discharged from the cell and enter blood vessels.

So could gut mucosal cells continue to produce spike proteins, which reach the blood vessels and pathologically interact with fibrinogen? How long would this last? Surely not indefinitely, as the mucosal cells will be turned over eventually.

Nevertheless, from the Austrian IBD biopsy paper —

Notably, only patients who displayed viral RNA expression in the gut reported symptoms compatible with post-acute COVID-19 sequelae

 

[Snow Leopard]

Assuming this research is accurate, this seems like a pretty significant finding.
What I don't understand is why it causes major, even deadly, clotting in some individuals, and seemingly has no ongoing ill effect in others.

Because in some people, the virus gets a much stronger foothold before the adaptive immune response kicks in. The big spill-over of all of the virion/spike protein doesn't occur until things are already very bad.

Some of which were suggested from the very start (due to research on SARS and other Coronaviruses). Namely the suppression of type I interferon responses of infected cells and syncitia of the cells lining the respiratory tract. This evasion of the immune system is likely the reason why the virus is more deadly for older men in particular.

 

[SNT Gatchaman]

What I don't understand is why it causes major, even deadly, clotting in some individuals, and seemingly has no ongoing ill effect in others.

Because in some people, the virus gets a much stronger foothold before the adaptive immune response kicks in. The big spill-over of all of the virion/spike protein doesn't occur until things are already very bad. [...] This evasion of the immune system is likely the reason why the virus is more deadly for older men in particular.

That presumably relates to acute, severe disease — in esp older men. But then there are those previously young and fit, esp female, with truly mild or even asymptomatic infection, who weeks or months down the line are going on to massive PE, stroke, MI etc. However, many (like me) are not having those severe post-acute venous or arterial thrombotic outcomes.

If glycated fibrinogen makes poor clots [1, 2] then perhaps fibrinogen+spike might also be less effective and macrothrombogenic. Perhaps from a purely coagulation viewpoint, it's "OK" to have this abnormal fibrinogen circulating around for most people. However it may be doing bad things to inflammation, autoantibodies, metabolism etc while only causing major thromboembolic disease in a minority.

1. Fibrinogen Glycation and Presence of Glucose Impair Fibrin Polymerization—An In Vitro Study of Isolated Fibrinogen and Plasma from Patients with Diabetes Mellitus (2020)
2. Glucose Concentration Affects Fibrin Clot Structure and Morphology as Evidenced by Fluorescence Imaging and Molecular Simulations (2018)
3. Determinants of Increased Fibrinogen in COVID-19 Patients With and Without Diabetes and Impaired Fasting Glucose (2021)

 

[Snow Leopard]

That presumably relates to acute, severe disease — in esp older men. But then there are those previously young and fit, esp female, with truly mild or even asymptomatic infection, who weeks or months down the line are going on to massive PE, stroke, MI etc.

The mechanism for that is likely different (autoimmunity, or unknown pre-conditions) and also cannot be explained simply by a generic pro-thombotic mechanism like that described in the pre-print.

 

[SNT Gatchaman]

Tragically these catastrophic thrombotic events are not just affecting adults in the Long Covid timeframe, also children, including in the few weeks post recovery.