SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19, 2021, Grobbelaar et al

[SNT Gatchaman]

SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19
Lize M. Grobbelaar, Chantelle Venter, Mare Vlok, Malebogo Ngoepe, Gert Jacobus Laubscher, Petrus Johannes Lourens, Janami Steenkamp, Douglas B. Kell and Etheresia Pretorius

Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients.

Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using platelet-poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin.

These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients.

This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.

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[SNT Gatchaman]

The study compared moderate to severe acute COVID patients with normal controls. Selected quotes.

We also determine if the spike protein may interfere with blood flow, by comparing naıve healthy platelet-poor plasma (PPP) samples, with and without added spike protein, to PPP samples from COVID-19 positive patients (before treatment).

Blood was collected from healthy volunteers (n=11; 3 males and 8 females; mean age: 43.4 +− 11.7 years) to serve as controls. Individuals who smoke, who were diagnosed with cardiovascular diseases, clotting disorders (coagulopathies), and/or any known inflammatory conditions (e.g. Type 2 Diabetes Mellitus (T2DM), rheumatoid arthritis, tuberculosis, asthma, etc.) could not serve as control volunteers. Furthermore, pregnancy, lactation, hormonal therapy, oral contraceptive usage, and/or using anticoagulants, were also factors that resulted in exclusion.

Microfluidics analysis included a preliminary analysis using PPP samples from two COVID-19 positive patients, on the day of first diagnosis and before any treatment was given. Both patients were diagnosed with moderate to severe COVID-19 symptoms (1 male and 1 female, mean age: 78.5 +− 7.7 years).

They analysed with fluorescence microscopy, scanning electron microscopy, microfluidics, proteomics (liquid chromatography, mass spectroscopy)

Scanning electron microscopy (SEM) was used to view healthy WB samples, with and without the addition of spike protein.

Microfluidic analysis was performed using healthy PPP and healthy pooled PPP samples (three pooled PPP samples), with and without spike protein, and two COVID-19 PPP samples. Pooled samples were used due to the volume required for this experiment.

Proteomics - Four healthy PPP samples were analyzed before and after addition of spike protein.

Average amyloid area
Healthy PPP vs healthy PPP + added thrombin p = 0.2
Healthy PPP + spike protein vs healthy PPP + spike protein + added thrombin p = 0.3
Healthy PPP vs healthy PPP + spike protein p = 0.004
Healthy PPP + added thrombin vs healthy PPP + spike protein + added thrombin p = 0.0036

In this laboratory analysis, we provide evidence that spike protein does indeed play a major role in hypercoagulability seen in COVID-19 patients. It causes anomalous clotting in both purified fluorescent fibrinogen and in PPP from healthy individuals, where the nature of the clots were shown to be amyloid

In healthy PPP exposed to spike protein, followed by incubation with ThT, there was a significant increase in anomalous clots with an amyloid nature

Spike protein also caused major ultrastructural changes in WB (as viewed with the SEM), where platelet hyperactivation was noted (Figure 6C,D). Increased in spontaneously formed fibrin network, as well as anomalous clot formation were also observed in SEM micrographs (Figure 6E–H). Interestingly, extensive spontaneous fibrin network formation was noted, without the addition of thrombin.

The clots that were observed in the healthy PPP with added spike protein, were of particular interest as they demonstrated a bridge between healthy PPP clots and COVID-19 clots. As described in the ‘Results’ section, the healthy PPP clots were relatively small and orderly, while COVID-19 PPP clots were large, disorderly masses that formed rapidly and disrupted PPP flow in the channel. The healthy PPP clots with added spike protein, were a combination of the two, demonstrating disorderly clumped clot areas, coexisting with laminar fibrous PPP clots (which were larger than the healthy PPP clots).

One of the obvious differences, which was inadvertently observed while trying to clean the channels with high-speed water flow (i.e. by mechanical means), was the ease of healthy PPP and healthy PPP with added spike protein clot dissolution.

While the protocol enables the study of fibrin microclots, which are of interest in COVID-19, it excludes the influence of RBCs, which are known to heavily influence the non-Newtonian flow behavior of blood at that scale

In the current study, mass spectrometry confirmed that spike protein causes structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins become less resistant to trypsinization and changes the conformation, in such a way that there is a significant difference in peptide structure before and after spike protein addition.

 

[Wonko]

This spike protein, that would presumbly be basically the same thing as we are injecting onto people as vaccines, to train the immune system to recognise covid 19?

Otherwise the vaccines wouldn't work?

 

[SNT Gatchaman]

Yes.

I think broadly speaking it doesn't matter what the origin of the spike protein is - it has the potential to cause trouble.

The spike protein as coded by mRNA vaccines is slightly modified (and protein subunit spike may also be modified) but I think that feature is unlikely to be of relevance to propensity for anomalous clot formation. However, even if we accept the above demonstration in vivo at scale, vaccine-derive S1 is in a controlled amount and (nominally) in upper arm soft tissues. Virus spike is uncontrolled in number and accompanied by S2 and other viral components and is far more likely to be systemic, rather than confined.

The risk/benefit is still strongly in favour of vaccine - though that is likely of academic comfort only to those affected. Still an unknown what the long-term effects of Omicron will be - but the default assumption should not be "mild acute in most / no long-term disease".