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Covid-19 spike protein and microclots

I consult for a client that has an interest in the Covid-19 spike protein, the mRNA, and microclots. These clots are not ordinary clots, as we are about to learn. These clots contain amyloid structures. The Medical Dictionary defines an amyloid as an insoluble, fibrous structure consisting chiefly of an aggregation of proteins arranged in beta sheets, forming extracellular deposits in organs or tissues and characteristic of certain diseases such as Alzheimer’s disease and Parkinson’s disease. Thioflavin T and Congo Red are two fluorescent dyes used to detect amyloids. PubChem acknowledges Thioflavin T as compound used to detect amyloids. RCSB.org is a good place to lookstructures. Beta-sheet rich peptides were engineered to form amyloids to better understand Thioflavin T binding. This particular structure gives us and understanding as to what an amyloid is. Some lay readers might enjoy watching some fun videos with first class sound tracks and animation first.

An image of ThioFlavin T from PubChem and two thioflavin T in the X-ray crystal structure 3EC5  The crystal structure of Thioflavin-T (ThT) binding OspA mutant pepides from rscb.org.
The image of a blood clot was obtained from Höök et al 2017. Calcium had been added back to platelet rich plasma. The clotting cascade image is from Bonis 2018. Fibrin is tan and platelets are blue. The PacMan circles are activated proteases, enzymes that cleave proteins. The tadpole things are protease inhibitors that stick in the “mouths” of proteases. The PacMan proteases with closed mouths area waiting activation by an upstream protease.

The role of platelets in clotting is discussed elsewhere. The point is that plasma used in this Grobbelear (2021) study was processed to be plate poor plasma (PPP).

Grobbelaar LM, Venter C, Vlok M, Ngoepe M, Laubscher GJ, Lourens PJ, Steenkamp J, Kell DB, Pretorius E. SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19. Biosci Rep. 2021 Aug 27;41(8):BSR20210611 PMC Free article

The authors prepared blood plasma from a healthy donor. This post is going to assume that PPP means that the plasma was spun down at a force sufficeent to pellet platelets as well as as blood cells. The S1 segment of the spike protein was purchased from Sino Biologicals and resuspended in endotoxin free distilled water.

3 a negative control with Alexafluor 88

Fluorescent dyes tend to be hydrophobic and therefore like to bind to hydrophobic surfaces of proteins. The Kell and Pretolius groups wanted to know how thrombin treated fibrinogen looked in presence of a sticky fluorophore like Alexafluor 88  ±spike protein. Yes, sticky and fluorescent Alexafluor 88 could be seen binding to both mixtures. IThe staining was diffuse and looked rather like images of clots. In the images shown, more binding was seen in the samle with spike protein.

4 smears of platelet poor plasma

In this study healthy was defined as patients not having any sort of condition that could alter blood coagulation. The authors took 5 μl PPP and exposed it to 1 ng.ml−1 (final concentrations) of spike protein. This mixture was smeared on a glass slide and covered with a coverslip. Somewhere in this process there was prior exposure to thioflavin T at a final concentration of 0.005 mM. for 30 minutes at room temperature. ) (Sigma–Aldrich, St. Louis, MO, U.S.A.) for 30 min at room temperature. The protease thrombin (7 U. per ml was added to the to 5 μl PPP and placed on a glass slide and covered with a coverslip. The excitation wavelength for ThT was set at 450–488 nm and the emission at 499–529 nm.

Figure 4 from Grobbelaar (2021)

One control that is lacking is spike protein without PPP and with thioflavin T.

5 Activated platelets

CD62P-PE is another name For P-selectin. P-selectin is stored in secretory α-granules in platelets and is relocated to the plasma membrane upon activation. What wasn’t really discussed is how spike protein might be activating platelets. What receptors do platelets have that bind the spike protein. This post won’t discuss platelet integrins, cell surface molecules that attach cells to the extracellular matrix proteins that contain Arginene-Glycine- Aspartic acid (RGD) motifs. Spike protein may use an intregrin binding motif for invasion. adhesion molecules.

Figure 5 from Grobellar 2021 with a cartoon adapted from a Creative Commons search

6 Some scanning electron microscopy work

Figure 6 from Grobellar 2021. All information from the figure legend has been added to the figure.

Grobellar and coauthors present some microfluidic studies in Figure 7 and some mass spectrometry data in Figure 8. Figure 9 is a proposed mechanism. My concern that this is time to stop representing their data in a hopefully more lay friendly way and direct lay readers to some Youtube videos showing actual amyloid formations

What are amyloids? Fun Videos

Amyloidosis- formation into beta-pleated sheets, fibrils, congo red staining, and green birefringence under polarized light. To a little Avenged Sevenfold.

This video discusses proteins that form large amyloid aggregates due to gene mutations. It is kind of interesting in the multi organ nature of amyloidosis. We just have to remember that thre are no mutations that we know of in OUR fibrin when it forms amyloids in response to SARS-CoV2 spike protein.
This video pertains to the cause of Creutzfeldt Jakob. We humans get Creutzfeldt Jakob Disease by consuming productrso f animals infected with the Scrapies prion form. Watch carefully as our own PrPc prion protein normal form makes contact with the mis-folded Scrapie prion protein. A chain reaction of protein mis folding ensues.

Just thinking about stuff now

Back in the first part of this post I introduced the tPA protease. tPA cleaves normal fibrin, and in this case fibrinogen, clots to D-dimer. Can tPA still cleave Spike protein catalyzed micro clots into D-dimer? Recall that fibrinogen is normally an inert protein until the thrombin protease comes along and turns it into a clotting protein. Now are these Spike Protein microclots bigger if the spike protein first binds an integrin cell surface or ACE2 receptor?

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