SARS-CoV-2 Spike Proteins Interacts With Platelet Integrins To Cause Deformities That Results In Coagulopathies

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SARS-CoV-2 Infections are typically associated with abnormalities in blood coagulation in severe cases and even in those with mild infections, clotting issues that causes strokes seems to be a lingering issue in Long COVID.

The study findings were published on a preprint server and are currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2022.11.22.517574v1

SARS-CoV-2 has shown unique pathological symptoms that can lead to a wide range of coagulopathic events in severe cases. In our study, we probed the direct effect of S protein to the change in morphology of platelets at a molecular level, and for the first time, we directly visualized the binding of S protein to the platelet surface (summarized in Fig. 6).

We hypothesized that the binding of the SARS-CoV-2 is mediated by integrin receptors based on the following reasons;

1) the activation of platelets is governed by filopodia formation,

2) filopodia formation is initiated by integrin receptors,

3) the major receptors on the platelets are integrin receptors

4) SARS-CoV-2 S protein contains a “RGD” sequence in the RBD, which is recognized by a subtype of integrin, and therefore we tested the interaction of platelet-expressed integrins with S protein.

Our integrin inhibition experiment using cilengitide and in vitro solid-phase binding assays support this hypothesis, particularly with the possibility that S protein recognizes integrin αvβ3. The binding of S protein to integrin was much lower compared to the interaction of integrins with their physiological ligands, and interestingly, we did not detect the binding to the major platelet integrin αIIbβ3.

Previously, an increased binding of the activated integrin αIIbβ3 antibody PAC-1 to platelets was observed in the presence of S protein 21. This may be due to an inside-out effect, in which the outside-in signaling is activated by the direct binding of S protein to integrin αvβ3 and in turn, αIIbβ3 would get activated through the intracellular signaling (inside-out).

We surmise that the weak affinity of S protein to platelet integrin receptors and the reversible binding, may reflect the fact that blood clotting defects observed in patients are rare complications and occur in severe cases of COVID-19.

However, here we should also note that there are other receptors on platelets that may also be accountable for the interaction with S protein 33,53 and combinatory effects of the binding of S protein to multiple receptors may also occur.

SARS-CoV-2 is found in the blood stream of COVID-19 patients 10, and an open question is how it can lead to rare but severe coagulation defects. We showed that the deformation of platelets itself does not always alter their intracellular signaling (Fig. S1), or induces activation.

It rather appears that platelets exposed to S protein are primed for the activation upon further stimuli, such as the attachment to an adhesion surface. Based on this observation, we speculate that the combination of the direct binding of S protein to platelets and other identified coagulation factors may induce a synergistic and irreversible activation of platelets, leading to coagulation.

During SARS-CoV-2 infection, several other procoagulant players are active, for example the formation of neutrophil extracellular traps18, the release of TF23, elevated fibrinogen levels54 and dysregulated release of cytokines 55, creating a hypercoagulative environment in the context of COVID-19.

In our study, we visualized the adaptable attachment of S protein to the platelet plasma membrane with a high degree of flexibility for the engagement to continuously curved membrane surfaces (Fig. 4D and 4E). Similarly, it has been reported that the stalk domain of S protein proximal to the viral membrane surface contains three hinges, presumably allowing the flexible motion of individual S protein on the viral surface to adapt to curved host cell surfaces 50.

This dual flexibility likely increases the probability for S protein to attach to a host cell receptor, thus, allowing an efficient action of S protein to the membrane surface.

Data availability
Tomograms of platelets in the presence of SARS-CoV-2 S protein used in the figures were deposited to the Electron Microscopy Data Bank (EMDB) with accession codes EMD-26794 (platelet protrusion shown in Fig. 3) and EMD-26796 (platelet protrusion shown in Fig. 4).

Additional tomograms used for subtomogram averaging were deposited to the Electron Microscopy Public Image Archive (EMPIAR) with accession code EMPIAR-11038. The 3D map of the single particle reconstructed S protein has been deposited with the accession code EMD-26798.

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