The US, Spain, and Japan researchers investigated the direct effect of the SARS-CoV-2 spike (S) protein on platelet morphology. They have discovered that the S protein changes the morphology of platelets and binds directly to the platelet integrin α5β1 and αvβ3 receptors.
One of the major pathological symptoms in severe cases of SARS-CoV-2 infection is abnormal platelet behavior. During SARS-CoV-2 infection, several other procoagulant players, such as the release of tissue factor, elevated fibrinogen levels and dysregulated release of cytokines create a hypercoagulative environment.
COVID-19 patients have a low platelet count (thrombocytopenia), microvascular thrombosis and coagulation, suggesting that SARS-CoV-2 can directly cause platelet dysfunction. Platelets isolated from COVID-19 patients also show abnormalities such as hyperactivity and increased spreading behavior.
Cytokines, antiphospholipid antibodies, interactions with other immune cells, and direct interaction between SARS-CoV-2 and platelets are potential causes of these abnormalities. The isolated platelets from healthy donors mixed with SARS-CoV-2 or the SARS-CoV-2 spike (S) protein show a faster thrombin-dependent clot retraction, and activate platelets independent of thrombin.
The S protein is crucial for understanding the molecular mechanisms of SARS-CoV-2 infection. The SARS-CoV-2 S protein consists of an S1 and an S2 subunit that are separated by host cell proteases. The receptor-binding domain in the S1 subunit is responsible for attachment to host cells. The S protein is thought to interact not only with the angiotensin-converting enzyme 2 (ACE2), but also with several other host receptors, including neuropilin-1 and CD147.
The results on the presence of ACE2, the main receptor for the S protein, on the surface of the platelets are not conclusive. On the contrary, integrin receptors are the main class of receptors expressed in platelets.
About the study
The researchers investigated the direct effect of the SARS-CoV-2 S protein on the change in platelet morphology. They isolated platelets from healthy human blood donors, tested their morphological changes in the presence of S protein, and visualized its effect on platelet morphology using live imaging and cryo-electron tomography.
The results showed that the S protein triggers the dynamic deformation of platelet morphology, leading in some cases to their irreversible activation. Cryo-electron tomography showed the formation of actin-rich filopodia at the end of the elongated platelets. Orientation analysis showed that the S protein binds to the membrane surface via different angular distributions. To gain molecular insights into the morphological changes of platelets in the presence of S protein, the research team performed a cryo-ET analysis of platelets in the presence and absence of the S protein. Platelets incubated with the S protein showed extensive deformation, while intact platelets showed their typical disc-shaped morphology.
To test the effects of the S protein on platelet activation, the researchers performed the ELISA test and a Western blotting. ELISA test showed an increase in platelet factor 4 secretion in platelet samples in the presence of the S protein.
Given the possibility that ACE2 is not widely expressed on the platelets, the authors speculated that the S protein could recognize integrin receptors directly. They tested the binding of the S protein to the platelet integrin receptors αIIbβ3, αvβ3, and α5β1, which are enriched in the tissue, but also expressed on platelets, and all recognize the RGD ligand motif. SARS-CoV-2 is the only beta-coronavirus containing an RGD (Arg-Gly-Asp) tripeptide motif in the receptor-binding domain, which is typically recognized by several members of the integrin membrane receptor family.
The results showed a weak but direct binding of integrin α5β1 and αvβ3 to the S protein, while integrin αIIbβ3 did not interact with it. According to researchers, 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 and 4) SARS-CoV-2 S protein contains a “RGD” sequence in the receptor binding domain, which is recognized by a subtype of integrin.
In this study, binding of the S protein to the platelet plasma membrane was directly visualized for the first time, and it was shown that the S protein alters platelet morphology and binds directly to the platelet integrin receptors α5β1 and αvβ3.
These results shed light on the coagulopathic events and microthrombosis caused by SARS-CoV-2 infection. According to the authors, a weak affinity of the S protein to platelet integrin receptors and the reversible binding may reflect the fact that coagulopathic events are rare complications and occur in severe cases of COVID-19. However, the authors suggested that other platelet receptors could also be responsible for the interaction with the S-protein.
The results of the study have been published in the scientific journal Nature Communications.
Kuhn, C.C. et al. Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 spike protein. Nat Commun 14, 620 (2023). https://doi.org/10.1038/s41467-023-36279-5 (Open Access). https://www.nature.com/articles/s41467-023-36279-5
The other studies also discussed recent findings suggesting that many mechanisms of hypercoagulability persist in post-acute COVID-19 syndrome, including persistent inflammation and endotheliopathy, accompanied by abnormal fibrin formation and impaired fibrinolysis. https://discovermednews.com/persistent-thromboinflammation-in-post-acute-covid-19-syndrome/
In addition, previous data based on the pharmacological approach investigated the pathways leading to excessive platelet activation, and showed that plasma from individuals with post-acute COVID-19 syndrome activates platelets through mechanisms dependent on purinergic receptors and integrin αIIbβ3 engagement. (Martins-Gonçalves et al., 2022. Circ Res 131, 944–947. https://doi.org/10.1161/CIRCRESAHA.122.321659