The Clot Thickens: Investigation of the Mechanism Behind Hypercoagulation In COVID-19 Patients

Since its emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with cardiovascular complications, which correlate with illness severity and increased mortality. It was initially thought that this hypercoagulable state was induced by endothelial dysfunction or cytokine storm because of SARS-CoV-2 infection. Although platelets have been reported to express the SARS-CoV-2 primary target receptor, angiotensin-converting enzyme 2 (ACE2), this remains controversial due to conflicting findings that suggest minimal to no ACE2 expression on platelets. This ambiguity has led to exploring alternative entry pathways, notably via the virus’s RGD (arginine-glycine-aspartic acid) sequence within its spike protein receptor-binding domain (RBD). The RGD motif is widely recognized as a cell-binding sequence that facilitates adhesion by engaging RGD-binding integrins. This sequence can also be found in viruses where it is used as a ligand to hijack integrins, repurposing them as receptors for infection. The ability of SARS-CoV-2 to use integrin-mediated entry provides an alternative mechanism for infecting cells with little to no ACE2 expression, such as platelets. Platelets’ involvement in SARS-CoV-2-induced thrombotic disorders has recently been investigated as a potential contributor to hypercoagulation. While studies suggest a direct interaction between the spike protein and platelets, they often do not specifically examine the precise ligand-receptor dynamics of this interaction. Platelets express three RGD-binding integrins—αIIbβ3, ανβ3, and α5β1. Although the ανβ3 and α5β1 integrins have been identified as RGD receptors for the virion’s adhesion to epithelial and endothelial cells, their involvement in platelet interaction with the RBD remains ii unexamined. Meanwhile, the dominant platelet integrin, αIIbβ3, has been reported to bind the spike protein. However, whether this direct interaction involves the αIIbβ3 integrin’s adhesion to the spike protein RGD sequence is unclear. Further research is essential to determine whether the SARS-CoV-2 RGD sequence interacts with platelets integrin αIIbβ3 and if this causes changes in platelet reactivity. This study investigates the platelet receptor and SARS-CoV-2 spike protein-ligand involved in their direct interaction. These results will introduce pertinent information for mitigating changes in platelet reactivity due to exposure to the SARS-CoV-2 RBD. Translationally, this novel information could be used to develop anti-platelet therapies to prevent the hypercoagulation exhibited in COVID-19 patients. This work is accomplished through two aims; the first is to determine if the spike protein RGD sequence is the SARS-CoV-2 motif that binds platelets and if this increases platelet activation. This is done by exposing platelets to RBDs with either inhibited or accessible RGD sequences. Platelets were characterized by acoustic flow cytometry. It was determined that the spike protein RGD sequence promoted increased platelet activation when uninhibited in both whole blood and isolated platelets compared to RBD with inhibited RGD. The second aim investigated the ligand and receptor involved in the spike protein’s interaction with platelets. This was done by exposing platelets with inhibited α5β1, ανβ3, and αIIbβ3 integrins to RBD. Additionally, a separate set of platelet samples without integrin inhibitors were introduced to RBDs with and without inhibited RGD sequences to determine if the differences in platelet activation involve interaction with the RGD. It was determined that the RBD significantly increases platelet activation in samples with accessible αIIbβ3 and/or α5β1 integrins. Platelets with uninhibited αIIbβ3 iii integrins showed the most significant increase in activation between samples exposed to the RBD versus controls, suggesting that the αIIbβ3 integrin plays a crucial role in platelet reactivity caused by the RBD. Additionally, the RGD sequence appears to mediate the SARS-CoV-2 virion’s interaction with the platelet αIIbβ3 integrin, causing enhanced platelet activation. These results suggest that the SARS-CoV-2 RBD amplifies platelet reactivity through direct engagement with platelet αIIbβ3 integrin and that this mechanism involves the RGD sequence.