Adenovirus holds the platelet in flow Free

In this issue of Blood, Chen et al¹ report a focused microfluidic study demonstrating the association of an adenovirus vector-based vaccine with platelets under arterial shear flow. They pinpointed the RGD (arginine-glycine-aspartate) sequence in the engineered adenoviral capsid as the main site binding platelet integrin αIIbβ3, mediating platelet adhesion and accumulation to the immobilized capsid. These findings carry significant mechanistic and therapeutic implications.

The COVID-19 pandemic’s onslaught on the world population has been largely brought under control thanks to rapid development and sweeping deployment of vaccines targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unfortunately, adverse effects have been associated with the vaccines. One example is the ChAdOx1 nCoV-19 vaccine, which has been linked to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but serious condition.^2,3 It is likely that the vaccine induces development of autoantibodies against platelet factor 4 (PF4), perhaps in a similar manner to that in heparin-induced thrombocytopenia. These PF4-autoantibody complexes activate platelets via FcγRIIa receptors, leading to excessive platelet aggregation, consumption, and thrombus formation.^2,4 In addition to VITT, increased risk of thrombotic events, including cerebral venous thrombosis, ischemic stroke, and myocardial infarction, have been associated with ChAdOx1.^5,6 The study of Chen et al helps shed new insights on the underlying mechanism.

ChAdOx1 is an adenovirus vector-based vaccine. It enters host cells through receptor-mediated endocytosis, primarily targeting cells expressing the coxsackievirus and adenovirus receptor, which is found on many cell types, including epithelial and immune cells. Once inside, the adenovirus vector delivers the genetic sequence encoding the SARS-CoV-2 spike protein into the nucleus, where it is transcribed into mRNA and then translated into the spike protein, thereby triggering immune recognition and antibody production. Integrins on host cells help facilitate viral entry and enhance the efficiency of adenoviral transduction by engaging with the RGD sequence in the adenoviral capsid, more specifically, the adenovirus penton base proteins.⁷ 

The most abundant glycoprotein receptor on the platelet surface is the integrin αIIbβ3. Once stimulated, the platelet undergoes drastic changes and induces αIIbβ3 into an activated conformation that can bind its natural ligand fibrinogen as well as ligands containing the RGD sequence. The RGD sequence can also bind integrin αIIbβ3 and induce outside-in signaling.⁸ These links between RGD and the platelet integrin and the central role platelets play in thrombus formation prompted Chen et al to investigate the association of the adenoviral capsid and the platelet.

In their study, Chen et al constructed a microfluidic device in which round posts are placed in the chamber to emulate stenosed vessel geometry and create disturbed flow. Once ChAdOx1 was coated inside the chamber and on the posts, they observed the adhesion of platelets to the posts under arterial flow rates but not at lower rates. The adhesion pattern was distinct from that mediated by von Willebrand factor and its platelet receptor glycoprotein Ib. Numerous control experiments, including the use of well-documented blocking antibodies as well as a mutant capsid in which the RGD sequence in the penton protein was replaced by the Ala-Ala-Ala sequence, were conducted to demonstrate that such adhesion under flow was mediated by the RGD sequence in the ChAdOx1 capsid and platelet integrin αIIbβ3. Furthermore, the platelets that adhered on the immobilized ChAdOx1 appeared capable of propagating platelet activation and further thrombus formation. Overall, their results support a new mechanism by which the ChAdOx1capsid may bind platelets under flow and initiate thrombus formation. It helps provide a plausible explanation for the increased risk of thrombosis that is independent of VITT following ChAdOx1 vaccination.

The adenovirus vector has been used to produce not just COVID-19 vaccines, such as ChAdOx1, and the RGD sequence is present in the penton proteins of many adenovirus vectors. Thus, the study of Chen et al suggests that, once entering circulation, all adenovirus-based vaccines that have the RGD sequence may bind platelets and pose an increased risk of thrombotic events. The RGD sequence in the adenoviral capsid facilitates viral entry into the host cells by binding to their resident integrins, such as αVβ3. To reduce the risk of thrombosis and to maintain the transduction efficiency of the adenovirus vector, the RGD sequence in the capsid may need focused revision to minimize interaction with the platelet αIIbβ3 and at the same time maintain binding to integrins on the intended host cells.

Conflict-of-interest disclosure: The author declares no competing financial interests.