A RBD without a tag was generated after TEV protease cleavage using an N-terminal His-tag RBD construct. Constructs RBD were transfected in Expi293F cells (3×10^6 viable cells/ml) using FectoPRO in Expi293 Expression Medium at 37 °C, 8% CO2 under 120 rpm shaking conditions for 3 to 4 day. RBD proteins were purified using Protein A agarose or HisPur nickel nitrilotriacetic acid resin. Proteins were buffer exchanged with PBS (10 mM sodium phosphate buffer, pH 7.4, 2.7 mM KCl, and 137 mM NaCl) and stored at −80 °C until use.

The COVID-19 pandemic caused by SARS-CoV-2 virus is an ongoing global health burden. Severe cases of COVID-19 and the rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are both associated with thrombosis and thrombocytopenia; however, the underlying mechanisms remain inadequately understood. Both infection and vaccination utilize the spike protein receptor-binding domain (RBD) of SARS-CoV-2. We found that intravenous injection of recombinant RBD caused significant platelet clearance in mice. Further investigation revealed the RBD could bind platelets, cause platelet activation, and potentiate platelet aggregation, which was exacerbated in the Delta and Kappa variants. The RBD-platelet interaction was partially dependent on the β3 integrin as binding was significantly reduced in β3^-/- mice. Furthermore, RBD binding to human and mouse platelets was significantly reduced with related αIIbβ3 antagonists and mutation of the RGD (arginine-glycine-aspartate) integrin binding motif to RGE (arginine-glycine-glutamate). We developed anti-RBD polyclonal and several monoclonal antibodies (mAbs) and identified 4F2 and 4H12 for their potent dual inhibition of RBD-induced platelet activation, aggregation, and clearance in vivo, and SARS-CoV-2 infection and replication in Vero E6 cells. Our data show that the RBD can bind platelets partially though αIIbβ3 and induce platelet activation and clearance, which may contribute to thrombosis and thrombocytopenia observed in COVID-19 and VITT. Our newly developed mAbs 4F2 and 4H12 have potential not only for diagnosis of SARS-CoV-2 virus antigen but also importantly for therapy against COVID-19.