SARS-CoV-2 RBD and its variants can induce platelet activation and clearance: Implications for vaccinations and antibody therapy against COVID-19

ABSTRACT

Background: Severe COVID-19 is associated with platelet activation, thrombosis, and thrombocytopenia, but the mechanisms remain unclear. Similarly, very rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are also poorly understood. Both infection and vaccination utilize the receptor-binding domain (RBD) of the spike protein for virus-host cell entry and to elicit an immune response, respectively. Interestingly, the RBD contains an RGD integrin-binding motif that may facilitate platelet binding. Aim(s) To determine whether the RBD binds platelets and causes platelet activation/clearance. Method(s) We intravenously injected different doses (0.25, 0.5, 1.0mug/g) of recombinant RBD into mice and measured platelet counts post-injection using a Z2 Series Coulter. Flow cytometry detected RBD/RBD variants binding to platelets and associated platelet activation, apoptosis, and desialylation. Human gel-filtered platelet aggregation was induced by ADP, Collagen and Thrombin. Six anti-RBD monoclonal antibodies (mAbs) were generated and tested in a SARS-CoV-2 Vero cell infection model with the envelop gene quantified by RT-qPCR to determine the virus replication. Result(s) RBD injection caused platelet clearance in a dose-dependent manner. The RBD could also bind to platelets, induce activation and potentiate platelet aggregation in vitro. Our preliminary data also showed the RBD Delta variant has greater potential in inducing platelet activation. Interestingly, the RBD bound beta3-/-platelets ~50% less relative than wildtype mice. Consistently, mutating the RGD motif to RGE, and preincubating platelets with the beta3 inhibitor Eptifibatide also reduced RBD binding to platelets. Our novel anti-RBD mAbs 4F2 and 4H12 inhibited RBD-induced platelet activation and RBD-potentiated platelet aggregation in vitro, and prevented RBD-induced platelet clearance in vivo. Importantly, these mAbs also inhibited SARS-CoV-2 viral replication in a dose-dependent manner. Conclusion(s) Our data demonstrate that the RBD could directly bind to platelets partially via beta3 integrin. RBD-induced platelet activation and clearance may contribute to thrombosis and thrombocytopenia observed in clinical cases of COVID-19 and VITT.