A Subset of Antibodies to the Spike Protein's Receptor-Binding Domain in Severe COVID-19 Activate Platelets

Severe COVID-19 is a multisystem condition with thromboinflammation as a significant driver of morbidity and mortality. Cellular components involved in inflammatory and coagulation responses include platelets, endothelium, monocytes, and neutrophils. Thrombotic manifestations, such as arterial thrombosis, venous thromboembolism, and tissue microthrombosis, occur in at least 20% of ICU COVID-19 patients, possibly more based on postmortem studies. This study investigates the presence of prothrombotic antibodies similar to those found in heparin-induced thrombocytopenia (HIT), a condition caused by platelet-activating antibodies targeting platelet factor 4 (PF4) when complexed with heparin (PF4/H). In a study of samples from 130 hospitalized patients collected on average 3.6 days after COVID-19 diagnosis, 80% were found to have IgG antibodies recognizing PF4/H. This percentage is significantly lower than in 22 HIT patients (100%) but higher than in 51 non-COVID-19 patients with acute respiratory symptoms (ARS) (25%) and 12 healthy subjects (0%). Additionally, 41% of the 130 patients had antibodies inducing PF4-dependent P-selectin expression in CpG-treated normal platelets, a rate significantly lower than in 12 HIT patients (100%) but higher than in 51 non-COVID-19 ARS patients (20%) and 12 healthy subjects (0%). Significantly, platelet-activating antibodies in COVID-19 patients were associated with a specific elevation of platelet α-granule proteins in the plasma and correlated with inflammation and tissue damage markers, suggesting these antibodies' functionality in patients. Significantly, unlike HIT, both PF4/H-reactive and platelet-activating antibodies were found in COVID-19 patients regardless of recent heparin exposure, indicating different antigenic stimuli for these antibodies in COVID-19. Notably, certain antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 possessed sequence motifs, RKH and Y5, in the heavy-chain complementarity-determining region 3 (HCDR3) resembling those in pathogenic HIT antibodies identified in our previous studies, suggesting that these antibodies can activate platelets. Introducing exogenous RBD to COVID-19 plasma or IgG purified from COVID-19 plasma significantly reduced their ability to activate platelets. Furthermore, immunodepleting RBD-reactive antibodies from COVID-19 plasma or IgG purified from COVID-19 plasma using RBD-coated beads significantly reduced their ability to activate platelets. These findings demonstrate that some RBD-specific plasma IgG antibodies can activate platelets. To demonstrate the presence of RBD-specific, platelet-activating antibodies at the clonal level, we produced recombinant antibodies from 42 RBD-binding IgG+ B cells, tested their ability to activate platelets, and identified four that could do so. Interestingly, three antibodies possess HCDR3 sequences resembling RKH or Y5 motifs. Additionally, we identified RKH or Y5 motifs in RBD-specific antibodies reported by other groups, produced 15 such antibodies recombinantly, tested their ability to activate platelets, and identified four more that could do so. IgG+ B cells with these HCDR3 motifs were markedly expanded in severe COVID-19 patients, suggesting an expansion of B cells producing platelet-activating antibodies. The demonstration of functional and structural similarities between certain RBD-specific antibodies in COVID-19 patients and pathogenic HIT antibodies suggests a novel mechanism by which RBD-specific antibodies might contribute to thrombosis in COVID-19.