NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia

Abstract Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of adenoviral vector vaccines (AstraZeneca and Johnson & Johnson) against COVID-191. Anti-platelet factor 4 (PF4) antibodies are present in VITT patients2,3. Although the current view suggests that platelet activation by anti-PF4 antibodies is the cause of thrombosis there is as yet no direct evidence that the antibodies induce clot formation and thrombocytopenia (reduction in platelet counts) in VITT and the mechanisms involved remain unknown4. Here we show that VITT antibodies induce thrombosis and thrombocytopenia, and that thrombus formation is mediated by neutrophil extracellular traps (NETs). We found markers of NETosis, abundance of neutrophil/platelet aggregates and presence of neutrophils undergoing NETosis in patients with active VITT. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 using an in vitro blood flow microfluidic system. In transgenic mice expressing human PF4 and FcγRIIa, VITT antibodies lead to thrombosis, thrombocytopenia and formation of low density granulocytes. Pharmacological and genetic inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting they are distinct processes. Our findings indicate that VITT antibodies activate cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia. This study identifies NETosis as a pathogenic mechanism for thrombus formation in VITT. We anticipate our findings will motivate future development of NETosis and FcγRIIa inhibitors as potential specific therapies for VITT and consequently better patient outcomes.