Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. 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 these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and FcγRIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.

Background The notable discrepancy between platelet count and bleeding manifestations in immune thrombocytopenia (ITP) patients with acquired Glanzmann thrombasthenia (GT) has been described. Objectives We aimed to examine the mechanisms responsible for thrombocytopenia and the bleeding phenotype in a patient with acquired GT. Patient, methods, and results A patient with primary ITP underwent splenectomy due to steroid intolerance. Despite platelet count normalization, bleeding continued. Platelet aggregometry was abnormal with all agonists except for ristocetin. Flow cytometry demonstrated the presence of antiplatelet antibody, which caused dose‐dependent inhibition of fibrinogen and PAC‐1 binding, induction of neuraminidase‐1 expression as well as platelet desialylation in donor platelets. Indirect monoclonal antibody immobilization of platelet specific antigen assay (MAIPA) confirmed specificity to αIIbβ3 only, corroborated by binding on Chinese hamster ovary (CHO) cells expressing human glycoprotein αIIbβ3 but not GP Ib/IX. Both desialylation and neuraminidase expression were observed with plasma adsorbed on Ib/IX CHO cells and with the immunoglobulin G (IgG) fraction. Desialylation was inhibited in the presence of anti‐Fc‐gamma receptor IIa (FcγRIIa) antibody. A nonobese diabetic/severe combined immunodeficient ITP murine model was established, which showed rapid hepatic donor platelet clearance in the presence of patient IgG. Treatment of mice with the neuraminidase inhibitor oseltamivir significantly reduced antibody‐induced platelet destruction. Conclusions We report the first case of a patient with acquired GT due to ITP with FcγRIIa mediated platelet desialylation, independent of platelet activation. Treatment with neuraminidase inhibitor may prevent platelet clearance by anti‐αIIbβ3 antibodies.

Immune thrombocytopenia (ITP) is a bleeding disorder caused by dysregulated B and T cell functions, which lead to platelet destruction. A well-recognised mechanism of ITP pathogenesis involves anti-platelet and anti-megakaryocyte antibodies recognising membrane glycoprotein (GP) complexes, mainly GPIb/IX and GPIIb/IIIa. In addition to the current view of phagocytosis of the opsonised platelets by splenic and hepatic macrophages via their Fc gamma receptors, antibody-induced platelet desialylation and apoptosis have also been reported to contribute to the ITP pathogenesis. Nevertheless, the relationship between the specific thrombocytopenic mechanisms and various types of antiplatelet antibodies has not been established. To ascertain such association, we used sera from 61 ITP patients and assessed the capacity of antiplatelet antibodies to induce neuraminidase 1 (NEU1) surface expression, RCA-1 lectin binding and loss of mitochondrial inner membrane potential on donors’ platelets. Sera from ITP patients with detectable antibodies caused significant platelet desialylation and apoptosis. Anti-GPIIb/IIIa antibodies appeared more capable of causing NEU1 surface translocation while anti-GPIb/IX complex antibodies resulted in a higher degree of platelet apoptosis. In ITP patients with anti-GPIIb/IIIa antibodies, both desialylation and apoptosis were dependent on Fc-gamma RIIa signalling rather than platelet activation. Finally, we confirmed in a murine model of ITP that destruction of human platelets induced by anti-GPIIb/IIIa antibodies can be prevented with the NEU1 inhibitor oseltamivir. A collaborative clinical trial is warranted to investigate the utility of oseltamivir in the treatment of ITP.