Glycoprotein Ibα Clustering Induces Macrophage-Mediated Platelet Clearance in the Liver

Abstract

Objective: Immune thrombocytopenia (ITP) is an autoimmune bleeding disease in which autoantibodies are directed against the patient’s own platelets, leading to platelet destruction and bleeding. Currently, platelet destruction in ITP is thought to be mediated by monocytic and phagocytic cells, and the antibody-coated platelets are removed from circulation by the spleen in an Fc-dependent manner. Conventional treatments for ITP therefore aim at reducing platelet destruction, either by immunosuppression or splenectomy. However, many ITP patients, particularly patients with anti-glycoprotein (GP) Ib-IX autoantibodies, do not respond to conventional treatments such as intravenous immunoglobulin G (IVIG). However, the underlying mechanism remains unclear.Methods: Anti-GPIb-IX antibodies-induced platelet aggregation was detected by an optical aggregometer. GPIbα clustering was measured by Fluorescence Resonance Energy Transfer (FRET) by flow cytometry. Antibodies-induced platelet depletion and rescue experiments were performed in guinea pigs and cynomolgus macaques. Platelet distribution after antibody-mediated depletion was detected by in vivo Imaging System (IVIS), immunohistochemistry and immunofluorescence microscopy.Results: We found that anti-GPIbα N-terminus antibody AN51, but not other anti-GPIbα antibodies (AK2, HIP1, VM16d, or WM23), induced integrin αⅡbβ3-dependent platelet aggregation. FRET result showed that AN51 induced obvious GPIbα clustering but not VM16d. After intravenous injection, AN51 dose-dependently induced thrombocytopenia in guinea pigs, and the platelets were mainly removed by macrophages in the liver. N-acetyl-D-glucosamine (GlcNAc), previously shown to inhibited αMβ2-mediated phagocytosis of refrigerated platelets, showed dose-dependent inhibition of AN51-induced platelet destruction. Furthermore, AN51 but not VM16d, induced rapid platelet clearance in the liver of cynomolgus macaques. Five of twenty-two chronic ITP patients had anti-GPIbα autoantibodies, and the autoantibodies from four of the five patients competed with AN51 for binding to platelets.Conclusion: GPIbα clustering induced by anti-GPIbα N-terminus antibody causes integrin αⅡbβ3-dependent platelet aggregation, phagocytosis, and rapid platelet clearance in the liver. Our findings reveal a novel Fc-independent mechanism underlying the pathogenesis of ITP, and suggest new therapeutic strategies for ITP patients with anti-GPIbα autoantibodies. DisclosuresNo relevant conflicts of interest to declare.