HIT immune complexes activate the inflammasome pathway in a complement-dependent manner

Abstract

Abstract Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction to heparin caused by pathological immunoglobulin G (IgG) antibodies targeting platelet factor 4(PF4)/heparin antigenic complexes. Immune complexes (ICs) consisting of IgG antibodies and PF4/heparin bind to cellular FcγRIIA receptors to initiate prothrombotic cell signaling, including cytokine production and release. Aims This study sought to investigate the role of inflammasome cytokine production and release of IL-1β following HIT IC challenge. Methods Inflammasome cytokine release of IL-1β and cellular procoagulant activity was measured in a whole blood assay, where healthy donor whole blood was incubated with PF4/heparin antigen and ICs consisting of either the monoclonal HIT-like antibody, KKO, or HIT patient plasma. Corresponding studies were conducted in peripheral blood mononuclear cells (PBMCs). Requirements for cellular FcγRIIA, complement pathway and inflammasome were determined with respective inhibitors. Results Whole blood or PBMCs incubated with HIT ICs elicited a significant increase in IL-1β secretion, which was dependent on FcγRIIA. IL-1β secretion required complement, as heat inactivated plasma or incubation with terminal complement inhibitors prevented cytokine release. Pretreatment of whole blood or PBMCs with the NLRP3 inhibitor MCC950 markedly reduced IL-1β secretion. Furthermore, inhibiting the NLRP3 inflammasome reduced FXa cellular procoagulant activity following HIT IC exposure. Conclusions These data demonstrate that HIT ICs activate the inflammasome pathway in a FcγR/complement-dependent manner leading to IL-1β secretion and cellular procoagulant activity. Our findings indicate a major role for the inflammasome pathway in mediating the thrombotic predisposition of HIT.