Prevention of thrombocytopenia and thrombosis in heparin-induced thrombocytopenia (HIT) using deglycosylated KKO: A novel therapeutic?

Abstract

Heparin-induced thrombocytopenia (HIT) is characterized by mild thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human (h) platelet factor 4 (PF4) complexed with various polyanions. While non-heparin anticoagulants and intravenous immunoglobulin (IVIG) are the mainstay of care, bleeding may develop, and risk of new thromboembolic events remain. We had described a mouse IgG{kappa}2b antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4:polyanion complexes. KKO, like HIT IgGs, activates platelets through Fc{gamma}RIIA and induces complement activation. We now asked whether Fc-modified KKO can be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). DGKKO bound to PF4-polyanion complexes, and blocked Fc{gamma}RIIA-dependent activation of PF4 treated platelets by KKO, 5B9 (another HIT-like monoclonal antibody), and isolated IgGs from HIT patients. DGKKO also decreased complement activation and deposition of C3c on platelets. Injection of DGKKO into "HIT mice" lacking mouse PF4, but transgenic for hPF4 and Fc{gamma}RIIA, prevented and reversed thrombocytopenia when injected before or after KKO, 5B9 or HIT IgG, respectively, in a microfluidic system. DGKKO reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis by IgG from a patient with the HIT-related disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT. Key PointsO_LIDeglycosylated (DG) KKO can reverse thrombocytopenia in a HIT murine model. C_LIO_LIDGKKO can prevent/reverse thrombosis in vitro and in a HIT murine model. C_LI