Epitope-Targeted Strategies for Inhibiting Pathogenic Immune Complex Formation in Heparin-Induced Thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse drug reaction to the anticoagulant heparin.1,2 HIT is characterized by pathogenic antibodies that form immune complexes with platelet factor 4 (PF4) and heparin, which can cause platelet activation and thrombosis.1,3-6 Prompt diagnosis is crucial because HIT patients experience a 5-10% daily increased risk of thrombotic episodes, which are fatal in up to 30% of patients.2,6-10 The immune response in HIT is polyspecific, resulting in the production of anti-PF4/heparin antibodies that either can (pathogenic) and/or cannot (non-pathogenic) activate platelets.10-12 Differentiating between pathogenic and non-pathogenic antibodies remains a significant diagnostic challenge in HIT, exacerbated by the low specificity and limited availability of current laboratory assays.10,14,15 Previously it was shown that platelet-activating immune complexes form when pathogenic antibodies bind PF4 at a localized region that is distinct from non-pathogenic antibodies.13 In this work, high affinity inhibitors of pathogenic anti-PF4/heparin antibodies were designed using a murine monoclonal antibody (named KKO) that binds to a region on PF4 overlapping with pathogenic antibodies.16 These inhibitors were used to develop a novel diagnostic assay that can detect pathogenic antibodies with improved specificity compared to most standard tests for HIT (specificity ~50-70%).2,15 Enzyme immunoassays (EIAs) are widely used for HIT diagnosis, but have high false-positive rates because they cannot differentiate between pathogenic and non-pathogenic antibodies.10,14,15 This study found that incorporating inhibitors to block clinically significant antibody binding sites on PF4 improved the specificity (90%) of a diagnostic EIA for HIT without sacrificing sensitivity (90-100%). Unlike most high-sensitivity and -specificity diagnostic tests for HIT, such as platelet-activation assays, this EIA can also be performed with technical ease in non-specialized laboratories.