Characterization and application of new materials for better detection of platelet activating antibodies

Heparin-induced thrombocytopenia (HIT) is a disease caused by the treatment of heparin as an anticoagulant after 5 to 10 days of heparin exposure. Heparin forms anitgenic complexes with platelet factor 4 (PF4/H), which trigger the immune system in around 2–5 % of patients to develop anti-PF4/H antibodies. Some of these anti-PF4/H antibodies (HIT antibody) bind to the PF4/H complexes through the Fab region, and the Fc region activates platelets via the FcγRIIa receptor, leading to thrombosis, platelet loss, and even stroke. Due to the lethal consequences, early detection of the HIT antibodies is crucial. Currently, there are two main detection methods: immunoassays and functional assays. Immunoassay possess high sensitivity and are easy to perform, making them one of the most commonly used methods. However, immunoassays have low specificity, leading to inaccurate diagnosis. Functional assays are highly accurate but require fresh platelets and more complex than immunoassays. Access to fresh platelets is not easy and platelets have a short storage time, making it difficult to be widely applied. Therefore, a method that combines the high sensitivity and easy accessibility of immunoassays and the high specificity of functional assays is highly demanded. In this study, I improved the current HIT detection enzyme linked-immunosorbent assay (ELISA) by developing a PF4 cell-based ELISA, inducing the accuracy from 50% of PF4/H ELISA up to 80%. Instead of typical PF4/H complexes, I used PF4 added the cancer cell immobilized on the ELISA plate. Furthermore, I investigated the interaction of HIT components with cancer cells. Platelets increased breast cancer cell survival, while HIT-like antibody KKO and PF4 reduced proliferation. Fondaparinux, a low-HIT-risk anticoagulant, was also investigated for forming antigenic complexes with PF4, which led to platelet aggregation in specific ratios.