Evaluating Biomolecular and Biochemical Characteristics of Anti-Platelet Factor 4/Heparin Antibodies in Serological Subtypes of Heparin-Induced Thrombocytopenia

Abstract

Heparin induced thrombocytopenia (HIT) is an adverse reaction to heparin (a commonly used anticoagulant), characterized by a low platelet count and potentially catastrophic thrombosis. Resembling HIT is vaccine-induced immune thrombocytopenia and thrombosis (VITT), which arose in response to the COVID-19 pandemic due to SARS-CoV-2 adenoviral vector-based vaccines. Both of these immune-mediated disorders are caused by antibodies against the chemokine, platelet-factor-4 (PF4/CXCL4), which activates platelets through the FcγIIa receptor, leading to the release of procoagulant platelet microparticles, reduction in platelet count level, and a high risk of venous and arterial thrombosis that can be severe and even fatal. There are two serological subtypes of HIT based on their platelet activation patterns in functional assays: classical heparin-dependent HIT (HD-HIT) antibodies that require heparin for platelet activation, and autoimmune heparin-independent HIT (HI-HIT) antibodies that do not require heparin for platelet activation. HI-HIT is a more severe subtype associated with higher rates of disseminated intravascular coagulation (DIC), more severe and prolonged thrombocytopenia (platelet count of <20 × 109/L), and persistence of symptoms after heparin cessation. Previous research in our laboratory found that HD-HIT and HI-HIT bound to different regions on PF4. Interestingly, in VITT, the precise anti-PF4 antibody epitopes correlated with clinical outcomes, such as whether the rare event of cerebral venous sinus thrombosis (CVST) occurred. Our research suggests that the precise antibody epitope targeted may be important for determining clinical outcomes in anti-PF4 disorders; by extension, we want to investigate whether this observation exists in HD-HIT and HI-HIT.

In this thesis, we conducted an in-depth comparison of HD-HIT (n=16) and HI-HIT (n=20) subtypes at the level of anti-PF4/heparin epitopes, antibody binding affinity, antibody titre, and clinical outcomes. We used established methods to determine the anti-PF4/heparin epitopes for a large cohort of HIT patients to expand upon earlier preliminary work. We found that the combined HIT binding site on PF4 (n=36) was distinct from previously identified epitopes of monoclonal anti-PF4 antibodies, including the KKO, 5B9, VITT, and 1E12, as well as the heparin binding site. By gaining a deeper understanding of anti-PF4/heparin binding sites in HIT, we can potentially develop diagnostics that can target this shared binding region. Additionally, we found that HD-HIT and HI-HIT antibodies bind to very similar regions on PF4, which was contrary to our expectations, but agrees with our recent finding that HIT is driven by a pathogenic monoclonal IgG antibody. Rather than differences in anti-PF4/heparin epitopes, we provide evidence that HI-HIT antibodies have higher antibody affinities and titres compared with HD-HIT. Additionally, we found one amino acid residue (P37) that was significantly different in HIT patients with a platelet nadir above (n=6) and below (n=7) 50 x109 platelets/L, and we found seven residues (D7, L8, T16, V29, A39, K50, I64) that were significantly different between HIT patients with (n=8) and without (n=5) thrombotic complications. Overall, this suggests that separating HIT patients based on their platelet activation patterns in functional assays may not be as important as separating HIT patients based on clinical outcomes.