The PF4-Centric Immunothrombotic Disorders, HIT, Vitt and APS, Lead to Complement Activation Responsive to Complement-Blocking Therapeutics
Authors: Sarkar Amrita , Field Conroy O , McCrae Keith R. , Ortel Thomas L. , Arepally Gow , Cofiell Roxanne , Kim Sung-Kwon , Rauova Lubica , Poncz Mortimer 
Published: December 2024

Background: Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia with thrombosis (VITT)are highly prothrombotic disorders mediated by platelet -activating anti-PF4 antibodies (Abs). Studies from our lab and others have shown that PF4 bound to von Willebrand factor (VWF) strings and neutrophil extracellular traps (NETs) creates a dense, antigenic target for pathogenic HIT and VITT Abs conducive for both FcgRIIA-mediated cell activation and complement activation. We hypothesize that in antiphospholipid syndrome (APS), b2 glycoprotein I (b2GPI) - known to bind to PF4 - also forms a similar antigenic target for APS Abs and that inhibition of the terminal complement pathway will be effective in ameliorating the thrombosis in all three PF4-mediated immunothrombotic disorders.
Aim: Toinvestigate the in vitro and in vivo effects of C5 inhibitor in microfluidic and animal models of thrombosis in HIT, VITT and APS.
Methods: Confluent-lined HUVECs were injured with hematoporphyrin to release VWF strings. Human whole blood from healthy donors was pre-incubated with eculizumab, a humanized monoclonal antibody that binds to C5 and prevents activation of complement terminal complex (10 µg/ml and 15 µg/ml) for 15 minutes. HIT, VITT or APS IgGs (100 µg/ml), each isolated from three distinct patients, along with PF4 (25 µg/ml) were added and incubated for another 15-20 minutes. Samples were then flowed through microfluidics channels and in vitro thrombus formation measured by epifluorescence microscopy of aggregated platelets every 5 minutes for 15 minutes. The channels were washed and stained for complement component C5b9 and imaged by confocal microscopy. In in vivo studies, FcgRIIA+/hPF4+/mPF4-/- (HIT) mice were injected with anti-mouseC5 Ab BB5.1 24h before injection of HIT-like moAb KKO to induce thrombocytopenia and thrombosis. Neutrophil rolling and platelet-fibrin clot were analyzed in the cremaster muscle injury model.
Results: In vitro data showed a dose-dependent inhibitory effect of eculizumab on thrombus formation and C5b9 deposition in HIT-, VITT- and APS-mediated microfluidic thrombosis models. Inhibition of the terminal complement complex by BB5.1 had a moderate effect on in vivo thrombocytopenia, but significantly inhibited neutrophil adhesion to the endothelium, as well as both venous and arterial thrombus formation in the HIT murine model.
Conclusions: These studies suggest that C5 inhibitors, eculizumab and BB5.1, can successfully inhibit complement and platelet activation in HIT-, VITT- and APS-mediated prothrombotic settings. Our data support the important role of complement activation in the progression of thrombosis in these related, antibody-mediated disorders. This concept provides a rationale for therapeutic interventions that act upstream of thrombin generation and might thereby amplify the benefits and reduce the risks of total reliance on anticoagulants for management of these diseases.