The PF4-Centric Immunothrombotic Disorders, HIT, Vitt and APS, Lead to Complement Activation Responsive to Complement-Blocking Therapeutics

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.

McCrae:Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ortel:Stago: Research Funding; Siemens: Research Funding; Up To Date: Honoraria; Instrumentation Laboratory: Consultancy, Research Funding. Arepally:Biokit: Research Funding; AstraZeneca: Research Funding; Annexon: Research Funding; Alexion Pharmaceuticals, Inc. a subsidiary of AstraZeneca: Research Funding; Veralox therapeutics: Research Funding; ABCAM PLC: Research Funding; Janssen Research $ Development: Research Funding; Sanofi: Research Funding. Rauova:NIH: Other: grant funding; Alexion: Other: grant funding; Astra Zeneca: Other: grant funding. Poncz:Alexion: Research Funding; Astra Zeneca: Research Funding.