PB0533 - New low molecular weight heparin antidote based on an antithrombin mutant

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

David Zaragoza Huesca, PhD student

PhD student
Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla
Murcia, Murcia, Spain

Co-Author(s)

JP

Julia Peñas Martínez

PhD student
Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla
Murcia, Murcia, Spain
SE

Salvador Espín

Technician
Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla
Murcia, Murcia, Spain
IZ

Isabel Zuazu

Haematologist
Centro Regional de Hemodonación, Hospital Universitario Morales Meseguer, IMIB-Pascual Parrilla
Murcia, Murcia, Spain
HC

Horacio Cano

MD
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, Murcia, Spain.
Murcia, Murcia, Spain
ML

María Luisa Lozano

Leader of Hematology Service in Murcia
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
Irene Martínez Martínez, PhD

Principal Investigator
Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla
Murcia, Murcia, Spain

Background: One of the main complications of low molecular weight heparins (LMWH) is the development of bleeding events that are difficult to manage, since to date, effective antidotes to neutralize the effect of the anticoagulant are not available in the clinic.

Aims: To design an antidote for LMWH capable of reducing their anticoagulant effect.

Methods: We expressed in HEK-EBNA cells a recombinant antithrombin with several mutations by site-directed mutagenesis. After protein purification, biochemical analyses were performed. In the in vivo study, C57BL/6J mice were injected with vehicle (PBS), 3mg/kg heparin, 20 µg/g antidote or a combination of the latter two (n=10 per group). Plasma was used to determine anti-FXa activity. In an ex vivo assay, plasma from patients (n=4) treated with LMWH (60 mg/12 hours of enoxaparin or 7500 IU/day of bemiparin) was used to measure anti-FXa activity in a coagulometer by adding different concentrations of the antidote.

Results: This antidote showed neither anti-FXa nor anti-FIIa activity, was expressed 2.7-fold higher than wild-type beta antithrombin, and also had an affinity for LMWH 2.6-fold higher than wild-type beta antithrombin. Plasma from mice injected with antidote or the heparin/antidote combination showed significantly higher FXa activity than mice injected with heparin. Additionally, the antidote completely reversed the anti-FXa activity at a dose of 2.14 µM in plasma of patients anticoagulated with LMWH.

Conclusion(s): Our results demonstrate that this recombinant molecule, by pleiotropic actions, could be effective in blocking the anticoagulant effect of LMWH.