PB0317 - Unravelling the disulfide-bonded states of antithrombin: Implications for anticoagulant activity

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

KL

Kenny Ningxin Lu

PhD Student
Centenary Institute, The University of Sydney, Sydney, Australia
Sydney, New South Wales, Australia

Co-Author(s)

Mark Schreuder, PhD

Research Officer
The Centenary Institute, University of Sydney
Sydney, New South Wales, Australia
DB

Diego Butera

Postdoctoral Scientist
The Centenary Institute, University of Sydney
Sydney, New South Wales, Australia
María Eugenia de la Morena-Barrio, PhD

PhD
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
CB

Carlos Bravo-Pérez

PhD MD
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
Javier Corral

Professor
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
JC

Joyce Chiu

Lecturer
The Centenary Institute, University of Sydney
Sydney, New South Wales, Australia
PH

Philip J. Hogg

Professor
The Centenary Institute, University of Sydney
sydney, New South Wales, Australia

Background: Antithrombin is a key anticoagulant protein that regulates the proteolytic activity of various procoagulant proteases, and its deficiency is associated with thrombosis. The redox state of antithrombin’s three disulfide bonds and role in coagulation are not fully understood.

Aims: Determine the disulfide-bonded state of antithrombin and functionally characterize an antithrombin mutant comprising a disulfide bond (C21A,C95A) ablation.

Methods: Disulfide-bonded states of plasma antithrombin were quantified using differential cysteine labelling and mass spectrometry. Recombinant antithrombin wild-type and disulfide bond C21A,C95A mutant were expressed in mammalian cells and purified. The second order rate constants of factor IXa, Xa and thrombin inhibition by antithrombin or heparin-antithrombin were determined in competitive chromogenic substrate assays.

Results: The C21-C95 disulfide bond was unformed in ~1 in 3 antithrombin molecules isolated from healthy donor plasma, whereas the C247-C430 and C8-C128 bonds were unformed in ~1 in 10 and ~1 in 20 antithrombin molecules, respectively. Recombinant antithrombin from HEK293 cells displayed an identical redox state as plasma-derived antithrombin. Mutant C21A,C95A antithrombin exhibited two-fold increase in rate of factor Xa inhibition compared to wild-type, whereas no significant difference was observed for inhibition of thrombin. C21A,C95A antithrombin in the presence of unfractionated heparin showed no difference in rate of factor Xa inhibition compared to wild-type, whereas rate of thrombin inhibition decreased two-fold. In the presence of low molecular weight heparin, C21A,C95A antithrombin exhibited a four-fold decrease in rate of factor Xa inhibition and two-fold reduced rate of thrombin inhibition relative to wild-type.

Conclusion(s): Antithrombin circulates in plasma in multiple disulphide-bonded states. The unformed C21,C95 bond in antithrombin enhances factor Xa inhibition, whereas the heparin-catalyzed inhibition is reduced for both factor Xa and thrombin. Potentially, changes in the antithrombin disulfide bond states can modulate the physiological and pathological role of this anticoagulant potentially leading to a bleeding or thrombotic phenotype.