OC 09.1 - Epitope Specificity of Anti-prothrombin Antibodies That Express Lupus Anticoagulant Activity

Saturday, June 24, 2023

13:00 – 13:15 ET

Room: 519

Session Available As: On-Demand

Presenting Author(s)

Rachel Gehlen, Master of Science (she/her/hers)

PhD-student
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands

Co-Author(s)

Tessa Noordermeer, MSc

PhD-candidate
Center for Benign Haematology, Thrombosis and Haemostasis, Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
Utrecht, Utrecht, Netherlands
WC

Walid Chayoua

PhD
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
Marisa Ninivaggi, PhD

Research Project Leader
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
Romy De Laat-Kremers, PhD

Post-doctoral Research Project Leader
Department of Data Analysis and Artificial Intelligence, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
Mark Roest, PhD (he/him/his)

Clinical Director
Department of Platelet pathophysiology, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
PG

Philip G. de Groot

Emeritus Professor
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
MAASTRICHT, Limburg, Netherlands
Rolf T. Urbanus, PhD

Associate professor
Center for Benign Haematology, Thrombosis and Haemostasis, Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
Utrecht, Utrecht, Netherlands
Bd

Bas de Laat, MD, PhD

General Director
Department of Functional Coagulation, Synapse Research Institute, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands

Background: The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the persistent presence of antiphospholipid antibodies. Some of these autoantibodies exert lupus anticoagulant (LAC) activity, a laboratory phenomenon characterized by the prolongation of phospholipid-dependent coagulation tests. Recently, our group identified that LAC-positive anti-prothrombin antibodies activate platelets via the platelet receptor, FcγRIIA. However, it is unknown where these antibodies bind on prothrombin.

Aims: To investigate the binding epitope of monoclonal and APS patient anti-prothrombin antibodies with LAC activity.

Methods: Recombinant fragments of prothrombin were designed and produced in Chinese hamster ovary cells. Prothrombin fragment 1, fragment 2, and fragment 1.2 were coated on ELISA plates to study epitope specificity. LAC-positive monoclonal anti-prothrombin antibodies (3B1 and 28F4), LAC-negative monoclonal anti-prothrombin antibodies (11H2 and 9C6), and plasma from APS patients were studied. A dilute Russell's viper venom time (DRVVT) test was used to evaluate whether prothrombin constructs could neutralize the effect of LAC-positive anti-prothrombin antibodies. Furthermore, a fragment 1.2 nanobody was produced by immunizing Lama’s followed by phage display. The ability of this nanobody to compete with LAC-positive anti-prothrombin antibodies for the prothrombin binding site was tested using the DRVVT test.

Results: The LAC-positive monoclonal anti-prothrombin antibodies recognized prothrombin fragment 2. Interestingly, LAC-negative monoclonal anti-prothrombin antibodies bound exclusively to fragment 1. LAC-ratios of patient samples correlated significantly with anti-fragment 2 antibody levels (p < 0.05, ρ=0.38), but not with anti-fragment 1 antibody levels. Furthermore, incubation with prothrombin fragment 2 or the F1.2 nanobody almost completely reversed the prolonged DRVVT in plasma containing LAC-positive anti-prothrombin antibodies, but not in plasma containing anti-β2GP1 antibodies. In contrast, this neutralizing effect was not observed when incubated with prothrombin fragment 1.

Conclusion(s): Our data show that LAC-positive anti-prothrombin antibodies recognize prothrombin fragment 2. We were able to neutralize a prothrombin-dependent LAC with fragment 2 and with a novel nanobody directed against fragment 1.2.

Learning Objectives:

Recognize the binding site of anti-prothrombin antibodies that have lupus anticoagulant activity.
Interpret Lupus anticoagulant results and understand the methods used to test lupus anticoagulant.
Define different anti-prothrombin antibody subpopulations and discuss the clinical relevance of each subpopulation.