OC 22.3 - Prothrombin Fragment 2: A Potential Therapeutic Target to Inhibit Antiprothrombin-induced Platelet Activation

Sunday, June 23, 2024

15:15 – 15:30 ICT

Room: Ballroom B2

Presenting Author(s)

Rachel Gehlen

PhD student
Synapse Research Institute
Maastricht, Limburg, Netherlands

Co-Author(s)

WC

Walid Chayoua

Resident in Clinical Chemistry
Medisch Spectrum Twente/Medlon BV
Enschede, Overijssel, Netherlands
FS

Frauke Swieringa

Senior Scientist
Synapse Research Institute
Maastricht, Limburg, Netherlands
Ilaria De Simone, PhD

PostDoc
Synapse Research Institute
Maastricht, Limburg, Netherlands
Marisa Ninivaggi

Research Project Leader
Synapse Research Institute
maastricht, Limburg, Netherlands
Mark Roest

PhD, Clinical Director
Synapse Research Institute
Maastricht, Limburg, Netherlands
JH

Johan W.M. Heemskerk

Advisor
Synapse Research Institute
Maastricht, Limburg, Netherlands
Pd

Philip G. de Groot

Professor Emeritus, advisor
Synapse Research Institute
Maastricht, Limburg, Netherlands
Bd

Bas de Laat

General Director
Synapse Research Institute
Maastricht, Limburg, Netherlands

Background: Antiprothrombin antibodies are frequently found in patients with antiphospholipid syndrome, however, their clinical relevance and mechanisms of action remain largely unknown. Recently, our group identified that antiprothrombin antibodies with lupus anticoagulant (LA)-activity can activate platelets via the platelet receptor, FcγRIIA. Nevertheless, experimental data on the binding site of these pathogenic antiprothrombin antibodies are still lacking.

Aims: To investigate the binding site of pathogenic antiprothrombin antibodies and uncover potential therapeutic targets and approaches.

Methods: Recombinant prothrombin constructs 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 the epitope specificity of monoclonal (28F4, 3B1 and 11H2) and patient-derived (P003) antiprothrombin antibodies. Antiprothrombin-induced platelet activation was assessed using Light transmission aggregometry and Flow cytometry. In short, washed platelets from healthy volunteers were activated using monoclonal and patient-derived antiprothrombin antibodies in the presence of Ca2+ and prothrombin. A nanobody directed against prothrombin fragment 2 was added prior to activation to evaluate its ability to inhibit antiprothrombin-induced platelet activation. The nanobody was produced by immunizing Lama’s followed by phage display.

Results: Antiprothrombin antibodies that were directed against prothrombin fragment 2 (28F4, 3B1, and P003) could activate platelets, while antibodies targeting fragment 1 (11H2) did not have this ability. This suggests that the capability to induce platelet activation is linked to the binding site of the antiprothrombin antibody. Furthermore, a nanobody directed against prothrombin fragment 2 capable of inhibiting antiprothrombin-induced platelet activation, emphasizing the role of prothrombin fragment 2 in antiprothrombin-induced platelet activation.

Conclusion(s): Only antiprothrombin antibodies which targeted prothrombin fragment 2 could induce platelet activation, highlighting prothrombin fragment 2 as a potential novel therapeutic target and strategy to inhibit antiprothrombin-induced platelet activation.