PB0797 - Comparison of platelet activation between established and novel FcγRIIA ligands

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Samantha J. Montague, PhD (she/her/hers)

Postdoctoral Researcher
University of Birmingham
Birmingham, England, United Kingdom

Co-Author(s)

YD

Ying Di

Research Technician
University of Birmingham
Birmingham, England, United Kingdom
EM

Eleyna Martin

Postdoctoral Research Fellow
University of Birmingham
Birmingham, England, United Kingdom
RB

Richard Buka

Clinical research fellow
University of Birmingham
Birmingham, England, United Kingdom
RL

Rachel Lamerton

PhD student
University of Birmingham
Birmingham, England, United Kingdom
AC

Adam Cunningham

Professor
University of Birmingham
Birmingham, England, United Kingdom
Jérôme Rollin, PhD

University of Tours
Tours, Centre, France
CV

Caroline Vayne

Academic researcher
Regional University Hospital Centre Tours
Tours, Centre, France
Claire Pouplard (she/her/hers)

Professor
University Hospital of Tours
Tours, Centre, France
YG

Yves Gruel

Professor of Haematology
Regional University Hospital Centre Tours
Tours, Centre, France
PN

Phillip LR Nicolson

Clinical Lecturer
University of Birmingham
Birmingham, England, United Kingdom
SW

Steve Watson

Professor
University of Birmingham
Birmingham, England, United Kingdom

Background: Platelet activation via the low affinity immune receptor FcγRIIA underlies thrombosis and thrombocytopenia in immune-complex driven pathologies, including heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT). Immune complexes comprising of platelet-factor-4 (PF4) and anti-PF4 antibodies in VITT, and also heparin in HIT, cluster FcγRIIA inducing activation. Despite similar exposure to heparin or vaccine, only a low proportion of patients, however, develop HIT/VITT. The monoclonal antibodies (mAbs) to PF4; 5B9 (HIT-like antibody) and 1E12 (VITT-like antibody), have been generated to help in diagnosis and establish the molecular mechanisms underlying HIT and can also be used in the study of VITT.

Aims: To explore if platelet activation in individuals varies in response to different FcγRIIA stimuli and generate novel FcγRIIA nanobodies.

Methods: We compared the ability of the mAbs 5B9 and 1E12 alongside a purified VITT patient antibody (6Ab) to stimulate aggregation and P-selectin expression in washed platelets. We have generated and characterised 20 nanobodies to FcγRIIA, and produced dimers, trimers and tetramers of the most potent one.

Results: 1E12 stimulated full aggregation and increased CD62P expression of washed platelets in all donors tested. 5B9 stimulated aggregation and CD62P expression in 75% of donors in the presence of 0.5 U/mL heparin. 6Ab in presence of PF4, stimulated aggregation in 75% of donors and CD62P expression in 83% of donors (n=8-12).
Nanobodies raised against the tandem Ig domains of FcγRIIA were tested for their binding to platelets in whole blood, in FcγRIIA-transfected DT40 cells, and in an FcγRIIA ELISA. Four out of twenty nanobodies showed strong binding, with the strongest, Nb17, used for generation of multivalent FcγRIIA ligands. These ligands are being compared to the above stimuli for platelet activation.

Conclusion(s): A high but not complete degree of similarity in platelet activation between the FcγRIIA stimuli in individuals indicates ligand- and platelet-specific variables in governing responsiveness.