PB0538 - A monoclonal antibody against human factor V impairs its activation and procoagulant function

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Francis Ayombil, PhD

Research Fellow
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Co-Author(s)

SQ

Sean M. Quinn

Post-doctoral Research Fellow
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
EC

Emily A. Cid

Research Technician
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
MB

Matthew W. Bunce

Research Laboratory Director
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia
Spring House, Pennsylvania, United States
Rodney M. Camire, PhD

Professor of Pediatrics
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia. Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Philadelphia, Pennsylvania, United States

Background: The activation of factor V (FV) to FVa is a crucial step in coagulation. FVa acts as a cofactor for FXa in the prothrombinase complex, significantly enhancing thrombin generation (TG). Developing methods to modulate FV/FVa function could be useful for limiting unwanted clot formation.

Aims: Develop FV-specific monoclonal antibodies that inhibit FV function and investigate mechanism of action.

Methods: Mice were immunized with FV, and hybridoma clones were screened to identify antibodies that inhibited clotting. Clone HA2 was purified and studied in plasma and purified assay systems. Its affinity for FV, and binding epitope on FV were determined.

Results: HA2 bound FV, FV-short, and FVa with high affinity (Kd ~ 2 nM). Immunoblotting revealed HA2 binds the C-terminus of the heavy chain (698-709). In clotting assays, HA2 prolonged the PT and APTT with complete inhibition (>95%) at 5 nM. In a TG assay, HA2 impaired TG in normal human plasma by >90% with an impact on peak thrombin and the lag phase. However, it only had a modest impact in the TGA when FV-deficient plasma was reconstituted with FVa or FV-short. In a continuous purified prothrombinase assay, FV was more susceptible to inhibition by HA2 compared to FVa, indicating an impact on FV activation. This was verified by western blotting, which showed that HA2 significantly delayed the cleavage of FV by thrombin or FXa. Using FV cleavage site variants, it was found that HA2 completely blocked cleavage at Arg709 on FV and FV-short.

Conclusion(s): HA2 is a FV-specific monoclonal antibody that binds the C-terminal end of the heavy chain and blocks cleavage at Arg709. It inhibits FV activation, delaying the initiation of coagulation. Additionally, HA2 has a modest impact on the function of FVa within the prothrombinase complex. This antibody could be useful for dampening coagulation and reducing clot formation.