PB1325 - A novel recombinant factor VII D60A may potentially reduce the risk of thrombotic complications

Tuesday, June 27, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Karnsasin Seanoon, PhD

Post Doc
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand
Lum Lukka, Pathum Thani, Thailand

Co-Author(s)

PP

Panwajee Payongsri

Dr.
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
Ratchathewi, Krung Thep, Thailand
Nongnuch Sirachainan, MD

Prof.
Faculty of Medicine Ramathibodi hospital, Mahidol University, Bangkok, Thailand
Bangkok, Krung Thep, Thailand
Ampaiwan Chuansumrit

Professor
Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
Bangkok, Krung Thep, Thailand
SH

Suradej Hongeng

Professor
Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
Ratchathewi, Krung Thep, Thailand
Pansakorn Tanratana, MD, PhD

Assistant Professor
Department of Pharmacology, Faculty of Science, Mahidol University
Bangkok, Krung Thep, Thailand

Background: Recombinant FVIIa (rFVIIa) has been approved by the FDA for treating patients with hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the off-label uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications.

Aims: To design a new rFVII molecule with lower coagulant activity that could be used as a novel hemostatic agent with less thrombogenicity.

Methods: D60A was introduced into F7 sequence by PCR-based mutagenesis. Wild-type (WT) and D60A were generated in HEK293T by stable transfection. The forms and antigen levels of secreted FVII proteins in culture supernatant were determined by Western blot (WB) analysis and ELISA, respectively. In addition, FVII coagulant activities were determined by amidolytic, chromogenic, thrombin generation (TG), and modified prothrombin time (PT) assays.

Results: WT and D60A only expressed the zymogen form (Fig 1A). WT secreted higher amount of FVII than D60A (Fig 1B). WT and D60A demonstrated similar FVIIa amidolytic activity (Fig 1C). However, D60A demonstrated about 50% activity compared to WT on FX activation (Fig 1D) and significantly longer lag time (Fig 1E) than that of WT in TG assay. Finally, the clotting time produced by D60A was significantly prolonged than that of WT (Fig 1F).

Conclusion(s): Given that FVII level of 15-25% (approximately 0.075-0.125 µg/mL) is considered adequate for normal hemostasis. The D60A molecule demonstrated lower coagulant activities because this point mutation could weaken FX binding, leading to impaired FX activation, delayed thrombin generation, and ultimately decreased fibrin formation. Therefore, D60A could be used as a safe prohemostatic agent for the treatment of bleeding with less thrombogenecity.