Site-directed mutagenesis of tissue factor pathway inhibitor–binding exosite D60A on factor VII results in a new factor VII variant with lower coagulant activity
Issued Date
2024-01-01
Resource Type
eISSN
24750379
Scopus ID
2-s2.0-85183490365
Journal Title
Research and Practice in Thrombosis and Haemostasis
Volume
8
Issue
1
Rights Holder(s)
SCOPUS
Bibliographic Citation
Research and Practice in Thrombosis and Haemostasis Vol.8 No.1 (2024)
Suggested Citation
Seanoon K., Kitiyanant V., Payongsri P., Sirachainan N., Angchaisuksiri P., Chuansumrit A., Hongeng S., Tanratana P. Site-directed mutagenesis of tissue factor pathway inhibitor–binding exosite D60A on factor VII results in a new factor VII variant with lower coagulant activity. Research and Practice in Thrombosis and Haemostasis Vol.8 No.1 (2024). doi:10.1016/j.rpth.2023.102309 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/95574
Title
Site-directed mutagenesis of tissue factor pathway inhibitor–binding exosite D60A on factor VII results in a new factor VII variant with lower coagulant activity
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Abstract
Background: Recombinant factor (F)VIIa (rFVIIa) has been approved by the US Food and Drug Administration for the treatment of hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the investigational uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence showing that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications. Objectives: To design a new rFVII with lower coagulant activity that could potentially be used as an alternative hemostatic agent aiming to minimize the risk of thrombogenicity. Methods: D60A was introduced into the F7 sequence by polymerase chain reaction–based mutagenesis. Wild type (WT) and D60A were generated in human embryonic kidney 293T cells by stable transfection. FVII coagulant activities were determined by amidolytic cleavage of the FVIIa-specific substrate, 2-step FXa generation, thrombin generation (TG), and clot-based assays. Results: WT and D60A demonstrated similar FVIIa amidolytic activity. However, D60A showed approximately 50% activity on FX activation and significantly longer lag time in the TG assay than that shown by WT. The clotting time produced by D60A spiked in FVII-deficient plasma was significantly prolonged than that of WT. Additionally, the ex vivo plasma half-lives of WT and D60A were comparable. Conclusion: D60A demonstrated lower coagulant activities, most likely due to the weakening of FX binding, leading to impaired FX activation and delayed TG and fibrin formation. Considering that a plasma FVII level of 15% to 25% is adequate for normal hemostasis, D60A is a molecule of interest for future development of an rFVII with a lesser extent of thrombogenicity.