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.

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.

Fusigen Reduces Intracellular Reactive Oxygen Species and Nitric Oxide Levels.

BACKGROUND/AIM: Oxidative stress caused by the production of excessive cellular reactive oxygen species (ROS) and high levels of nitric oxide contribute to several human pathologies. This study aimed to examine the anti-oxidant effects of fusigen, a compound produced from Aureobasidium melanogenum. MATERIALS AND METHODS: Extracts of A. melanogenum were selected as a source for the isolation of fusigen. The anti-oxidant, nitric oxide suppression, as well as the free radical scavenging activities of fusigen were tested in BEAS-2B human bronchial epithelial cell line (BEAS-2B cells) and human dermal papilla cells (DP cells) using specific fluorescence dyes and flow cytometry analysis. Cell viability was determined by the MTT assay. RESULTS: Fusigen did not exert cytotoxicity in the human normal BEAS-2B and DP cells at concentrations up to 100 µM. Fusigen decreased basal levels of cellular ROS, as well as the levels of ROS induced by hydrogen peroxide and ferrous ion enrichment. ROS decreasing effect was confirmed in DP cells. In addition, fusigen treatment suppressed intracellular NO levels in both BEAS-2B and DP cells. CONCLUSION: The optimal process of production of purified fusigen from A. melanogenum was determined. Fusigen exhibited a low cytotoxic effect and the potential to suppress ROS and NO. These results demonstrated that fusigen may be used for the treatment or prevention of human diseases.