Abstract
PURPOSE: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here. METHODS: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline. RESULTS: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 μg/mL), (super)FVa did not (6.7 μg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 μg/mL) or saline (5.6 μg/mL). CONCLUSION: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.