Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant.

Abstract

BACKGROUND:

Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia.

OBJECTIVES:

To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A.

METHODS:

Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice.

RESULTS AND CONCLUSIONS:

FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). '(super) FVa,' a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.

• Authors: A von Drygalski, T J Cramer, V Bhat, J H Griffin, A J Gale, L O Mosnier
• Journal: Journal of thrombosis and haemostasis : JTH (J Thromb Haemost) Vol. 12 Issue 3 Pg. 363-72 ( 2014) ISSN: 1538-7836 [Electronic] England
• PMID: 24818532 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: © 2013 International Society on Thrombosis and Haemostasis.

Chemical References

• Disulfides
• Protein C
• Recombinant Proteins
• Factor Va
• Thromboplastin
• Thrombin

Topics

• Animals
• Blood Coagulation
• Disulfides (chemistry)
• Factor Va (genetics, metabolism)
• Hemophilia A (genetics)
• Hemostasis (genetics)
• Humans
• Mice
• Mice, Transgenic
• Mutation
• Partial Thromboplastin Time
• Protein C (chemistry)
• Protein Engineering (methods)
• Prothrombin Time
• Recombinant Proteins (chemistry)
• Thrombin (chemistry)
• Thromboplastin (chemistry, genetics)