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To cite this article: Callan MB, Aljamali MN, Margaritis P, Griot-Wenk ME, Pollak ES, Werner P, Giger U, High KA. A novel missense mutation responsible for factor VII deficiency in research Beagle colonies. J Thromb Haemost 2006; 4: 2616-22.Summary. Background: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. Objective: Our aim was to identify and characterize the mutation causing cFVII deficiency. Methods: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVIIdeficient Beagles and obligate carriers were utilized. Results: In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of £ 4% in affected Beagles. In vitro expression indicated that the majority (96%) of cFVII-G96E protein was retained intracellularly. In addition, analysis of purified recombinant wild-type and mutant cFVII proteins demonstrated reduced activity of the mutant (< 2%) compared with wild-type. Rotational thromboelastometry revealed a severe impairment of clotting activity in affected Beagles, and heterozygotes also exhibited changes in coagulation-based assays. Using a mutation-specific polymerase chain reaction/ restriction digest that allows rapid identification of the G96E mutation, we surveyed a US research Beagle colony and identified a mutant allelic frequency of 31%. Conclusions: We have identified a single causative mutation for cFVII deficiency that may have implications for pharmacotoxicologic research, because reduced FVII coagulant activity may alter hemostatic and/or cardiovascular endpoints in this commonly used animal species.

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We describe a surprisingly useful approach to improve hemostasis in vivo using a variant of coagulation factor Xa (FXaI16L). This conformationally pliant derivative is partially inactive due to a defect in transitioning from zymogen to protease 1,2. Using mouse models of hemophilia, we show that FXaI16L has a prolonged half-life, relative to wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXaI16L is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXaI16L is more efficacious than FVIIa which is used to treat bleeding in hemophilia inhibitor patients3. Because of its underlying mechanism of action, FXaI16L may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

Hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation Factor VIII (FVIII) or FIX. Current treatment is through intravenous infusion of the missing protein. The major complication of treatment is the development of neutralizing Ab's to the clotting factor. Infusion of recombinant activated human Factor VII (rhFVIIa), driving procoagulant reactions independently of human FVIII (hFVIII) or hFIX, has been successful in such patients and could in theory provide hemostasis in all hemophilia patients. However, its high cost and short half-life have limited its use. Here, we report a novel treatment strategy with a recombinant adeno-associated virus vector delivering a modified FVII transgene that can be intracellularly processed and secreted as activated FVII (FVIIa). We show long-term expression, as well as phenotypic correction of hemophilia B mice following gene transfer of the murine FVIIa homolog, with no evidence of thrombotic complications at these doses. These data hold promise for a potential treatment for hemophilia and other bleeding disorders.