Extravascular Factor IX Pool Fed By Prophylaxis Is a True Hemostatic Barrier Against Bleeding

Factor IX (FIX) can bind to type-IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. However, the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds have not been demonstrated. The capacity of 4 different FIX molecules (plasma derived [pd] and recombinants) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knock-out (KO) mice. A specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50-100 IU/kg, while pdFIX generated a FIX signal at all doses including 50 IU/kg. After five 100 IU/kg daily infusions of rFIX, a clear accumulation of FIX was seen in the liver and joints of animals having received 5-day daily FIX infusions with all FIX molecules, with a higher FIX labelling intensity compared to that observed after a single infusion of FIX 100 IU/kg or a single infusion of FIX 500 IU/kg.

The extravascular procoagulant activity of FIX was assessed in saphenous vein bleeding assays performed 7 days after a single infusion of FIX 100 IU/kg. An additional group of FIX KO mice received intravenously for 5 days a single daily dose of 100 IU/Kg of one the four types of FIX concentrate in order to mimic prophylaxis. The number of clots formed in 15 minutes was significantly greater in mice having received prophylaxis during 5 days with 100 IU/kg FIX compared to untreated FIX KO mice (p=0.009) and to mice having received a single infusion of FIX 100 IU/kg (p<0.0001) (Figure 1).

Taken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection towards bleeding.