Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B

Abstract

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibodyantigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the ␥-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX. (Blood. 2001;98:1416-1423)

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

“…First, domain swap mutants were used in which individual domains were exchanged by their counterparts from human FIX. 29 Second, fine mapping within the protease domain was performed using purified hFIX chimeras with FX replacements in surface loops 223 to 227, 258 to 267, or 315 to 323. 30 Together these loops comprise the human/rhesus differences in positions 226, 227, 261, 315 and 321.…”

Role of the α-Helix 163-170 in Factor Xa Catalytic Activity

“…Recombinant FX Derivatives-The mammalian expression plasmid pKG5 containing human FX cDNA and encoding wildtype (wt) FX (25) (Fig. 2).…”

“…All constructs were transfected into Madin-Darby canine kidney cells using calcium phosphate precipitation, and cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Stable cell lines producing recombinant FX derivatives and wt-FX were prepared as already described (25) and maintained in 300-cm 2 flasks for protein production in serum-free Dulbecco's modified Eagle's medium/F-12 supplemented with 800 g/ml Geneticin G-418, 100 units/ml penicillin, 100 g/ml streptomycin, 5 g/ml vitamin K 1 , and 1% insulin-transferrin-selenium-X. FXcontaining medium was harvested every 48 h. Benzamidine and phenylmethylsulfonyl fluoride were added to a final concentration of 10 and 2 mM, respectively, and the medium was centrifuged (6000 ϫ g), passed over cellulose acetate membranes (0.45 m) to eliminate cell debris, and stored at Ϫ80°C.…”

Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

“…Recombinant FX Derivatives-Plasmids encoding wt-FX, FX/FIX-(Gla), and FX/FIX(EGF1) have been described previously (36) as well as the domain borders of the chimeras. The FIX and FX domains are expressed by the chimera names, i.e.…”