Abstract

Activated human factor IX (FIXa) exhibits very low catalytic activity towards its physiologic substrate factor X (FX), unless the cofactors, activated factor VIII (FVIIIa), procoagulant phospholipid membranes and Ca2+ are present. We were interested in the elucidation of the structural basis for the FVIIIa dependency of FIXa activity. The surface loop 99 of FIXa is important for regulation of FIXa activity. In FIXa not bound to FVIIIa, this loop is stabilized in an inactive conformation and limits access of substrate to the catalytic cleft. Aiming to improve the catalytic activity in the absence of FVIIIa, amino acid exchanges supposedly influencing the 99-loop were introduced into FIX’s catalytic domain (

Hopfner KP et al.
EMBO J
1997
;
16
:
6626
–35
;
Sichler K et al.
JBC
2003
;
278
:
4121
–26
). One of the 3 most promising mutants generated was FIX-Y94F/K98T (FIX-C), with 2 residues exchanged in the 99-loop. Both residues are known to restrict access of FX to the S2 and S4 substrate binding pockets. In FIX-Y94F/K98T/Y177F/I213V/E219G (FIX-L) two additional sites were targeted. By hydrogen bond formation with Asn97 and Asn100, Y177 locks the 99-loop in an inactive conformation, which is released by binding of FVIIIa to FIXa. I213 and E219 at the S1 site are conserved in FIX whereas V213 and G219 are conserved among most other trypsin-like proteases. In FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIX-M) the mutation A95aK is thought to support the interaction between loop 99 and 60, so that conformations similar to equivalent loops of FXa can be adopted. All full-length mutants and wild type FIX were expressed in HEK293 cells, purified and preactivated FIX was removed by chromatographic steps. The resulting proteins were tested for their amidolytic activity and further assessed in FX activation assays in the absence and the presence of FVIIIa. Activated partial thromboplastin time (aPTT) assays were carried out, using plasmas depleted of FVIII or FIX, and FVIII-inhibitor patient plasma. The rationally designed proteins showed not only improved catalytic properties but also some FVIIIa independent activity (table 1). In an aPTT assay, FIX-M activity was equivalent to 1.6% FVIII in FVIII-depleted plasma and to 162 mU/ml FEIBA in FVIII inhibitor patient plasma whereas the combination of mutations Y94F/K98T as exemplified in FIX-C resulted in a molecule with ∼2.5-fold higher specific activity than plasma derived FIX when assayed in FIX-depleted plasma.

Characterization of rationally designed FIX proteins.

Amidolytic ActivityFX activationaPTT
+FVIIIa−FVIIIaRatioFIX-DPFVIII-DPFVIII-IP
kcat / KMkcat / KM+FVIIIa / −FVIIIa
mM−1min−1 ×10−3mM−1min−1mM−1min−1 ×10−4×103mU/mgmU FVIII /5μg FIXmU FEIBA / 5μg FIX
(Abbreviations: DP, depleted plasma; IP, inhibitor patient plasma) 
pdFIX 10 52 194 
FIX-WT 37 216 15 
FIX-C 22 12 29 498 15 114 
FIX-L 15 32 33 10 180 12 115 
FIX-M 11 208 16 162 
Amidolytic ActivityFX activationaPTT
+FVIIIa−FVIIIaRatioFIX-DPFVIII-DPFVIII-IP
kcat / KMkcat / KM+FVIIIa / −FVIIIa
mM−1min−1 ×10−3mM−1min−1mM−1min−1 ×10−4×103mU/mgmU FVIII /5μg FIXmU FEIBA / 5μg FIX
(Abbreviations: DP, depleted plasma; IP, inhibitor patient plasma) 
pdFIX 10 52 194 
FIX-WT 37 216 15 
FIX-C 22 12 29 498 15 114 
FIX-L 15 32 33 10 180 12 115 
FIX-M 11 208 16 162 

Author notes

Disclosure:Employment: All five authors work for a company (Baxter AG) whose business area includes treatment of Hemophilia.