The Interface between the EGF2 Domain and the Protease Domain in Blood Coagulation Factor IX Contributes to Factor VIII Binding and Factor X Activation

Abstract

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

“…FVIII light chain variants were purified using CLBCAg117 coupled to CNBr-Sepharose 4B as an affinity matrix followed by concentration using Q-Sepharose (31) (Amersham Biosciences, Roosendaal, The Netherlands). HEK293 cell lines stably expressing FVIIIdB variants were produced as described (34) and grown in DMEM-F12 medium supplemented with 10% FCS. Recombinant FVIIIdB variants were purified and analyzed as described using CLB-VK34 IgG1 coupled to CNBrSepharose 4B as an affinity matrix followed by concentration using Q-Sepharose (21).…”

Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

“…Sequence reactions were performed with a BigDye terminator sequencing kit (Applied Biosystems, Foster City, CA). HEK293 cell lines stably expressing B domaindeleted FVIII and the R2159N variant were produced as described (23) and grown in DMEM-F12 medium supplemented with 10% FCS. Recombinant FVIII-WT and the R2159N variant were purified and analyzed as described using CLB-VK34 IgG1 coupled to CNBr-Sepharose 4B as an affinity matrix (16,24).…”

Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice

“…HEK293 stable cell lines expressing recombinant proteins were prepared as described previously. 19 All proteins were purified by immunoaffinity chromatography with the use of anti-FVIII Ab VK34 coupled to CNBr Sepharose 4B as described previously. 20 Protein concentration was measured by Bradford protein assay.…”