Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.
BACKGROUND
The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa.


OBJECTIVE
Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII).


METHODS
In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex.


RESULTS
Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different.


CONCLUSIONS
Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.
Gale
A J
AJ
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. agale@scripps.edu
Radtke
K-P
KP
Cunningham
M A
MA
Chamberlain
D
D
Pellequer
J-L
JL
Griffin
J H
JH
D015949:Factor IXa / Q000378:metabolism*; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D015944:Factor VIIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D006467:Hemophilia A / Q000188:drug therapy; D009154:Mutation; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
D011486:Protein C; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D015944:Factor VIIIa; C078147:F8 protein, human; D005169:Factor VIII; D015949:Factor IXa; D013917:Thrombin