Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII.
Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3-C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA-VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.
Genetics Institute, Inc., Cambridge, MA 02140.
D D
DD
Pittman
E M
EM
Alderman
K N
KN
Tomkinson
J H
JH
Wang
A R
AR
Giles
R J
RJ
Kaufman
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004285:Dogs; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007108:Immune Tolerance; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000378:metabolism
D006023:Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005169:Factor VIII