Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays.
BACKGROUND
Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa.


OBJECTIVE
The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood.


METHODS
Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum.


RESULTS
Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa.


CONCLUSIONS
C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.
Radtke
K-P
KP
Bayer HealthCare, Biological Products, Research Triangle Park, NC, USA.
Griffin
J H
JH
Riceberg
J
J
Gale
A J
AJ
D001777:Blood Coagulation / Q000187:drug effects; D002460:Cell Line; D003029:Coagulants / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015944:Factor VIIIa / Q000378:metabolism*; D006207:Half-Life; D006467:Hemophilia A / Q000097:blood* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013916:Thrombelastography; D013917:Thrombin / Q000378:metabolism; D013918:Thrombin Time; D014162:Transfection; D014914:Whole Blood Coagulation Time
D003029:Coagulants; D004220:Disulfides; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005169:Factor VIII; D013917:Thrombin