Amino acid residues 721-729 are required for full factor VIII activity.
Recombinant two-chain factor VIII, from which the B domain had been deleted, was expressed in Chinese hamster ovary cells. In addition to the major product, three minor factor VIII forms were isolated. The A2 domains generated by thrombin cleavage showed different electrophoretic mobilities. Peptide mapping of the A2 domains showed that two of the factor VIII forms had the expected C-terminus of the heavy chain at Arg740 [FVIII-(1-740)] and that the other factor VIII forms had C-termini at Tyr729 [FVIII-(1-729)] or Glu720 [FVIII-(1-720)]. The major FVIII-(1-740) form, FVIII-(1-729), and FVIII-(1-720) contained sulfated tyrosine residues at Tyr718, Tyr719 and Tyr723. The minor FVIII-(1-740) form was shown to lack sulfation at these positions. The specific clotting activity was approximately 1 x 10(4) U/mg for FVIII-(1-740) (both forms) and FVIII-(1-729), but twofold lower for FVIII-(1-720). A time study of thrombin activation showed that FVIII-(1-720) was activated slower than FVIII-(1-740), FVIII-(1-729) and plasma-derived factor VIII. Partially sulfated FVIII-(1-740) was activated at the same rate as the fully sulfated FVIII-(1-740). The equilibrium dissociation constant for binding of factor VIII to inactivated immobilized thrombin was the same for all factor VIII forms, showing that the slower activation of FVIII-(1-720) was not due to a lower affinity for the anion-binding exosite in thrombin.
Kjalke
M
M
Dept. of Hemostasis, Novo Nordisk A/S, Gentofte, Denmark.
Heding
A
A
Talbo
G
G
Persson
E
E
Thomsen
J
J
Ezban
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation / Q000235:genetics*; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D010446:Peptide Fragments; D011994:Recombinant Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D005169:Factor VIII; D013917:Thrombin