AMINO-ACID-RESIDUES 721-729 ARE REQUIRED FOR FULL FACTOR-VIII ACTIVITY

Recombinant two-chain factor VIII, from which the B domain had been de leted, was expressed in Chinese hamster ovary cells. In addition to th e major product, three minor factor VIII forms were isolated. The A2 d omains 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 Ar g740 [FVIII-(1-740)] and that the other factor VIII forms had C-termin i 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 tyr osine residues at Tyr718, Tyr719 and Tyr723. The minor FVIII-(1-740) f orm was shown to lack sulfation at these positions. The specific clott ing activity was approximately 1X10(4) U/mg for FVIII-(1-740) (both fo rms) and FVIII-(1-729), but twofold lower for FVIII-(1-720). A time st udy of thrombin activation showed that FVIII-(1-720) was activated slo wer 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 f or 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 ex osite in thrombin.