Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII Δ II

Abstract

Factor VIII Δ II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771 – Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N‐terminal amino acid sequencing. Thrombin first cleaves factor VIII Δ II at Arg740‐Ser741 to generate the 90‐kDa heavy chain and an 80‐kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73‐kDa light chain. The 90‐kDa fragment is further cleaved, giving rise to 50‐kDa and 40‐kDa fragments while the 80‐kDa fragment generates a 71/73‐kDa doublet. The 71/73‐kDa doublet, 50‐kDa and 40‐kDa fragments were further analysed by N‐terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII Δ II, the essential structural elements required for thrombin activation are conserved. Copyright © 1991, Wiley Blackwell. All rights reserved