Interacting regions in the A1 and A2 subunits of factor VIIIa identified by zero-length cross-linking

Factor Villa is a heterotrimer of A1. A2, and A3-C1-C2 subunits, the activity of which is labile due to a weak affinity interaction of the A2 subunit with the A1/A3-C1-C2 dimer. We have used the zero-length cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), to localize regions of interaction within the A1 and A2 subunits. Reaction of factor Villa with EDC resulted in the formation of a crosslinked product of approximately 90 kD consisting of the A1 and A2 subunits as judged by Western blotting. Alkaline resistance of this product indicated an amide rather than ester linkage, Factor Villa activity decreased as the concentration of cross-linked product increased, suggesting that flexibility in the inter-subunit interaction may be required for proper cofactor function. This product was not formed in the contiguous A1-A2 domains of factor VIII, suggesting that, upon cofactor activation, a conformational change occurs that leads to the formation of a new interdomainal salt bridge(s). Reaction of the EDC-treated factor VIIIa with activated protein C (APC), which cleaves the A1 subunit at Arg(336) and bisects the A2 subunit at Arg(562), resulted in the formation of an approximately 30 kD product that contains the C-terminus region of Al covalently linked to the N-terminal half of the A2. The approximately 90 kD cross-linked product was generated after reaction of A2 subunit with A1/A3-C1-C2 dimer but not with A1(336)/A3-C1-C2, a form of the dimer produced by APC cleavage and lacking the C-terminal acidic region of A1. A synthetic peptide corresponding to this acidic region (Met(337)-Arg(372)) was found to covalently cross-link to the isolated A2 subunit in 1:1 stoichiometry, suggesting that this region is both necessary and sufficient for the interaction of the Al and A2 subunits. Sequence analysis of this product suggested that Glu(344) in the Al peptide may contribute to the cross-linkage. These results indicate that activation of factor VIII results in formation of a new ionic linkage(s) localized to the acidic C-terminal region of Al and the N-terminal half of A2. (C) 1997 by The American Society of Hematology.