INTERACTION OF THE A1 SUBUNIT OF FACTOR VIIIA AND THE SERINE-PROTEASEDOMAIN OF FACTOR-X IDENTIFIED BY ZERO-LENGTH CROSS-LINKING

We have previously used a solid phase binding assay to localize a Fact or X (FX) interactive site to the acidic C-terminus of the Al subunit of FVIIIa (Lapan KA. Fay PJ. J Biol Chem 1997; 272: 2082-2088). The co mplex of FVIII-FX was made covalent following reaction with the zero-l ength cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodii mide hydrochloride (EDC). Western blotting of the thrombin-cleaved com plex showed that the AI subunit of FVIIIa associated with FX heavy cha in. The FX-AI product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1(336)/A3 -C1-C2 form, indicating that a residue(s) in the region spanning Met(3 37)-Arg(372) contributed to the intermolecular ion pair(s). A syntheti c peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) we re formed. Sequence analysis of the FX-FVIII337-372 adduct suggested t hat the first 12 NH2-terminal residues of the FX and peptide do not pa rticipate in cross-link formation. Conversion of the cross-linked prod uct to FXa by RW-X showed that the peptide was associated with the ser ine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) f ormed between residues of the Al acidic C-terminus of the cofactor (wi thin residues 349-372) and the serine protease-forming domain of the s ubstrate.