The protein cofactor, factor (F) VIIIa, is required for the efficient conversion of the substrate FX to FXa by the serine protease FIXa. The interaction between human FVIII (and its constituent subunits) and FX was characterized using a solid phase binding assay performed in the absence of phospholipid and FIXa. Saturable binding of FX to heterodimeric FVIII, the FVIII heavy chain (contiguous A1-A2 domains), the FVIIIa-derived A1/A3-C1-C2 dimer, and the isolated A1 subunit was observed with estimated Kd values ranging from approximately 1 to 3 microM. The interaction of FX with FVIII was inhibited by moderate ionic strength and was Ca2+-dependent, consistent with the salt sensitivity observed in a phospholipid-independent FXa generation assay. Negligible binding to FX was observed for the isolated A2 and A3-C1-C2 subunits of FVIIIa, suggesting that the A1 subunit of FVIII contains a primary binding site for FX. A synthetic peptide to the COOH-terminal acidic region of the A1 subunit, designated FVIII337-372, bound FX and effectively competed with A1 for FX binding (Ki = approximately 16 microM). Cross-linking between the FVIII337-372 peptide and the FX heavy chain was observed following reaction with 1-ethyl-3-[(diethylamino)propyl]carbodiimide. The presence of FX reduced the rate of activated protein C-catalyzed cleavage at Arg336 by approximately 5-fold. These results identify a primary FX interactive site on the cofactor of the intrinsic FXase.

We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC 50 ‫؍‬ 11 M) and partially blocked cleavage at the A2-B junction (IC 50 ‫؍‬ 100 M). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of ϳ50% and an activation rate constant of ϳ30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.Factor VIII, a plasma protein deficient of defective in individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa (1). Factor VIII is synthesized as a multi-domain single chain molecule (A1-A2-B-A3-C1-C2) consisting of 2332 amino acid residues with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B-domain linked to a light chain consisting of the A3, C1, and C2 domains (2-4).Factor VIII is converted into an active form, factor VIIIa, following limited proteolysis catalyzed by either thrombin or factor Xa (5). Cleavages at Arg 372 and Arg 740 of the heavy chain produce the 50-kDa A1 and 40-kDa A2 subunits. Cleavage of the 80-kDa light chain at Arg 1689 produces a 72-kDa A3-C1-C2 subunit. Additionally, cleavage by factor Xa at Arg 1721 produces a 67-kDa A3-C1-C2 subunit. Proteolysis at Arg 372 and Arg 1689 is essential for generating factor VIIIa cofactor activity (see for review see Ref. 6). Cleavage at the former site ...

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

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