Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Abstract

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 ...

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

“…Thus, whereas similar exosite interactions facilitate formation of the enzyme-substrate complexes, the sites for tethering appear to differ significantly. This contention is further supported by our observation that the 373-395 peptide blocked thrombin-catalyzed cleavage of isolated heavy chain, whereas this peptide showed no effect on factor Xa-catalyzed proteolysis (32). Furthermore, we observed that thrombin and factor Xa demonstrated markedly different reactivities toward cleaving the Arg 740 -Ser 741 scissile bond in the factor VIII D392A/D394A mutant.…”

“…Mechanistic studies of factor Xa-catalyzed proteolysis of factor VIII heavy chain indicated that the proteinase interacts with acidic residues localized within the 337-372 region separating the A1 and A2 domains to facilitate cleavages at both Arg 372 and Arg 740 (32). Furthermore, this binding utilizes the heparin-binding exosite of factor Xa (63).…”

“…Recombinant Factor VIII and Thrombin-Recombinant wild type factor VIII and the mutant factor VIII D392A/D394A, in which Asp 392 and Asp 394 were replaced by Ala, were constructed, expressed, and purified using methods described previously (32). The activity and antigen levels of factor VIII proteins were determined by a one-stage clotting assay and a sandwich enzyme-linked immunosorbent assay, respectively, and the latter used two anti-factor VIII monoclonal antibodies as described previously (33).…”

Mechanisms of Interactions of Factor X and Factor Xa with the Acidic Region in the Factor VIII A1 Domain

“…The synthetic peptide corresponding to factor VIII residues 337-372 was prepared by Quality Controlled Biochemicals, Inc. (Hopkinton, MA). Biotinylated peptide was prepared using sulfo-NHSbiotin as described previously (17). The B-domainless factor VIII expression construct RENeo factor VIII and baby hamster kidney cells were gifts kindly provided by Dr. Pete Lollar.…”

“…In addition, we demonstrated recently that the 337-372 acidic region following the A1 domain, in particular a cluster of amino acid residues 361-363, contributes to a unique factor Xa-interactive site within the factor VIII heavy chain (17). Factor Xa also interacts with these residues in the A1 subunit, possibly via the heparin-binding exosite of this protease (18), and this binding modulates cleavage at Lys 36 (11).…”