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

Abstract

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zerolength cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys 36 cleavage site, generating the A1 However, similar K m values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.Factor VIII, a plasma protein deficient or 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 multidomain, single chain molecule (A1-A2-B-A3-C1-C2) consisting of 2,332 amino acid residues with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of divalent metal ion-dependent heterodimers following cleavage at the B-A3 junction, generating a heavy chain consisting of the A1, A2, and heterogeneous fragments of partially proteolyzed B domains, together with a light chain consisting of the A3, C1, and C2 domains (2-4).Factor Xa and thrombin convert factor VIII into an active form, factor VIIIa, following limited proteolysis at Arg 372 , Arg 740 , and Arg 1689 (5). Proteolysis at Arg 372 and Arg 1689 are essential for generating factor VIIIa cofactor activity (6). Cleavage at the former site exposes a functional factor IXa-interactive site within the A2 domain that is cryptic in the unactivated molecule (7). Cleavage at the latter site liberates the cofactor from its carrier protein, von Willebrand factor (8), as well as contributes to overall specific activity of the cofactor (9, 10). Factor Xa also inactivates factor VIIIa following cleavages at Arg 336 (5) and Lys 36 (11) within the A1 subunit. Inactivation after cleavage at Arg 336 likely occurs by altered interaction of the A2 subunit with the truncated A1 (12) coupled with an increase in the K m for substrate factor X (13), the latter reflecting loss of a factor X-interactive site within an acidic residuerich region defined by residues 337-372 (14). Other proteases including activated protein C (5) and factor IXa (15) have been shown also to attack this site. Furthermore, cleavage at Lys 36 by factor Xa has been suggested to alter the conformation of A1 limiting productive interaction with the A2 ...

The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

“…Factor VIII (30 nM) was activated by thrombin and incubated with factor IXa (0.5 nM)/peptide 2228 -2240 mixtures together with various concentrations of factor X in the presence of phospholipid (20 M). Since rC2-factor IXa interaction was optimal at relatively low concentrations of Ca 2ϩ (ϳ1 mM), under these circumstances, the V max was ϳ20-fold lower than that previously reported (31). Nevertheless, in the presence of peptide 2228 -2240, the K m value remained unchanged, whereas the V max was decreased, dependent on the concentration of the peptide (Fig.…”

“…Factor Xa Generation Assays-The rate of conversion of factor X to factor Xa was monitored in a purified system (18,31). Factor Xa was generated at 22°C in HBS buffer containing 1 mM CaCl 2 and 0.1% BSA.…”

Targeting exosites on blood coagulation proteases

“…Interestingly, as seem with thrombin exosite 1, the FXa heparin-binding exosite seems to be in a precursor state in FX, consisting in a recognition site for the protein cofactor FVIIIa (Nogami et al 2004). Complex formation between ixolaris and FX strongly decreases the zymogen activation by the intrinsic tenase complex (FIXa/FVIIIa).…”

Mechanisms of Plasmin-catalyzed Inactivation of Factor VIII

“…This inactivation appears to be the result of altered interaction with the A2 subunit and an increased K m value of the truncated A1 for the substrate factor X (12,13), the latter reaction reflecting loss of a factor X-interactive site within residues 337-372 (14). Factor Xa and APC also cleave at Lys 36 (13) and at Arg 562 (15), respectively.…”

“…It is well known that serine proteases, including APC and factor Xa, inactivate factor VIII(a) following cleavage at Arg 336 (15,35). Inactivation occurs because of an altered interaction between the A2 subunit and the truncated A1 and results in the loss of a factor X-interactive site within residues 337-372 (14) and an increase in the K m value for substrate factor X (12, 13). Our data using a clotting-based assay and SDS-PAGE supported the concept that the degree of factor VIII inactivation is likely to be dependent on the proportion of unactivated molecules, activated molecules, and decay following subunit dissociation, the more rapid the cleavage at Arg 336 in the A1 subunit, the more rapid the inactivation of factor VIII(a).…”