Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Abstract

Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type.N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar MichaelisMenten constant (K m , apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (V max ) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P 1 Arg, and this property likely reflects the severity of the hemophilia A phenotype. IntroductionFactor 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 2332 amino acid residues with a molecular mass of approximately 300 kDa. 2,3 Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a variable heavy chain (90-210 kDa) consisting of the A1, A2, and heterogeneous fragments of partially proteolyzed B domains, together with a light chain (80 kDa) consisting of the A3, C1, and C2 domains. [2][3][4] Activation of factor VIII proceeds by limited proteolysis catalyzed by thrombin or factor Xa, with the former likely representing the physiologic activator. 5 Both proteases convert factor VIII into the active cofactor form, factor VIIIa, following cleavage of 3 P 1 positions at Arg372 between the A1-A2 domainal junction and at Arg740 between the A2-B domainal junction to generate the 50-kDa A1 and 40-kDa A2 subunits, and at Arg1689 to release a 40 acidic residue-rich peptide from the 80-kDa light chain to generate the 70-kDa A3-C1-C2 fragment. 6 Proteolysis at Arg372 and Arg1689 appears essential for generating factor VIIIa cofactor activity (see Fay 7 for review). Cleavage at the former site exposes a functional factor IXainteractive site within the A2 domain that is cryptic in the unactivated molecule. 8 Cleavage at the latter site libera...

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

“…The cleavage rate for thrombin at this site was reduced ϳ340-fold for the R1689H variant compared with wild type (Table 1). This value reflects a greater reduction in cleavage rate than the ϳ80-fold reduction that was observed in a prior study when His replaced Arg at the 372 site (25). However, this result is consistent with accommodation of His in the specificity binding pocket of the enzyme, allowing for scissile bond cleavage.…”

“…To evaluate whether cleavage at Arg-1689 contributes to the thrombin-catalyzed activation of factor VIII and in particular the slow step of cleaving Arg-372, we prepared and stably expressed two recombinant factor VIII mutants, R1689H and R1689Q. Selection of these mutations was based on a previous study evaluating the hemophilia A missense mutation R372H (25). That study demonstrated an ϳ80-fold reduction in the rate of thrombin cleavage at His-372 as compared with Arg-372 in wild-type protein.…”

Sequences Flanking Arg336 in Factor VIIIa Modulate Factor Xa-catalyzed Cleavage Rates at this Site and Cofactor Function

“…Samples were subjected to SDS-PAGE using 8% polyacrylamide gels and Western blotting was performed as described previously (22). Blotting used the 58.12 (anti-A1) monoclonal antibody and binding was detected with a goat anti-mouse alkaline phosphatase-linked antibody (Sigma).…”

Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII

“…The specific activity of R740H was 50% that of wild-type factor VIII and would yield a normal phenotype, while a specific activity of ϳ20% for the R740Q mutant would yield a mild hemophilia A phenotype. A recent study showed that replacement of Arg 372 with His yielded a factor VIII variant possessing a severe hemophilia A phenotype (1% wild type) and showed an ϳ80-fold reduced rate for thrombin catalyzed cleavage at His 372 (24). While the His 740 mutation resulted in a similar fold reduction in the rate of thrombin cleavage at this site as well as Arg 372 , as judged by A2 subunit generation data, these impairments were not reflected in the specific activity values.…”

“…To evaluate thrombin cleavage at Arg 740 , two recombinant factor VIII mutants, R740H and R740Q were prepared and stably expressed. These residues were selected based upon a prior study evaluating cleavage at the Arg 372 site (24). In that study, the thrombin-catalyzed cleavage rate at His 372 site was reduced ϳ80-fold relative to Arg at this position, whereas no detectable cleavage was observed for the P1 Gln 372 mutation.…”