Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

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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 ...

Section: Discussionmentioning

confidence: 85%

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

Nogami

Lapan

Zhou

et al. 2004

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“…Transfer between Fl-FPR-thrombin and Ac-A1 or Ac-A2 Subunit-Whereas a thrombin-interactive site that contributes to enzyme docking and facilitates catalysis of cleavage of factor VIII light chain at Arg 1689 has been localized within the C2 domain (25), analogous sites within factor VIII heavy chain have not been identified. To address this question, an initial series of experiments was performed to assess thrombin binding to the isolated A1 and A2 subunits of factor VIIIa.…”

Section: Energymentioning

confidence: 99%

“…An interactive site for thrombin that facilitates cleavage at Arg 1689 has been located within the C2 domain in the light chain of factor VIII (25). However, analogous site(s) for tethering interactions with factor VIII heavy chain remain unknown.…”

mentioning

confidence: 99%

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

Nogami

Zhou

Myles

et al. 2005

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Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser 205 3 Ala mutant indicated a high affinity site in the A1 subunit (K d ϳ5 nM) that was dependent upon the Na ؉ -bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K d ϳ100 nM) was observed for both Na ؉ -bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719 -740, blocked thrombin cleavage of the isolated heavy chain at Arg 372 and Arg 740 and inhibited A2 binding to thrombin Ser 205 3 Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp 392 3 Ala/Asp 394 3 Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg 740 , whereas cleavage at Arg 372 was not affected. These data suggest the acidic region comprising residues 389 -394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg 740 during procofactor activation.

“…These mutations might exhibit defects in other activities that cannot be easily tested for the isolated domain, such as binding to factor Xa or thrombin (24,25). Interactions between the C2 domain and adjacent domains within factor VIII (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…The vWF and membrane binding activities of the C2 domain appear to be competitive and mutually exclusive (18,19,22,23). The C2 domain of factor VIII has also been shown to participate in binding to factor Xa and thrombin, further illustrating its importance (24,25). Additionally many surface epitopes for both alloimmune and autoimmune antibodies as well as monoclonal antibodies are located within the C2 domain, indicating that the C2 domain might be an antigenic "hotspot" (22, 26 -29).…”

mentioning

confidence: 99%

Surface-exposed Hemophilic Mutations across the Factor VIII C2 Domain Have Variable Effects on Stability and Binding Activities

Spiegel

Murphy

Stoddard

2004

Factor VIII (fVIII) is a plasma glycoprotein that functions as an essential cofactor in blood coagulation. Its carboxyl-terminal "C2" domain is responsible for binding to both activated platelet surfaces and von Willebrand factor. We characterized the effect of 20 hemophilia-associated missense mutations across this domain (that all occur in patients in vivo) on its stability and its binding activities. At least six of these mutations were severely destabilizing, and another four caused moderate destabilization and corresponding reductions in both binding functions. One mutant (A2201P) displayed a significant reduction in its membrane binding activity but normal von Willebrand factor binding, while two others (P2300S and R2304H) caused the opposite effect. Several mutations (including L2210P, V2223M, M2238V, and R2304C) displayed near wild-type stabilities and binding activities and may instead affect mRNA splicing or alternative properties or functions of the protein. This study demonstrated that von Willebrand factor and membrane binding activities can be uncoupled and uniquely disrupted by different mutations and that either effect can lead to similar reductions in clotting activity. It also illustrated how a heterogeneous genetic disorder causes diverse molecular phenotypes that result in similar disease states.