Altered Interactions between the A1 and A2 Subunits of Factor VIIIa following Cleavage of A1 Subunit by Factor Xa

Self Cite

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: 99%

Section: From the Departments Of ‡Biochemistry And Biophysics And ¶Mementioning

confidence: 99%

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

Nogami

Lapan

Zhou

et al. 2004

Self Cite

“…Fluorophore Labeling of Proteins-Acrylodan-labeled (Ac-) isolated A1 and A2 subunits were prepared as described previously (35). Approximately 0.9 -1.1 mol of acrylodan was incorporated per mol of A1 or A2 subunit.…”

Section: Methodsmentioning

confidence: 99%

“…Thrombin was modified at its active site using ATA-FPR-CH 2 Cl and was subsequently labeled at the reactive ATA moiety using fluorescein 5-maleimide (fluorescence acceptor). The use of this fluorophore pairing was based upon an earlier study examining inter-subunit affinity following reconstitution of factor VIIIa using fluorophore-labeled subunits (35). Ac-A1 and Ac-A2 subunits retained Ͼ80% specific activity compared with unlabeled subunits based upon reconstitution of factor VIIIa (data not shown).…”

Section: Energymentioning

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

Self Cite

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.

“…APC cleaves both procofactor factor VIII and factor VIIIa, although the latter represents the more relevant, physiologic substrate, at Arg 336 (A1 subunit) and at Arg 562 (A2 subunit) (9,10). Cleavage at the former site alters the interactions between A1 and A2 subunits yielding reduced k cat values for factor Xase (11), as well as an increased K m for factor X (12). Cleavage at the latter site occurs within an important factor IXa-interactive site, the 558-loop (13).…”

mentioning

confidence: 99%

Residues Surrounding Arg336 and Arg562 Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C

Varfaj¹,

Wakabayashi

Fay

2007

Self Cite

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg 336 and Arg 562 within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate ϳ25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3 sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. variant showed a modest increase in cleavage rate (ϳ4-fold) at Arg 562 compared with WT, whereas these rates were increased by ϳ27-and 6-fold for 562(P4-P3)336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3 residues surrounding Arg 336 and Arg 562 make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg 336 by APC is attributed to favorable P4-P3 residues at this site, whereas cleavage at Arg 562 can be accelerated following replacement with more optimal P4-P3 residues.Factor VIII plays a critical role in the coagulation cascade evident from hemophilia A occurring in individuals with deficient or defective factor VIII. Factor VIII is synthesized as a single chain precursor (1, 2) consisting of three A domains homologous to the A domains in factor V, a unique B domain, and two homologous C domains (1, 3). A region rich in acidic amino acid residues designated a1 (residues 337-372), a2 (residues 711-740), and a3 (residues 1649 -1689) borders each A domain. Thus, the factor VIII domain organization is represented as NH 2 -A1-a1-A2-a2-B-a3-A3-C1-C2-COOH. Intracellular proteolytic processing generates the factor VIII procofactor composed of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains) that are associated via a metal ion-dependent linkage. Thrombin activates the procofactor by limited proteolysis generating the active cofactor, factor VIIIa, a heterotrimer composed of A1, A2, and A3-C1-C2 subunits (4, 5). Factor VIIIa associates with the serine protease factor IXa in a phospholipid membrane-dependent interaction, forming the intrinsic factor Xase complex that efficiently activates factor X during the propagation phase of coagulation (see Ref. 6 for a review).Down-regulation of the intrinsic factor Xase complex is largely due to inactivation of factor VIIIa, which is thought to occur by two independent mechanisms. The first reflects dissociation of the A2 subunit from the A1/A3-C1-C2 dimer, a result of a weak affinity electrostatic interaction (5, 7). The second mechanism results from proteolytic inactivation of the cofactor catalyzed by APC.3 APC is a potent anticoagulant that proteolytically inactivates factor Va and factor VIIIa, and deficiencies in this proteinase are linked to thrombosis (see Ref. 8 for a review). APC cleaves both procofactor factor VIII and factor VIIIa, although the latter represents the more relevant, physiologic substrate, at Arg 336 (A1 subunit) and at Arg 562 (A2 subunit) (9, 10). Cleavage at the former site alters the interactions ...