Localization of a Factor X Interactive Site in the A1 Subunit of Factor VIIIa

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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: 99%

Section: Discussionmentioning

confidence: 99%

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

confidence: 99%

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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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“…Phospholipid vesicles containing 20% phosphatidylserine (PS), 40% phosphatidylcholine (PC), and 40% phosphatidylethanolamine (PE) were prepared using octyl glucoside as described previously (22). The A1/A3C1C2 dimer was purified by Mono S chromatography following activation of WT factor VIII by thrombin as described (23). The A2 domain-specific monoclonal antibody R8B12 was obtained from Green Mountain Antibodies (Burlington, VT).…”

Section: Methodsmentioning

confidence: 99%

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

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Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cellexpressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K d for A2. The second assay determined the K d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K d and k cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.Factor VIIIa is an essential blood coagulation protein that acts as a cofactor for the serine protease factor IXa in the conversion of factor X to Xa during the propagation phase of coagulation. Defects or deficiencies in factors VIII and IX result in hemophilia A and B, respectively. Factor VIII is synthesized as a multidomain, single chain precursor with a molecular mass of ϳ300 kDa and domain structure A1-A2-B-A3-C1-C2. It circulates primarily as a heterodimer resulting from proteolysis at the B-A3 junction wherein the heavy chain (A1A2B) and light chain (A3C1C2) are associated by metal ion-dependent and independent linkages (see Ref. 1 for review). Factor VIII is activated by limited proteolysis catalyzed by thrombin or factor Xa to the active cofactor, factor VIIIa. These cleavages convert the heterodimer or single chain precursor to the heterotrimer (2, 3) with the heavy chain-derived A1 subunit and light chain-derived A3C1C2 subunit maintaining stable association, whereas the A2 subunit is weakly associated with the A1/A3C1C2 dimer through electrostatic interactions (4, 5). Factor VIIIa can be reconstituted from the isolated A2 subunit and A1/A3C1C2 dimer to regenerate high levels of cofactor activity (3, 5).Association of factors VIIIa and IXa on a phospholipid surface forms the intrinsic factor Xase complex, which increases the cataly...

“…The exact mechanism by which FVIIIa enhances the catalytic activity of FIXa toward FX is still unknown. It is generally believed that FVIIIa has three functions within the intrinsic FX-activating complex: (i) FVIIIa stabilizes a conformation of FIXa that has strongly increased protease activity toward FX, (ii) FVIIIa acts as a receptor for FIXa on activated platelets (2), which in vivo provide the procoagulant phospholipid surface (21), and (iii) more recent data indicate that FVIIIa directs the cleavage sites in FX toward the active site of FIXa (22). We identified a murine monoclonal antibody, 224AE3, that specifically binds to human FIXa and that enhances the catalytic activity of the FVIIIa-FIXa-Ca 2ϩ -phospholipid complex.…”

Section: Discussionmentioning

confidence: 99%

An Antibody Specific for Coagulation Factor IX Enhances the Activity of the Intrinsic Factor X-activating Complex

Kerschbaumer

Riedrich

Kral

et al. 2004

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (k cat ) of the enzyme complex increased 2-to 3-fold whereas the K m for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.One of the key events during normal hemostasis is the conversion of the zymogen factor X (FX) 1 into its enzymatically active form factor Xa (FXa) via the intrinsic coagulation pathway, a process that subsequently leads to activation of prothrombin and the formation of a fibrin clot (1). The FX-activating protease factor IXa (FIXa) assembles (together with FVIIIa as well as Ca 2ϩ ions) on a phospholipid surface to form the intrinsic FX-activating complex (2). In this complex FVIIIa serves as a cofactor for the enzyme FIXa and increases the turnover number (k cat ) of FIXa by 4 to 5 orders of magnitude (3, 4). Binding of the stoichiometric FVIIIa-FIXa complex and the substrate FX to a phospholipid surface strongly reduces the K m for the substrate (3, 4), probably because the protein-protein interactions are limited to two dimensions (5).The physiological importance of the intrinsic FX-activating complex is well defined because the severe bleeding disorders hemophilia A and hemophilia B are characterized by very low plasma levels (Ͻ 5%) or by the absence of the pro-cofactor FVIII and the pro-enzyme FIX, respectively. Treatment of hemophilia is successfully achieved by supplementing the blood of patients with either human FVIII or human FIX. Both proteins can be derived from human plasma or produced as recombinant protein in mammalian cell culture (6, 7). Although progress in the production of coagulation factors to ensure their purity, efficacy, and viral safety has been made over the last 20 years, treatment of hemophilia is still beset with several difficulties. First, production of recombinant and plasma-derived, therapeutic coagulation factors is expensive, an...