Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

AbstractFactor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.

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Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽

10.1182/blood.v110.11.2686.2686 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2686-2686

Author(s):

Jennifer Newell ◽

Qian Zhou ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Specific Activity ◽

Factor Xa ◽

Factor X ◽

Wild Type ◽

Factor Viiia ◽

N Terminus ◽

Acidic Residues ◽

Type Factor ◽

A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

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Partial Reconstitution of Factor VIII Activity from a Mild Crm+ Hemophilia A Patient by Replacement of the Defective A2 Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615099 ◽

1998 ◽

Vol 79 (05) ◽

pp. 943-948 ◽

Cited By ~ 1

Author(s):

W. C. Pieneman ◽

P. Fay ◽

E. Briët ◽

P. H. Reitsma ◽

R. M. Bertina

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Wild Type ◽

Coding Region ◽

Factor Viii Activity ◽

Factor Ixa ◽

Factor Viiia ◽

Factor Viii Antigen ◽

A2 Domain ◽

Reduced Activity

SummaryWe further characterised the abnormal factor VIII molecule (factor VIII Leiden) of a Crm+, mild hemophilia A patient with a factor VIII activity of 0.18 IU/ml and a factor VIII antigen of 0.95 IU/ml. Mutation analysis of the coding region, promoter and 3’ untranslated region of the factor VIII gene revealed the presence of a C to T substitution at codon 527. This nucleotide change predicts the replacement of an arginine to tryptophan in the A2 domain close to a suggested binding site for factor IXa. Since a previous study of this mutant factor VIII protein suggested that this protein had a reduced affinity for factor IXa, position 527 in the protein might be involved in the interaction with factor IXa.In this study we gathered evidence for our hypothesis that the Arg to Trp mutation at position 527 is the cause of the reduced activity of factor VIII Leiden. Replacement of the mutated A2 domain by wild type A2 domain partially corrected the defect.Factor VIII from normal and factor VIII Leiden plasma was concentrated by cryoprecipitation, activated with thrombin and incubated with excess wild type A2 domain. Competition with excess isolated human A2 domain resulted in a partial reconstitution of the factor VIIIa activity of thrombin treated factor VIII Leiden. This supports the hypothesis that the mutation in the A2 domain is the cause of the reduced factor VIII activity.

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Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽

10.1182/blood.v108.11.1692.1692 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1692-1692

Author(s):

Jennifer Newell ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Time Course ◽

Specific Activity ◽

Factor Xa ◽

Factor X ◽

Wild Type ◽

Thrombin Cleavage ◽

Factor Ixa ◽

Factor Viiia ◽

Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

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Enhancement of Factor VIII Stability by Replacing Multiple Charged Residues at the A2 Domain Interface

Blood ◽

10.1182/blood.v112.11.1016.1016 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1016-1016

Author(s):

Hironao Wakabayashi ◽

Amy E Griffiths ◽

Philip Fay

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Specific Activity ◽

Factor Xa ◽

Fold Increase ◽

Decay Rates ◽

Single Mutation ◽

Factor Viiia ◽

Group A ◽

Group B

Abstract Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665, or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 2008, in press). These mutations did not appreciably affect factor VIII specific activity or thrombin generation parameters. Factor VIII structure studies suggest D519 is buried at the A1/A2 domain interface, while E665 and E1984 localize at the A2/A3 domain interface. In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effect of these mutations. Factor VIII variants generated included double mutants with a mutation at D519 and mutation at either E665 or E1984 (Group A), double mutants with a mutation at E665 and mutation at E1984 (Group B), and triple mutants with a mutation at D519 and mutations at both E665 and E1984 (Group C). Most of the mutants retained normal specific activity values compared to wild type (WT) with exceptions noted for E665A/E1984A, E665A/E1984V and D519V/E665V/E1984V which showed ~2-fold reductions in this parameter. Studies assessing factor VIII stability involved monitoring the rates of loss of factor VIII activity by factor Xa generation assay following incubation of factor VIII (4 nM) at 55ºC. The rate of decay of factor VIIIa was monitored over time at 23ºC using the factor Xa generation assay following activation of factor VIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. The Group A variants D519A/E665A, D519A/D665V, and D519V/E665V showed significant enhancement (up to ~1.3-fold for the D519A/D665V variant) in factor VIII thermal stability as compared with the best single mutation in that pairing, and representing actual decay rates that approached 45% the WT value. On the other hand, the relative factor VIII decay rates for three of the four of the Group B mutants were somewhat increased compared with the best single mutation in the pairing. No significant changes were observed for the Group C mutants. Evaluation of factor VIIIa stability revealed large enhancements of up to ~4-fold compared with the single mutants for all of the Group A variants. Group B variants yielded poorer results when compared with the better individual mutation in the pairing. The triple mutations (Group C) showed the largest factor VIIIa stability enhancements with maximal stability observed for D519V/E665V/E1984A, which showed a decay rate that was ~10% the WT value. A calibrated thrombin generation assay using a fluorogenic substrate was performed on selected mutants using a sub-physiologic factor VIII concentration (0.2 nM). Enhancements in selected parameter values were observed for the D519V/E665V variant (~2.3-fold increase in the peak height and ~1.5-fold increase in endogenous thrombin potential compared with WT), while the D519A/E665V, D519V/E1984A, and D519V/E665V/E1984A variants showed 1.2 to 1.7-fold increases in these parameter values. These observations may suggest a greater capacity for thrombin generation per unit concentration factor VIII for these variants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield factor VIII reagents with improved stability parameters.

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The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII

Blood ◽

10.1182/blood.v91.2.538 ◽

1998 ◽

Vol 91 (2) ◽

pp. 538-548 ◽

Cited By ~ 31

Author(s):

Kagehiro Amano ◽

Rita Sarkar ◽

Susan Pemberton ◽

Geoffrey Kemball-Cook ◽

Haig H. Kazazian ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Specific Activity ◽

Genetic Alterations ◽

Published Data ◽

Missense Mutations ◽

Wild Type ◽

Factor Ixa ◽

Fviii Activity ◽

A2 Domain

Abstract Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

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A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage

Blood ◽

10.1182/blood.v79.2.389.389 ◽

1992 ◽

Vol 79 (2) ◽

pp. 389-397 ◽

Cited By ~ 26

Author(s):

DD Pittman ◽

M Millenson ◽

K Marquette ◽

K Bauer ◽

RJ Kaufman

Keyword(s):

Factor Viii ◽

Light Chain ◽

Deletion Mutant ◽

Specific Activity ◽

Active Form ◽

Procoagulant Activity ◽

Thrombin Cleavage ◽

Factor Viiia ◽

Human Factor Viii ◽

A2 Domain

Abstract Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site- directed DNA-mediated mutagenesis. Thrombin-activated human recombinant- derived factor VIII was isolated in an active form by passage over Mono- S fast protein liquid chromatography. The peak fractions had a specific activity of 60,000 U/mg. The subunit composition in the peak fraction contained the 50-Kd A1 domain from the heavy chain, the 73-Kd light chain fragment, and trace amounts of the 43-Kd A2 domain. The requirement of domain A2 for functional activity was shown in several ways. First, the addition of an inhibitory monoclonal antibody that recognizes domain A2 destroyed factor VIIIa activity. Second, addition of a Mono-S FPLC fraction that contained the A2 domain polypeptide back to the peak activity fraction increased activity of the factor VIIIa by 22-fold. The maximum specific activity achieved was 180,000 U/mg. Finally, expression of an A2 domain deletion mutant did not yield procoagulant activity, although the mutant was effectively secreted from the cell, exhibited appropriate heavy and light chain association, and was susceptible to thrombin cleavage. Cotransfection of this A2 domain deletion mutant with an A2 domain expression vector yielded a secreted complex and restored procoagulant activity in the conditioned medium. This result shows that the A2 domain can fold and assemble with A2-deleted factor VIII to yield a functional molecule. We conclude that the A2 domain is required for functional factor VIIIa activity and loss of activity in activated factor VIII may result from dissociation of A2 from the thrombin-activated heterotrimer.

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A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage

Blood ◽

10.1182/blood.v79.2.389.bloodjournal792389 ◽

1992 ◽

Vol 79 (2) ◽

pp. 389-397 ◽

Cited By ~ 1

Author(s):

DD Pittman ◽

M Millenson ◽

K Marquette ◽

K Bauer ◽

RJ Kaufman

Keyword(s):

Factor Viii ◽

Light Chain ◽

Deletion Mutant ◽

Specific Activity ◽

Active Form ◽

Procoagulant Activity ◽

Thrombin Cleavage ◽

Factor Viiia ◽

Human Factor Viii ◽

A2 Domain

Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site- directed DNA-mediated mutagenesis. Thrombin-activated human recombinant- derived factor VIII was isolated in an active form by passage over Mono- S fast protein liquid chromatography. The peak fractions had a specific activity of 60,000 U/mg. The subunit composition in the peak fraction contained the 50-Kd A1 domain from the heavy chain, the 73-Kd light chain fragment, and trace amounts of the 43-Kd A2 domain. The requirement of domain A2 for functional activity was shown in several ways. First, the addition of an inhibitory monoclonal antibody that recognizes domain A2 destroyed factor VIIIa activity. Second, addition of a Mono-S FPLC fraction that contained the A2 domain polypeptide back to the peak activity fraction increased activity of the factor VIIIa by 22-fold. The maximum specific activity achieved was 180,000 U/mg. Finally, expression of an A2 domain deletion mutant did not yield procoagulant activity, although the mutant was effectively secreted from the cell, exhibited appropriate heavy and light chain association, and was susceptible to thrombin cleavage. Cotransfection of this A2 domain deletion mutant with an A2 domain expression vector yielded a secreted complex and restored procoagulant activity in the conditioned medium. This result shows that the A2 domain can fold and assemble with A2-deleted factor VIII to yield a functional molecule. We conclude that the A2 domain is required for functional factor VIIIa activity and loss of activity in activated factor VIII may result from dissociation of A2 from the thrombin-activated heterotrimer.

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The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII

Blood ◽

10.1182/blood.v91.2.538.538_538_548 ◽

1998 ◽

Vol 91 (2) ◽

pp. 538-548 ◽

Cited By ~ 2

Author(s):

Kagehiro Amano ◽

Rita Sarkar ◽

Susan Pemberton ◽

Geoffrey Kemball-Cook ◽

Haig H. Kazazian ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Specific Activity ◽

Genetic Alterations ◽

Published Data ◽

Missense Mutations ◽

Wild Type ◽

Factor Ixa ◽

Fviii Activity ◽

A2 Domain

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

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Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.

Blood ◽

10.1182/blood.v104.11.1725.1725 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1725-1725 ◽

Cited By ~ 1

Author(s):

Hironao Wakabayashi ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Specific Activity ◽

High Specific Activity ◽

Factor Xa ◽

Saturation Mutagenesis ◽

Single Amino Acid ◽

Single Point Mutation ◽

Wild Type ◽

Activity Increase ◽

Wide Range

Abstract We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

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Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Blood ◽

10.1182/blood.v104.11.1711.1711 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1711-1711 ◽

Cited By ~ 1

Author(s):

Keiji Nogami ◽

Qian Zhou ◽

Hironao Wakabayashi ◽

Timothy Myles ◽

Lawrence L. Leung ◽

...

Keyword(s):

Factor Viii ◽

Heavy Chain ◽

Active Site ◽

Specific Activity ◽

Solid Phase ◽

Factor Xa ◽

Site Directed Mutagenesis ◽

Marginal Effect ◽

Thrombin Cleavage ◽

A2 Domain

Abstract Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

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