scholarly journals Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

2008 ◽  
Vol 283 (17) ◽  
pp. 11645-11651 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Structural Stability ◽  
Factor Viiia ◽  
A2 Domain

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

Blood ◽  
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.

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

1998 ◽  
Vol 79 (05) ◽  
pp. 943-948 ◽  
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.

Identification of a Protein S-Interactive Site on the Factor VIII A2 Domain.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2684-2684
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Tetsuhiro Soeda ◽  
Akira Yoshioka ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Activated Protein C ◽  
Protein S ◽  
Cross Linking ◽  
Factor V ◽  
Dependent Manner ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Mutant Forms ◽  
A2 Domain

Abstract Protein S functions as a cofactor of activated protein C that inactivates factor VIII(a) and factor V(a). We recently have reported a new regulatory mechanism that protein S interacted with both the A2 and A3 domains in factor VIII, and consequently this cofactor directly impaired the factor Xase complex by competing the interaction of factor IXa to factor VIIIa (Blood2006; 108, 487a). Since factor IXa blocked the binding of A2 subunit to protein S, we attempted several approaches to localize the protein S-interactive site(s) on the factor VIII A2 domain. An anti-A2 monoclonal antibody (mAb413) with the 484–509 epitope, recognizing a factor IXa-interactive site on the A2, inhibited the A2 binding to immobilized protein S up to approximately 90% in a dose-dependent manner in a surface plasmon resonance-based assay. Furthermore, ELISA-based assay showed that a synthetic peptide corresponding to residues 484–509 directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. The cross-linked product formed with EDC was consistent with 1:1 stoichiometry of reactants, suggesting specificity in the 484–509 peptide and protein S interaction. This cross-linking formation was blocked by the addition of the 484–497 peptide, whilst not by the 498–509 peptide, supporting the presence of protein S-interactive site within residues 484–497. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S product showed that three sequential basic residues (S488, R489 and R490) could not be detected, supporting that three residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for protein S-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in baculovirus system and purified. Compared with wild type A2 (Kd: ∼9 nM), each binding affinity of S488A, R489A, or R490A A2 mutant for protein S was decreased by 4∼5-fold (32, 40 and 40 nM, respectively). These results indicate that the 484–509 region in the factor VIII A2 domain, and in particular a cluster of basic amino acids at residues 488–490, contributes to a unique protein S-interactive site.

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

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2761-2769 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Fatbardha Varfaj ◽  
Jennifer DeAngelis ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Fold Increase ◽  
Covalent Modification ◽  
Stability Factor ◽  
Wild Type ◽  
Factor Viiia ◽  
Charged Residues ◽  
A2 Domain ◽  
Enhanced Stability

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.

scholarly journals Cleavage of Factor VIII Heavy Chain Is Required for the Functional Interaction of A2 Subunit with Factor IXa

2001 ◽  
Vol 276 (15) ◽  
pp. 12434-12439 ◽  
Author(s):  
Philip J. Fay ◽  
Maria Mastri ◽  
Mary E. Koszelak ◽  
Hironao Wakabayashi
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Fluorescence Anisotropy ◽  
Factor Xa ◽  
Factor X ◽  
Functional Interaction ◽  
Factor Ixa ◽  
Factor Viiia ◽  
A2 Domain ◽  
Stimulation Of

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

Factor VIII A2 Domain Residues 497–510 and 584–593 Comprise the R8B12 Epitope.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4028-4028
Author(s):  
Charles Ansong ◽  
Stephen Miles ◽  
Philip J. Fay
Keyword(s):  
Mass Spectrometry ◽  
Factor Viii ◽  
Direct Sequencing ◽  
Homology Model ◽  
Database Search ◽  
Peptide Fragments ◽  
Mass Peak ◽  
Factor Viiia ◽  
The Masses ◽  
A2 Domain

Abstract The murine monoclonal antibody R8B12 recognizes the C-terminal region (residues 563–740) of the A2 subunit of factor VIIIa (Fay et al. J. Biol. Chem. 266:20139, 1991), as judged by western blotting. The location of the R8B12 epitope within the A2 subunit is not known. In the present study we used affinity-directed mass spectrometry (Zhao et al. Proc. Natl. Acad. Sci. USA.93:4020, 1996) to map the R8B12 epitope. Purified A2 subunit was subjected to proteolytic digestion with trypsin. The digest was then subjected to immunoprecipitation (IP) using R8B12 IgG, in which peptide(s) are selected that bind the antibody. Subsequently the masses of the affinity-selected peptides were determined directly from the immune complex by MALDI-TOF mass spectrometry. Proteolysis of A2 with trypsin generated a pre-IP peptide fingerprint that covered ~40% of the A2 domain sequence (Figure, left). Analysis of the post-IP peptide fingerprint showed two masses, 1309 and 1654, as representing affinity-selected peptide fragments (Figure, right). A theoretical database search identified the 1309 mass peak as A2 domain residues 584–593 and the 1654 mass peak as A2 domain residues 497–510. Direct sequencing of both mass peaks confirmed the results of the theoretical database search. These residues mapped to regions on the A2 domain of the factor VIII A domain homology model that are surface exposed and proximal to each other. Taken together, the above results suggest that A2 domain residues 497–510 and 584–593 represent a discontinous epitope for R8B12. Furthermore, based upon blotting specificity of R8B12 to the C-terminal portion of A2, we speculate that the latter sequence that forms this epitope makes a substantially greater contribution to the binding energy. Figure Figure

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

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 389-397 ◽  
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.

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

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 389-397 ◽  
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.

Localization of Factor VIII Residues Contributing to Factor VIII and/or VIIIa Structural Stability as Detected by Rates of Activity Decay.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1763-1763
Author(s):  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Hydrogen Bonding ◽  
Factor Viii ◽  
Decay Rate ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Xa ◽  
Decay Rates ◽  
Factor Viiia ◽  
Activity Decay ◽  
A2 Domain

Abstract Factor VIII circulates as a heterodimer composed of a heavy chain and light chain. Thrombin converts factor VIII into the active cofactor, factor VIIIa, by cleaving heavy chain into A1 and A2 subunits. While the A1 subunit maintains a stable interaction with the light chain-derived A3C1C2 subunit, the A2 subunit is weakly associated in the trimer and this low affinity interaction accounts for the instability of factor VIIIa activity. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bond pairings based upon spatial separations of <2.8Å were found between side chains of the A2 domain residues and residues in the A1 or A3 domain represented by residues D27, R282, E287, D302, S313, H317, Y476, T522, R531, N538, E540, S650, Y664, N684, N694, S695, D696, Y1786, S1791, Y1792, D1795, E1829, and S1949. Since hydrogen bonds at an interactive site contribute to structural stability, we performed a scanning mutagenesis study where these residues were individually replaced with Ala, except Tyr residues were replaced with Phe, in order to examine the contribution of each site to the stability of the factor VIII/VIIIa forms. Factor VIII activity decay was followed by incubating factor VIII (5 nM) at 55°C, and at indicated times removing an aliquot, activating with thrombin and measuring residual activity by a factor Xa generation assay. Factor VIIIa activity decay was measured by mixing factor VIII (5 nM) with factor IXa (40 nM), activating factor VIII with thrombin, and following factor Xase activity at 23°C by factor Xa generation. Non-linear least squares regression using a single exponential decay equation of activity versus time was performed to obtain rates for factor VIII/VIIIa activity decay. Eleven out of 23 factor VIII mutants showed increases in either or both decay rates by >2-fold compared to wild type (WT) (Figure). Of these mutants, R282A showed the largest increase in both factor VIII and VIIIa decay rates (∼30-fold compared to WT). Interestingly, 5 mutants (T522A, D1795A, Y1792F, Y1786F, and E1892A) showed >2-fold increased rates in factor VIIIa decay compared with the rates for factor VIII decay, whereas 2 mutants (N694A and Y664F) showed >2-fold increased rates in factor VIII decay compared with rates for factor VIIIa decay. These results suggest that several residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein through hydrogen bonding and that mutation at these sites result in loss of stability as determined by enhanced rates of activity decay. Furthermore, these results permit discrimination between stabilizing hydrogen bonding in the procofactor from active cofactor, where bonding in the latter appears to make a more significant contribution to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions for the A2 domain following factor VIII activation. Factor VIII/FVIIIa Decay Rate Factor VIII/FVIIIa Decay Rate

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

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