Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

Pipe, Steven W.; Saenko, Evgueni L.; Eickhorst, Angela N.; Kemball‐Cook, Geoffrey; Kaufman, Randal J.

doi:10.1182/blood.v97.3.685

Cited by 91 publications

(112 citation statements)

References 35 publications

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“…Several FVIII point mutations have been shown to facilitate the rate of dissociation of A2 relative to WT, and these residues localize to either the A1-A2 domain interface (23,24) or the A2-A3 domain interface (25). Previously, we reported that among several charged residues located at the interface between A1 and A2 domains or between A2 and A3 domains Asp-519, Glu-665, and Glu-1984 were found to be detrimental to FVIII/FVIIIa stability (20,26).…”

Section: Discussionmentioning

confidence: 99%

Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Wakabayashi

Griffiths

Fay

2011

Journal of Biological Chemistry

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Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ϳ3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57°C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23°C following activation by thrombin. Both R121C/ L2302C and A108I variants showed up to ϳ4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ϳ4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability. Factor F (FVIII)2 is a plasma protein that is decreased or defective in individuals with hemophilia A. FVIII is expressed as both single chain and heterodimer forms in heterologous cells. The latter consists of a heavy chain composed of A1(a1)A2(a2)B domains and a light chain composed of (a3)A3C1C2 domains, with the lowercase "a" representing short segments rich in acidic residues (see Ref. 1 for review). FVIII is activated following limited proteolysis catalyzed by thrombin or FXa at Arg-372 (a1A2 junction), Arg-740 (a2B junction), and Arg-1689 (a3A3 junction). The resulting product, FVIIIa, is a heterotrimer composed of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for FIXa in the membrane-dependent activation of FX to FXa (see Ref 1 for review).Earlier structural studies identified the C2 domain as making a primary contribution to anionic phospholipid membrane binding through a combination of electrostatic and hydrophobic interactions (2). More recently, the intermediate resolution x-ray structures of FVIII (3, 4) showed that the C1 and C2 domains are aligned such that both domains may interact with the phospholipid membrane surface. In addition, these structures show close contact between A1 (heavy chain) and C2 (light chain) domains. We recently reported that an FVIII variant lacking the C2 domain retained the capacity to bind phospholipid membranes, ...

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Section: Discussionmentioning

confidence: 99%

Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Wakabayashi

Griffiths

Fay

2011

Journal of Biological Chemistry

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“…In vitro experiments have estabilished that vWF is required for stable association of F8C heavy and light chains, efficient F8C production and protection from enzymatic degradation, for example by activated protein C and FXa. vWF also prevents F8C binding onto phospholipids (Pipe et al, 2001). Two F8C regions are involved in F8C binding to vWF: the aminoterminal region of the A3 domain and the C2 domain.…”

Section: Resultsmentioning

confidence: 99%

Identification of seven novel mutations of F8C by DHPLC

et al. 2002

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Hemophilia A is an X-linked recessive disorder resulting from deficiency of Factor VIII (F8C), an important protein in blood coagulation. A large number of disease producing mutations have been reported in the F8C gene. However, a comprehensive analysis of mutations is difficult to conduct due to the large gene size, its many scattered exons, and the high frequency of de novo mutations. In this study, we performed analysis using PCR, Conformation Sensitive Gel Electrophoresis (CSGE), Denaturing High Performance Liquid Chromatography (DHPLC) and direct sequencing. We found seven novel mutations causing severe, moderate and mild Hemophilia

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“…12,13 Limited information is available regarding the association of the A2 subunit in factor VIIIa, and residues in both the A1 and A3 domains appear to make contributions to the retention of this subunit. Several factor VIII point mutations have been shown to facilitate the dissociation of A2 relative to wild type (WT), and these residues localize to either the A1-A2 domain interface 14,15 or the A2-A3 domain interface. 16 These factor VIII variants demonstrate a characteristic 1-stage/2-stage assay discrepancy, 17,18 with significant reductions in activity values determined by the latter assay as a result of increased rates of A2 subunit dissociation.…”

Section: Introductionmentioning

confidence: 99%

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

Wakabayashi

Varfaj

DeAngelis

et al. 2008

Blood

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Factor 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. (Blood. 2008;112:2761-2769) IntroductionFactor VIII, a plasma protein that is decreased or defective in patients with hemophilia A, circulates as a noncovalent, metal ion-dependent heterodimer. This procofactor form of the protein consists of a heavy chain (HC) composed of A1(a1)A2(a2)B domains and a light chain (LC) composed of (a3)A3C1C2 domains, with the lower case "a" representing short (ϳ 30-40 residue) segments rich in acidic residues (for review, see Fay 1 ). Factor VIII is activated by proteolytic cleavages at the a1A2, a2B, and a3A3 junctions catalyzed by thrombin or factor Xa. The product of this reaction, factor VIIIa, is a heterotrimer composed of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (for review, see Fay 1 ).Reconstitution studies have shown that the factor VIII heterodimeric structure is supported by both electrostatic and hydrophobic interactions, 2,3 and the interchain affinity is further strengthened by factor VIII binding von Willebrand factor. 2,4 Metal ions also contribute to the interchain affinity and activity parameters. 5 Calcium is required to yield the active factor VIII conformation. Mutagenesis studies mapped a calcium-binding site to a segment rich in acidic residues within the A1 domain (residues 110-126) and identified specific residues within this region prominent in the coordination of the ion. 6 A recent intermediate resolution X-ray structure 7 confirmed this calcium-binding site as well as suggested a second potential site within the A2 domain. This structure also showed occupancy of the 2 type 1 copper ion sites within...

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Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

Cited by 91 publications

References 35 publications

Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Identification of seven novel mutations of F8C by DHPLC

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

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