RSS-Feed abonnieren
DOI: 10.1160/TH14-01-0086
Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit
Financial support: This work was supported by NIH Grant HL38199 (to P. J. F.) and NIH predoctoral trainee-ship GM068411 from the Institutional Ruth L. Kirschstein National Research Award (to M. M.).Publikationsverlauf
Received:
27. Januar 2014
Accepted after major revision:
10. April 2014
Publikationsdatum:
20. November 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/201403/lookinside/thumbnails/10-1160-th14-01-0086-1.jpg)
Summary
Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.
† Dr. Philip Fay has recently passed away.
-
References
- 1 Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18: 1-15.
- 2 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J Biol Chem 1991; 266: 8957-8962.
- 3 Lollar P, Parker CG. pH-dependent denaturation of thrombin-activated porcine factor VIII. J Biol Chem 1990; 265: 1688-1692.
- 4 Pipe SW, Eickhorst AN, McKinley SH. et al. Mild haemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. Blood 1999; 93: 176-183.
- 5 Pipe SW, Saenko EL, Eickhorst AN. et al. Haemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa. Blood 2001; 97: 685-691.
- 6 Hakeos WH, Miao H, Sirachainan N. et al. Haemophilia A mutations within the factor VIII A2-A3 subunit interface destabilize factor VIIIa and cause onestage/two-stage activity discrepancy. Thromb Haemost 2002; 88: 781-787.
- 7 Duncan EM, Duncan BM, Tunbridge LJ. et al. Familial discrepancy between the one-stage and two-stage factor VIII methods in a subgroup of patients with haemophilia A. Br J Haematol 1994; 87: 846-848.
- 8 Rudzki Z, Duncan EM, Casey GJ. et al. Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII:C methods. Br J Haematol 1996; 94: 400-406.
- 9 Pemberton S, Lindley P, Zaitsev V. et al. A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. Blood 1997; 89: 2413-2421.
- 10 Wakabayashi H, Varfaj F, Deangelis J. et al. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface. Blood 2008; 112: 2761-2769.
- 11 Wakabayashi H, Griffiths AE, Fay PJ. Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations. J Thromb Haemost 2009; 07: 438-444.
- 12 Goodeve AC, Reitsma PH, McVey JH. Working Group on Nomenclature of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Nomenclature of genetic variants in haemostasis. J Thromb Haemost 2011; 09: 852-855.
- 13 Mimms LT, Zampighi G, Nozaki Y. et al. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry 1981; 20: 833-840.
- 14 Fay PJ, Smudzin TM. Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa. J Biol Chem 1992; 267: 13246-13250.
- 15 Doering C, Parker ET, Healey JF. et al. Expression and characterization of recombinant murine factor VIII. Thromb Haemost 2002; 88: 450-458.
- 16 Wakabayashi H, Freas J, Zhou Q. et al. Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity. J Biol Chem 2004; 279: 12677-12684.
- 17 Fay PJ, Mastri M, Koszelak ME. et al. Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA. J Biol Chem 2001; 276: 12434-12439.
- 18 Wakabayashi H, Griffiths AE, Fay PJ. Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability. J Biol Chem 2011; 286: 25748-25755.
- 19 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.
- 20 Jagannathan I, Ichikawa HT, Kruger T. et al. Identification of residues in the 558-loop of factor VIIIa A2 subunit that interact with factor IXa. J Biol Chem 2009; 284: 32248-32255.
- 21 Ansong C, Fay PJ. Factor VIII A3 domain residues 1954-1961 represent an A1 domain-interactive site. Biochemistry 2005; 44: 8850-8857.
- 22 Pipe SW, Kaufman RJ. Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc Natl Acad Sci USA 1997; 94: 11851-11856.
- 23 Greene TK, Wang C, Hirsch JD. et al. In vivo efficacy of platelet-delivered, high specific activity factor VIII variants. Blood 2010; 116: 6114-6122.
- 24 Gale AJ, Pellequer JL. An engineered interdomain disulfide bond stabilizes human blood coagulation factor VIIIa. J Thromb Haemost 2003; 01: 1966-1971.
- 25 Radtke KP, Griffin JH, Riceberg J. et al. Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays. J Thromb Haemost 2007; 05: 102-108.
- 26 Leong L, Thompson T, Tran K. et al. Reduction of A2 Subunit Dissociation Increases Factor VIII Potency and Efficacy in HemA Mice. J Thromb Haemost 2013; 11 s2 145.