Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/ Two-stage Activity Discrepancy

Summary

Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.

Keywords

Factor VIII - one-stage - two-stage - discrepancy - mutations

• References

• 1 van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256 (07) 3433-42.
• 2 Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD. et al. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312 5992 342-7.
• 3 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM. et al. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84 (14) 4846-50.
• 4 Koschinsky ML, Funk WD, van Oost BA, MacGillivray RT. Complete cDNA sequence of human preceruloplasmin. Proc Natl Acad Sci USA 1986; 83 (14) 5086-90.
• 5 Weiss HJ, Sussman II, Hoyer LW. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest 1977; 60 (02) 390-404.
• 6 Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25 (02) 505-12.
• 7 Fay PJ, Beattie TL, Regan LM, O'Brien LM, Kaufman RJ. Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis. J Biol Chem 1996; 271 (11) 6027-32.
• 8 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 (19) 13246-50.
• 9 Fay PJ, Haidaris PJ, Huggins CF. Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa. J Biol Chem 1993; 268 (24) 17861-6.
• 10 Hathaway WE, Christian MJ, Jacobson LJ. Variant mild haemophilia. Discrepancy in one stage and two stage factor VIII assays. Thrombosis and Haemostasis 1983; 50: 1123a.
• 11 Over J. Methodology of the one-stage assay of Factor VIII (VIII: C). Scand J Haematol Suppl 1984; 41: 13-24.
• 12 Barrowcliffe TW. Comparisons of one-stage and two-stage assays of Factor VIII:C. Scand J Haematol Suppl 1984; 41: 39-54.
• 13 Rosen S. Assay of factor VIII:C with a chromogenic substrate. Scand J Haematol Suppl 1984; 40: 139-45.
• 14 Duncan EM, Duncan BM, Tunbridge LJ, Lloyd JV. 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 (04) 846-8.
• 15 Rudzki Z, Duncan EM, Casey GJ, Neumann M, Favaloro EJ, Lloyd JV. 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 (02) 400-6.
• 16 Kemball-Cook G, Tuddenham EGD, Wacey AI. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res 1998; 26 (01) 216-9.
• 17 Keeling DM, Sukhu K, Kemball-Cook G, Waseem N, Bagnall R, Lloyd JV. Diagnostic importance of the two-stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954→Leu substitution in the factor VIII A3 domain. Br J Haematol 1999; 105 (04) 1123-6.
• 18 Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ. Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. Blood 1999; 93 (01) 176-83.
• 19 Pipe SW, Saenko EL, Eickhorst AN, Kemball-Cook G, Kaufman RJ. 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. Blood 2001; 97 (03) 685-91.
• 20 Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EG, Kemball-Cook G. A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. Blood 1997; 89 (07) 2413-21.
• 21 Kaufman RJ. Vectors used for expression in mammalian cells. Methods Enzymol 1990; 185: 487-511.
• 22 Pittman DD, Marquette KA, Kaufman RJ. Role of the B domain for factor VIII and factor V expression and function. Blood 1994; 84 (12) 4214-25.
• 23 Pittman DD, Kaufman RJ. Site-directed mutagenesis and expression of coagulation factors VIII and V in mammalian cells. Methods Enzymol 1993; 222: 236-60.
• 24 Michnick DA, Pittman DD, Wise RJ, Kaufman RJ. Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage. J Biol Chem 1994; 269 (31) 20095-102.
• 25 Johnsson B, Lofas S, Lindquist G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem 1991; 198 (02) 268-77.
• 26 O’Shannessy DJ, Brigham-Burke M, Soneson KK, Hensley P, Brooks I. Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods. Anal Biochem 1993; 212 (02) 457-68.
• 27 Schwaab R, Oldenburg J, Kemball-Cook G, Albert T, Juhler C, Hanfland P. et al. Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family. Br J Haematol 2000; 109 (03) 523-8.
• 28 Lollar P, Parker ET, Fay PJ. Coagulant properties of hybrid human/porcine factor VIII molecules. J Biol Chem 1992; 267 (33) 23652-7.
• 29 Youssoufian H, Kazazian Jr. HH, Phillips DG, Aronis S, Tsiftis G, Brown VA. et al. Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature 1986; 324 6095 380-2.
• 30 Pipe SW, Kaufman RJ. Factor VIII C2 domain missense mutations exhibit defective trafficking of biologically functional proteins. J Biol Chem 1996; 271 (41) 25671-6.
• 31 Roelse JC, De Laaf RT, Timmermans SM, Peters M, Van Mourik JA, Voorberg J. Intracellular accumulation of factor VIII induced by missense mutations Arg593→Cys and Asn618→Ser explains cross-reacting materialreduced haemophilia A. Br J Haematol 2000; 108 (02) 241-6.
• 32 Voorberg J, de Laaf RT, Koster PM, van Mourik JA. Intracellular retention of a factor VIII protein with an Arg2307→Gln mutation as a cause of haemophilia A. Biochem J 1996; 318 (03) 931-7.
• 33 Arai M, Inaba H, Higuchi M, Antonarakis SE, Kazazian Jr. HH, Fujimaki M. et al. Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372–histidine). Proc Natl Acad Sci USA 1989; 86 (11) 4277-81.
• 34 O’Brien DP, Pattinson JK, Tuddenham EG. Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A. Blood 1990; 75 (08) 1664-72.
• 35 Johnson DJ, Pemberton S, Acquila M, Mori PG, Tuddenham EG, O’Brien DP. Factor VIII S373L: mutation at P1' site confers thrombin cleavage resistance, causing mild haemophilia A. Thromb Haemost 1994; 71 (04) 428-33.
• 36 Aly AM, Higuchi M, Kasper CK, Kazazian Jr. HH, Antonarakis SE, Hoyer LW. Hemophilia A due to mutations that create new N-glycosylation sites. Proc Natl Acad Sci USA 1992; 89 (11) 4933-7.
• 37 Aly AM, Hoyer LW. Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand factor. J Clin Invest 1992; 89 (05) 1382-7.
• 38 Amano K, Sarkar R, Pemberton S, Kemball-Cook G, Kazazian Jr. HH, Kaufman RJ. The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII. Blood 1998; 91 (02) 538-48.
• 39 Roelse JC, de Laaf RTM, Mertens K, van Mourik JA, Oldenburg J, Voorberg J. Altered sensitivity towards factor IXa explains assay discrepancies in plasma of haemophilia A patients with a GLU720 LYS substitution in factor VIII. Thromb Haemost 1999; 82: 5a.
• 40 Goodeve AC, Hinks JL, Nesbitt IM, Sampson B, Burgess C, Khair K. et al. Unusual discrepant factor VIII:C assays in haemophilia a patients with TYR346CYS and GLU321LYS FVIII gene mutations. In: Proceedings of the XVIIIth ISTH Congress. 2001; Thromb Haemost; 2001 P1370.
• 41 Mumford AD, Laffan M, O’Donnell J, McVey JH, Johnson DJD, Manning RA. et al. A Tyr346→Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII:C assay discrepancy. Br J Haematol 2002; 118 (02) 589-94.
• 42 Nesbitt IM, Hinks JL, Sampson B, Kitchen S, Peake IR, Makris M. et al. Discrepant one and two stage FVIII assays are very common in mild haemophilia A: correlation with FVIII gene mutations. In: Proceedings of the XVIIIth ISTH Congress. 2001; July: Thromb Haemost 2001 OC195
• 43 Celie PH, Van Stempvoort G, Jorieux S, Mazurier C, Van Mourik JA, Mertens K. Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function. Br J Haematol 1999; 106 (03) 792-800.
• 44 Lollar P, Parker ET. Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J Biol Chem 1991; 266 (19) 12481-6.
• 45 Pipe SW, Kaufman RJ. Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc Natl Acad Sci USA 1997; 94 (22) 11851-6.
• 46 Gale AJ, Pellequer J-L, Getzoff ED, Griffin JH. Mechanism of factor Va inactivation by activated protein C clarified using engineered disulfide bond between A2 and A3 domains in Va. In: Proceedings of the XVIIIth ISTH Congress. 2001; Thromb Haemost 2001 OC869.