Thrombosis and Haemostasis, Inhaltsverzeichnis Thromb Haemost 2003; 89(05): 788-794DOI: 10.1055/s-0037-1613463 Blood Coagulation, Fibrinolysis, and Cellular Haemostasis Schattauer GmbH The preparation and phospholipid binding property of the C2 domain of human factor VIII Authors Institutsangaben Kazuya Takeshima 1 Departments of Biochemistry, Seattle Washington, USA 3 Present address; Research Division, Mitsubishi Pharma Co., Hirakata Osaka, Japan Christina Smith 2 Laboratory Medicine University of Washington, Seattle Washington, USA Jonathan Tait 2 Laboratory Medicine University of Washington, Seattle Washington, USA Kazuo Fujikawa 1 Departments of Biochemistry, Seattle Washington, USA Artikel empfehlen Abstract als PDF herunterladen(opens in new window) Artikel einzeln kaufen(opens in new window) Summary The C2 domain of human factor VIII was expressed in a yeast secretion system and its binding properties were studied. A cDNA coding the C2 domain sequence of human factor VIII with a N-terminal six amino acids extension (C-C2) was constructed, transformed into Pichia pastoris cells and expressed. The product was purified by ammonium sulfate fractionation and anion exchange chromatography. It emerged as a single peak from both ion exchange and gel filtration columns, indicating C-C2 is a homogenous monomer. The binding activity of C-C2 to phosphatidylserine-containing phospholipid vesicles was measured by competitive binding with annexin V. The values of IC50were approximately 70nM for both factor VIII and its light chain, but were about 7000nM for C-C2. These results indicated C-C2 has 100-fold less binding affinity than factor VIII or the light chain. Direct binding to solidified phosphatidylserine-containing phospholipids also showed that C-C2 has ~50-fold less binding affinity than does the light chain. C-C2 poorly inhibited Xase activity. These results together clearly show that the C2 domain alone does not have full membrane binding activity, and suggest that the other light chain domains, A3 and/or C1, are also involved in the phospholipid binding activity of factor VIII. Keywords KeywordsFactor VIII - C2 domain - phospholipid binding - annexin V PDF (143 kb) Referenzen References 1 Kaufman RS, Antonarakis SE, Fay PJ.. Factor VIII and Hemophilia A in Hemostasis and Thrombosis, Basic Principles & Clinical Practice, 2001, 4th edition. Colman RW, Hirsh J, Marder VJ, Clowes AW, George JN.. eds Loppincott Williams; 135-56. 2 Tuddenham EGD... Factor VIII in Molecular Basis of Thrombosis and Hemostasis. 1995 High KA, Robert HR.. eds. Marcel Dekker 147. 3 Vehar GA, Keyt B, Eaton D.. et al. Structure of human factor VIII. Nature 1984; 312: 337-42. 4 Toole JJ, Knopf JL, Wozney JM.. et al. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312: 342-7. 5 Kane WH, Davie EW.. Blood coagulation factor V and VIII: structure and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 1988; 71: 539-55. 6 Eaton DL, Wood WI, Eaton D.. et al. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry 1986; 25: 8343-7. 7 Nordfang O, Ezban M.. Generation of active coagulation factor VIII from isolated subunits. J Biol Chem 1988; 263: 1115-8. 8 Gilbert GE.. et al. Platelet-derived microparticles express high affinity receptors for factor VIII. J Biol Chem 1991; 266: 17261-8. 9 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: 3433-42. 10 Kemball-Cook G, Edwards SJ, Sewerin K, Andersson LO, Barriwcliffe TW.. Factor VIII procoagulant protein interacts with phospholipid vesicles via its 80 kDa light chain. Thromb Haemost 1988; 60: 442-6. 11 Arai M, Scandella D, Hoyer LW.. Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid. J Clin Invest 1989; 83: 1978-84. 12 Krishnaswamy S, Mann KG.. The binding of factor Va to phospholipid vesicles. J Biol Chem 1988; 263: 5714-23. 13 Kalafatis M, Jenny RJ, Mann KG.. Identification and characterization of a phospholipid-binding site of bovine factor Va. J Biol Chem 1990; 265: 21580-9. 14 Ortel T, Devore-Carter D, Quinn-Allen M, Kane WH.. Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain. J Biol Chem 1992; 267: 4189-98. 15 Pratt KP, Shen BW, Takeshima K, Davie EW, Fujikawa K, Stoddard BL.. Structure of the C2 domain of human factet VIII at 1.5 A resolution. Nature 1999; 402: 439-42. 16 Macedo-Ribeiro S, Bode W, Huber R.. et al. Crystal structures of the membrane-binding C2 domain of human coagulation factor V. Nature 1999; 25: 434-9. 17 Tait JF, Gibson D, Fujikawa K.. Phospholipid binding properties of human placental antocoagulant protein-I, a member of the lipocortin family. J Biol Chem 1989; 264: 7944-9. 18 Sanger F, Nicklen S, Coulson A.. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci, USA 1977; 74: 5463-7. 19 Scorer CA, Clare JJ, McCombie WR.. et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology (NY) 1994; 12: 181-4. 20 Bloom JW.. The interaction of rDNA factor VIII, factor VIIIdes-797-1562 and factor VIIIdes-797-1562-derived peptides with phospholipid. Thromb Res 1987; 48: 439-48. 21 Gabriel NE, Roberts MF.. Spontaneous formation of stable unilamellar vesicles. Biochemistry 1984; 23: 4011-5. 22 Chen PS, Toribara TY, Warner H.. Micro-determination of phosphorus. Aanal Chem 1956; 28: 1756-8. 23 Laemmli UK.. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5. 24 Okabayashi K.. et al. Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris. Gene 1996; 177: 69-76. 25 Saenko EL, Scandella D, Yakhyaev AV, Greco NJ.. Activation of factor VIII by thrombin increases its affinity for binding to synthetic phospholipid membranes and activated platelets. J Biol Chem 1998; 273: 27918-26. 26 Lenting PJ.. et al. Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII. J Biol Chem 1994; 269: 7150-5. 27 Srivastava A.. et al. Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va. Biochemistry 2001; 40: 8246-55. 28 Ortel TL, Quinn-Allen MA, Kane WH.. Purification and characterization of the second C-type domain of human factor V expressed in Baculovius. Blood 1994; 84: 387a (Abst No. 1553) 29 Takeshima K, Fujikawa K.. The phospholipid binding property of the C2 domain of human factor VIII. Thromb Haemost 1999. Supplement: 234 (Abstr N0. 737). 30 Spiegel Jr PC. et al. Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII. Blood 2001; 98: 13-9.
