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Thromb Haemost 1998; 80(03): 418-422
DOI: 10.1055/s-0037-1615223
Rapid Communications
Schattauer GmbH

Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking

Kirsty A. Lapan
1   From the Department of Biochemistry and Biophysics, New York, USA
,
Philip J. Fay
1   From the Department of Biochemistry and Biophysics, New York, USA
2   From the Department of Medicine University of Rochester School of Medicine and Dentistry Rochester, New York, USA
› Author Affiliations
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Publication History

Received 02 March 1998

Accepted after revision 20 May 1998

Publication Date:
08 December 2017 (online)

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Summary

We have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

 

  • References

  • 1 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
    PubMedGoogle Scholar
  • 2 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.
    PubMedGoogle Scholar
  • 3 Wood WI, Capon DJ, Simonsen CC, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EDG, Vehar GA, Lawn RM. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-7.
    CrossrefPubMedGoogle Scholar
  • 4 Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC, Amphlett GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312: 342-7.
    CrossrefPubMedGoogle Scholar
  • 5 Vehar GA, Keyt B, Eaton D, Rodriguez H, O’Brien DP, Rotblat F, Opper-mann H, Keck R, Wood WI, Harkins RN. et al. Structure of human factor VIII. Nature 1984; 312: 337-42.
    CrossrefPubMedGoogle Scholar
  • 6 Fass DN, Knutson GJ, Katzmann JA. Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein. Blood 1982; 59: 594-600.
    PubMedGoogle Scholar
  • 7 Andersson LO, Forsman N, Huang K, Larsen K, Lundin A, Pavlu B, Sand-berg H, Sewerin K, Smart J. Isolation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipi-tate, and plasma. Proc Natl Acad Sci ( USA) 1986; 83: 2979-83.
    CrossrefPubMedGoogle Scholar
  • 8 Fay PJ, Anderson MT, Chavin SI, Marder VJ. The size of human factor VIII heterodimers and the effects produced by thrombin. Biochim Biophs Acta 1986; 871: 268-78.
    PubMedGoogle Scholar
  • 9 Eaton DL, Vehar GA. Factor VIII structure and proteolytic processing. Prog Hem Throm 1986; 8: 47-70.
    PubMedGoogle Scholar
  • 10 Lollar P, Parker CG. Subunit structure of thrombin-activated porcine factor VIII. Biochemistry 1989; 28: 666-74.
    CrossrefPubMedGoogle Scholar
  • 11 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: 8957-62.
    PubMedGoogle Scholar
  • 12 Lollar P, Parker ET, Fay PJ. Coagulant properties of hybrid human/porcine factor VIII molecules. J Biol Chem 1992; 267: 23652-7.
    PubMedGoogle Scholar
  • 13 Lollar P, Parker CG. pH-dependent denaturation of thrombin-activated porcine factor VIII. J Biol Chem 1990; 265: 1688-92.
    PubMedGoogle Scholar
  • 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: 13246-50.
    PubMedGoogle Scholar
  • 15 Di Scipio RG, Hermodson MA, Davie EW. Activation of human factor X (Stuart Factor) by a protease from Russell‘s viper venom. Biochemistry 1977; 6: 5253-60.
    PubMedGoogle Scholar
  • 16 Fujikawa K, Legaz ME, Davie EW. Bovine factor X 1 (Stuart factor). Mechanism of activation by protein from Russell‘s viper venom. Biochemistry 1972; 11: 4892-9.
    CrossrefPubMedGoogle Scholar
  • 17 Hertzberg M. Biochemistry of factor X. Blood Rev 1994; 8: 56-62.
    CrossrefPubMedGoogle Scholar
  • 18 Lapan KA, Fay PJ. Localization of a factor X interactive site in the A1 subunit of factor VIIIa. J Biol Chem 1997; 272: 2082-8.
    CrossrefPubMedGoogle Scholar
  • 19 Carraway KL, Koshland DE. Carbodiimide modification of proteins. Meth Enzym 1972; 25: 616-21.
    PubMedGoogle Scholar
  • 20 O’Brien LM, Medved LV, Fay PJ. Localization of factor IXa and factor VIIIa interactive sites. J Biol Chem 1995; 270: 27087-92.
    CrossrefPubMedGoogle Scholar
  • 21 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: 17861-6.
    PubMedGoogle Scholar
  • 22 Foster PA, Fulcher CA, Houghten RA, de Graaf Mahoney S, Zimmerman TS. Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine 351-serine365 of the factor VIII heavy chain. J Clin Invest 1988; 82: 123-8.
    CrossrefPubMedGoogle Scholar
  • 23 Regan LM, O’Brien LM, Beattie TL, Sudhakar K, Walker FJ, Fay PJ. Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase. J Biol Chem 1996; 271: 3982-7.
    CrossrefPubMedGoogle Scholar
  • 24 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedGoogle Scholar
  • 25 Nesheim M, Pittman DD, Giles AR, Fass DN, Wang JH, Slonosky D, Kaufman RJ. The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets. J Biol Chem 1991; 266: 17815-20.
    PubMedGoogle Scholar
  • 26 Iino M, Takeya H, Nishioka J, Nakagaki T, Tamura K, Suzuki K. The role of human factor X activation peptide in activation of factor X by factor IXa. J Biochem 1994; 116: 335-40.
    CrossrefPubMedGoogle Scholar
  • 27 Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Huber R, Blankenship DT, Cardin AD, Kisiel W. Structure of human des(1-45) factor Xa at 2.2 A resolution. J Mol Biol 1993; 232: 947-66.
    CrossrefPubMedGoogle Scholar
  • 28 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: 505-12.
    CrossrefPubMedGoogle Scholar
  • 29 Fay PJ, Smudzin TM, Walker FJ. Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity. J Biol Chem 1991; 266: 20139-45.
    PubMedGoogle Scholar
  • 30 O’Brien DP, Johnson D, Byfield P, Tuddenham EG. Inactivation of factor VIII by factor IXa. Biochemistry 1992; 31: 2805-12.
    CrossrefPubMedGoogle Scholar
  • 31 Lamphear BJ, Fay PJ. Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa. Blood 1992; 80: 3120-6.
    PubMedGoogle Scholar
  • 32 Neuenschwander PF, Jesty J. Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate. Arch Biochem Biophys 1992; 296: 426-34.
    CrossrefPubMedGoogle Scholar
  • 33 Brandstetter H, Bauer M, Huber R, Lollar P, Bode W. X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B. Proc Natl Acad Sci (USA) 1995; 92: 9796-800.
    CrossrefPubMedGoogle Scholar
  • 34 Husten EJ, Esmon CT, Johnson AE. The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex. J Biol Chem 1987; 262: 12953-61.
    PubMedGoogle Scholar
  • 35 Mutucumarana VP, Duffy EJ, Lollar P, Johnson AE. The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study. J Biol Chem 1992; 267: 17012-21.
    PubMedGoogle Scholar
  • 36 Yegneswaran S, Wood G, Esmon C, Johnson AE. Protein S alters the active site location of activated protein C above the membrane surface. J Biol Chem 1997; 272: 25103-21.
    PubMedGoogle Scholar
  • 37 Guinto ER, Esmon CT. Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C. J Biol Chem 1984; 259: 13986-92.
    PubMedGoogle Scholar
  • 38 Luckow EA, Lyons DA, Ridgeway TM, Esmon CT, Laue TM. Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation. Biochemistry 1989; 28: 2348-54.
    CrossrefPubMedGoogle Scholar
  • 39 Esmon CT, Jackson CM. The conversion of prothrombin to thrombin. The function of the fragment 2 region during activation in the presence of factor V. J Biol Chem 1974; 249: 7791-7.
    PubMedGoogle Scholar
  • 40 Kotkow KJ, Deithcher SR, Furie B, Furie BC. The second kringle domain of prothrombin promotes FVa-mediated prothrombin activation by prothrombinase. J Biol Chem 1995; 270: 4551-7.
    CrossrefPubMedGoogle Scholar