Download PDF
Thromb Haemost 1998; 79(03): 557-563
DOI: 10.1055/s-0037-1614944
Review Articles
Schattauer GmbH

Mutation at either Arg336 or Arg562 in Factor VIII Is Insufficient for Complete Resistance to Activated Protein C (APC)-mediated Inactivation: Implications for the APC Resistance Test

Kagehiro Amano
1   Howard Hughes Medical Institute
2   Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI, USA
,
Donna A. Michnick
3   The Genetics Institute Inc., Cambridge, MA, USA
,
Micheline Moussalli
1   Howard Hughes Medical Institute
,
Randal J. Kaufman
1   Howard Hughes Medical Institute
2   Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI, USA
› Author AffiliationsSupported by NIH grants R01 HL53777 and R01 HL52173 (RJK) and the Tokyo Medical College (KA)
Further Information

Publication History

Received 17 March 1997

Accepted 09 October 1997

Publication Date:
07 December 2017 (online)

    Permissions and Reprints

Summary

Activated protein C (APC)-mediated inactivation of factor VIII (FVIII) correlates with cleavage at either Arg336 and/or Arg562. To elucidate the APC cleavage requirements for inactivation of FVIII, APC cleavage site mutants in FVIII (R336I, R562K and R336I/R562K) were made by site-directed mutagenesis. Analysis of these FVIII mutants expressed in COS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was resistant to APC cleavage at residue 562 but not at Arg336 and the thrombin cleaved mutant R336I was mostly resistant to APC cleavage at residue 336, but was sensitive to APC cleavage at Arg562. The double mutant R336I/R562K was mostly resistant to cleavage at residue 336 and completely resistant to cleavage at residue 562. Thus, APC cleavage of FVIII does not require a specific order of cleavage at either residue. The functional inactivation by APC was studied using partially purified preparations of FVIII expressed in Chinese hamster ovary cells. Both single mutants were inactivated at similar rates but slower than wild-type FVIII, whereas the double mutant R336I/R562K was resistant to inactivation. The ability of a commercially available APC-resistance assay kit to detect APC resistant FVIII was tested by reconstituting FVIII deficient plasma with the APC resistant mutants. Only the R336I/R562K demonstrated a reduced APC-resistance ratio, indicating that this assay can not detect the single APC cleavage site mutant of FVIII. These results suggest that APC-mediated cleavage at either Arg336 or Arg562 partially inactivate FVIII.

 

  • References

  • 1 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-70.
    CrossrefPubMedGoogle Scholar
  • 2 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Annu Rev Biochem 1988; 57: 915-56.
    CrossrefPubMedGoogle Scholar
  • 3 Kane WH, Davie EW. Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin. Proc Natl Acad Sci USA 1986; 83: 6800-4.
    CrossrefPubMedGoogle Scholar
  • 4 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-50.
    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, Tuddenham EGD, Lawn RM, Capon DJ. Structure of human factor VIII. Nature 1984; 312: 337-42.
    CrossrefPubMedGoogle Scholar
  • 6 Toole JJ, Knoph 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 antihemophilic factor. Nature 1984; 312: 342-7.
    CrossrefPubMedGoogle Scholar
  • 7 Ortel TL, Takahashi N, Putnamm FW. Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains. Proc Natl Acad Sci USA 1984; 81: 4761-5.
    CrossrefPubMedGoogle Scholar
  • 8 Stubbs JD, Lekutis C, Singer KL, Bui A, Yuzuki D, Srinivasan U, Parry G. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like. Proc Natl Acad Sci USA 1990; 87: 8417-21.
    CrossrefPubMedGoogle Scholar
  • 9 Guinto ER, Esmon CT, Mann KG, MacGillivray RTA. The complete cDNA sequence of bovine coagulation factor V. J Biol Chem 1992; 267: 2971-8.
    PubMedGoogle Scholar
  • 10 Eaton D, Rodriguez H, Vehar GA. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry 1986; 25: 505-12.
    CrossrefPubMedGoogle Scholar
  • 11 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: 12481-6.
    PubMedGoogle Scholar
  • 12 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIII a subunit structure. J Biol Chem 1991; 266: 8957-62.
    PubMedGoogle Scholar
  • 13 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-9.
    PubMedGoogle Scholar
  • 14 Walker FJ, Chavin SI, Fay PJ. Inactivation of factor VIII by activated protein C and protein S. Arch Bioch Biophys 1987; 252: 322-8.
    CrossrefPubMedGoogle Scholar
  • 15 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
  • 16 Dahlbäck B, Carlsson M, Svenson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-8.
    CrossrefPubMedGoogle Scholar
  • 17 Dahlbäck B, Hilderbrand B. Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V. Proc Natl Acad Sci USA 1994; 91: 1396-400.
    CrossrefPubMedGoogle Scholar
  • 18 Sun X, Evatt B, Griffin JH. Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia. Blood 1994; 83: 3120-5.
    PubMedGoogle Scholar
  • 19 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Driven RJ, de Ronde H, van der Veiden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
    CrossrefPubMedGoogle Scholar
  • 20 Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular defect in factor VR506Q. J Biol Chem 1995; 270: 4053-7.
    CrossrefPubMedGoogle Scholar
  • 21 Nicolaes GAF, Tans G, Thomassen MCLGD, Hemker HC, Pabinger I, Varadi K, Schwarz HP, Rosing J. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 270: 21158-66.
    PubMedGoogle Scholar
  • 22 Pittman DD, Kaufman RJ. Site-directed mutagenesis and expression of coagulation factors VIII and V in mammalian cells. Methods in Enzymology. vol. 222 Academic Press, INC.; San Diego, CA: 1993: 236-60.
    Google Scholar
  • 23 Toole JJ, Pittman DD, Orr EC, Murtha P, Wasley LC, Kaufman RJ. A large region (~95kDA) of human factor VIII is dispensable for in vitro activity. Proc Natl Acad Sci USA 1986; 83: 5939-42.
    CrossrefPubMedGoogle Scholar
  • 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: 20095-102.
    PubMedGoogle Scholar
  • 25 Fay PJ, Beattle T, 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: 6027-32.
    CrossrefPubMedGoogle Scholar
  • 26 Zatloukal K, Cotten M, Berger M, Schmidt W, Wagener E, Birnstiel ML. In vivo production of human factor VIII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor-mediated, adenovirus-augmented gene delivery. Proc Natl Acad Sci USA 1994; 91: 5148-52.
    CrossrefPubMedGoogle Scholar
  • 27 Pittman DD, Kaufman RJ. Proteolytic requirements for activation and inactivation of antihemophilic factor (Factor VIII). Proc Natl Acad Sci USA 1988; 85: 2429-33.
    CrossrefPubMedGoogle Scholar
  • 28 Pittman DD, Wang JH, Kaufman RJ. Identification and functional importance of tyrosine-sulfate residues within recombinant factor VIII. Biochemistry 1992; 31: 3315-25.
    CrossrefPubMedGoogle Scholar
  • 29 Koedam JA, Meijers JCM, Sixma JJ, Bouma BN. Inactivation of human factor VIII by activated protein C: Cofactor activity of protein S and protective effect of von Willebrand factor. J Clin Invest 1988; 82: 1236-43.
    CrossrefPubMedGoogle Scholar
  • 30 Fay PJ, Coumans JV, Walker FJ. von Willebrand factor mediates protection of factor VIII from activated protein C-catalyzed inactivation. J Biol Chem 1991; 266: 2172-7.
    PubMedGoogle Scholar
  • 31 Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis [see comments]. N Engl J Med 1994; 330: 517-22.
    CrossrefPubMedGoogle Scholar
  • 32 Fay PJ, Beattie T, Huggins CF, Regan LM. Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site. J Biol Chem 1994; 269: 20522-7.
    PubMedGoogle Scholar
  • 33 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
  • 34 Pittman DD, Millenson M, Marquette K, Bauer K, Kaufman RJ. The A2 domain of human recombinant derived factor VIII is required for procoagulant activity but not for thrombin cleavage. Blood 1992; 79: 389-97.
    PubMedGoogle Scholar
  • 35 Walker FJ, Scandella D, Fay PJ. Identification of the binding site for activated protein C on the light chain of factors V and VIII. J Biol Chem 1990; 265: 1484-9.
    PubMedGoogle Scholar
  • 36 Lenting PJ, Donath MJSH, van Mourik JA, Mertens K. 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.
    PubMedGoogle Scholar
  • 37 Roelse JC, Koopman MMW, Buller HR, ten Cate JW, Montaruli B, van Mourik JA, Voorberg J. Absence of mutations at the activated protein C cleavage sites of factor VIII in 125 patients with venous thrombosis. Brit J Haematol 1996; 92: 740-3.
    CrossrefPubMedGoogle Scholar