Download PDF
Thromb Haemost 1983; 50(02): 547-551
DOI: 10.1055/s-0038-1665252
Original Article
Schattauer GmbH Stuttgart

Dissociation of Factor VIII Procoagulant Antigen VIII : CAg and Factor VIII Related Antigen VIIIR : Ag by EDTA - Influence of Divalent Cation on the Binding of VIII: CAg and VIIIR :Ag

T H Tran
The Coagulation and Fibrinolysis Laboratory, Kantonsspital, Basle, Switzerland
,
F Duckert
The Coagulation and Fibrinolysis Laboratory, Kantonsspital, Basle, Switzerland
› Author Affiliations
Further Information

Publication History

Received 15 March 1983

Accepted 27 May 1983

Publication Date:
18 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

Assuming 1 U/ml in titrated plasma, the VIII: CAg concentration was found 1.66 U/ml in EDTA-plasma, 1.09 U/ml in heparinized plasma and 0.67 U/ml in serum. Addition of 10 mmol/1 EDTA to titrated and heparinized plasmas increased VIII: CAg 1.5fold. There was no increase of VIII: CAg in serum. Gel filtration of plasmas on different anticoagulants showed an elution of VIII: CAg in the void volume Vo and in the later fractions. The VIII: CAg amount detected in the internal volume increased following the series heparin < citrate < EDTA. Serum VIII: CAg was eluted at 2.2 Vo. Presence of EDTA in the elution buffer or incubation of plasma with EDTA prior to chromatography caused a displacement of practically all VIII: CAg amount in the internal volume with a peak at 2.2-2.3 Vo. VIIIR: Ag was exclusively detected in the void volume.

Removal of divalent cation by chelation likely exposes more antigenic determinants of VIII: CAg, which are otherwise masked by steric hindrance due to VIIIR: Ag in citrated and heparinized milieu. Moreover gel filtration of plasma in the presence of EDTA completely dissociates VIII: CAg from VIIIR :Ag. The VIII: CAg fragment, having an estimated molecular weight of 70,000, might also be present in serum.

Keywords

Immuunoradiometric assay of VIII: CAg - VIII: CAg increase by EDTA - Dissociation of factor VIII by gel filtration - Serum procoagulant antigen
 

  • References

  • 1 Bouma BN, Wiegerinck Y, Sixma JJ, van Mourik JA, Mochtar IA. Immunological characterization of purified anti-haemophilic factor A (factor VIII) which corrects abnormal platelet retention in von Willebrand’s disease. Nature 1972; 236: 104-106
    PubMedGoogle Scholar
  • 2 Weiss HJ, Rogers J, Brand H. Defective ristocetin-induced platelet aggregation in von Willebrand’s disease and its correction by factor VIII. J Clin Invest 1973; 52: 2697-2707
    CrossrefPubMedGoogle Scholar
  • 3 Weiss HJ, Kochwa S. Molecular forms of antihaemophilic globulin in plasma, cryoprecipitate and after thrombin activation. Br J Haematol 1970; 18: 89-100
    CrossrefPubMedGoogle Scholar
  • 4 Owen WG, Wagner RH. Antihemophilic factor: separation of an active fragment following dissociation by salts and detergents. Thrombos Diathes Haemorrh 1972; 27: 502-515
    PubMedGoogle Scholar
  • 5 Rick ME, Hoyer LW. Activation of low molecular weight fragment of antihaemophilic factor (factor VIII) by thrombin. Nature 1974; 252: 404-405
    CrossrefPubMedGoogle Scholar
  • 6 Cooper HA, Reisner FF, Hall M, Wagner RH. Effects of thrombin treatment on preparations of factor VIII and the Ca2+-dissociated active fragment. J Clin Invest 1975; 56: 751-760
    CrossrefPubMedGoogle Scholar
  • 7 Poon MC, Ratnoff OD. Evidence that functional subunits of antihaemophilic factor (factor VIII) are linked by non covalent bonds. Blood 1976; 48: 87-94
    PubMedGoogle Scholar
  • 8 Lavergne JM, Meyer D, Jenkins CS P, Obert B, Larrieu MJ. Influence of high ionic strength buffers on factor VIII - von Willebrand factor from different species. Thromb Res 1978; 13: 409-418
    CrossrefPubMedGoogle Scholar
  • 9 Tuddenham EG D, Trabold NC, Collins SA, Hoyer LW. The properties of factor VIII coagulant activity prepared by immunoadsorbent chromatography. J Lab Clin Med 1979; 93: 40-53
    PubMedGoogle Scholar
  • 10 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochem 1980; 19: 401-410
    CrossrefPubMedGoogle Scholar
  • 11 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
    CrossrefPubMedGoogle Scholar
  • 12 Tran TH, Marbet GA, Duckert F. Rabbit antibodies against the procoagulant activity (VIII: C) of human factor VIII. Some theoretical and practical considerations on the human factor VIII molecule using heterologous antibodies. Thromb Haemostas 1981; 46: 699-705
    PubMedGoogle Scholar
  • 13 Laurell CB. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966; 15: 45-52
    CrossrefPubMedGoogle Scholar
  • 14 Ruggeri ZM, Mannucci PM, Jeffcoate SL, Ingram GI. Immunoradiometric assay of factor VIII related antigen, with observations in 32 patients with von Willebrand’s disease. Br J Haematol 1976; 33: 221-232
    CrossrefPubMedGoogle Scholar
  • 15 Peake IR, Bloom AL, Giddings JC, Ludlam CA. An immunoradiometric assay for procoagulant factor VIII antigen: results in haemophilia, von Willebrand’s disease and fetal plasma and serum. Br J Haematol 1979; 42: 269-281
    CrossrefPubMedGoogle Scholar
  • 16 Davies BL, Furlong RA, Peake IR. Studies on the relationship between factor VIII related antigen (VIIIR:Ag) and factor VIII clotting antigen (VIII :CAg) by immunoelectrophoresis and autoradiography using 125I-anti-VIII: CAg. Thromb Res 1981; 22: 87-96
    CrossrefPubMedGoogle Scholar
  • 17 Lavergne JM, Meyer D, Girma JP, Larrieu MJ. Precipitating anti- VIII: C antibodies in two patients with hemophilia A. Br J Haematol 1982; 50: 135-146
    CrossrefPubMedGoogle Scholar