The In Vitro Association of Antihemophilic Factor and von Willebrand Factor

Summary

Considerable evidence, including results of gel chromatography, indicates that antihemophilic factor (AHF; factor VIII: C) is associated with von Willebrand factor (vWF; factor VIIIR: RC or VTIIR: Ag) in citrated plasma. The present study was undertaken to determine whether these factors are also associated in plasma with a physiologic calcium ion concentration, or in an artificial medium using purified antihemophilic factor and plasma as a source of vWF. When fresh BaS0₄-treated native normal plasma was passed through a column of Sepharose CL-4B that was equilibrated and eluted with fresh BaS0₄-treated plasma from a patient with severe von Willebrand’s disease, the AHF and vWF activities were found in the void volume. Thus, AHF remains associated with vWF on gel chromatography in the presence of physiological concentrations of all plasma constituents except the vitamin-K-dependent clotting factors. On the other hand, when to 200,000 X purified “vWF-free” AHF was chromatographed in buffered 4% albumin with 2 mM CaCl₂, virtually all of it appeared in the included volume of the column with an apparent molecular weight between that of fibrinogen or factor V (340,000) and gamma globulin (160,000). The combination of the “vWF-free” AHF with the vWF in plasma was studied by adding the AHF to BaS0₄-treated plasma from normal subjects or patients with severe hemophilia or von Willebrand’s disease and chromatographing the mixture. The AHF activity appeared in the void volume in an amount that was inversely related to the ratio of the AHF to vWF activity. Thus, with 1-12 U of AHF per unit of vWF, virtually all of the AHF eluted in the void volume, with 30 and 500 units of AHF per unit of vWF, only about 50% and 10% of the AHF, respectively, eluted in the void volume, and in the absence of vWF, none of the AHF activity eluted in the void volume.

Keywords

Antihemophilic factor - von Willebrand factor - Factor VIII - Radioimmunoassay

• References

• 1 Hoyer LW. The factor VIII complex: structure and function. Blood 1981; 58: 01-13
• 2 Ratnoff OD, Kass L, Lang PD. Studies on the purification of antihemophilic factor (Factor VIII). II. Separation of partially purified antihemophilic factor by gel filtration of plasma. J Clin Invest 1969; 48: 957-962
• 3 Weiss HJ, Sussman II, Hoyer LW. Stabilization of factor VIII in plasma by the von Willebrand factor. J Clin Invest 1977; 60: 390-404
• 4 Hoyer LW, Trabold NC. The effect of thrombin on human factor VIII. Cleavage of the factor VIII procoagulant protein during activation. J Lab Clin Med 1981; 97: 50-64
• 5 Rock GA, Palmer DS, Tackaberry ES, Cruickshank WH. The presence of high and low molecular weight forms of factor VIII in heparinized plasma. Thromb Res 1978; 13: 85-96
• 6 Seghatchian MJ, Nilsson IM, Holmberg L, Miller-Anderson M. Molecular size distribution of factor VIII in native plasma. Thromb Res 1979; 14: 589-598
• 7 Newman J, Harris RB, Johnson AJ. Molecular weights of anti-haemophilic factor and von Willebrand factor proteins in human plasma. Nature 1976; 263: 612-613
• 8 Zucker MB, Owen J. Non-decalcified barium sulfate-adsorbed plasma – a potentially useful reagent for studying blood clotting, platelets or complement. Thromb Haemostas 1982; 47: 182-184
• 9 Johnson AJ, MacDonald VE, Semar M, Fields JE, Schuck J, Lewis C, Brind J. Preparation of the major plasma fractions by solid-phase polyelectrolytes. J Lab Clin Med 1978; 92: 194-210
• 10 Johnson AJ, Soberano ME, Kowalski S, Fulton AJ, MacDonald VE, Semar S. Separation of AHF from vWF by solid-phase polyelectrolytes (PE); further evidence for their separate identity. Thromb Haemostas 1981; 46: 255 (Abstr.)
• 11 Harris JU, Soberano ME, Kowalski S, Fulton AJ, Johnson AJ. The electroblot identification of VIII :C antigen in partially purified VIII:C preparations following SDS-PAGE. Fed Proc 1982; 41: 769 (Abstr.)
• 12 Newman J, Johnson AJ, Karpatkin MH, Puszkin S. Method for the production of clinically effective intermediate-and high-purity factor-VIII concentrates. Br J Haematol 1971; 21: 1-20
• 13 Macfarlane DE, Stibbe J, Kirby EP, Zucker MB, Grant RA, McPherson J. A method for assaying von Willebrand factor (ristocetin cofactor). Thrombos Diathes Haemorrh 1975; 34: 306-308
• 14 Hoyer LW. Immunologic studies of antihemophilic factor (AHF, factor VIII).IV. Radioimmunoassay of AHF antigen. J Lab Clin Med 1972; 80: 822-832
• 15 McPherson J, Zucker MB. Platelet retention in glass bead columns: adhesion to glass and subsequent platelet-platelet interactions. Blood 1976; 47: 55-67
• 16 Engvall E, Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 1977; 20: 1-5
• 17 Bloom JW, Nesheim ME, Mann KG. A rapid technique for the preparation of factor V deficient plasma. Thromb Res 1979; 15: 595-599
• 18 Bolhuis PA, Beeser-Visser NH, Sixma JJ. Molecular weight of human plasma factor VIII. Thromb Res 1979; 16: 497-506
• 19 Laurent TC, Killander J. A theory of gel filtration and its experimental verification. J Chromatogr 1964; 14: 317-330
• 20 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
• 21 Cooper HA, Wagner RH. The defect in hemophilic and von Willebrand’s disease plasmas studied by a recombination technique. J Clin Invest 1974; 54: 1093-1099
• 22 Benson RE, Johnson GS, Dodds WJ. Binding of low-molecular-weight canine factor VIII coagulant from von Willebrand plasma to canine factor-Vlll-related antigen. Br J Haematol 1981; 49: 541-550