The In Vitro Association of Antihemophilic Factor and von Willebrand Factor

Abstract

SummaryConsiderable 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.