FACTOR VIII/VON WILLEBRAND FACTOR COMPLEX: ONLY THE 440 000 SUBUNIT OF ENDOTHELIAL CELL-DERIVED VON WILLEBRAND FACTOR FORMS A COMPLEX WITH PURIFIED PLASMA FACTOR VIIIC

Abstract

Von Willebrand Factor (vWF) circulates in plasma as a series of multimers with moleculag weight ranging from M = 0.44 x 106 up to more than 20 x 106 . Besides the mediation of platelet adhesion to exposed subendothelium, the protein plays an important role in the stabilization and the transport of Factor VIIIC (FVIIIC). In the present study the interaction between FVIIIC and vWF was studied by recombination experiments. vWF was isolated from cultured human umbilical vein endothelial cells by immunoprécipitation. This source of vWF ascertained, that it was free of FVIIIC as indicated by the absence of FVIIIC activity as well as FVIIIC antigen. FVIIIC was prepared by immunoabsorption from human plasma yielding an activity of 1600 IJ/mg. SDS-PAGE analysis showed two main bands at Mr= 0.28 x 106 and 0.18 x 106 , respectively. vWF-multimers were separated by SDS agarose gel electrophoresis and were electrophoretically transferred onto nitrocellulose sheets. After extensive washing, the sheets were incubated for 12 h with 20 U/ml FVIIIC in PBS, pH 7.4, containing 2.5 mM calcium chloride. Subsequently, associated FVIIIC was detected by autoradiography with a 125-I-labelled monoclonal mouse anti-(human FVIIIC) antibody. The results of recombination experiments exclusively showed prominent staining of the Mr= 0.44 x 106 vWF band in the autoradiography. However, proteolytically degraded FVIIIC with partly retained procoagulant activity did not show a positive stain. The results indicate that an intact FVIIIC molecule and the smallest multimer of vWF are required for the formation of a stable FVIII/vWF complex.