TRIPLET STRUCTURE OF HUMAN VON-WILLEBRAND-FACTOR

Human von Willebrand factor (hp-vWF) is a high-molecular-mass protein found in plasma as a series of multimers. It consists of subunits comp rising 2050 amino acids linked by disulphide bonds into multimers of v arious size ranging in molecular mass up to greater than 10000 kDa. Pa rtial proteolysis at position Tyr(842)-Mer(843) Of the subunit [Dent e t al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6306-6310] by a vWF-spe cific protease [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] results in the generation of an N-terminal and a C-termina l fragment and the appearance of hp-vWF triplet bands. It has been sug gested [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-75 07] that (i) the intermediate triplet band of the primary dimer repres ents a dimer of two C-terminal fragments, (ii) the slower migrating sa tellite band of the primary dimer represents an asymmetric structure c omposed of a mature subunit to which one N-terminal and one C-terminal fragment are linked by disulphide bonds, and (iii) the faster migrati ng satellite band of the primary dimer contains two N-terminal fragmen ts. Here we used recombinant vWF (r-vWF) for structural analysis of hp -vWF multimers. r-vWF exhibited no proteolytic degradation and all mul timers contained mature subunits. High-resolution agarose-gel electrop horesis and two-dimensional electrophoresis demonstrated that (i) r-vW F multimers and hp-vWF intermediate triplet bands exhibited identical molecular mass and electrophoretic mobilities, (ii) the faster and slo wer migrating satellite bands of hp-vWF differ by less than the molecu lar mass of one subunit from the corresponding intermediate triplet ba nd, and (iii) the triplet bands of hp-vWF are composed of mature and d egraded subunits. The results support a structural model of hp-vWF tri plet bands according to which the intermediate triplet bands represent multiple numbers of symmetric and/or asymmetric dimers, the slower mi grating satellite bands have one extra N-terminal fragment, and the fa ster migrating satellite band lacks one N-terminal fragment respective ly in comparison with the corresponding intermediate triplet band.