Subunit Flexibility of Multimeric von Willebrand Factor/Factor VIII Complexes

Von Willebrand factor (VWF) is a plasma glycoprotein that participates in platelet adhesion and aggregation and serves as a carrier for blood coagulation factor VIII (fVIII). Plasma VWF consists of a population of multimers that range in molecular weight from similar to 0.55 MDa to greater than 10 MDa. The VWF multimer consists of a variable number of concatenated disulfide-linked similar to 275 kDa subunits. We fractionated plasma-derived human VWF/fVIII complexes by size-exclusion chromatography at a pH of 7.4 and subjected them to analysis by sodium dodecyl sulfate agarose gel electrophoresis, sedimentation velocity analytical ultracentrifugation (SV AUC), dynamic light scattering (DLS), and multi-angle light scattering (MALS). Weight-average molecular weights, M-w, were independently measured by MALS and by application of the Svedberg equation to SV AUC and DLS measurements. Estimates of the Mark-Houwink-Kuhn-Sakurada exponents alpha(Rg), alpha(s), and alpha(D) describing the functional relationship between the z-average radius of gyration, < R-g >(z), weight-average sedimentation coefficient, s(w), z-average diffusion coefficient, D-z, and M-w were consistent with a random coil conformation of the VWF multimer. Ratios of < R-g >(z) to the z-average hydrodynamic radius, < R-h >(z), estimated by DLS, were calculated across an M-w, range from 2 to 5 MDa. When compared to values calculated for a semi-flexible, wormlike chain, these ratios were consistent with a contour length over 1000-fold greater than the persistence length. These results indicate a high degree of flexibility between domains of the VWF subunit.