Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow

Abstract

Von Willebrand factor (VWF) is a plasma glycoprotein that circulates noncovalently bound to blood coagulation factor VIII (fVIII). VWF is a population of multimers composed of a variable number of ∼280 kDa monomers that is activated in shear flow to bind collagen and platelet glycoprotein Ibα. Electron microscopy, atomic force microscopy, small-angle neutron scattering, and theoretical studies have produced a model in which the conformation of VWF under static conditions is a compact, globular “ball-of-yarn,” implying strong, attractive forces between monomers. We performed sedimentation velocity (SV) analytical ultracentrifugation measurements on unfractionated VWF/fVIII complexes. There was a 20% per mg/ml decrease in the weight-average sedimentation coefficient, s _w , in contrast to the ∼1% per mg/ml decrease observed for compact globular proteins. SV and dynamic light scattering measurements were performed on VWF/fVIII complexes fractionated by size-exclusion chromatography to obtain s _w values and z-average diffusion coefficients, D _z . Molecular weights estimated using these values in the Svedberg equation ranged from 1.7 to 4.1 MDa. Frictional ratios calculated from D _z and molecular weights ranged from 2.9 to 3.4, in contrast to values of 1.1–1.3 observed for globular proteins. The Mark–Houwink–Kuhn–Sakurada scaling relationships between s _w , D _z and molecular weight, $s=k^{\prime }{M}^{a_s}$ and $D=k^{″}{M}^{a_D}$ , yielded estimates of 0.51 and –0.49 for a _s and a _D , respectively, consistent with a random coil, in contrast to the a _s value of 0.65 observed for globular proteins. These results indicate that interactions between monomers are weak or nonexistent and that activation of VWF is intramonomeric.