Effect of von Willebrand Factor and Its Proteolytic Fragments on Kinetics of Interaction between the Light and Heavy Chains of Human Factor VIII

Abstract

It was previously shown that vWF increases the rate of divalent cation-mediated fVIII reconstitution from isolated light chain (LCh) and heavy chain (HCh) subunits. We examined the effect of vWF on kinetic parameters for interaction between LCh and HCh in the presence of Ca2+ and Mn2+ ions, the most effective mediators of fVIII reconstitution from isolated subunits, and determined the minimal structural portion of vWF able to enhance fVIII formation. We found that affinity (Kd) for LCh/HCh binding mediated by Ca2+ and Mn2+ was 91 and 34.9 nM in the absence of vWF and 15.5 and 5.6 nM in its presence. This decrease of Kd resulted from a sixfold increase of the association rate constant (kon) for this interaction. The value of the dissociation rate constant (koff) for LCh/HCh complex was lower in the presence of Mn2+ (koff 4.6×10−6 s−1) than Ca2+ (koff 8.4×10−6 s−1) but in both cases vWF had no effect on koff. This indicates that at physiological concentration of 1 nM the rate of fVIII inactivation via dissociation to subunits would be entirely determined by the koff value, and it should not depend on the presence of vWF. Indeed, our experiments demonstrated that vWF did not have any effect on the rate of fVIII inactivation resulting from its dissociation to subunits at the physiological concentrations of the fVIII and vWF proteins. We identified the minimal portion of the vWF molecule, able to enhance reconstitution of fVIII from isolated subunits. Only vWF large proteolytic N-terminal homodimeric fragment SPIII (vWF residues 1–1365), but not small monomeric N-terminal fragment SPIII-T4 (1–272), both of which are known to contain a major fVIII binding site, was able to support reconstitution of fVIII activity from isolated LCh and HCh subunits in the presence of Mn2+ or Ca2+. The effect of SPIII on the LCh/HCh association was similar to that of vWF, because both proteins identically increased of the value of kon and did not alter the koff value.