Von Willebrand Factor: clearance as regulator of plasma levels

Von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are plasma proteins that circulate in a tight, noncovalent complex. Binding to VWF is required to stabilize the FVIII heterodimer and to prevent its premature clearance. The half-life of FVIII mimics that of the FVIII/VWF complex, suggesting that the complex is cleared as a single entity. The objectives of studies presented in this thesis were to identify parameters that affect VWF plasma levels and to elucidate elements that contribute to the VWF clearance mechanism. Little is known about the clearance mechanism of VWF. However, a number of parameters have been identified that influence VWF plasma levels. One of these factors is its glycosylation profile. VWF contains both N- and O-linked glycosylation. The blood group sugars on VWF all belong to the N-linked glycans and average VWF plasma levels are decreased in persons with blood group O. We investigated the influence of O-linked glycans on VWF plasma levels. Therefore we set up an assay that could determine the level O-linked glycans present on plasma VWF. We showed an inverse correlation between the extend of O-linked glycosylation and VWF plasma levels. Moreover, the amount of O-linked glycosylation is proportional to the VWF/ propeptide ratio. Because increased ratios may be indicative for increased clearance of VWF, the possibility exists that also modifications in O-linked glycosylation affect clearance of VWF. Also mutation in VWF may affect its plasma levels. We investigated the in vivo survival of three cysteine mutations, expressed in patients suffering from Von Willebrand Disease (VWD). We observed decreased survival of the VWF mutants in both patients an in an experimental mouse model. We explored the possibility that FVIII half-life can be predicted using VWF plasma level concentrations. Therefore we determined FVIII half-life and VWF antigen levels in severe haemophiliacs. VWF antigen levels correlated well with FVIII half-life in blood-group non-O, but poorly in severe haemophiliacs. This poor correlation for blood-group O patients could be considerably improved when VWF/propeptide ratios were used. Thus determination of VWF antigen levels and propeptide allows prediction of FVIII half-life. In order to gain more insight into the clearance mechanism of FVIII and VWF, VWF clearance was investigated in mice deficient for VWF. VWF was mainly targeted to the liver and spleen. Analysis of liver and spleen sections of VWF-deficient mice infused with FVII, VWF or the VWF/FVIII complex revealed co-localisation of all proteins with macrophages in both tissues. We also tested the contribution of macrophages to the in vivo clearance of VWF. Treatment of mice with gadolinium chloride (GdCl3) prior to VWF infusion resulted in decreased macrophage cell numbers. This resulted in a 2-fold increase of endogenous VWF in wild-type mice pre-treated with GdCl3. In addition, the survival of infused VWF was prolonged almost 2-fold in VWF-deficient mice. To study the uptake of the VWF by macrophages, we developed an in vitro model using macrophages and showed binding of VWF and FVIII to human macrophages. Moreover, VWF bound to these cells in a dose-dependent and saturable manner, which was followed by a rapid uptake and subsequent degradation of the internalised protein