title: Storage of factor VIII variants with impaired von Willebrand factor binding in Weibel-Palade bodies in endothelial cells.
abstract: BACKGROUND
  Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood.
  
  
  RESULTS
  We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH.
  
  
  CONCLUSIONS
  Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo.
authors: Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands. m.vandenbiggelaar@sanquin.nl
authors: Maartje
authors: M
authors: van den Biggelaar
authors: Eveline A M
authors: EA
authors: Bouwens
authors: Jan
authors: J
authors: Voorberg
authors: Koen
authors: K
authors: Mertens
mesh_terms: D019943:Amino Acid Substitution; D003594:Cytoplasmic Granules / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D005169:Factor VIII