title: Factor VIII/V C-domain swaps reveal discrete C-domain roles in factor VIII function and intracellular trafficking.
abstract: Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells forin vitrocharacterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function.
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Eduard H T M
authors.initials: EH
authors.lastname: Ebberink
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Eveline A M
authors.initials: EA
authors.lastname: Bouwens
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: Bloem
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Mariëtte
authors.initials: M
authors.lastname: Boon-Spijker
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Maartje
authors.initials: M
authors.lastname: van den Biggelaar
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.|Landsteiner Laboratory of AMC and Sanquin, University of Amsterdam, the Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.|Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, the Netherlands.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands k.mertens@sanquin.nl.|Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, the Netherlands.
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
mesh_terms: D042783:Endothelial Cells / Q000378:metabolism; D005165:Factor V / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D005165:Factor V; D005169:Factor VIII