Factor VIII/V C-domain swaps reveal discrete C-domain roles in factor VIII function and intracellular trafficking

Factor VIII C-domains are believed to contain specific function in co-factor activity and in interaction 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 furthermore addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and co-factor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, VIII and YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, 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 factor IXa whereas the factor VIII/V-C2 chimera showed a small reduction in von Willebrand factor binding and normal interaction with factor IXa. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand Factor and factor IXa. 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, which disrupts the natural C-domain tandem pair orientation. This affects intracellular trafficking more than extracellular function.