The Role of Factor VIII C Domains in Sorting to Weibel-Palade Bodies

Abstract 2241

Recent studies have shown that factor VIII (FVIII) expressed in endothelial cells sorts with von Willebrand factor (VWF) to secretory Weibel-Palade bodies (WPBs). The sorting mechanism remains controversial although VWF is thought to be essential. However, mutations that lead to impaired FVIII-VWF complex assembly do not reduce the sorting efficiency of FVIII. As factor V (FV) and FVIII are highly homologous in structure, we addressed the possibility that FV sorts to WPBs as well. Our study was designed to identify domains in FVIII that are needed for sorting to WPBs by means of domain deletions and FVIII-FV domain exchange. As the C domains of FVIII contain membrane and VWF binding sites, we particularly focused on comparing the C domains of FVIII and FV. Blood outgrowth endothelial cells (BOECs) were transduced with lentiviral vectors encoding FV, FVIII deletion mutants, or FVIII-FV chimeras. We found by confocal microscopy and subcellular fractionations that FV displays a strong reduction in sorting efficiency (2% sorting efficiency) compared to FVIII (20% sorting efficiency). This indicates that sorting to WPBs is mediated by FVIII-specific structural elements. As the C domains of FVIII are implicated in membrane and VWF binding, these domains could drive sorting to WPBs. Therefore, we constructed FVIII variants lacking C domains to establish their role in WPB sorting. Quantitative determination of the sorting efficiencies demonstrates that the C1 domain is not of major importance for sorting to WPBs (10% sorting efficiency), whereas the C2 domain is (not detectable in WPB fractions). Moreover, exchanging the FVIII C domains for corresponding domains of FV also suggests that the C2 domain drives WPB sorting (3% sorting efficiency). This leads to the conclusion that FVIII sorting to WPBs is driven by FVIII-specific structural elements in both C domains, but in particular the C2 domain.