ER-to-Golgi transport of coagulation factors V and VIII

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DESCRIPTION (provided by applicant): ER-to-Golgi transport receptor in regulating coagulation factors V and VIII Coagulation factor V (FV) and factor VIII (FVIII) are both secreted glycoproteins that share pivotal roles in both hemostasis and thrombosis. Although many secreted proteins (referred to as cargo) are believed to require transport receptors for efficient ER-to-Golgi transport, only a limited number of such receptors have been described, mostly in yeast. Evidence for the existence of mammalian cargo receptors came unexpectedly from studies of the human genetic disorder combined deficiency of FV and FVIII, which identified mutations in LMAN1 and MCFD2 as the cause of the disorder. Combined deficiency of FV and FVIII is a rare bleeding disorder characterized by the reduction of both FV and FVIII to 5-30% of normal. LMAN1 and MCFD2 form a Ca2+-dependent protein complex in the ER-Golgi intermediate compartment that interacts with FV and FVIII, suggesting that the LMAN1-MCFD2 complex is a cargo receptor required for efficient transport of FV and FVIII from the ER to the Golgi. The requirement of both a transmembrane component (LMAN1) and a soluble cofactor (MCFD2) suggests a more sophisticated mechanism for cargo trafficking in higher eukaryotes, and could represent a paradigm for the organization of other mammalian cargo receptors. We hypothesize that dual sorting signals on FV and FVIII are separately recognized by LMAN1 and MCFD2. The proposed studies will identify these sorting signals that regulate cargo binding and release, investigate the ER-to-Golgi transport deficiency as a novel mechanism of hemophilia A and use mouse models to investigate LMAN1-MCFD2 mediated secretion of FV and FVIII in vivo. Results will not only answer fundamental questions regarding the mechanism of LMAN1-MCFD2 receptor-mediated secretion of FV and FVIII, but will also provide fundamental new insight into general mechanism of mammalian ER-to-Golgi protein transport. Genetic deficiency of FVIII results in hemophilia A, which affects ~1 in 5000 males. On the other hand, a gain-of-function mutation in FV (FV Leiden) and increased FVIII activity are major risk factors for venous thrombosis, which affects ~1:1,000 individuals in the US per year. The findings will have practical importance for improving FVIII expression and may expedite the eventual goal of somatic cell gene therapy for hemophilia A, as well as new approaches to limiting FV and FVIII production in prothrombotic states.