Plenary Paper THROMBOSIS AND HEMOSTASIS Murine coagulation factor VIII is synthesized in endothelial cells

Plasma coagulation factor VIII (FVIII) is tightly associated with von Willebrand factor (VWF). Though it is well established that VWF is synthesized exclusively in endothelial cells andmegakaryocytes, the cellular source of FVIII has been debated for decades. The delayed rise of FVIII in von Willebrand disease patients infused with VWF demonstrates that VWF and FVIII do not require synthesis in the same cell for complex formation. Dozens of conflicting reports have implicated numerous cell and tissue types as the source of FVIII, though themajority of evidence points to either an endothelial and/or hepatocyte origin of FVIII synthesis. Multiple curative organ transplantation studies in hemophilia A patients and experimental dog models have shown that the liver contributes significantly to FVIII biosynthesis, though the failure of FVIII levels in hemophilia A liver transplant recipients to respond to desmopressin suggested FVIII localization to a different compartment than VWF. As early as 1971, Webster et al demonstrated extrahepatic FVIII synthesis by showing that FVIII synthesis was maintained after replacing the livers of normal dog recipients with hemophilic livers. Similarly, extrahepatic FVIII production was shown in humans after transplantation of a hemophilia A donor liver into a nonhemophilic recipient. Spleen and lung transplantation has also been shown to restore FVIII plasma levels in several animal models of hemophilia, and FVIII production also has been documented by human purified microvascular endothelial cells from lung in vitro. Immunoradiometric assays localized FVIII antigen to guinea pig liver, spleen, lung, kidney, and isolated hepatocytes and to human lymph nodes, lung, liver, and spleen, whereas microscopy studies suggested synthesis in human hepatocytes or human liver sinusoidal endothelial cells (LSECs). F8 messenger RNA (mRNA) transcripts have been detected in purified murine LSECs and hepatocytes at comparable levels, but not in Kupffer cells, and by others in human spleen, lymph nodes, kidney, and isolated hepatocytes, but not in white blood cells or cultured umbilical vein endothelial cells. F8 mRNA transcripts have also been shown to be synthesized in hemophilia A mouse livers by hepatocytes and LSECs that were derived from transplanted wild-type bone marrow progenitor cells. We took advantage of a genetically modifiedmouse model that is deficient for lectin,mannose-binding protein 1 (LMAM1), alsoknown as endoplasmic reticulum/Golgi intermediate compartment (ERGIC)53). LMAN1 is part of a ubiquitously expressed cargo receptor that cycles between the ERGIC in the early secretory pathway. LMAN1 is required for the efficient secretion of FVIII as well as coagulation FV to the plasma. Mutations in LMAN1 cause an autosomalrecessive bleeding disorder known as combined deficiency of coagulation FV and FVIII (F5F8D). Although FV and FVIII are synthesized at markedly different levels (human plasma FV ;7.0 mg/mL and FVIII ;200 ng/mL) and potentially in different tissues, loss of the LMAN1 cargo receptor in F5F8D leads to parallel