Normal hemostasis requires the functional interaction of multiple coagulation factors to culminate in the final conversion of prothrombin to thrombin. Two essential coagulation cofactors are factors VII (FVIII) and V (FV). These proteins are structurally related and serve similar functions to accelerate the activation of factor X and prothrombin, respectively. Deficiency in either or both o these proteins leads to severe bleeding disorders that require therapeutic intervention. Individuals with FVIII deficiency require replacement therapy in order to maintain hemostasis. The use of recombinant- derived FVIII has minimized potential therapeutic complications associated with plasma-derived FVIII, although significant limitations with FVIII expression remain. Whereas FVIII protein secretion is inhibited through interaction with protein chaperones in the lumen of the endoplasmic reticulum (ER), FV is efficiently and rapidly transported out of the ER. Our preliminary data support that FVIII secretion, and not FV secretion, requires glucose trimming on oligosachardes, a modification likely required for FVIII to bind two recently identified protein chaperones calnexin (CNX) and calreticulin (CRT). The hypothesis that CNX and/or CRT interaction is essential for FVIII secretion and is defective in a subset of patients with hemophilia A will be tested in Specific aim 1 by elucidating the role for CNX/CRT interaction with FVIII. Combined FV/FVIII deficiency is a recessive autosomal chromosome disorder for which the molecular basis is unknown. Based on the structural homologies between FVIII and FV, we propose that the combined deficiency results from a defect in a common protein synthesis and/or medication requirement. This hypothesis will be tested in Specific Aim 2 through isolation of the gene that is defective in combined FV/FVIII deficiency. Our exciting preliminary Studies using homozygosity mapping have identified a locus on human chromosome 18 that is tightly linked to the gene for combined FV/FVIII deficiency (LOD>12). Using recently developed molecular genetic techniques we will isolate the defective gene, identify mutations within affected patients, and create a mouse model for the human disease. This research will provide tools for the diagnosis and potential treatment of FV/VIII deficiency and will elucidate the mechanism of this rare disorder. The overall research program should provide fundamental new insight into the biosynthesis and/or processing of FVIII and FV that will be essential in considering avenues for gene therapy for FVIII and/or FV deficiency.
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