Factor VIII (fVIII) and factor IX (fIX) are two key plasma proteins that are deficient in the bleeding disorders Hemophilia A and Hemophilia B, respectively. Factor VIII circulates as a complex with its carrier protein von Willebrand factor (vWf). Upon injury within the vasculature, this cofactor is activated to factor VIIIa (fVIIIa) by limited proteolysis resulting in its dissociation from vWf and subsequent assembly onto the membrane surface with an enzymatically active form of fIX (fIXa) to form a macromolecular Xase complex. This complex activates factor X, the next proenzyme in the coagulation cascade. Recent work has suggested that the functional activity of the Xase complex may be regulated by the low-density lipoprotein receptor-related protein (LRP-1 or LRP), a large endocytic receptor that plays an important role in the catabolism of several ligands including apoE-enriched lipoproteins, plasminogen activators, and serpin-enzyme complexes. LRP along with the LDL receptor (LDLR) functions in vivo to mediate the hepatic clearance of fVIII. Elucidation of molecular mechanisms associated with fVIII catabolism is important, as fVIII is used in replacement therapy for Hemophilia A, and bioengineering of a recombinant fVIII molecule which would stay in the circulation for longer time would enhance therapy. The central hypothesis of this application is that LDL receptor family members, especially LRP, play an important role in regulating the blood coagulation process by mediating the cellular catabolism of two key players, fVIII(a) and fIXa. The specific hypotheses to be tested are: 1) that heparan sulfate proteoglycans (HSPG) modulate the dissociation of fVIII from vWF leading to enhanced uptake of fVIII by LRP/LDLR;2) that we can engineer a fVIII molecule with delayed clearance properties that would be effective for therapeutic use;and 3) that LDL receptor family members (i.e. LRP, LDLR and VLDLR) modulate localized thrombosis by promoting the rapid cellular internalization of the macromolecular Xase complex. These hypotheses will be tested in three specific aims. In the first aim, the initial step in the catabolic pathway of fVIII will be explored and two mechanisms which may modulate dissociation of fVIII from vWf leading to enhanced uptake of fVIII by LRP/LDLR will be tested - a non-proteolytic mechanism mediated by HSPG and an activation-dependent mechanism. In the second aim, functionally active recombinant fVIII with minimized in vivo LRP/LDLR-mediated clearance properties will be generated. This will be accomplished by site-directed mutagenesis of key regions on the fVIII molecule that contribute to LRP-, LDLR- and HSPG-binding. In the third aim, we will explore the ability of the LDL receptor family members to modulate activity of the intrinsic Xase complex. The assembly, function and clearance of the Xase complex on macrophages genetically deficient in LRP will be investigated. Overall, these studies will give insight into how the LDL receptor family members modulate the blood coagulation process, and should lead to the development of a recombinant fVIII molecule with enhanced properties for therapeutic use.
The bleeding disorder Hemophilia A is associated with deficiency of coagulation factor VIII (fVIII) and is treated by repeated injections of exogenous fVIII. Replacement therapy remains expensive and is estimated as up to $200,000/adult patient/year. The goals of this project are to define molecular mechanisms involved in the catabolism of fVIII. Accomplishment of this project will result in generation of functionally-active recombinant fVIII with a significantly prolonged lifetime in the circulation, which would improve efficacy and reduce the cost of replacement therapy of Hemophilia A.
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