Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein, factor VIII. There is no cure for hemophilia A and treatment consists of replacement therapy using preparations of the (purified) protein. During the last funding period the applicant has made significant strides in identifying and characterizing sequences and ligands important for factor VIII/factor VIIIa subunit structure and function. The applicant will use this information as a platform for study, applying physical and biochemical approaches and utilizing molecular biological methods, to elucidate fine point structural details of intra-protein interactions that will define mechanisms for the regulation of this critical plasma protein.
The first aim will study the relatively the weak affinity interaction between the Al and A2 subunits in factor VIIIa which governs subunit structure and function of the active protein. The goal is to identify and determine the roles of residues that participate in this inter-subunit interaction. To this end the applicant will (i) use zero-length cross-linking to identify interactive residues, (ii) construct and analyze point mutations in factor VIII and subunits to assess these interactions, (iii) examine the role of critical histidine residues in the Ph-dependent association of the subunits, (iv) evaluate naturally occurring inhibitors in the inter-subunit interaction, (v) examine changes in A2 conformation upon its binding to the primary interactive site in Al and (vi) probe for interactions independent of that site.
The second aim studies interactions between factor VIII light chain and heavy chain. The goal is to define and model the association of these two chains. Proposed studies will (i) compare Ca(II) and Mn(II) as effectors of reconstitution and determine their influence on subunit structure and conformation, (ii) characterize Me2+-independent interactions of the heavy and light chains, and (iii) model the association of the subunits.The underlying theme of this program is to identify sequences and mechanisms responsible for the integrity and stability of molecular structure. Definition of these issues will yield valuable insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information for the design of superior therapeutics.
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